Complexin and Ca2+ stimulate SNARE-mediated membrane fusion

PubMed Central

Yoon, Tae-Young; Lu, Xiaobind; Diao, Jiajie; Lee, Soo-Min; Ha, Taekjip; Shin, Yeon-Kyun

2008-01-01

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release. PMID:18552825

[Clinical utility of real-time fluorescent PCR for combined detection of anaplastic lymphoma kinase and c-ros oncogene 1 receptor tyrosine kinase in non-small cell lung cancer].

PubMed

Bai, D Y; Zhang, H P; Zhong, S; Suo, W H; Gao, D H; Ding, Y; Tu, J H

2016-12-23

Objective: To investigate the clinical application value of combined detection of ALK fusion gene and c-ros oncogene 1 receptor tyrosine kinase (ROS1) fusion gene in non-small cell lung cancer (NSCLC) using real-time fluorescent PCR. Methods: A kit for combined detection of ALK fusion gene and ROS1 fusion gene based on fluorescent PCR was used to simultaneously detect the two fusion genes in 302 cases of NSCLC specimens. The results were validated through Sanger sequencing. The consistency of the two detection methods was analyzed. Results: All 302 cases of NSCLC specimens were successfully analyzed through fluorescent PCR (302/302). 12 cases (4.0%) were found to contain ALK fusion gene, including 3 cases with ALK-M1, 3 with ALK-M2, 3 with ALK-M3, 1 with ALK-M4, and 2 with ALK-M6 fusion gene.12 cases (4.0%) were found to contain ROS1 fusion gene, including 1 case with ROS1-M7, 8 cases with ROS1-M8, 1 case with ROS1-M12, 1 case with ROS1-M14, and 1 case with double-positive ROS1-M3 and ROS1-M8 fusion genes. The total detection rate of ALK fusion gene and ROS1 fusion gene was 7.9% (24/302) and 278 cases showed to be negative for ALK fusion gene and ROS1 fusion gene. The successful detection rates for Sanger DNA sequencing were also 100%. The positive, negative and total coincidence rates obtained by real-time fluorescent PCR and by Sanger DNA sequencing were all 100%. Conclusions: The results of Sanger DNA sequencing demonstrate that the real-time fluorescent PCR assay is equally effective in detecting ALK and ROS1 fusion genes in NSCLC tissues. Furthermore, real-time fluorescent PCR assay can be used to detect trace ALK and ROS1 fusion gene simultaneously in tiny samples, and can save time and avoid repeated sampling. It is worthy of recommendation as a rapid and reliable detection technique.

Salicylic acid interferes with GFP fluorescence in vivo

PubMed Central

de Jonge, Jennifer; Hofius, Daniel

2017-01-01

Abstract Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue‐specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP‐fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP‐derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP‐tagged proteins upon SA treatment should therefore be evaluated with caution. PMID:28369601

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

PubMed

Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

2016-03-18

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

Laser-induced tissue fluorescence in radiofrequency tissue-fusion characterization.

PubMed

Su, Lei; Fonseca, Martina B; Arya, Shobhit; Kudo, Hiromi; Goldin, Robert; Hanna, George B; Elson, Daniel S

2014-01-01

Heat-induced tissue fusion is an important procedure in modern surgery and can greatly reduce trauma, complications, and mortality during minimally invasive surgical blood vessel anastomosis, but it may also have further benefits if applied to other tissue types such as small and large intestine anastomoses. We present a tissue-fusion characterization technology using laser-induced fluorescence spectroscopy, which provides further insight into tissue constituent variations at the molecular level. In particular, an increase of fluorescence intensity in 450- to 550-nm range for 375- and 405-nm excitation suggests that the collagen cross-linking in fused tissues increased. Our experimental and statistical analyses showed that, by using fluorescence spectral data, good fusion could be differentiated from other cases with an accuracy of more than 95%. This suggests that the fluorescence spectroscopy could be potentially used as a feedback control method in online tissue-fusion monitoring.

Salicylic acid interferes with GFP fluorescence in vivo.

PubMed

de Jonge, Jennifer; Hofius, Daniel; Hennig, Lars

2017-03-01

Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue-specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP-fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP-derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP-tagged proteins upon SA treatment should therefore be evaluated with caution. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase.

PubMed

Zhang, Chong; Liu, Min-Sheng; Xing, Xin-Hui

2009-09-01

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60 degrees C in either single or fusion form can be completely recovered by treatment at 0 degrees C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15 degrees C and 37 degrees C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15 degrees C is ten times of that at 37 degrees C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.

ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic β Cells and Functions in Insulin Exocytosis: Interaction of ELKS with Exocytotic Machinery Analyzed by Total Internal Reflection Fluorescence MicroscopyV⃞

PubMed Central

Ohara-Imaizumi, Mica; Ohtsuka, Toshihisa; Matsushima, Satsuki; Akimoto, Yoshihiro; Nishiwaki, Chiyono; Nakamichi, Yoko; Kikuta, Toshiteru; Nagai, Shintaro; Kawakami, Hayato; Watanabe, Takashi; Nagamatsu, Shinya

2005-01-01

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic β cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic β cells. PMID:15888548

Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

PubMed Central

2015-01-01

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein–protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein–protein interactions within whole animals. PMID:25265085

Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

PubMed

Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

2015-05-15

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

Progress in understanding the neuronal SNARE function and its regulation.

PubMed

Yoon, T-Y; Shin, Y-K

2009-02-01

Vesicle budding and fusion underlies many essential biochemical deliveries in eukaryotic cells, and its core fusion machinery is thought to be built on one protein family named soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). Recent technical advances based on site-directed fluorescence labelling and nano-scale detection down to the single-molecule level rapidly unveiled the protein and the lipid intermediates along the fusion pathway as well as the molecular actions of fusion effectors. Here we summarize these new exciting findings in context with a new mechanistic model that reconciles two existing fusion models: the proteinaceous pore model and the hemifusion model. Further, we attempt to locate the points of action for the fusion effectors along the fusion pathway and to delineate the energetic interplay between the SNARE complexes and the fusion effectors.

[Development of a Surgical Navigation System with Beam Split and Fusion of the Visible and Near-Infrared Fluorescence].

PubMed

Yang, Xiaofeng; Wu, Wei; Wang, Guoan

2015-04-01

This paper presents a surgical optical navigation system with non-invasive, real-time, and positioning characteristics for open surgical procedure. The design was based on the principle of near-infrared fluorescence molecular imaging. The in vivo fluorescence excitation technology, multi-channel spectral camera technology and image fusion software technology were used. Visible and near-infrared light ring LED excitation source, multi-channel band pass filters, spectral camera 2 CCD optical sensor technology and computer systems were integrated, and, as a result, a new surgical optical navigation system was successfully developed. When the near-infrared fluorescence was injected, the system could display anatomical images of the tissue surface and near-infrared fluorescent functional images of surgical field simultaneously. The system can identify the lymphatic vessels, lymph node, tumor edge which doctor cannot find out with naked eye intra-operatively. Our research will guide effectively the surgeon to remove the tumor tissue to improve significantly the success rate of surgery. The technologies have obtained a national patent, with patent No. ZI. 2011 1 0292374. 1.

Glycosylatable GFP as a compartment-specific membrane topology reporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hunsang; Min, Jisoo; Heijne, Gunnar von

2012-11-02

Highlights: Black-Right-Pointing-Pointer An N-linked glycosylation site is introduced near the GFP fluorophore. Black-Right-Pointing-Pointer gGFP is not glycosylated and is fully fluorescent in the cytosol. Black-Right-Pointing-Pointer gGFP is glycosylated and non-fluorescent in the lumen of the ER. Black-Right-Pointing-Pointer gGFP is fused to membrane proteins of known topology. Black-Right-Pointing-Pointer Its applicability as a membrane topology reporter is demonstrated. -- Abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present amore » robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.« less

Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

PubMed

Liu, J; Wang, Y; Szalay, A A; Escher, A

2000-01-01

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells. Copyright 2000 John Wiley & Sons, Ltd.

Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion

PubMed Central

Gallagher, John R.; Atanasiu, Doina; Saw, Wan Ting; Paradisgarten, Matthew J.; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.

2014-01-01

Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy. PMID:25233449

Design and Study of Efflux Function of EGFP Fused MexAB-OprM Membrane Transporter in Pseudomonas aeruginosa Using Fluorescence Spectroscopy

PubMed Central

Ding, Feng; Lee, Kerry J.; Vahedi-Faridi, Ardeschir; Yoneyama, Hiroshi; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2014-01-01

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in ΔMexB (MexB deletion) strain of Pseudomonas aeruginosa to create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined their expression in live cells by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy. Efflux function of the new strain was studied by measuring its accumulation kinetics of ethidium bromide (EtBr, a pump substrate) using fluorescence spectroscopy, which was compared with the cells (WT, ΔMexM, ΔABM, and nalB1) with various expression levels of MexAB-OprM. The new strain shows 6-fold lower accumulation rates of EtBr (15 μM) than ΔABM, 4-fold lower than ΔMexB, but only 1.1-fold higher than WT. As EtBr concentration increases to 40 μM, the new strain has nearly the same accumulation rate of EtBr as ΔMexB, but 1.4-fold higher than WT. We observed the nearly same level of inhibitory effect of CCCP (carbonyl cyanide-m-chlorophenylhydrazone) on the efflux of EtBr by the new strain and WT. Antibiotic susceptibility study shows that the minimum inhibitory concentrations (MICs) of aztreonam (AZT) and chloramphenicol (CP) for the new strain are 6-fold or 3-fold lower than WT, respectively, and 2-fold higher than those of ΔMexB. Taken together, the results suggest that the fusion protein partially retains the efflux function of MexAB-OprM. Modeled structure of the fusion protein shows that the position and orientation of the N-terminal fused EGFP domain may either partially block the translocation pore or restrict the movement of the individual pump domains, which leads to partially restrict efflux activity. PMID:24781334

Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

PubMed

Huang, S K; Cheng, M; Hui, S W

1990-11-01

Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values.

Calcium accelerates SNARE-mediated lipid mixing through modulating α-synuclein membrane interaction.

PubMed

Zhang, Zeting; Jiang, Xin; Xu, Danrui; Zheng, Wenwen; Liu, Maili; Li, Conggang

2018-04-04

α-Synuclein is involved in Parkinson's disease, and its interaction with cell membrane is vital to its pathological and physiological functions. We have shown that Ca 2+ can regulate α-synuclein membrane interaction, but the physiological role of Ca 2+ in modulating α-synuclein membrane interaction is still unexplored. Based on the previous findings that α-synuclein inhibits membrane fusion and its inhibitory effect is highly related to its membrane binding, here we employed solution state Nuclear Magnetic Resonance (NMR) spectroscopy and the ensemble fluorescence fusion assay to show that Ca 2+ can modulate the inhibitory effect of α-synuclein on SNARE-mediated membrane fusion through disrupting α-synuclein membrane interaction, resulting in acceleration of SNARE-mediated membrane fusion. These results suggest a modulatory effect of Ca 2+ on membrane mediated normal function of α-synuclein, which of importance for the study of the Parkinson's disease. Copyright © 2018 Elsevier B.V. All rights reserved.

A fusion-spliced near-field optical fiber probe using photonic crystal fiber for nanoscale thermometry based on fluorescence-lifetime measurement of quantum dots.

PubMed

Fujii, Takuro; Taguchi, Yoshihiro; Saiki, Toshiharu; Nagasaka, Yuji

2011-01-01

We have developed a novel nanoscale temperature-measurement method using fluorescence in the near-field called fluorescence near-field optics thermal nanoscopy (Fluor-NOTN). Fluor-NOTN enables the temperature distributions of nanoscale materials to be measured in vivo/in situ. The proposed method measures temperature by detecting the temperature dependent fluorescence lifetimes of Cd/Se quantum dots (QDs). For a high-sensitivity temperature measurement, the auto-fluorescence generated from a fiber probe should be reduced. In order to decrease the noise, we have fabricated a novel near-field optical-fiber probe by fusion-splicing a photonic crystal fiber (PCF) and a conventional single-mode fiber (SMF). The validity of the novel fiber probe was assessed experimentally by evaluating the auto-fluorescence spectra of the PCF. Due to the decrease of auto-fluorescence, a six- to ten-fold increase of S/N in the near-field fluorescence lifetime detection was achieved with the newly fabricated fusion-spliced near-field optical fiber probe. Additionally, the near-field fluorescence lifetime of the quantum dots was successfully measured by the fabricated fusion-spliced near-field optical fiber probe at room temperature, and was estimated to be 10.0 ns.

Characterization of protein-protein interaction domains within the baculovirus Autographa californica multiple nucleopolyhedrovirus late expression factor LEF-3.

PubMed

Downie, Kelsey; Adetola, Gbolagade; Carstens, Eric B

2013-11-01

Autographa californica nucleopolyhedrovirus late expression factor 3 (LEF-3) is required for late viral gene expression probably through its numerous functions related to DNA replication, including nuclear localization of the virus helicase P143 and binding to ssDNA. LEF-3 appears to interact with itself as a homo-oligomer, although the details of this oligomeric structure are not yet known. To examine LEF-3-LEF-3 interactions, a bimolecular fluorescent protein complementation assay was used. Pairs of recombinant plasmids expressing full-length LEF-3 fused to one of two complementary fragments (V1 or V2) of a variant of yellow fluorescent protein named 'Venus' were constructed. Plasmids expressing fusions with complementary fragments of Venus were co-transfected into Sf21 cells and analysed by fluorescence microscopy. Co-transfected plasmids expressing full-length V1-LEF-3 and V2-LEF-3 showed positive fluorescence, confirming the formation of homo-oligomers. A series of truncated V1/V2-LEF-3 fusions was constructed and used to investigate interactions with one another as well as with full-length LEF-3.

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

NASA Astrophysics Data System (ADS)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

PubMed

Wolters, Manuel; Zobiak, Bernd; Nauth, Theresa; Aepfelbacher, Martin

2015-10-13

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.

PubMed

Duellman, Tyler; Burnett, John; Yang, Jay

2015-03-15

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles

PubMed Central

2012-01-01

Background It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. Results N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Biochemical and initial imaging analysis indicated that productive fusion events occur predominantly within 4–6 h after virus attachment. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Quantitative monitoring of the fraction of individual viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. Conclusions The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques. PMID:22935135

Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

PubMed

Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

2015-10-29

HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-07-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-11-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Expression and purification of RHC-EGFP fusion protein and its application in hyaluronic acid assay.

PubMed

Duan, Ningjun; Lv, Wansheng; Zhu, Lingli; Zheng, Weijuan; Hua, Zichun

2017-03-16

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM-enhanced green fluorescence protein (RHC-EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC-EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.

Human adipose tissue-derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes.

PubMed

Metzele, Roxana; Alt, Christopher; Bai, Xiaowen; Yan, Yasheng; Zhang, Zhi; Pan, Zhizhong; Coleman, Michael; Vykoukal, Jody; Song, Yao-Hua; Alt, Eckhard

2011-03-01

Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell-based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue-derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant DiI and then treated with fusion-inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte-like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole-cell voltage-clamp analysis demonstrated action potentials in beating fused cells. RT-PCR analysis using rat- or human-specific myosin heavy chain primers revealed that the myosin heavy-chain expression in fused cells was derived from rat cardiomyocytes. Real-time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory that fusion, even if artificially induced in our study, could indeed be a mechanism for cardiomyocyte renewal in the heart.

A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

PubMed

Wu, Tsung-Meng; Lin, Ke-Chun; Liau, Wei-Shiang; Chao, Yun-Yang; Yang, Ling-Hung; Chen, Szu-Yun; Lu, Chung-An; Hong, Chwan-Yang

2016-01-01

In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

ETS Gene Fusions as Predictive Biomarkers of Resistance to Radiation Therapy for Prostate Cancer

DTIC Science & Technology

2013-08-01

confocal microscopy to characterize the timing, location, and order of recruitment of the ERG-DNAPK interaction in relation to radiation delivery. In the...DNAPK linked to different fluorescent proteins, and Subtask #5B was to overexpress these constructs in the VCaP cell line and perform real-time...first half of year 3, we generated fusion constructs of ERG to various fluorescent proteins [green fluorescent protein (GFP), yellow fluorescent

[Construction and expression of six deletion mutants of human astrovirus C-terminal nsP1a/4 protein].

PubMed

Zhao, Wei; Niu, Ke; Zhao, Jian; Jin, Yi-ming; Sui, Ting-ting; Wang, Wen

2013-09-01

Human astrovirus (HAstV) is one of the leading causes of actue virual diarrhea in infants. HAstV-induced epithdlial cell apoptosis plays an important role in the pathogenesis of HAstV infection. Our previous study indicated that HAstV non-structural protein nsPla C-terminal protein nsPla/4 was the major apoptosis functional protein and probably contained the main apoptosis domains. In order to screen for astrovirus encoded apoptotic protien, nsPla/4 and six turncated proteins, which possessed nsPla/4 protein different function domain ,were cloned into green fluorescent protein (GFP) vector pEG-FP-N3. After 24-72 h transfection, the fusion protein expression in BHK21 cells, was analysis by fluorescence microscope and Western blot. The results indicated seven fusion proteins were observed successfully in BHK21 cell after transfected for 24 h. Western blot analysis showed that the level of fusion protein expressed in BHK21 cells was increased significantly at 72h compared to 48h in transfected cells. The successful expression of deletion mutants of nsPla/4 protein was an important foundation to gain further insights into the function of apoptosis domains of nsPla/4 protein and it would also provide research platform to further confirm the molecule pathogenic mechanism of human astrovirus.

The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao Maofu; Kielian, Margaret

2005-02-05

The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residuesmore » showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion.« less

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum

PubMed Central

Kurian, Smija M.; Di Pietro, Antonio

2018-01-01

Fusarium oxysporum exhibits conidial anastomosis tube (CAT) fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging. PMID:29734342

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum.

PubMed

Kurian, Smija M; Di Pietro, Antonio; Read, Nick D

2018-01-01

Fusarium oxysporum exhibits conidial anastomosis tube (CAT) fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging.

Engineering of living cells for the expression of holo-phycobiliprotein-based constructs

DOEpatents

Glazer, Alexander N.; Tooley, Aaron J.; Cai, Yuping

2004-05-25

Recombinant cells which express a fluorescent holo-phycobiliprotein fusion protein and methods of use are described. The cells comprises a bilin, a recombinant bilin reductase, an apo-phycobiliprotein fusion protein precursor of the fusion protein comprising a corresponding apo-phycobiliprotein domain, and a recombinant phycobiliprotein domain-bilin lyase, which components react to form the holo-phycobiliprotein fusion protein. Also described are holo-phycobiliprotein based transcription reporter cells and assays, which cells conditionally express a heterologous-to-the-cell, fluorescent, first holo-phycobiliprotein domain.

Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae.

PubMed

Hama, Shinji; Tamalampudi, Sriappareddy; Shindo, Naoki; Numata, Takao; Yamaji, Hideki; Fukuda, Hideki; Kondo, Akihiko

2008-07-01

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.

Quantifying the kinetics and morphological changes of the fusion of spheroid building blocks.

PubMed

Susienka, Michael J; Wilks, Benjamin T; Morgan, Jeffrey R

2016-10-10

Tissue fusion, whereby two or more spheroids coalesce, is a process that is fundamental to biofabrication. We have designed a quantitative, high-throughput platform to investigate the fusion of multicellular spheroids using agarose micro-molds. Spheroids of primary human chondrocytes (HCH) or human breast cancer cells (MCF-7) were self-assembled for 24 h and then brought together to form an array comprised of two spheroids (one doublet) per well. To quantify spheroid fusogenicity, we developed two assays: (1) an initial tack assay, defined as the minimum amount of time for two spheroids to form a mechanically stable tissue complex or doublet, and (2) a fusion assay, in which we defined and tracked key morphological parameters of the doublets as a function of time using wide-field fluorescence microscopy over a 24 h time-lapse. The initial tack of spheroid fusion was measured by inverting the micro-molds and centrifuging doublets at various time points to assess their connectedness. We found that the initial tack between two spheroids forms rapidly, with the majority of doublets remaining intact after centrifugation following just 30 min of fusion. Over the course of 24 h of fusion, several morphological changes occurred, which were quantified using a custom image analysis pipeline. End-to-end doublet lengths decreased over time, doublet widths decreased for chondrocytes and increased for MCF-7, contact lengths increased over time, and chondrocyte doublets exhibited higher intersphere angles at the end of fusion. We also assessed fusion by measuring the fluorescence intensity at the plane of fusion, which increased over time for both cell types. Interestingly, we observed that doublets moved and rotated in the micro-wells during fusion and this rotation was inhibited by ROCK inhibitor Y-27632 and myosin II inhibitor blebbistatin. Understanding and optimizing tissue fusion is essential for creating larger tissues, organs, or other structures using individual microtissues as building parts.

Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karttunen, Jenni; Mäntynen, Sari; Ihalainen, Teemu O.

2015-08-15

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of themore » P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.« less

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

PubMed Central

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins. PMID:23957834

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

PubMed

Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

2013-08-20

Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.

Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells.

PubMed

Sun, Qing; Zhao, Lixiang; Song, Qingqing; Wang, Zheng; Qiu, Xusheng; Zhang, Wenjun; Zhao, Mingjun; Zhao, Guo; Liu, Wenbo; Liu, Haiyan; Li, Yunsen; Liu, Xiufan

2012-03-01

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli.

PubMed

Xiao, Wenjun; Jiang, Li; Wang, Weiyu; Wang, Ruyue; Fan, Jun

2018-02-01

Fusion of solubility-enhancing tag is frequently used for improving soluble production of target protein in Escherichia coli. The Arabidopsis tetraticopeptide domain-containing thioredoxin (TDX) has been documented to exhibit functions of disulfide reductase, foldase chaperone, and holdase chaperone. Here, we identified that fusion of rice TDX with the smaller size increased soluble expression levels of three fluorescent proteins with different fluorophores in the E. coli strain BL21(DE3) or the Rosetta (DE3) strain with coexpression of six rare tRNAs, but decreased conformational quality of certain fluorescent proteins, as comparison with the His6-tagged ones. Among five maize proteins, the rice TDX fusion carrier displayed higher solubility-enhancing activity than the yeast SUMO3 tag toward three proteins in both E. coli strains. Five fusion constructs were cleaved with the co-expressed TEV protease variant, but the released target proteins were partly insolubly aggregated in vivo. Attachment of the His6-tag to the TDX tagged proteins had little impact on protein solubility. After Ni-NTA purification, five His6-TDX tagged proteins displayed different apparent purities. Taken together, our work presents that rice TDX tag is a novel solubility enhancer. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Novel fusion for hybrid optical/microcomputed tomography imaging based on natural light surface reconstruction and iterated closest point

NASA Astrophysics Data System (ADS)

Ning, Nannan; Tian, Jie; Liu, Xia; Deng, Kexin; Wu, Ping; Wang, Bo; Wang, Kun; Ma, Xibo

2014-02-01

In mathematics, optical molecular imaging including bioluminescence tomography (BLT), fluorescence tomography (FMT) and Cerenkov luminescence tomography (CLT) are concerned with a similar inverse source problem. They all involve the reconstruction of the 3D location of a single/multiple internal luminescent/fluorescent sources based on 3D surface flux distribution. To achieve that, an accurate fusion between 2D luminescent/fluorescent images and 3D structural images that may be acquired form micro-CT, MRI or beam scanning is extremely critical. However, the absence of a universal method that can effectively convert 2D optical information into 3D makes the accurate fusion challengeable. In this study, to improve the fusion accuracy, a new fusion method for dual-modality tomography (luminescence/fluorescence and micro-CT) based on natural light surface reconstruction (NLSR) and iterated closest point (ICP) was presented. It consisted of Octree structure, exact visual hull from marching cubes and ICP. Different from conventional limited projection methods, it is 360° free-space registration, and utilizes more luminescence/fluorescence distribution information from unlimited multi-orientation 2D optical images. A mouse mimicking phantom (one XPM-2 Phantom Light Source, XENOGEN Corporation) and an in-vivo BALB/C mouse with implanted one luminescent light source were used to evaluate the performance of the new fusion method. Compared with conventional fusion methods, the average error of preset markers was improved by 0.3 and 0.2 pixels from the new method, respectively. After running the same 3D internal light source reconstruction algorithm of the BALB/C mouse, the distance error between the actual and reconstructed internal source was decreased by 0.19 mm.

Early steps of supported bilayer formation probed by single vesicle fluorescence assays.

PubMed Central

Johnson, Joseph M; Ha, Taekjip; Chu, Steve; Boxer, Steven G

2002-01-01

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles. PMID:12496104

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.

PubMed

Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie

2013-05-01

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE PAGES

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick; ...

2017-11-24

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Nuclear dynamics during germination, conidiation, and hyphal fusion of Fusarium oxysporum.

PubMed

Ruiz-Roldán, M Carmen; Köhli, Michael; Roncero, M Isabel G; Philippsen, Peter; Di Pietro, Antonio; Espeso, Eduardo A

2010-08-01

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.

Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors.

PubMed

Roderick, H L; Campbell, A K; Llewellyn, D H

1997-03-24

The multi-functional protein calreticulin (CRT) is normally found within the lumen of the endoplasmic reticulum (ER). However, some of its proposed functions require it to be located within the nucleus, where its presence is contentious. We have investigated this in live COS7, HeLa and LM(TK-) cells using green fluorescent protein (GFP)-fusion proteins. GFP-CRT, and GFP, with an ER signal peptide and a KDEL sequence (ER-GFP), were localised to the ER. In addition, GFP-CRT was located in the nucleus of all the cell types at low levels. The higher levels of nuclear fluorescence in LM(TK-) and HeLa cells suggested that glucocorticoid receptors might enhance nuclear localisation of calreticulin. Dexamethasone treatment of LM(TK-) cells doubled the amount of nuclear GFP-CRT, but did not affect the localisation of a GFP-CRT fusion in which the glucocorticoid receptor-binding N-domain of calreticulin had been deleted. Thus, despite ER targeting and retention signals, calreticulin is also located within the nucleus where its presence increases due to its interaction with glucocorticoid receptors.

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci.

PubMed

Milbredt, Sarah; Waldminghaus, Torsten

2017-06-07

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci. Copyright © 2017 Milbredt and Waldminghaus.

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci

PubMed Central

Milbredt, Sarah; Waldminghaus, Torsten

2017-01-01

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci. PMID:28450375

Some properties of human neuronal alpha 7 nicotinic acetylcholine receptors fused to the green fluorescent protein.

PubMed

Palma, Eleonora; Mileo, Anna M; Martinez-Torres, Ataulfo; Eusebi, Fabrizio; Miledi, Ricardo

2002-03-19

The functional properties and cellular localization of the human neuronal alpha7 nicotinic acetylcholine (AcCho) receptor (alpha7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutalpha7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtalpha7 receptors decay much faster than those elicited by the mutalpha7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated alpha7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable "run-down" of the AcCho currents generated by mutalpha7-GFP receptors, whereas those of the wtalpha7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutalpha7-GFP oocytes was accompanied by a marked decrease of alpha-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtalpha7 and mutalpha7 receptors provides powerful tools to study the distribution and function of alpha7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins.

Selective cell-surface labeling of the molecular motor protein prestin

PubMed Central

McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

2011-01-01

Prestin, a multipass transmembrane protein whose N- an C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity. PMID:21651892

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots.

PubMed

Wang, Yuh-Shuh; Motes, Christy M; Mohamalawari, Deepti R; Blancaflor, Elison B

2004-10-01

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins. Copyright 2004 Wiley-Liss, Inc.

Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice.

PubMed

Homma, S; Toda, G; Gong, J; Kufe, D; Ohno, T

2001-11-01

The prevention of recurrence of hepatocellular carcinoma (HCC) after treatment is very important for improvement of the prognosis of HCC patients. Dendritic cells (DCs) are potent antigen-presenting cells that can prime naive T cells to induce a primary immune response. We attempted to induce preventive antitumor immunity against HCC by immunizing BALB/c mice with fusions of DCs and HCC cells. Murine bone marrow-derived DCs and a murine HCC cell line. BNL cells, were fused by treatment with 50% polyethyleneglvcol (PEG). Fusion efficacy was assessed by the analysis of fusions of BNL cells stained with red fluorescent dye and DCs stained with green fluorescent dye. Mice injected intravenously with DC/BNL fusions were challenged by BNL cell inoculation. About 30% of the PEG-treated non-adherent cells with both fluorescences were considered to be fusion cells. The cell fraction of DC/BNL fusions showed phenotypes of DCs, MHC class II, CD80, CD86, and intercellular adhesion molecule (ICAM)-1, which were not expressed on BNL cells. Mice immunized with the fusions were protected against the inoculation of BNL tumor cells, whereas injection with a mixture of DCs and BNL cells not treated with PEG did not provide significant resistance against BNL cell inoculation. Splenocytes from DC/BNL fusion-immunized mice showed lytic activity against BNL cells. These results demonstrate that immunization with fusions of DCs and HCC cells is capable of inducing preventive antitumor immunity against HCC.

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

NASA Astrophysics Data System (ADS)

Heuvingh, J.; Pincet, F.; Cribier, S.

2004-07-01

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of “hemifusion” is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

Subcellular localization of transiently expressed fluorescent fusion proteins.

PubMed

Collings, David A

2013-01-01

The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

Two-photon imaging in living brain slices.

PubMed

Mainen, Z F; Maletic-Savatic, M; Shi, S H; Hayashi, Y; Malinow, R; Svoboda, K

1999-06-01

Two-photon excitation laser scanning microscopy (TPLSM) has become the tool of choice for high-resolution fluorescence imaging in intact neural tissues. Compared with other optical techniques, TPLSM allows high-resolution imaging and efficient detection of fluorescence signal with minimal photobleaching and phototoxicity. The advantages of TPLSM are especially pronounced in highly scattering environments such as the brain slice. Here we describe our approaches to imaging various aspects of synaptic function in living brain slices. To combine several imaging modes together with patch-clamp electrophysiological recordings we found it advantageous to custom-build an upright microscope. Our design goals were primarily experimental convenience and efficient collection of fluorescence. We describe our TPLSM imaging system and its performance in detail. We present dynamic measurements of neuronal morphology of neurons expressing green fluorescent protein (GFP) and GFP fusion proteins as well as functional imaging of calcium dynamics in individual dendritic spines. Although our microscope is a custom instrument, its key advantages can be easily implemented as a modification of commercial laser scanning microscopes. Copyright 1999 Academic Press.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

PubMed Central

Shenoy, Dinesh; Fu, Wei; Li, Jane; Crasto, Curtis; Jones, Graham; DiMarzio, Charles; Sridhar, Srinivas; Amiji, Mansoor

2006-01-01

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site. PMID:16467923

Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer

PubMed Central

Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

2015-01-01

A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. PMID:26269359

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

PubMed Central

Masuda, Isao; Igarashi, Takao; Sakaguchi, Reiko; Nitharwal, Ram G.; Takase, Ryuichi; Han, Kyu Young; Leslie, Benjamin J.; Liu, Cuiping; Gamper, Howard; Ha, Taekjip; Sanyal, Suparna

2017-01-01

Abstract Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering. PMID:27956502

Cellular Transfection to Deliver Alanine-Glyoxylate Aminotransferase to Hepatocytes: A Rational Gene Therapy for Primary Hyperoxaluria-1 (PH-1)

PubMed Central

Koul, Sweaty; Johnson, Thomas; Pramanik, Saroj; Koul, Hari

2005-01-01

Background: Primary hyperoxaluria-type 1 (PH-1) is a rare autosomal recessive disorder of glyoxalate metabolism caused by deficiency in the liver-specific peroxisomal enzyme alanine-glyoxalate transaminase 1 (AGT) resulting in the increased oxidation of glyoxalate to oxalate. Accumulation of oxalate in the kidney and other soft tissues results in loss of renal function and significant morbidity. The present treatment options offer some relief in the short term, but they are not completely successful. In the present study, we tested the feasibility of corrective gene therapy for this metabolic disorder. Methods: A cDNA library was made from HepG2 cells. PCR primers were designed for the AGT sequence with modifications to preclude mistargeting during gene delivery. Amplified AGT cDNA was cloned as a fusion protein with green fluorescent protein (GFP) using the vector EGFP-C1 (Clontech) for monitoring subcellular distribution. Sequence and expression of the fusion protein was verified. Fusion protein vectors were transfected into hepatocytes by liposomal transfection. AGT expression and subcellular distribution was monitored by GFP fluorescence. Results: HepG2 cells express full-length mRNA coding for AGT as confirmed by insert size as well as sequence determination. Selective primers allowed us to generate a modified recombinant GFP-AGT fusion protein. Cellular transfections with Lipofectamine resulted in transfection efficiencies of 60–90%. The recombinant AGT did localize to peroxisomes as monitored by GFP fluorescence. Conclusions: The results demonstrate preliminary in vitro feasibility data for AGT transfection into the hepatocytes. To the best of our knowledge, this is the first study to attempt recombinant AGT gene therapy for treatment of primary hyperoxaluria-1. PMID:15849465

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

PubMed Central

Sapoznik, Etai; Niu, Guoguang; Zhou, Yu; Prim, Peter M.; Criswell, Tracy L.

2018-01-01

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening. PMID:29444187

Chemometric classification of Chinese lager beers according to manufacturer based on data fusion of fluorescence, UV and visible spectroscopies.

PubMed

Tan, Jin; Li, Rong; Jiang, Zi-Tao

2015-10-01

We report an application of data fusion for chemometric classification of 135 canned samples of Chinese lager beers by manufacturer based on the combination of fluorescence, UV and visible spectroscopies. Right-angle synchronous fluorescence spectra (SFS) at three wavelength difference Δλ=30, 60 and 80 nm and visible spectra in the range 380-700 nm of undiluted beers were recorded. UV spectra in the range 240-400 nm of diluted beers were measured. A classification model was built using principal component analysis (PCA) and linear discriminant analysis (LDA). LDA with cross-validation showed that the data fusion could achieve 78.5-86.7% correct classification (sensitivity), while those rates using individual spectroscopies ranged from 42.2% to 70.4%. The results demonstrated that the fluorescence, UV and visible spectroscopies complemented each other, yielding higher synergic effect. Copyright © 2015 Elsevier Ltd. All rights reserved.

Segmentation and classification of cell cycle phases in fluorescence imaging.

PubMed

Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

2009-01-01

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors.

PubMed

Chen, Rong; Zhou, Jingjing; Qin, Lingyun; Chen, Yao; Huang, Yongqi; Liu, Huili; Su, Zhengding

2017-06-27

In nearly half of cancers, the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many potent Mdm2-specific inhibitors have already undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 μM. The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library, including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p-N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.

Fluorescent Proteins: A Cell Biologist's User Guide

PubMed Central

Snapp, Erik Lee

2009-01-01

Fluorescent Proteins (FPs) have revolutionized cell biology. The value of labeling and visualizing proteins in living cells is evident from thousands of publications since the cloning of Green Fluorescent Protein (GFP). Biologists have been flooded with a cornucopia of FPs; however, the FP toolbox has not necessarily been optimized for cell biologists. Common FP plasmids are suboptimal for FP-fusion protein construction. More problematic are commercial and investigator-constructed FP-fusion proteins that disrupt important cellular targeting information. Even when cell biologists correctly construct FP-fusion proteins, it is rarely self-evident which FP should be used. Important FP information, such as oligomer formation or photostability, is often unsearchable or anecdotal. This brief guide is offered to assist in correctly exploiting FPs in cells. PMID:19819147

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

PubMed Central

Higuchi-Sanabria, Ryo; Garcia, Enrique J.; Tomoiaga, Delia; Munteanu, Emilia L.; Feinstein, Paul; Pon, Liza A.

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging. PMID:26727004

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

PubMed Central

Nikolaus, Joerg; Karatekin, Erdem

2016-01-01

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. PMID:27585113

Quantification of Coffea arabica and Coffea canephora var. robusta concentration in blends by means of synchronous fluorescence and UV-Vis spectroscopies.

PubMed

Dankowska, A; Domagała, A; Kowalewski, W

2017-09-01

The potential of fluorescence, UV-Vis spectroscopies as well as the low- and mid-level data fusion of both spectroscopies for the quantification of concentrations of roasted Coffea arabica and Coffea canephora var. robusta in coffee blends was investigated. Principal component analysis was used to reduce data multidimensionality. To calculate the level of undeclared addition, multiple linear regression (PCA-MLR) models were used with lowest root mean square error of calibration (RMSEC) of 3.6% and root mean square error of cross-validation (RMSECV) of 7.9%. LDA analysis was applied to fluorescence intensities and UV spectra of Coffea arabica, canephora samples, and their mixtures in order to examine classification ability. The best performance of PCA-LDA analysis was observed for data fusion of UV and fluorescence intensity measurements at wavelength interval of 60nm. LDA showed that data fusion can achieve over 96% of correct classifications (sensitivity) in the test set and 100% of correct classifications in the training set, with low-level data fusion. The corresponding results for individual spectroscopies ranged from 90% (UV-Vis spectroscopy) to 77% (synchronous fluorescence) in the test set, and from 93% to 97% in the training set. The results demonstrate that fluorescence, UV, and visible spectroscopies complement each other, giving a complementary effect for the quantification of roasted Coffea arabica and Coffea canephora var. robusta concentration in blends. Copyright © 2017 Elsevier B.V. All rights reserved.

Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.

PubMed

Wang, Y; Yu, Y A; Shabahang, S; Wang, G; Szalay, A A

2002-10-01

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.

Membrane Topology Mapping of the Na+-Pumping NADH: Quinone Oxidoreductase from Vibrio cholerae by PhoA- Green Fluorescent Protein Fusion Analysis▿

PubMed Central

Duffy, Ellen B.; Barquera, Blanca

2006-01-01

The membrane topologies of the six subunits of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na+-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed. PMID:17041063

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells.

PubMed

Hanamata, Shigeru; Kurusu, Takamitsu; Okada, Masaaki; Suda, Akiko; Kawamura, Koki; Tsukada, Emi; Kuchitsu, Kazuyuki

2013-01-01

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells

PubMed Central

Hanamata, Shigeru; Kurusu, Takamitsu; Okada, Masaaki; Suda, Akiko; Kawamura, Koki; Tsukada, Emi; Kuchitsu, Kazuyuki

2013-01-01

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli. PMID:23123450

Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer.

PubMed

Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

2015-10-01

A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast.

PubMed

Hori, Akiko; Yoshida, Minoru; Ling, Feng

2011-05-01

Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

ERIC Educational Resources Information Center

Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

2015-01-01

Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

Selective cell-surface labeling of the molecular motor protein prestin.

PubMed

McGuire, Ryan M; Silberg, Jonathan J; Pereira, Fred A; Raphael, Robert M

2011-06-24

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity. Copyright © 2011 Elsevier Inc. All rights reserved.

Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heo, Paul; Kong, Byoungjae; Jung, Young-Hun

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regardingmore » the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening. - Highlights: • SNARE proteins drive membrane fusion and open a pore for cargo release. • Biotinylated GFP and DyStrp was used as the reporter pair of fusion pore opening. • Procedure for efficient SNARE reconstitution and reporter encapsulation was established. • The FRET pair reported opening of a large fusion pore bigger than 5 nm. • The assay was robust and provided information of stoichiometry of vesicle fusion.« less

High efficiency and brightness fluorescent organic light emitting diode by triplet-triplet fusion

DOEpatents

Forrest, Stephen; Zhang, Yifan

2015-02-10

A first device is provided. The first device further comprises an organic light emitting device. The organic light emitting device further comprises an anode, a cathode, and an emissive layer disposed between the anode and the cathode. The emissive layer may include an organic host compound and at least one organic emitting compound capable of fluorescent emission at room temperature. Various configurations are described for providing a range of current densities in which T-T fusion dominates over S-T annihilation, leading to very high efficiency fluorescent OLEDs.

The distribution of RNA polymerase in Escherichia coli is dynamic and sensitive to environmental cues | Center for Cancer Research

Cancer.gov

Despite extensive genetic, biochemical and structural studies on Escherichia coli RNA polymerase (RNAP), little is known about its location and distribution in response to environmental changes. To visualize the RNAP by fluorescence microscopy in E. coli under different physiological conditions, we constructed a functional rpoC-gfp gene fusion on the chromosome.

A Hierarchical Convolutional Neural Network for vesicle fusion event classification.

PubMed

Li, Haohan; Mao, Yunxiang; Yin, Zhaozheng; Xu, Yingke

2017-09-01

Quantitative analysis of vesicle exocytosis and classification of different modes of vesicle fusion from the fluorescence microscopy are of primary importance for biomedical researches. In this paper, we propose a novel Hierarchical Convolutional Neural Network (HCNN) method to automatically identify vesicle fusion events in time-lapse Total Internal Reflection Fluorescence Microscopy (TIRFM) image sequences. Firstly, a detection and tracking method is developed to extract image patch sequences containing potential fusion events. Then, a Gaussian Mixture Model (GMM) is applied on each image patch of the patch sequence with outliers rejected for robust Gaussian fitting. By utilizing the high-level time-series intensity change features introduced by GMM and the visual appearance features embedded in some key moments of the fusion process, the proposed HCNN architecture is able to classify each candidate patch sequence into three classes: full fusion event, partial fusion event and non-fusion event. Finally, we validate the performance of our method on 9 challenging datasets that have been annotated by cell biologists, and our method achieves better performances when comparing with three previous methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

An efficient transgenic system by TA cloning vectors and RNAi for C. elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gengyo-Ando, Keiko; CREST, JST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012; Yoshina, Sawako

2006-11-03

In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusionmore » gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.« less

Engineering tumor cell targeting in nanoscale amyloidal materials

NASA Astrophysics Data System (ADS)

Unzueta, Ugutz; Seras-Franzoso, Joaquin; Virtudes Céspedes, María; Saccardo, Paolo; Cortés, Francisco; Rueda, Fabián; Garcia-Fruitós, Elena; Ferrer-Miralles, Neus; Mangues, Ramon; Vázquez, Esther; Villaverde, Antonio

2017-01-01

Bacterial inclusion bodies are non-toxic, mechanically stable and functional protein amyloids within the nanoscale size range that are able to naturally penetrate into mammalian cells, where they deliver the embedded protein in a functional form. The potential use of inclusion bodies in protein delivery or protein replacement therapies is strongly impaired by the absence of specificity in cell binding and penetration, thus preventing targeting. To address this issue, we have here explored whether the genetic fusion of two tumor-homing peptides, the CXCR4 ligands R9 and T22, to an inclusion body-forming green fluorescent protein (GFP), would keep the interaction potential and the functionality of the fused peptides and then confer CXCR4 specificity in cell binding and further uptake of the materials. The fusion proteins have been well produced in Escherichia coli in their full-length form, keeping the potential for fluorescence emission of the partner GFP. By using specific inhibitors of CXCR4 binding, we have demonstrated that the engineered protein particles are able to penetrate CXCR4+ cells, in a receptor-mediated way, without toxicity or visible cytopathic effects, proving the availability of the peptide ligands on the surface of inclusion bodies. Since no further modification is required upon their purification, the biological production of genetically targeted inclusion bodies opens a plethora of cost-effective possibilities in the tissue-specific intracellular transfer of functional proteins through the use of structurally and functionally tailored soft materials.

Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level

PubMed Central

Baubet, Valérie; Le Mouellic, Hervé; Campbell, Anthony K.; Lucas-Meunier, Estelle; Fossier, Philippe; Brûlet, Philippe

2000-01-01

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca2+ stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca2+ increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals. PMID:10860991

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

PubMed

Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

2009-01-01

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

Exchange of rotor components in functioning bacterial flagellar motor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuoka, Hajime; Inoue, Yuichi; Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai 980-8577

2010-03-26

The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP,more » and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s{sup -1}, meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.« less

Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two typesmore » of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.« less

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

PubMed Central

Warner, Katherine Deigan; Chen, Michael C.; Song, Wenjiao; Strack, Rita L.; Thorn, Andrea; Jaffrey, Samie R.; Ferré-D’Amaré, Adrian R.

2014-01-01

Green fluorescent protein (GFP) and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro evolved RNA mimic of GFP, which as genetically encoded fusions, makes possible live-cell, real-time imaging of biological RNAs, without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we have solved its co-crystal structure bound to its cognate exogenous chromophore, revealing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex, and an unpaired guanine. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs, and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure has guided the design of a miniaturized 'Baby Spinach', and provides the foundation for structure-driven design and tuning of fluorescent RNAs. PMID:25026079

Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

PubMed Central

Palma, Eleonora; Mileo, Anna M.; Martínez-Torres, Ataúlfo; Eusebi, Fabrizio; Miledi, Ricardo

2002-01-01

The functional properties and cellular localization of the human neuronal α7 nicotinic acetylcholine (AcCho) receptor (α7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated α7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable “run-down” of the AcCho currents generated by mutα7-GFP receptors, whereas those of the wtα7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutα7-GFP oocytes was accompanied by a marked decrease of α-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtα7 and mutα7 receptors provides powerful tools to study the distribution and function of α7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. PMID:11891308

Application to processing system using intra-molecular BRET

NASA Astrophysics Data System (ADS)

Otsuji, Tomomi; Okuda-Ashitaka, Emiko; Kojima, Satoshi; Akiyama, Hidehumi; Ito, Seiji; Ohmiya, Yoshihiro

2003-07-01

Luciferases are used as the reporter gene for promoter activity, whereas a green fluorescent protein (GFP) is used as marker for cellular function and localization. Recently, bioluminescence resonance energy transfer (BRET) between luciferase and YFP is used for analysis of inter-molecular reaction such as ligand-receptor in the living cells. The neuropeptides nocistatin (NST) and nociceptin/orphanin FQ (Noc/OFQ) are derived from the same precursor protein, while NST exhibits antagonism against Noc/OFQ-actions. In this study, we attempt an intra-molecular BRET system for monitoring dynamic biological process of the production of NST and Noc/OFQ in the living cells. At first, we constructed a fusion protein (Rluc-GFP) covalently linking luciferase (Renilla luciferase; Rluc) to Aequorea GFP as an intra-molecular BRET partner. Furthermore, we inserted constructs of mouse NST and Noc/OFQ (Rluc-m-GFP) or bovine NST and Noc/OFQ (Rluc-b-GFP) containing a proteolytic cleavage motif (Lys-Arg) within Rluc-GFP. When these constructions were transfected into Cos7 cells, all fusion proteins had luciferase activity and specific fluorescence. Luminescence spectra of Rluc-GFP, Rluc-m-GFP and Rluc-b-GFP fusion proteins with DeepBlueC as a substrate showed two peaks centered at 400 nm and 510 nm, whereas Rluc showed one peak centered at 400 nm. These results indicate that the proteolytic cleavage motif inserted fusion proteins between luciferase and GFP are available for intra-molecular BRET systems at first step.

Development of a novel set of Gateway-compatible vectors for live imaging in insect cells.

PubMed

Maroniche, G A; Mongelli, V C; Alfonso, V; Llauger, G; Taboga, O; del Vas, Mariana

2011-10-01

Insect genomics is a growing area of research. To exploit fully the genomic data that are being generated, high-throughput systems for the functional characterization of insect proteins and their interactomes are required. In this work, a Gateway-compatible vector set for expression of fluorescent fusion proteins in insect cells was developed. The vector set was designed to express a protein of interest fused to any of four different fluorescent proteins [green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry] by either the C-terminal or the N-terminal ends. Additionally, a collection of organelle-specific fluorescent markers was assembled for colocalization with fluorescent recombinant proteins of interest. Moreover, the vector set was proven to be suitable for simultaneously detecting up to three proteins by multiple labelling. The use of the vector set was exemplified by defining the subcellular distribution of Mal de Río Cuarto virus (MRCV) outer coat protein P10 and by analysing the in vivo self-interaction of the MRCV viroplasm matrix protein P9-1 in Förster resonance energy transfer (FRET) experiments. In conclusion, we have developed a valuable tool for high-throughput studies of protein subcellular localization that will aid in the elucidation of the function of newly described insect and virus proteins. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

PubMed

Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

2005-10-14

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Didier, P.; Weiss, E.; Sibler, A.-P.

2008-02-22

Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the samemore » scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.« less

Secretagogue stimulation of neurosecretory cells elicits filopodial extensions uncovering new functional release sites.

PubMed

Papadopulos, Andreas; Martin, Sally; Tomatis, Vanesa M; Gormal, Rachel S; Meunier, Frederic A

2013-12-04

Regulated exocytosis in neurosecretory cells relies on the timely fusion of secretory granules (SGs) with the plasma membrane. Secretagogue stimulation leads to an enlargement of the cell footprint (surface area in contact with the coverslip), an effect previously attributed to exocytic fusion of SGs with the plasma membrane. Using total internal reflection fluorescence microscopy, we reveal the formation of filopodia-like structures in bovine chromaffin and PC12 cells driving the footprint expansion, suggesting the involvement of cortical actin network remodeling in this process. Using exocytosis-incompetent PC12 cells, we demonstrate that footprint enlargement is largely independent of SG fusion, suggesting that vesicular exocytic fusion plays a relatively minor role in filopodial expansion. The footprint periphery, including filopodia, undergoes extensive F-actin remodeling, an effect abolished by the actomyosin inhibitors cytochalasin D and blebbistatin. Imaging of both Lifeact-GFP and the SG marker protein neuropeptide Y-mCherry reveals that SGs actively translocate along newly forming actin tracks before undergoing fusion. Together, these data demonstrate that neurosecretory cells regulate the number of SGs undergoing exocytosis during sustained stimulation by controlling vesicular mobilization and translocation to the plasma membrane through actin remodeling. Such remodeling facilitates the de novo formation of fusion sites.

IFITM Proteins Restrict Viral Membrane Hemifusion

PubMed Central

Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

2013-01-01

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection. PMID:23358889

The dynamics and regulation of mesenchymal cell fusion in the sea urchin embryo.

PubMed

Hodor, P G; Ettensohn, C A

1998-07-01

Cell-cell fusion occurs in a wide variety of developmental contexts, yet the mechanisms involved are just beginning to be elucidated. In the sea urchin embryo, primary mesenchyme cells (PMCs) fuse to form syncytial filopodial cables within which skeletal spicules are deposited. Taking advantage of the optical transparency and ease of micromanipulation of sea urchin embryos, we have developed methods for directly observing the dynamics of PMC fusion in vivo. A fraction of the PMCs was labeled with fluorescent dextran and transfer of the dye to unlabeled PMCs was followed by time-lapse, fluorescence microscopy. Fusion was first detected about 2 h after PMCs began to migrate within the blastocoel. Fusion proceeded in parallel with the assembly of the PMC ring pattern and was complete by the early gastrula stage. The formation of a single, extensive PMC syncytium was confirmed by DiI labeling of fixed embryos. When single micromeres were isolated and cultured in unsupplemented seawater, they divided and their progeny underwent fusion. This shows that the capacity to fuse is autonomously programmed in the micromere-PMC lineage by the 16-cell stage. PMC transplantations at late embryonic stages revealed that these cells remain fusion-competent long after their fusion is complete. At late stages, other mesenchyme cells (blastocoelar cells) are also present within the blastocoel and are migrating and fusing with one another. Fusion-competent blastocoelar cells and PMCs come into contact but do not fuse with one another, indicating that these two cell types fuse by distinct mechanisms. When secondary mesenchyme cells convert to a skeletogenic fate they alter their fusogenic properties and join the PMC syncytium, as shown by transfer of fluorescent dextran. Our analysis has provided a detailed picture of the cellular basis and regulation of mesodermal cell fusion and has important implications regarding molecular mechanisms that underlie fusion.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging.

PubMed

Zhang, Nan; Membreno, Edward; Raj, Susan; Zhang, Hongjie; Khan, Liakot A; Gobel, Verena

2017-10-03

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics

PubMed Central

Pieknik, Julianna R.; Tang, Shuang

2018-01-01

Fluorescent herpes simplex viruses (HSV) are invaluable tools for localizing virus in cells, permitting visualization of capsid trafficking and enhancing neuroanatomical research. Fluorescent viruses can also be used to study virus kinetics and reactivation in vivo. Such studies would be facilitated by fluorescent herpes simplex virus recombinants that exhibit wild-type kinetics of replication and reactivation and that are genetically stable. We engineered an HSV-2 strain expressing the fluorescent mNeonGreen protein as a fusion with the VP26 capsid protein. This virus has normal replication and in vivo recurrence phenotypes, providing an essential improved tool for further study of HSV-2 infection. PMID:29738431

ReAsH/FlAsH labeling and image analysis of tetracysteine sensor proteins in cells.

PubMed

Irtegun, Sevgi; Ramdzan, Yasmin M; Mulhern, Terrence D; Hatters, Danny M

2011-08-31

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

PubMed Central

Marks, Kevin M.; Braun, Patrick D.; Nolan, Garry P.

2004-01-01

Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF′). A fluorescein conjugate to the engineered ligand (FL-SLF′) retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF′ labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF′ was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF′ was proportional to the FKBP12(F36V) expression level. This FL-SLF′-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF′ mediated FALI of a β-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF′ also mediated FALI of a β-galactosidase fusion expressed in living NIH 3T3 cells, where β-galactosidase activity was reduced in 15 s. Thus, FL-SLF′ can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI. PMID:15218100

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis

PubMed Central

Maes, Margaret E.; Schlamp, Cassandra L.

2017-01-01

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies. PMID:28880942

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis.

PubMed

Maes, Margaret E; Schlamp, Cassandra L; Nickells, Robert W

2017-01-01

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.

Fusion of GFP and phase contrast images with complex shearlet transform and Haar wavelet-based energy rule.

PubMed

Qiu, Chenhui; Wang, Yuanyuan; Guo, Yanen; Xia, Shunren

2018-03-14

Image fusion techniques can integrate the information from different imaging modalities to get a composite image which is more suitable for human visual perception and further image processing tasks. Fusing green fluorescent protein (GFP) and phase contrast images is very important for subcellular localization, functional analysis of protein and genome expression. The fusion method of GFP and phase contrast images based on complex shearlet transform (CST) is proposed in this paper. Firstly the GFP image is converted to IHS model and its intensity component is obtained. Secondly the CST is performed on the intensity component and the phase contrast image to acquire the low-frequency subbands and the high-frequency subbands. Then the high-frequency subbands are merged by the absolute-maximum rule while the low-frequency subbands are merged by the proposed Haar wavelet-based energy (HWE) rule. Finally the fused image is obtained by performing the inverse CST on the merged subbands and conducting IHS-to-RGB conversion. The proposed fusion method is tested on a number of GFP and phase contrast images and compared with several popular image fusion methods. The experimental results demonstrate that the proposed fusion method can provide better fusion results in terms of subjective quality and objective evaluation. © 2018 Wiley Periodicals, Inc.

Exploiting the synergistic effect of concurrent data signals: Low-level fusion of liquid chromatographic with dual detection data.

PubMed

Teglia, Carla M; Azcarate, Silvana M; Alcaráz, Mirta R; Goicoechea, Héctor C; Culzoni, María J

2018-08-15

A low-level data fusion strategy was developed and implemented for data processing of second-order liquid chromatographic data with dual detection, i.e. absorbance and fluorescence monitoring. The synergistic effect of coupling individual information provided by two different detectors was evaluated by analyzing the results gathered after the application of a series of data preprocessing steps and chemometric resolution. The chemometric modeling involved data analysis by MCR-ALS, PARAFAC and N-PLS. Their ability to handle the new data block was assessed through the estimation of the analytical figures of merits achieved in the prediction of a validation set containing fifteen fluorescent and non-fluorescent veterinary active ingredients that can be found in poultry litter. Eventually, the feasibility of the application of the fusion strategy to real poultry litter samples containing the studied compounds was verified. Copyright © 2018 Elsevier B.V. All rights reserved.

A new microcolumn-type microchip for examining the expression of chimeric fusion genes using a nucleic acid sandwich hybridization technique.

PubMed

Ohnishi, Michihiro; Sasaki, Naoyuki; Kishimoto, Takuya; Watanabe, Hidetoshi; Takagi, Masatoshi; Mizutani, Shuki; Kishii, Noriyuki; Yasuda, Akio

2014-11-01

We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

PubMed

Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

2015-07-01

Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.

Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane▿

PubMed Central

Kaserer, Wallace A.; Jiang, Xiaoxu; Xiao, Qiaobin; Scott, Daniel C.; Bauler, Matthew; Copeland, Daniel; Newton, Salete M. C.; Klebba, Phillip E.

2008-01-01

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB+ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins. PMID:18390658

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

PubMed

Adamus, Tomasz; Konieczny, Paweł; Sekuła, Małgorzata; Sułkowski, Maciej; Majka, Marcin

2014-01-01

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

PubMed

Mullaney, J M; Black, L W

1998-11-13

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range

Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

PubMed Central

Kohl, Tobias; Haustein, Elke; Schwille, Petra

2005-01-01

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

Tracking fusion of human mesenchymal stem cells after transplantation to the heart.

PubMed

Freeman, Brian T; Kouris, Nicholas A; Ogle, Brenda M

2015-06-01

Evidence suggests that transplanted mesenchymal stem cells (MSCs) can aid recovery of damaged myocardium caused by myocardial infarction. One possible mechanism for MSC-mediated recovery is reprogramming after cell fusion between transplanted MSCs and recipient cardiac cells. We used a Cre/LoxP-based luciferase reporter system coupled to biophotonic imaging to detect fusion of transplanted human pluripotent stem cell-derived MSCs to cells of organs of living mice. Human MSCs, with transient expression of a viral fusogen, were delivered to the murine heart via a collagen patch. At 2 days and 1 week later, living mice were probed for bioluminescence indicative of cell fusion. Cell fusion was detected at the site of delivery (heart) and in distal tissues (i.e., stomach, small intestine, liver). Fusion was confirmed at the cellular scale via fluorescence in situ hybridization for human-specific and mouse-specific centromeres. Human cells in organs distal to the heart were typically located near the vasculature, suggesting MSCs and perhaps MSC fusion products have the ability to migrate via the circulatory system to distal organs and engraft with local cells. The present study reveals previously unknown migratory patterns of delivered human MSCs and associated fusion products in the healthy murine heart. The study also sets the stage for follow-on studies to determine the functional effects of cell fusion in a model of myocardial damage or disease. Mesenchymal stem cells (MSCs) are transplanted to the heart, cartilage, and other tissues to recover lost function or at least limit overactive immune responses. Analysis of tissues after MSC transplantation shows evidence of fusion between MSCs and the cells of the recipient. To date, the biologic implications of cell fusion remain unclear. A newly developed in vivo tracking system was used to identify MSC fusion products in living mice. The migratory patterns of fusion products were determined both in the target organ (i.e., the heart) and in distal organs. This study shows, for the first time, evidence of fusion products at sites distal from the target organ and data to suggest that migration occurs via the vasculature. These results will inform and improve future, MSC-based therapeutics. ©AlphaMed Press.

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

PubMed Central

Masante, Cyril; El Najjar, Farah; Chang, Andres; Jones, Angela; Moncman, Carole L.

2014-01-01

ABSTRACT Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection. IMPORTANCE Human metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified. PMID:24672047

Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times

PubMed Central

Schmidt, Florian I.; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S.

2013-01-01

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements. PMID:23870263

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic-Targeting and Imaging.

PubMed

Sun, Lu; Xie, Shuping; Qi, Jing; Liu, Ergang; Liu, Di; Liu, Quan; Chen, Sunhui; He, Huining; Yang, Victor C

2017-11-15

Matrix metalloproteinases (MMPs) activatable imaging probe has been explored for tumor detection. However, activation of the probe is mainly done in the extracellular space without intracellular uptake of the probe for more sensitivity. Although cell-penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we have developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling a hexahistidine-tagged (His-tagged) fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the low-molecular-weight protamine peptide as the CPP, and the MMP-2 cleavage sequence fused with the hexahistidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain nonpermeable to normal tissues, thereby offsetting the nonselective cellular uptake, but was also suppressed of fluorescent signals during magnetic tumor-targeting in the circulation, primarily because of the masking of the CPP activity and quenching of the fluorophore by the associated NiFe 2 O 4 nanoparticles. However, these properties were recovered or "turned on" by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching, and wide-spread.

Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine

NASA Astrophysics Data System (ADS)

Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

2010-01-01

Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by ~300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

[Analysis of EML4-ALK gene fusion mutation in patients 
with non-small cell lung cancer].

PubMed

Wang, Xuzhou; Chen, Weisheng; Yu, Yinghao

2015-02-01

Non-small cell lung cancer (NSCLC) is the main type of lung cancer, and the related locus mutation detection research has become a hot direction of molecular targeted therapy, studying on gene mutation status of echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR), detecting the sensitivity of EML4-ALK gene fusion and gene mutation of EGFR. EML4-ALK gene fusion in 85 cases of paraffin embedded tumor tissue and adjacent lung tissue was detected with the application of immunohistochemistry (IHC), Scorpions amplification refractory mutation system (Scorpions ARMS) fluorescence quantitative PCR and fluorescence in situ hybridization (FISH) technology, and EGFR gene in 18, 19, 20 and 21 exon mutation status was detected with the application of ARMS method. In 115 cases of NSCLC, IHC showed 32 cases with ALK (D5F3) expression, the expression rate was 27.8%; ARMS showed 27 cases with EML4-ALK fusion gene mutation, the mutation detection rate was 23.5%; 53 cases were detected with EGFR mutation, the mutation rate was 46%. While FISH showed 23 cases with EML4-ALK fusion gene mutation, the detection rate was 20%, slightly lower than the ARMS detection results, suggesting that ARMS more sensitive. The application of IHC, ARMS fluorescence quantitative PCR and FISH technology can make a rapid and accurate evaluation of EML4-ALK gene fusion.

[Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

PubMed

Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

2012-08-01

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells, indicating a potential candidate method for clinical HER2 positive specimens detection.

Interactions between late acting proteins required for peptidoglycan synthesis during sporulation

PubMed Central

Fay, Allison; Meyer, Pablo; Dworkin, Jonathan

2010-01-01

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the later steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a non-essential, sporulation-specific homolog of the well-conserved and essential SEDS proteins, and SpoVD, a non-essential class B penicillin binding protein (PBP). We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and Fluorescence Resonance Energy Transfer experiments indicated that SpoVE and SpoVD interact and co-affinity purification in E. coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of a SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in a loss of function of the entire fusion that was not complemented by a wild type protein. Thus, SpoVE has a direct and functional interaction with SpoVD and this conclusion will facilitate understanding the essential function SpoVE and related SEDS proteins such as FtsW and RodA play in bacterial growth and division. PMID:20417640

Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-10-01

Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans.

PubMed

Mao, Yuxin; Zhang, Zimei; Wong, Brian

2003-12-01

Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.

Interaction of phospholipase C with liposome: A conformation transition of the enzyme is critical and specific to liposome composition for burst hydrolysis and fusion in concert

NASA Astrophysics Data System (ADS)

Patra, Samir Kumar; Sengupta, Dipta; Deb, Moonmoon; Kar, Swayamsiddha; Kausar, Chahat

2017-02-01

Phospholipase C (PLC)1 is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC activation and liposome fusion on the lipid composition.

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

PubMed Central

Irtegun, Sevgi; Ramdzan, Yasmin M.; Mulhern, Terrence D.; Hatters, Danny M.

2011-01-01

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations). PMID:21897361

Femtosecond laser surgery of two-cell mouse embryos: effect on viability, development, and tetraploidization

NASA Astrophysics Data System (ADS)

Osychenko, Alina A.; Zalessky, Alexandr D.; Kostrov, Andrey N.; Ryabova, Anastasia V.; Krivokharchenko, Alexander S.; Nadtochenko, Viktor A.

2017-12-01

The effect of the laser pulse energy and total expose of the energy incident on the embryo blastomere fusion probability was investigated. The probability of the four different events after laser pulse was determined: the fusion of two blastomeres with the following formation of tetraploid embryo, the destruction of the first blastomere occurs, the second blastomere conservation remains intact, the destruction and the death of both cells; two blastomeres were not fused, and no morphological changes occurred. We report on viability and quality of the embryo after laser surgery as a function of the laser energy incident. To characterize embryo quality, the probability of the blastocyst stage achievement was estimated and the blastocyst cells number was calculated. Blastocoel formation is the only event of morphogenesis in the preimplantation development of mammals, so we assumed it as an indicator of the time of embryonic "clocks" and observed it among fused and control embryos. The blastocoel formation time is the same for fused and control embryos. It indicates that embryo clocks were not affected due to blastomere fusion. Thus, the analysis of the fluorescence microscopic images of nuclei in the fused embryo revealed that nuclei fusion does not occur after blastomere fusion.

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

PubMed

Liang, Xing-xiang; Wang, Bei-bei; Sun, Yu-fei; Lin, Ying; Han, Shuang-yan; Zheng, Sui-ping; Cui, Tang-bing

2013-03-01

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu Guo; Institute of Neuroscience, Department of Neurobiology, Second Military Medical University, Shanghai 200433; Yang Huayan

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to {beta}{sub 2} subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasmamore » membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively.« less

Fusion-independent expression of functional ACh receptors in mouse mesoangioblast stem cells contacting muscle cells

PubMed Central

Grassi, Francesca; Pagani, Francesca; Spinelli, Gabriele; Angelis, Luciana De; Cossu, Giulio; Eusebi, Fabrizio

2004-01-01

Mesoangioblasts are vessel-associated fetal stem cells that can be induced to differentiate into skeletal muscle, both in vitro and in vivo. Whether this is due to fusion or to transdifferentiation into bona fide satellite cells is still an open question, for mesoangioblasts as well as for other types of stem cells. The early steps of satellite cell myogenic differentiation involve MyoD activation, membrane hyperpolarization and the appearance of ACh sensitivity and gap junctional communication. If mesoangioblasts differentiate into satellite cells, these characteristics should be observed in stem cells prior to fusion into multinucleated myotubes. We have investigated the functional properties acquired by mononucleated green fluorescent protein (GFP)-positive mesoangioblasts co-cultured with differentiating C2C12 myogenic cells, using the patch-clamp technique. Mesoangioblasts whose membrane contacted myogenic cells developed a hyperpolarized membrane resting potential and ACh-evoked current responses. Dye and electrical coupling was observed among mesoangioblasts but not between mesoangioblasts and myotubes. Mouse MyoD was detected by RT-PCR both in single, mononucleated mesoangioblasts co-cultured with C2C12 myotubes and in the total mRNA from mouse mesoangioblasts co-cultured with human myotubes, but not in human myotubes or stem cells cultured in isolation. In conclusion, when co-cultured with muscle cells, mesoangioblasts acquire many of the functional characteristics of differentiating satellite cells in the absence of cell fusion, strongly indicating that these stem cells undergo transdifferentiation into satellite cells, when exposed to a myogenic environment. PMID:15319417

Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity.

PubMed

Humenik, Martin; Mohrand, Madeleine; Scheibel, Thomas

2018-04-18

The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allows the generation of fibrous biomaterials with catalytic or light emitting properties.

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

PubMed Central

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg

2003-01-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of ∼13 s. PMID:12719264

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

PubMed

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W; Knoch, Tobias A; Waldeck, Waldemar; Langowski, Jörg

2003-05-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

PAX3/7-FOXO1 fusion status in older rhabdomyosarcoma patient population by fluorescent in situ hybridization.

PubMed

Dumont, Sarah N; Lazar, Alexander J; Bridge, Julia A; Benjamin, Robert S; Trent, Jonathan C

2012-02-01

In pediatric alveolar rhabdomyosarcoma, the PAX3-FOXO1 and PAX7-FOXO1 gene fusions are prognostic indicators, while little is known concerning this disease in older patients. To determine whether PAX3/7-FOXO1 fusion gene status correlates with outcome in adolescent, young adult, and adult rhabdomyosarcoma patients, the histological, immunohistochemical, and clinical characteristics of 105 patients followed at The University of Texas MD Anderson Cancer Center from 1957 to 2001 were evaluated. The samples were assembled into a tissue microarray, and fusion gene status was determined by fluorescence in situ hybridization using PAX3, PAX7, and FOXO1 loci-specific probes. The disease characteristics and specific gene fusion were correlated with patient outcomes using the log-rank test. Fifty-two percent of the samples exhibited a PAX3-FOXO1 fusion, 15% the PAX7-FOXO1 fusion, and 33% were negative for a rearrangement of these loci. The presence of PAX3/7-FOXO1 translocation was significantly associated with a higher frequency of metastatic disease. Although a statistically significant correlation between the PAX3/7-FOXO1 fusion gene status and overall survival was not identified, there was a trend toward better outcomes for patients with fusion-negative RMS. Therefore, identification of a FOXO1 fusion appears to be an interesting tool for predicting outcomes in older rhabdomyosarcoma patients and is worth further investigations in this rare subgroup of RMS population.

Single-virus fusion experiments reveal proton influx into vaccinia virions and hemifusion lag times.

PubMed

Schmidt, Florian I; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S

2013-07-16

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Novel, fluorescent, SSB protein chimeras with broad utility

PubMed Central

Liu, Juan; Choi, Meerim; Stanenas, Adam G; Byrd, Alicia K; Raney, Kevin D; Cohan, Christopher; Bianco, Piero R

2011-01-01

The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use. PMID:21462278

Self-Assembled Materials Made from Functional Recombinant Proteins.

PubMed

Jang, Yeongseon; Champion, Julie A

2016-10-18

Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function. We have designed functional recombinant fusion proteins capable of self-assembling into materials with unique structures that maintain or improve the functionality of the protein. Fusion of either a functional protein or an assembly domain to a leucine zipper domain makes the materials design strategy modular, based on the high affinity between leucine zippers. The self-assembly domains, including elastin-like polypeptides (ELPs) and defined-sequence random coil polypeptides, can be fused with a leucine zipper motif in order to promote assembly of the fusion proteins into larger structures upon specific stimuli such as temperature and ionic strength. Fusion of other functional domains with the counterpart leucine zipper motif endows the self-assembled materials with protein-specific functions such as fluorescence or catalytic activity. In this Account, we describe several examples of materials assembled from functional fusion proteins as well as the structural characterization, functionality, and understanding of the assembly mechanism. The first example is zipper fusion proteins containing ELPs that assemble into particles when introduced to a model extracellular matrix and subsequently disassemble over time to release the functional protein for drug delivery applications. Under different conditions, the same fusion proteins can self-assemble into hollow vesicles. The vesicles display a functional protein on the surface and can also carry protein, small-molecule, or nanoparticle cargo in the vesicle lumen. To create a material with a more complex hierarchical structure, we combined calcium phosphate with zipper fusion proteins containing random coil polypeptides to produce hybrid protein-inorganic supraparticles with high surface area and porous structure. The use of a functional enzyme created supraparticles with the ability to degrade inflammatory cytokines. Our characterization of these protein materials revealed that the molecular interactions are complex because of the large size of the protein building blocks, their folded structures, and the number of potential interactions including hydrophobic interactions, electrostatic interactions, van der Waals forces, and specific affinity-based interactions. It is difficult or even impossible to predict the structures a priori. However, once the basic assembly principles are understood, there is opportunity to tune the material properties, such as size, through control of the self-assembly conditions. Our future efforts on the fundamental side will focus on identifying the phase space of self-assembly of these fusion proteins and additional experimental levers with which to control and tune the resulting materials. On the application side, we are investigating an array of different functional proteins to expand the use of these structures in both therapeutic protein delivery and biocatalysis.

A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

PubMed

Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

2018-01-01

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

Laser-induced fusion of human embryonic stem cells with optical tweezers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Shuxun; Wang Xiaolin; Sun Dong

2013-07-15

We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

PubMed Central

Marshall, Kathryn E.; Robinson, Errol W.; Hengel, Shawna M.; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation. PMID:22470473

FRET imaging of diatoms expressing a biosilica-localized ribose sensor.

PubMed

Marshall, Kathryn E; Robinson, Errol W; Hengel, Shawna M; Paša-Tolić, Ljiljana; Roesijadi, Guritno

2012-01-01

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.

[Method of fused sample preparation after nitrify-determination of primary and minor elements in manganese ore by X-ray fluorescence spectrometry].

PubMed

Song, Yi; Guo, Fen; Gu, Song-hai

2007-02-01

Eight components, i. e. Mn, SiO2, Fe, P, Al2O3, CaO, MgO and S, in manganese ore were determined by X-ray fluorescence spectrometer. Because manganese ore sample releases a lot of air bubbles during fusion which effect accuracy and reproducibility of determination, nitric acid was added to the sample to destroy organic matter before fusion by the mixture flux at 1000 degrees C. This method solved the problem that the flux splashed during fusion because organic matter volatilized brought out a lot of air bubbles, eliminated particle size effects and mineral effect, while solved the problem of volatilization of sulfur during fusion. The experiments for the selection of the sample preparation conditions, i. e. fusion flux, fusion time and volume of HNO3, were carried out. The matrix effects on absorption and enhancement were corrected by variable theoretical alpha coefficient to expand the range of determination. Moreover, the precision and accuracy experiments were performed. In comparison with chemical analysis method, the quantitative analytical results for each component are satisfactory. The method has proven rapid, precise and simple.

Red fluorescent protein eqFP611 and its genetically engineered dimeric variants.

PubMed

Wiedenmann, Jörg; Vallone, Beatrice; Renzi, Fabiana; Nienhaus, Karin; Ivanchenko, Sergey; Röcker, Carlheinz; Nienhaus, G Ulrich

2005-01-01

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four beta cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form. Copyright 2005 Society of Photo-Optical Instrumentation Engineers

[Experimental study of glioma stem cell-mediated immune tolerance in tumor microenvironment].

PubMed

Xie, T; Ma, J W; Liu, B; Dong, J; Huang, Q

2017-11-23

Objective: To investigate the tumor microenvironment of immune tolerance induced by glioma stem cells (GSC). Methods: Human GSC SU3 cells transfected with red fluorescent protein (SU3-RFP) gene were implanted into the brain, subcutis (armpit and foot), liver and abdominal cavity of transgenic green fluorescence protein (GFP) nude mice to establish RFP(+) /GFP(+) dual fluorescence solid tumor model. The re-cultured cells derived from implanted tumor tissues, SU3-RFP cells co-cultured with peritoneal fluid of transgenic GFP nude mice and malignant ascites of tumor-bearing mice were observed by fluorescence microscopy and real-time video image tracing to analyze the microenvironment of immune tolerance mediated by RFP(+) /GFP(+) implanted tumor. Results: Dual fluorescence labeled frozen section showed that all of cells in the tumor microenvironment were GFP(+) , while the pressed tissue-patch showed that the tumor blood vessels exhibited a RFP(+) /GFP(+) double-positioning yellow. In the GFP single fluorescence labeled tumor tissue, all of cells in the microenvironment were green, including tumor edge, necrotic foci and blood vessel. Among them, CD68(+) , F4/80(+) , CD11c(+) , CD11b(+) and CD80(+) cells were observed. In the dual fluorescence labeled co-cultured cells, the phagocytosis and fusion between green host cells and red tumor cells were also observed, and these fusion cells might transfer to the malignant dendritic cells and macrophages. Conclusions: The tumor microenvironment of immune tolerance induced by GSC is not affected by the tissue types of tumor-inoculated sites, and the immune tolerance mediated by inflammatory cells is associated with the inducible malignant transformation, which may be driven by cell fusion.

Core labeling of adenovirus with EGFP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le, Long P.; Le, Helen N.; Nelson, Amy R.

2006-08-01

The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expressionmore » vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.« less

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

PubMed

Li, Junhua; Zhang, Yang; Yang, Yanjun

2013-03-01

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

PAX3/7–FOXO1 fusion status in older rhabdomyosarcoma patient population by fluorescent in situ hybridization

PubMed Central

Lazar, Alexander J.; Bridge, Julia A.; Benjamin, Robert S.; Trent, Jonathan C.

2014-01-01

Purpose In pediatric alveolar rhabdomyosarcoma, the PAX3–FOXO1 and PAX7–FOXO1 gene fusions are prognostic indicators, while little is known concerning this disease in older patients. To determine whether PAX3/7–FOXO1 fusion gene status correlates with outcome in adolescent, young adult, and adult rhabdomyosarcoma patients, the histological, immunohistochemical, and clinical characteristics of 105 patients followed at The University of Texas MD Anderson Cancer Center from 1957 to 2001 were evaluated. Methods The samples were assembled into a tissue microarray, and fusion gene status was determined by fluorescence in situ hybridization using PAX3, PAX7, and FOXO1 loci-specific probes. The disease characteristics and specific gene fusion were correlated with patient outcomes using the log-rank test. Results Fifty-two percent of the samples exhibited a PAX3–FOXO1 fusion, 15% the PAX7–FOXO1 fusion, and 33% were negative for a rearrangement of these loci. The presence of PAX3/7–FOXO1 translocation was significantly associated with a higher frequency of metastatic disease. Although a statistically significant correlation between the PAX3/7–FOXO1 fusion gene status and overall survival was not identified, there was a trend toward better outcomes for patients with fusion-negative RMS. Conclusions Therefore, identification of a FOXO1 fusion appears to be an interesting tool for predicting outcomes in older rhabdomyosarcoma patients and is worth further investigations in this rare subgroup of RMS population. PMID:22089931

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca{sup 2+} concentration ([Ca{sup 2+}]{sub PM}) with the Ca{sup 2+} indicator Fura Red in rat {beta} cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca{sup 2+}]{sub PM} caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca{sup 2+}]{sub PM} induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked secondmore » phase release. These data suggest that the pattern of the [Ca{sup 2+}]{sub PM} rise directly determines the types of fusing granules.« less

In Vitro Reconstitution of Autophagosome-Lysosome Fusion.

PubMed

Diao, J; Li, L; Lai, Y; Zhong, Q

2017-01-01

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins are a highly regulated class of membrane proteins lying in the center of membrane fusion. In conjunction with accessory proteins, SNAREs drive efficient merger of two distinct lipid bilayers into one interconnected structure. This chapter describes our fluorescence resonance energy transfer (FRET)-based proteoliposome fusion assays for the roles of various SNARE proteins, accessory proteins, and effects of different lipid compositions on membrane fusion involved in autophagy. © 2017 Elsevier Inc. All rights reserved.

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

PubMed

Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

2017-01-01

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

Femtosecond laser surgery of two-cell mouse embryos: effect on viability, development, and tetraploidization.

PubMed

Osychenko, Alina A; Zalessky, Alexandr D; Kostrov, Andrey N; Ryabova, Anastasia V; Krivokharchenko, Alexander S; Nadtochenko, Viktor A

2017-12-01

The effect of the laser pulse energy and total expose of the energy incident on the embryo blastomere fusion probability was investigated. The probability of the four different events after laser pulse was determined: the fusion of two blastomeres with the following formation of tetraploid embryo, the destruction of the first blastomere occurs, the second blastomere conservation remains intact, the destruction and the death of both cells; two blastomeres were not fused, and no morphological changes occurred. We report on viability and quality of the embryo after laser surgery as a function of the laser energy incident. To characterize embryo quality, the probability of the blastocyst stage achievement was estimated and the blastocyst cells number was calculated. Blastocoel formation is the only event of morphogenesis in the preimplantation development of mammals, so we assumed it as an indicator of the time of embryonic "clocks" and observed it among fused and control embryos. The blastocoel formation time is the same for fused and control embryos. It indicates that embryo clocks were not affected due to blastomere fusion. Thus, the analysis of the fluorescence microscopic images of nuclei in the fused embryo revealed that nuclei fusion does not occur after blastomere fusion. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yoosoo; Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791; Kim, Se-Hyun

Highlights: • Membrane fusion driven by SNARE complex is hindered by several polyphenols. • Distinctive inhibitory effect of each polyphenol on SNARE zippering in neuron was examined. • FRET between fluorescence protein-tagged SNAREs probed well SNARE zippering in PC12 cells. • Delphinidin and cyanidin inhibit N-terminal SNARE nucleation in Ca{sup 2+}-independent manner. • Myricetin inhibits Ca{sup 2+}-dependent transmembrane association of SNARE complex. - Abstract: Fusion of synaptic vesicles with the presynaptic plasma membrane in the neuron is mediated by soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) proteins. SNARE complex formation is a zippering-like process which initiates at the N-terminus andmore » proceeds to the C-terminal membrane-proximal region. Previously, we showed that this zippering-like process is regulated by several polyphenols, leading to the arrest of membrane fusion and the inhibition of neuroexocytosis. In vitro studies using purified SNARE proteins reconstituted in liposomes revealed that each polyphenol uniquely regulates SNARE zippering. However, the unique regulatory effect of each polyphenol in cells has not yet been examined. In the present study, we observed SNARE zippering in neuronal PC12 cells by measuring the fluorescence resonance energy transfer (FRET) changes of a cyan fluorescence protein (CFP) and a yellow fluorescence protein (YFP) fused to the N-termini or C-termini of SNARE proteins. We show that delphinidin and cyanidin inhibit the initial N-terminal nucleation of SNARE complex formation in a Ca{sup 2+}-independent manner, while myricetin inhibits Ca{sup 2+}-dependent transmembrane domain association of the SNARE complex in the cell. This result explains how polyphenols exhibit botulinum neurotoxin-like activity in vivo.« less

Arabidopsis thaliana is a susceptible host plant for the holoparasite Cuscuta spec.

PubMed

Birschwilks, Mandy; Sauer, Norbert; Scheel, Dierk; Neumann, Stefanie

2007-10-01

Arabidopsis thaliana and Cuscuta spec. represent a compatible host-parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host-parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP-ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP-ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP-ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.

Fluorescence and absorption spectroscopy for warm dense matter studies and ICF plasma diagnostics

NASA Astrophysics Data System (ADS)

Hansen, S. B.; Harding, E. C.; Knapp, P. F.; Gomez, M. R.; Nagayama, T.; Bailey, J. E.

2018-05-01

The burning core of an inertial confinement fusion (ICF) plasma produces bright x-rays at stagnation that can directly diagnose core conditions essential for comparison to simulations and understanding fusion yields. These x-rays also backlight the surrounding shell of warm, dense matter, whose properties are critical to understanding the efficacy of the inertial confinement and global morphology. We show that the absorption and fluorescence spectra of mid-Z impurities or dopants in the warm dense shell can reveal the optical depth, temperature, and density of the shell and help constrain models of warm, dense matter. This is illustrated by the example of a high-resolution spectrum collected from an ICF plasma with a beryllium shell containing native iron impurities. Analysis of the iron K-edge provides model-independent diagnostics of the shell density (2.3 × 1024 e/cm3) and temperature (10 eV), while a 12-eV red shift in Kβ and 5-eV blue shift in the K-edge discriminate among models of warm dense matter: Both shifts are well described by a self-consistent field model based on density functional theory but are not fully consistent with isolated-atom models using ad-hoc density effects.

Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion.

PubMed

Hoekstra, D

1982-06-08

The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, which occurs when the local concentration of the probe increases upon Ca2+-induced phase separation in phosphatidylserine (PS) bilayers [Hoekstra, D. (1982) Biochemistry 21, 1055-1061]. Fusion was determined by using the resonance energy transfer fusion assay [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which monitors the mixing of fluorescent lipid donor and acceptor molecules, resulting in an increase in energy transfer efficiency. The results show that in the presence of Ca2+, fusion proceeds much more rapidly (t 1/2 less than 5 s) than the process of phase separation (T 1/2 congruent to 1 min). Mg2+ also induced fusion, albeit at higher concentrations than Ca2+. Mg2+-induced phase separation were not detected, however. Subthreshold concentrations of Ca2+ (0.5 mM) or Mg2+ (2 mM) induced extensive fusion of PS-containing vesicles in poly(ethylene glycol) containing media. This effect did not appear to be a poly(ethylene glycol)-facilitated enhancement of cation binding to the bilayer, and consequently Ca2+-induced phase separation was not observed. The results suggest that macroscopic phase separation may facilitate but does not induced the fusion process and is therefore, not directly involved in the actual fusion mechanism. The fusion experiments performed in the presence of poly(ethylene glycol) suggest that the degree of bilayer dehydration and the creation of "point defects" in the bilayer without rigorous structural rearrangements in the membrane are dominant factors in the initial fusion events.

High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

PubMed Central

Kreutzberger, Alex J.B.; Kiessling, Volker; Tamm, Lukas K.

2015-01-01

Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays. PMID:26200867

Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

NASA Astrophysics Data System (ADS)

Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

2015-11-01

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals. Electronic supplementary information (ESI) available: Schematic representation of PeryBPb1-fused DsRed-Monomer, fluorescence spectra of perylene crystals and DsRed-Monomer, and emission spectra of DsRed-Monomer at various excitation wavelengths. See DOI: 10.1039/c5nr06471f

Excess cholesterol inhibits glucose-stimulated fusion pore dynamics in insulin exocytosis.

PubMed

Xu, Yingke; Toomre, Derek K; Bogan, Jonathan S; Hao, Mingming

2017-11-01

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic β-cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from β-cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in β-cells and contribute to β-cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs β-cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single-granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose-stimulated fusion events, and modulated the proportion of full fusion and kiss-and-run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol-overloaded β-cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol-induced phosphatidylinositol 4,5-bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss-and-run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Aggregation of endosomal-vacuolar compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tatsumi, Akinori; Shoji, Jun-ya; Kikuma, Takashi

2007-10-19

Previously, we found that deletion of Aovps24, an ortholog of Saccharomyces cerevisiae VPS24, that encodes an ESCRT (endosomal sorting complex required for transport)-III component required for late endosomal function results in fragmented and aggregated vacuoles. Although defective late endosomal function is likely responsible for this phenotype, critical lack of our knowledge on late endosomes in filamentous fungi prevented us from further characterization. In this study, we identified late endosomes of Aspergillus oryzae, by expressing a series of fusion proteins of fluorescent proteins with orthologs of late endosomal proteins. Using these fusion proteins as markers, we observed late endosomes in themore » wild type strain and the Aovps24 disruptant and demonstrated that late endosomes are aberrantly aggregated in the Aovps24 disruptant. Moreover, we revealed that the aggregated late endosomes have features of vacuoles as well. As deletion of another ESCRT-III component-encoding gene, Aovps2, resulted in similar phenotypes to that in the Aovps24 disruptant, phenotypes of the Aovps24 disruptant are probably due to defective late endosomal function.« less

Warthin-like Mucoepidermoid Carcinoma: A Combined Study of Fluorescence In Situ Hybridization and Whole-slide Imaging.

PubMed

Ishibashi, Kenichiro; Ito, Yohei; Masaki, Ayako; Fujii, Kana; Beppu, Shintaro; Sakakibara, Takeo; Takino, Hisashi; Takase, Hiroshi; Ijichi, Kei; Shimozato, Kazuo; Inagaki, Hiroshi

2015-11-01

There has been some debate as to whether a subset of metaplastic Warthin tumors (mWTs) harbor the mucoepidermoid carcinoma (MEC)-associated CRTC1-MAML2 fusion. We analyzed 15 tumors originally diagnosed as mWT (mWT-like tumors), 2 of which had concurrent MECs. We looked for the CRTC1/3-MAML2 fusion transcripts and performed immunohistochemistry for p63 and fluorescence in situ hybridization (FISH) for the MAML2 split. To localize MAML2 split-positive cells at the cellular level, whole tumor tissue sections were digitalized (whole-slide imaging [WSI]). The CRTC1-MAML2, but not CRTC3-MAML2 was detected in 5/15 mWT-like tumors. FISH-WSI results showed that all epithelial cells harbored the MAML2 split in fusion-positive mWT-like tumors and were totally negative in fusion-negative mWT-like tumors. A review of the hematoxylin and eosin-stained slides showed that morphology of the "metaplastic" epithelium was virtually indistinguishable between fusion-positive and fusion-negative tumors. However, oncocytic bilayered tumor epithelium, characteristic to typical WT, was always found somewhere in the fusion-negative tumors but not in the fusion-positive tumors. This distinguishing histologic finding enabled 5 pathologists to easily differentiate the 2 tumor groups with 100% accuracy. The age and sex distribution of fusion-positive mWT-like tumor cases was similar to that of fusion-positive MEC cases and significantly different from those of fusion-negative mWT-like tumor and typical WT cases. In addition, only fusion-positive mWT-like tumors possessed concurrent low-grade MECs. In conclusion, a subset of mWT-like tumors were positive for the CRTC1-MAML2 fusion and had many features that are more in accord with MEC than with WT. The term Warthin-like MEC should be considered for fusion-positive mWT-like tumors.

Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelsey, D.R.; Flanagan, T.D.; Young, J.E.

1991-06-01

Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectivenessmore » of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.« less

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

PubMed

Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R

2003-06-24

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

Fixed-Cell Imaging of Schizosaccharomyces pombe.

PubMed

Hagan, Iain M; Bagley, Steven

2016-07-01

The acknowledged genetic malleability of fission yeast has been matched by impressive cytology to drive major advances in our understanding of basic molecular cell biological processes. In many of the more recent studies, traditional approaches of fixation followed by processing to accommodate classical staining procedures have been superseded by live-cell imaging approaches that monitor the distribution of fusion proteins between a molecule of interest and a fluorescent protein. Although such live-cell imaging is uniquely informative for many questions, fixed-cell imaging remains the better option for others and is an important-sometimes critical-complement to the analysis of fluorescent fusion proteins by live-cell imaging. Here, we discuss the merits of fixed- and live-cell imaging as well as specific issues for fluorescence microscopy imaging of fission yeast. © 2016 Cold Spring Harbor Laboratory Press.

Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry.

PubMed

Morell, Montse; Espargaro, Alba; Aviles, Francesc Xavier; Ventura, Salvador

2008-01-01

We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein-protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.

Novel push-pull fluorescent dyes - 7-(diethylamino)furo- and thieno[3,2-c]coumarins derivatives: structure, electronic spectra and TD-DFT study

NASA Astrophysics Data System (ADS)

Akchurin, Igor O.; Yakhutina, Anna I.; Bochkov, Andrei Y.; Solovjova, Natalya P.; Medvedev, Michael G.; Traven, Valerii F.

2018-05-01

Novel push-pull fluorescent dyes - 7-(diethylamino)furo- and 7-(diethylamino)thieno[3,2-c]coumarins derivatives have been synthesized using formyl derivatives of furo- and thieno[3,2-c]coumarins as starting materials. Electron absorption and fluorescent spectra of the dyes have been recorded in different solvents. Structure and solvent effects on the dyes spectral characteristics were analyzed. The fusion of five-membered heterocycle to coumarin provides a definite increase of Stokes shifts in all solvents and results in higher quantum yields of fluorescence. The absorption and emission bands of thieno[3,2-c] coumarin derivatives are definitely shifted to the red region (3-30 nm) compared to similar derivatives of furo[3,2-c]coumarin. TD-DFT calculations of some of the studied compounds have shown that hybrid DFT functionals and adequate representation of molecular environment are essential for obtaining accurate UV-Vis absorption spectra for the dyes with extended π-system. The longest-wave electron transitions in the studied compounds were computationally shown to be of push-pull nature.

Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

PubMed Central

Bakayan, Adil; Vaquero, Cecilia F.; Picazo, Fernando; Llopis, Juan

2011-01-01

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene. PMID:21589654

Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

DOEpatents

Waldo, Geoffrey S.

2007-09-18

The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background.

PubMed

Stöhr, Katharina; Siegberg, Daniel; Ehrhard, Tanja; Lymperopoulos, Konstantinos; Öz, Simin; Schulmeister, Sonja; Pfeifer, Andrea C; Bachmann, Julie; Klingmüller, Ursula; Sourjik, Victor; Herten, Dirk-Peter

2010-10-01

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

PubMed Central

Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

2012-01-01

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

Practical three color live cell imaging by widefield microscopy

PubMed Central

Xia, Jianrun; Kim, Song Hon H.; Macmillan, Susan

2006-01-01

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope. PMID:16909160

Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

PubMed Central

Kiessling, Volker; Liang, Binyong; Kreutzberger, Alex J. B.; Tamm, Lukas K.

2017-01-01

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

Long Term Non-Invasive Imaging of Embryonic Stem Cells Using Reporter Genes

PubMed Central

Sun, Ning; Lee, Andrew; Wu, Joseph C.

2013-01-01

Development of non-invasive and accurate methods to track cell fate following delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. Here we describe a protocol for the in vivo monitoring of stem cell survival, proliferation, and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes using lentiviral transduction. We used fluorescence activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography imaging to track them in vivo, and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. PMID:19617890

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

PubMed Central

Woo, Sang Su; James, Declan J.; Martin, Thomas F. J.

2017-01-01

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

PubMed Central

Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

2016-01-01

Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

Display of adenoregulin with a novel Pichia pastoris cell surface display system.

PubMed

Ren, Ren; Jiang, Zhengbing; Liu, Meiyun; Tao, Xinyi; Ma, Yushu; Wei, Dongzhi

2007-02-01

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo1p with its own secretion signal sequence or the alpha-factor secretion signal sequence, a polyhistidine (6xHis) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

PubMed

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

PubMed

Suetsugu, Atsushi; Matsumoto, Takuro; Hasegawa, Kosuke; Nakamura, Miki; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

2016-08-01

Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Identification of novel recurrent ETV6-IGH fusions in primary central nervous system lymphoma.

PubMed

Bruno, Aurélie; Labreche, Karim; Daniau, Maïlys; Boisselier, Blandine; Gauchotte, Guillaume; Royer-Perron, Louis; Rahimian, Amithys; Lemoine, Frédéric; de la Grange, Pierre; Guégan, Justine; Bielle, Franck; Polivka, Marc; Adam, Clovis; Meyronet, David; Figarella-Branger, Dominique; Villa, Chiara; Chrétien, Fabrice; Eimer, Sandrine; Davi, Frédéric; Rousseau, Audrey; Houillier, Caroline; Soussain, Carole; Mokhtari, Karima; Hoang-Xuan, Khê; Alentorn, Agusti

2018-02-08

Primary central nervous system lymphoma (PCNSL) represents a particular entity within non-Hodgkin lymphomas and is associated with poor outcome. The present study addresses the potential clinical relevance of chimeric transcripts in PCSNL discovered by using RNA-sequencing (RNA-Seq). Seventy-two immunocompetent and newly diagnosed PCNSL cases were included in the present study. Among them, six were analyzed by RNA-seq to detect new potential fusion transcripts. We confirmed the results in the remaining 66 PCNSL. The gene fusion was validated by fluorescence in situ hybridization (FISH) using formalin-fixed paraffin-embedded (FFPE) samples. We assessed the biological and clinical impact of one new gene fusion. We identified a novel recurrent gene fusion ETV6-IgH. Overall, ETV6-IgH was found in 13 out of 72 PCNSL (18%). No fusion conserved an intact functional domain of ETV6 and ETV6 was significantly underexpressed at gene level, suggesting an ETV6 haploinsufficiency mechanism. The presence of the gene fusion was also validated by FISH in FFPE samples. Finally, PCNSL samples harboring ETV6-IgH showed a better prognosis in multivariate analysis, p-value=0.03, HR=0.33, 95% interval confidence (IC95) [0.12-0.88]. The overall survival at 5 years was of 69% for PCNSL harboring ETV6-IgH vs 29% for samples without this gene fusion. ETV6-IgH is a new potential surrogate marker of PCNSL with favorable prognosis with ETV6 haploinsuffiency as a possible mechanism. The potential clinical impact of ETV6-IgH should be validated in larger prospective studies. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

PubMed Central

2013-01-01

Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient. PMID:23687945

Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement.

PubMed

Kallemeijn, Wouter W; Scheij, Saskia; Voorn-Brouwer, Tineke M; Witte, Martin D; Verhoek, Marri; Overkleeft, Hermen S; Boot, Rolf G; Aerts, Johannes M F G

2016-09-15

β-Glucoside-configured cyclophellitols are activity-based probes (ABPs) that allow sensitive detection of β-glucosidases. Their applicability to detect proteins fused with β-glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M-777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4-methylumbelliferyl β-d-lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre-blocking with conduritol β-epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β-glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high-resolution detection moieties) should assist further research in living cells and organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fusion-activated cation entry (FACE) via P2X₄ couples surfactant secretion and alveolar fluid transport.

PubMed

Thompson, Kristin E; Korbmacher, Jonas P; Hecht, Elena; Hobi, Nina; Wittekindt, Oliver H; Dietl, Paul; Kranz, Christine; Frick, Manfred

2013-04-01

Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X₄ receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (Isc) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in Isc. Overexpressing dominant-negative P2X₄ abrogated this effect by ∼50%, whereas potentiating P2X4 lead to ∼80% increase in Isc. Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae

PubMed Central

Higuchi-Sanabria, Ryo; Charalel, Joseph K.; Viana, Matheus P.; Garcia, Enrique J.; Sing, Cierra N.; Koenigsberg, Andrea; Swayne, Theresa C.; Vevea, Jason D.; Boldogh, Istvan R.; Rafelski, Susanne M.; Pon, Liza A.

2016-01-01

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud. PMID:26764088

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

PubMed Central

Provost, Christopher R.; Sun, Luo

2010-01-01

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells. PMID:20485262

Advantages of Data Fusion: First Multivariate Curve Resolution Analysis of Fused Liquid Chromatographic Second-Order Data with Dual Diode Array-Fluorescent Detection.

PubMed

Pellegrino Vidal, Rocío B; Ibañez, Gabriela A; Escandar, Graciela M

2017-03-07

For the first time, liquid chromatography-diode array detection (LC-DAD) and liquid-chromatography fluorescence detection (LC-FLD) second-order data, collected in a single chromatographic run, were fused and chemometrically processed for the quantitation of coeluting analytes. Two different experimental mixtures composed of fluorescent and nonfluorescent endocrine disruptors were analyzed. Adequate pretreatment of the matrices before their fusion was crucial to attain reliable results. Multivariate curve resolution-alternating least-squares (MCR-ALS) was applied to LC-DAD, LC-FLD, and fused LC-DAD-FLD data. Although different degrees of improvement are observed when comparing the fused matrix results in relation to those obtained using a single detector, clear benefits of data fusion are demonstrated through: (1) the obtained limits of detection in the ranges 2.1-24 ng mL -1 and 0.9-6.3 ng mL -1 for the two evaluated systems and (2) the low relative prediction errors, below 7% in all cases, indicating good recoveries and precision. The feasibility of fusing data and its advantages in the analysis of real samples was successfully assessed through the study of spiked tap, underground, and river water samples.

Paramyxovirus fusion: Real-time measurement of parainfluenza virus 5 virus-cell fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connolly, Sarah A.; Lamb, Robert A.

2006-11-25

Although cell-cell fusion assays are useful surrogate methods for studying virus fusion, differences between cell-cell and virus-cell fusion exist. To examine paramyxovirus fusion in real time, we labeled viruses with fluorescent lipid probes and monitored virus-cell fusion by fluorimetry. Two parainfluenza virus 5 (PIV5) isolates (W3A and SER) and PIV5 containing mutations within the fusion protein (F) were studied. Fusion was specific and temperature-dependent. Compared to many low pH-dependent viruses, the kinetics of PIV5 fusion was slow, approaching completion within several minutes. As predicted from cell-cell fusion assays, virus containing an F protein with an extended cytoplasmic tail (rSV5 F551)more » had reduced fusion compared to wild-type virus (W3A). In contrast, virus-cell fusion for SER occurred at near wild-type levels, despite the fact that this isolate exhibits a severely reduced cell-cell fusion phenotype. These results support the notion that virus-cell and cell-cell fusion have significant differences.« less

Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses

PubMed Central

Sinha, Raunak; Ahmed, Saheeb; Jahn, Reinhard; Klingauf, Jurgen

2011-01-01

Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission. PMID:21844343

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

PubMed

Srivastava, Preeti; Deb, J K

2002-07-02

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Red to far-red multispectral fluorescence image fusion for detection of fecal contamination on apples

USDA-ARS?s Scientific Manuscript database

This research developed a multispectral algorithm derived from hyperspectral line-scan fluorescence imaging under violet/blue LED excitation for detection of fecal contamination on Golden Delicious apples. Using a hyperspectral line-scan imaging system consisting of an EMCCD camera, spectrograph, an...

Analysis of the function of the photoreceptors phytochrome B and phytochrome D in Nicotiana plumbaginifolia and Arabidopsis thaliana.

PubMed

Fernández, Aurora Piñas; Gil, Patricia; Valkai, Ildiko; Nagy, Ferenc; Schäfer, Eberhard

2005-05-01

To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.

Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

PubMed

Bazl, M Rajabi; Rasaee, M J; Foruzandeh, M; Rahimpour, A; Kiani, J; Rahbarizadeh, F; Alirezapour, B; Mohammadi, M

2007-02-01

There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

Fluorescence and absorption spectroscopy for warm dense matter studies and ICF plasma diagnostics

DOE PAGES

Hansen, Stephanie B.; Harding, Eric C.; Knapp, Patrick F.; ...

2018-03-07

The burning core of an inertial confinement fusion (ICF) plasma produces bright x-rays at stagnation that can directly diagnose core conditions essential for comparison to simulations and understanding fusion yields. These x-rays also backlight the surrounding shell of warm, dense matter, whose properties are critical to understanding the efficacy of the inertial confinement and global morphology. In this work, we show that the absorption and fluorescence spectra of mid-Z impurities or dopants in the warm dense shell can reveal the optical depth, temperature, and density of the shell and help constrain models of warm, dense matter. This is illustrated bymore » the example of a high-resolution spectrum collected from an ICF plasma with a beryllium shell containing native iron impurities. Lastly, analysis of the iron K-edge provides model-independent diagnostics of the shell density (2.3 × 10 24 e/cm 3) and temperature (10 eV), while a 12-eV red shift in Kβ and 5-eV blue shift in the K-edge discriminate among models of warm dense matter: Both shifts are well described by a self-consistent field model based on density functional theory but are not fully consistent with isolated-atom models using ad-hoc density effects.« less

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

PubMed

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro translocation experiments with RxLR-reporter fusion proteins of Avr1b from Phytophthora sojae and AVR3a from Phytophthora infestans fail to demonstrate specific autonomous uptake in plant and animal cells.

PubMed

Wawra, Stephan; Djamei, Armin; Albert, Isabell; Nürnberger, Thorsten; Kahmann, Regine; van West, Pieter

2013-05-01

Plant-pathogenic oomycetes have a large set of secreted effectors that can be translocated into their host cells during infection. One group of these effectors are the RxLR effectors for which it has been shown, in a few cases, that the RxLR motif is important for their translocation. It has been suggested that the RxLR-leader sequences alone are enough to translocate the respective effectors into eukaryotic cells through binding to surface-exposed phosphoinositol-3-phosphate. These conclusions were primary based on translocation experiments conducted with recombinant fusion proteins whereby the RxLR leader of RxLR effectors (i.e., Avr1b from Phytophthora sojae) were fused to the green fluorescent protein reporter-protein. However, we failed to observe specific cellular uptake for a comparable fusion protein where the RxLR leader of the P. infestans AVR3a was fused to monomeric red fluorescent protein. Therefore, we reexamined the ability of the reported P. sojae AVR1b RxLR leader to enter eukaryotic cells. Different relevant experiments were performed in three independent laboratories, using fluorescent reporter fusion constructs of AVR3a and Avr1b proteins in a side-by-side comparative study on plant tissue and human and animal cells. We report that we were unable to obtain conclusive evidence for specific RxLR-mediated translocation.

Two-Photon Fluorescent Probe for Monitoring Autophagy via Fluorescence Lifetime Imaging.

PubMed

Hou, Liling; Ning, Peng; Feng, Yan; Ding, Yaqi; Bai, Lei; Li, Lin; Yu, Haizhu; Meng, Xiangming

2018-06-19

We reported the first lysosome targeted two-photon fluorescent probe (Lyso-NP) as a viscosity probe for monitoring autophagy. The fluorescence lifetime of Lyso-NP exhibited an excellent linear relationship with viscosity value ( R 2 = 0.99, x = 0.39). Lyso-NP also showed the specific capability for imaging lysosomal viscosity under two-photon excitation at 860 nm along with good biocompatibility. More importantly, Lyso-NP could be used to monitor the autophagy process in living cells by quantitatively detecting lysosomal viscosity changes during the membrane fusion process via two-photon fluorescence lifetime imaging.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

PubMed

Mas, Abraham; Amenós, Montse; Lois, L Maria

2016-01-01

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

Expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration.

PubMed

McCormick, Aleesha M; Jarmusik, Natalie A; Endrizzi, Elizabeth J; Leipzig, Nic D

2014-01-22

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

PubMed Central

McCormick, Aleesha M.; Jarmusik, Natalie A.; Endrizzi, Elizabeth J.; Leipzig, Nic D.

2014-01-01

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification. PMID:24513608

Ricin A chain reaches the endoplasmic reticulum after endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Qiong; Department of Biochemistry and Molecular Biology, Ningbo University Medical School, Ningbo 315211; Zhan Jinbiao

Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purifiedmore » by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.« less

Intracellular Localization of Arabidopsis Sulfurtransferases1

PubMed Central

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D.; Papenbrock, Jutta

2004-01-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism. PMID:15181206

Intracellular localization of Arabidopsis sulfurtransferases.

PubMed

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D; Papenbrock, Jutta

2004-06-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.

Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaturvedi, Sonali; Rao, A.L.N., E-mail: arao@ucr.edu

2014-09-15

In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase.more » Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER.« less

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

PubMed

Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

2009-05-01

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

PubMed

Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

2013-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms, which may play an important role in DR4-mediated redox signaling in CAECs and consequently endothelial dysfunction.

Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis

PubMed Central

Stannek, Lorena; Gunka, Katrin; Care, Rachel A.; Gerth, Ulf; Commichau, Fabian M.

2015-01-01

The Gram-positive model bacterium Bacillus subtilis contains two glutamate dehydro genase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (gudBCR) in the laboratory strain 168 due to a perfect 18 bp-long direct repeat that renders the GudB enzyme inactive and unstable. Although constitutively expressed the GudBCR protein can hardly be detected in B. subtilis as it is rapidly degraded within stationary growth phase. Its high instability qualifies GudBCR as a model substrate for studying protein turnover in B. subtilis. Recently, we have developed a visual screen to monitor the GudBCR stability in the cell using a GFP-GudBCR fusion. Using fluorescent microscopy we found that the GFP protein is simultaneously degraded together with GudBCR. This allows us to analyze the stability of GudBCR in living cells. By combining the visual screen with a transposon mutagenesis approach we looked for mutants that show an increased fluorescence signal compared to the wild type indicating a stabilized GFP-GudBCR fusion. We observed, that disruption of the arginine kinase encoding gene mcsB upon transposon insertion leads to increased amounts of the GFP-GudBCR fusion in this mutant. Deletion of the cognate arginine phosphatase YwlE in contrast results in reduced levels of the GFP-GudBCR fusion. Recently, it was shown that the kinase McsB is involved in phosphorylation of GudBCR on arginine residues. Here we show that selected arginine-lysine point mutations of GudBCR exhibit no influence on degradation. The activity of McsB and YwlE, however, are crucial for the activation and inhibition, respectively, of a proteolytic machinery that efficiently degrades the unstable GudBCR protein in B. subtilis. PMID:25610436

Kiss-and-Run Is a Significant Contributor to Synaptic Exocytosis and Endocytosis in Photoreceptors

PubMed Central

Wen, Xiangyi; Saltzgaber, Grant W.; Thoreson, Wallace B.

2017-01-01

Accompanying sustained release in darkness, rod and cone photoreceptors exhibit rapid endocytosis of synaptic vesicles. Membrane capacitance measurements indicated that rapid endocytosis retrieves at least 70% of the exocytotic membrane increase. One mechanism for rapid endocytosis is kiss-and-run fusion where vesicles briefly contact the plasma membrane through a small fusion pore. Release can also occur by full-collapse in which vesicles merge completely with the plasma membrane. We assessed relative contributions of full-collapse and kiss-and-run in salamander photoreceptors using optical techniques to measure endocytosis and exocytosis of large vs. small dye molecules. Incubation with small dyes (SR101, 1 nm; 3-kDa dextran-conjugated Texas Red, 2.3 nm) loaded rod and cone synaptic terminals much more readily than larger dyes (10-kDa Texas Red, 4.6 nm; 10-kDa pHrodo, 4.6 nm; 70-kDa Texas Red, 12 nm) consistent with significant uptake through 2.3–4.6 nm fusion pores. By using total internal reflection fluorescence microscopy (TIRFM) to image individual vesicles, when rods were incubated simultaneously with Texas Red and AlexaFluor-488 dyes conjugated to either 3-kDa or 10-kDa dextran, more vesicles loaded small molecules than large molecules. Using TIRFM to detect release by the disappearance of dye-loaded vesicles, we found that SR101 and 3-kDa Texas Red were released from individual vesicles more readily than 10-kDa and 70-kDa Texas Red. Although 10-kDa pHrodo was endocytosed poorly like other large dyes, the fraction of release events was similar to SR101 and 3-kDa Texas Red. We hypothesize that while 10-kDa pHrodo may not exit through a fusion pore, release of intravesicular protons can promote detection of fusion events by rapidly quenching fluorescence of this pH-sensitive dye. Assuming that large molecules can only be released by full-collapse whereas small molecules can be released by both modes, our results indicate that 50%–70% of release from rods involves kiss-and-run with 2.3–4.6 nm fusion pores. Rapid retrieval of vesicles by kiss-and-run may limit membrane disruption of release site function during ongoing release at photoreceptor ribbon synapses. PMID:28979188

Fluorescence correlation spectroscopy study of the complexation of DNA hybrids, IgG antibody, and a chimeric protein of IgG-binding ZZ domains fused with a carbohydrate binding module.

PubMed

Rosa, A M M; Prazeres, D M F; Paulo, P M R

2017-06-28

Fluorescence correlation spectroscopy (FCS) was used to characterize the molecular interactions between the four components of a DNA recognition system. A fluorescent DNA probe was used to assess: (i) the hybridization with a complementary biotin-labeled target, (ii) the complexation of the resulting hybrid and an anti-biotin antibody, and (iii) the binding of the latter complex to a ZZ-CBM fusion protein that combines small synthetic IgG Fc-binding Z domains with a carbohydrate binding module (CBM). These binding interactions were monitored by exposing the fluorescent DNA probe to different amounts and combinations of the other molecules in solution. Through the analysis of FCS autocorrelation curves, an association constant (K a ) of 2.9 × 10 7 M -1 was estimated for DNA·DNA hybridization, and the presence of (non-) complementary target DNA in solution could be discriminated. The specific capture of biotinylated DNA hybrids by anti-biotin IgG was verified, with an apparent K a of 2.5 × 10 6 M -1 . The increment in the diffusion time measured when the DNA·DNA:antibody complexes were in contact with the ZZ-CBM fusion protein suggested that the binding occurs at a stoichiometric ratio of DNA/antibody complex to fusion larger than 1 : 1. The FCS-derived information obtained is useful to gain insight into molecular interactions involved in diagnostic assays.

The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro.

PubMed

Byrne, Richard D; Larijani, Banafshé; Poccia, Dominic L

2016-01-01

FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.

Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.

PubMed

Norman, Christel; Liu, Zhen-Wei; Rigby, Paul; Raso, Albert; Petrov, Yevgeniy; Martinac, Boris

2005-07-01

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.

Embryonic Stem Cells Contribute to Mouse Chimeras in the Absence of Detectable Cell Fusion

PubMed Central

Kidder, Benjamin L.; Oseth, Leann; Miller, Shanna; Hirsch, Betsy; Verfaillie, Catherine

2008-01-01

Abstract Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras. PMID:18338954

Proteins on exocytic vesicles mediate calcium-triggered fusion.

PubMed Central

Vogel, S S; Zimmerberg, J

1992-01-01

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous. Images PMID:1584814

Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

PubMed

Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

2015-11-15

Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. Copyright © 2015 Elsevier Inc. All rights reserved.

Using a Split Luciferase Assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins

PubMed Central

Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

2015-01-01

Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. PMID:26022509

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Preparation of triple-negative breast cancer vaccine through electrofusion with day-3 dendritic cells.

PubMed

Zhang, Peng; Yi, Shuhong; Li, Xi; Liu, Ruilei; Jiang, Hua; Huang, Zenan; Liu, Yu; Wu, Juekun; Huang, Yong

2014-01-01

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) in human immune system. DC-based tumor vaccine has met with some success in specific malignancies, inclusive of breast cancer. In this study, we electrofused MDA-MB-231 breast cancer cell line with day-3 DCs derived from peripheral blood monocytes, and explored the biological characteristics of fusion vaccine and its anti-tumor effects in vitro. Day-3 mature DCs were generated from day-2 immature DCs by adding cocktails composed of TNF-α, IL-1β, IL-6 and PEG2. Day-3 mature DCs were identified and electofused with breast cancer cells to generate fusion vaccine. Phenotype of fusion cells were identified by fluorescence microscope and flow cytometer. The fusion vaccine was evaluated for T cell proliferation, secretion of IL-12 and IFN-γ, and induction of tumor-specific CTL response. Despite differences in morphology, day-3 and day-7 DC expressed similar surface markers. The secretion of IL-12 and IFN-γ in fusion vaccine group was much higher than that in the control group. Compared with control group, DC-tumor fusion vaccine could better stimulate the proliferation of allogeneic T lymphocytes and kill more breast cancer cells (MDA-MB-231) in vitro. Day-3 DCs had the same function as the day-7 DCs, but with a shorter culture period. Our findings suggested that day-3 DCs fused with whole apoptotic breast cancer cells could elicit effective specific antitumor T cell responses in vitro and may be developed into a prospective candidate for adoptivet immunotherapy.

Nuclear fusion during yeast mating occurs by a three-step pathway.

PubMed

Melloy, Patricia; Shen, Shu; White, Erin; McIntosh, J Richard; Rose, Mark D

2007-11-19

In Saccharomyces cerevisiae, mating culminates in nuclear fusion to produce a diploid zygote. Two models for nuclear fusion have been proposed: a one-step model in which the outer and inner nuclear membranes and the spindle pole bodies (SPBs) fuse simultaneously and a three-step model in which the three events occur separately. To differentiate between these models, we used electron tomography and time-lapse light microscopy of early stage wild-type zygotes. We observe two distinct SPBs in approximately 80% of zygotes that contain fused nuclei, whereas we only see fused or partially fused SPBs in zygotes in which the site of nuclear envelope (NE) fusion is already dilated. This demonstrates that SPB fusion occurs after NE fusion. Time-lapse microscopy of zygotes containing fluorescent protein tags that localize to either the NE lumen or the nucleoplasm demonstrates that outer membrane fusion precedes inner membrane fusion. We conclude that nuclear fusion occurs by a three-step pathway.

Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

PubMed Central

Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

2016-01-01

Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.

PubMed

Kerppola, Tom K

2006-01-01

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.

Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation.

PubMed

Muraki, Michiro

2016-01-01

Human Fas ligand extracellular domain has been investigated as an important target protein in the field of medical biotechnology. In a recent study, the author developed an effective method to produce biologically active human Fas ligand extracellular domain derivatives using site-specific chemical modifications. A human Fas ligand extracellular domain derivative containing a reactive cysteine residue within its N-terminal tag sequence, which locates not proximal to the binding interface between the ligand and the receptor in terms of the three-dimensional structure, was modified by Fluorescein-5-Maleimide without impairing the specific binding activity toward human Fas receptor extracellular domain. The purified protein sample free of low molecular-weight contaminants showed a characteristic fluorescence spectrum derived from the attached Fluorescein moieties, and formed a stable binding complex with human Fas receptor extracellular domain-human IgG1 Fc domain fusion protein in solution. The conjugation number of the fluorochrome was estimated to be 2.5 per a single human Fas ligand extracellular domain trimer from the ratio of the absorbance value at 280 nm to that at 495 nm. A functional fluorescent human Fas ligand extracellular domain derivative was prepared via a site-specific conjugation of fluorochrome, which was guided by the three-dimensional structure information on the ligand-receptor complex. Fluorescent derivatives created by this method may contribute to the development of an improved diagnosis system for the diseases related to Fas receptor.

Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.

PubMed

Poe, Jerrod A; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E

2014-04-01

Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pszon-Bartosz, Kamila; Hansen, Jesper S.; Technical University of Denmark, Department of Micro- and Nanotechnology, DK-2800 Kongens Lyngby

2011-03-04

Research highlights: {yields} We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). {yields} Maximal fusion obtained was almost 150,000 porin insertions during 20 min. {yields} Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a proteinmore » incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10{sup 5} FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm{sup 2} within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.« less

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

PubMed Central

Shaner, Nathan C.; Lambert, Gerard G.; Chammas, Andrew; Ni, Yuhui; Cranfill, Paula J.; Baird, Michelle A.; Sell, Brittney R.; Allen, John R.; Day, Richard N.; Israelsson, Maria; Davidson, Michael W.; Wang, Jiwu

2013-01-01

Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins. PMID:23524392

The SI strain of measles virus derived from a patient with subacute sclerosing panencephalitis possesses typical genome alterations and unique amino acid changes that modulate receptor specificity and reduce membrane fusion activity.

PubMed

Seki, Fumio; Yamada, Kentaro; Nakatsu, Yuichiro; Okamura, Koji; Yanagi, Yusuke; Nakayama, Tetsuo; Komase, Katsuhiro; Takeda, Makoto

2011-11-01

Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity.

The SI Strain of Measles Virus Derived from a Patient with Subacute Sclerosing Panencephalitis Possesses Typical Genome Alterations and Unique Amino Acid Changes That Modulate Receptor Specificity and Reduce Membrane Fusion Activity ▿ ‡

PubMed Central

Seki, Fumio; Yamada, Kentaro; Nakatsu, Yuichiro; Okamura, Koji; Yanagi, Yusuke; Nakayama, Tetsuo; Komase, Katsuhiro; Takeda, Makoto

2011-01-01

Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity. PMID:21917959

Oxygen transmittance correction for solar-induced chlorophyll fluorescence measured on proximal sensing: application to the NASA-GSFC fusion tower

USDA-ARS?s Scientific Manuscript database

Since oxygen (O2) absorption of light becomes more pronounced at higher pressure levels, even a few meters distance between the target and the sensor can strongly affect canopy leaving Solar-Induced chlorophyll Fluorescence (SIF) retrievals. This study was conducted to quantify the consequent error ...

[Effects of redox state of disulfide bonds on the intrinsic fluorescence and denaturation of Trx-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea].

PubMed

Zhang, Teng; Feng, Juan; Li, Yang; Chen, Rui; Tang, Li-Xia; Pang, Xiao-Feng; Ren, Zheng-Long

2010-02-01

In the present paper, thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea, desigated as Trx-GcGASA and expressed prokaryotically, was purified and identified by using Ni(2+) -NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol (DTT), oxidized glutathione (GSSG), peroxide and guanidine hydrochloride (GdnHCl) by means of steady-state fluorescence spectroscopic methods. It was found that (1) at the neutral pH Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine residues. (2) The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8. (3) Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol x L(-1) GSSG or 5 mmol x L(-1) peroxide. The latter was roughly consistent with the antioxidative activity reported in vivo. (4) No matter whether 1 mmol x L(-1) DTT was absent or present, the fusion protein could not be fully unfolded with lambda(max) < 350 nm following the treatment of 6 mol x L(-1) GdnHCl. (5) Fusion protein Trx-GcGASA experienced GdnHCl-induced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change (deltaG) of 3.7 kJ x mol(-1). However, it was not the case for reduced Trx-GcGASA protein. The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics, kinetics and refolding process of Trx-GcGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

NASA Technical Reports Server (NTRS)

Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

2002-01-01

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Costantini, Lindsey M.; Irvin, Susan C.; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFPmore » enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.« less

Thermal precipitation fluorescence assay for protein stability screening.

PubMed

Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis.

PubMed

Weber, Kristoffer; Bartsch, Udo; Stocking, Carol; Fehse, Boris

2008-04-01

Functional gene analysis requires the possibility of overexpression, as well as downregulation of one, or ideally several, potentially interacting genes. Lentiviral vectors are well suited for this purpose as they ensure stable expression of complementary DNAs (cDNAs), as well as short-hairpin RNAs (shRNAs), and can efficiently transduce a wide spectrum of cell targets when packaged within the coat proteins of other viruses. Here we introduce a multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors designed according to the "building blocks" principle. Using a wide spectrum of different fluorescent markers, including drug-selectable enhanced green fluorescent protein (eGFP)- and dTomato-blasticidin-S resistance fusion proteins, LeGO vectors allow simultaneous analysis of multiple genes and shRNAs of interest within single, easily identifiable cells. Furthermore, each functional module is flanked by unique cloning sites, ensuring flexibility and individual optimization. The efficacy of these vectors for analyzing multiple genes in a single cell was demonstrated in several different cell types, including hematopoietic, endothelial, and neural stem and progenitor cells, as well as hepatocytes. LeGO vectors thus represent a valuable tool for investigating gene networks using conditional ectopic expression and knock-down approaches simultaneously.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulnessmore » as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.« less

Irradiation-induced fusion between giant vesicles and photoresponsive large unilamellar vesicles containing malachite green derivative.

PubMed

Uda, Ryoko M; Yoshikawa, Yuki; Kitaba, Moe; Nishimoto, Noriko

2018-07-01

Light-initiated fusion between vesicles has attracted much attention in the research community. In particular, fusion between photoresponsive and non-photoresponsive vesicles has been of much interest in the development of systems for the delivery of therapeutic agents to cells. We have performed fusion between giant vesicles (GVs) and photoresponsive smaller vesicles containing malachite green (MG) derivative, which undergoes ionization to afford a positive charge on the molecule by irradiation. The fusion proceeds as the concentration of GV lipid increases toward equimolarity with the lipid of the smaller vesicle. It is also dependent on the molar percentage of photoionized MG in the lipid of the smaller vesicle. On the other hand, the fusion is hardly affected by the anionic component of the GV. The photoinduced fusion was characterized by two methods, involving the mixing of lipid membranes and of aqueous contents. Fluorescence microscopy revealed that irradiation triggered the fusion of a single GV with the smaller vesicles containing MG. Copyright © 2018 Elsevier B.V. All rights reserved.

A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon.

PubMed

Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

2015-01-01

The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting.

A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon

PubMed Central

Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

2016-01-01

The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting. PMID:26858693

Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

PubMed

Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

2014-01-09

The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching*

PubMed Central

Billen, Bert; Brams, Marijke; Debaveye, Sarah; Remeeva, Alina; Alpizar, Yeranddy A.; Waelkens, Etienne; Kreir, Mohamed; Brüggemann, Andrea; Talavera, Karel; Nilius, Bernd; Voets, Thomas; Ulens, Chris

2015-01-01

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies. PMID:25829496

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

PubMed Central

Taylor, Matthew P.; Kratchmarov, Radomir; Enquist, Lynn W.

2013-01-01

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. PMID:23978901

Development of a Multi-modal Tissue Diagnostic System Combining High Frequency Ultrasound and Photoacoustic Imaging with Lifetime Fluorescence Spectroscopy

PubMed Central

Sun, Yang; Stephens, Douglas N.; Park, Jesung; Sun, Yinghua; Marcu, Laura; Cannata, Jonathan M.; Shung, K. Kirk

2010-01-01

We report the development and validate a multi-modal tissue diagnostic technology, which combines three complementary techniques into one system including ultrasound backscatter microscopy (UBM), photoacoustic imaging (PAI), and time-resolved laser-induced fluorescence spectroscopy (TR-LIFS). UBM enables the reconstruction of the tissue microanatomy. PAI maps the optical absorption heterogeneity of the tissue associated with structure information and has the potential to provide functional imaging of the tissue. Examination of the UBM and PAI images allows for localization of regions of interest for TR-LIFS evaluation of the tissue composition. The hybrid probe consists of a single element ring transducer with concentric fiber optics for multi-modal data acquisition. Validation and characterization of the multi-modal system and ultrasonic, photoacoustic, and spectroscopic data coregistration were conducted in a physical phantom with properties of ultrasound scattering, optical absorption, and fluorescence. The UBM system with the 41 MHz ring transducer can reach the axial and lateral resolution of 30 and 65 μm, respectively. The PAI system with 532 nm excitation light from a Nd:YAG laser shows great contrast for the distribution of optical absorbers. The TR-LIFS system records the fluorescence decay with the time resolution of ~300 ps and a high sensitivity of nM concentration range. Biological phantom constructed with different types of tissues (tendon and fat) was used to demonstrate the complementary information provided by the three modalities. Fluorescence spectra and lifetimes were compared to differentiate chemical composition of tissues at the regions of interest determined by the coregistered high resolution UBM and PAI image. Current results demonstrate that the fusion of these techniques enables sequentially detection of functional, morphological, and compositional features of biological tissue, suggesting potential applications in diagnosis of tumors and atherosclerotic plaques. PMID:21894259

Development of a Multi-modal Tissue Diagnostic System Combining High Frequency Ultrasound and Photoacoustic Imaging with Lifetime Fluorescence Spectroscopy.

PubMed

Sun, Yang; Stephens, Douglas N; Park, Jesung; Sun, Yinghua; Marcu, Laura; Cannata, Jonathan M; Shung, K Kirk

2008-01-01

We report the development and validate a multi-modal tissue diagnostic technology, which combines three complementary techniques into one system including ultrasound backscatter microscopy (UBM), photoacoustic imaging (PAI), and time-resolved laser-induced fluorescence spectroscopy (TR-LIFS). UBM enables the reconstruction of the tissue microanatomy. PAI maps the optical absorption heterogeneity of the tissue associated with structure information and has the potential to provide functional imaging of the tissue. Examination of the UBM and PAI images allows for localization of regions of interest for TR-LIFS evaluation of the tissue composition. The hybrid probe consists of a single element ring transducer with concentric fiber optics for multi-modal data acquisition. Validation and characterization of the multi-modal system and ultrasonic, photoacoustic, and spectroscopic data coregistration were conducted in a physical phantom with properties of ultrasound scattering, optical absorption, and fluorescence. The UBM system with the 41 MHz ring transducer can reach the axial and lateral resolution of 30 and 65 μm, respectively. The PAI system with 532 nm excitation light from a Nd:YAG laser shows great contrast for the distribution of optical absorbers. The TR-LIFS system records the fluorescence decay with the time resolution of ~300 ps and a high sensitivity of nM concentration range. Biological phantom constructed with different types of tissues (tendon and fat) was used to demonstrate the complementary information provided by the three modalities. Fluorescence spectra and lifetimes were compared to differentiate chemical composition of tissues at the regions of interest determined by the coregistered high resolution UBM and PAI image. Current results demonstrate that the fusion of these techniques enables sequentially detection of functional, morphological, and compositional features of biological tissue, suggesting potential applications in diagnosis of tumors and atherosclerotic plaques.

Time-resolved measurements of the hot-electron population in ignition-scale experiments on the National Ignition Facility (invited)

NASA Astrophysics Data System (ADS)

Hohenberger, M.; Albert, F.; Palmer, N. E.; Lee, J. J.; Döppner, T.; Divol, L.; Dewald, E. L.; Bachmann, B.; MacPhee, A. G.; LaCaille, G.; Bradley, D. K.; Stoeckl, C.

2014-11-01

In laser-driven inertial confinement fusion, hot electrons can preheat the fuel and prevent fusion-pellet compression to ignition conditions. Measuring the hot-electron population is key to designing an optimized ignition platform. The hot electrons in these high-intensity, laser-driven experiments, created via laser-plasma interactions, can be inferred from the bremsstrahlung generated by hot electrons interacting with the target. At the National Ignition Facility (NIF) [G. H. Miller, E. I. Moses, and C. R. Wuest, Opt. Eng. 43, 2841 (2004)], the filter-fluorescer x-ray (FFLEX) diagnostic-a multichannel, hard x-ray spectrometer operating in the 20-500 keV range-has been upgraded to provide fully time-resolved, absolute measurements of the bremsstrahlung spectrum with ˜300 ps resolution. Initial time-resolved data exhibited significant background and low signal-to-noise ratio, leading to a redesign of the FFLEX housing and enhanced shielding around the detector. The FFLEX x-ray sensitivity was characterized with an absolutely calibrated, energy-dispersive high-purity germanium detector using the high-energy x-ray source at NSTec Livermore Operations over a range of K-shell fluorescence energies up to 111 keV (U Kβ). The detectors impulse response function was measured in situ on NIF short-pulse (˜90 ps) experiments, and in off-line tests.

De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

PubMed

Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

2012-04-01

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

Analysis and quality control of carbohydrates in therapeutic proteins with fluorescence HPLC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Kun; Huang, Jian; Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054

Conbercept is an Fc fusion protein with very complicated carbohydrate profiles which must be carefully monitored through manufacturing process. Here, we introduce an optimized fluorescence derivatization high-performance liquid chromatographic method for glycan mapping in conbercept. Compared with conventional glycan analysis method, this method has much better resolution and higher reproducibility making it excellent for product quality control.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

PubMed

Woo, Sang Su; James, Declan J; Martin, Thomas F J

2017-03-15

Munc13-4 is a Ca 2+ -dependent SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca 2+ -evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca 2+ -binding C2 domains functions as a Ca 2+ sensor for SG exocytosis. Unexpectedly, Ca 2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4 + /Rab7 + /Rab11 + endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4 + /Rab7 + SGs, followed by a merge with Rab11 + endosomes, and depended on Ca 2+ binding to Munc13-4. Munc13-4 promoted the Ca 2+ -stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca 2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. © 2017 Woo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum.

PubMed

Corral-Ramos, Cristina; Roca, M Gabriela; Di Pietro, Antonio; Roncero, M Isabel G; Ruiz-Roldán, Carmen

2015-01-01

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum

PubMed Central

Corral-Ramos, Cristina; Roca, M Gabriela; Di Pietro, Antonio; Roncero, M Isabel G; Ruiz-Roldán, Carmen

2015-01-01

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum. PMID:25560310

Comprehensive Analysis of CBFβ-MYH11 Fusion Transcripts in Acute Myeloid Leukemia by RT-PCR Analysis

PubMed Central

Kadkol, ShriHari S.; Bruno, Annette; Dodge, Carol; Lindgren, Valerie; Ravandi, Farhad

2004-01-01

CBFβ-MYH11 fusion transcripts are expressed in acute myeloid leukemias of the M4Eo subtype. Patients who express CBFβ-MYH11 fusion transcripts respond favorably to high-dose chemotherapy and are generally spared allogeneic bone marrow transplantation. Hence it is important to identify this fusion in all patients with acute myeloid leukemia M4Eo leukemia. The fusion can be detected by cytogenetics, fluorescence in-situ hybridization (FISH), or by molecular analysis with RT-PCR. Multiple fusion transcripts arising as a result of various breakpoints in the CBFβ and MYH11 have been identified. In this report we describe a comprehensive RT-PCR assay to identify all known fusion transcripts and provide an algorithm for molecular analysis of CBFβ-MYH11 fusions from patient specimens. Further, identification of the fusion transcript by such an assay would help in the diagnosis and follow up of patients with cryptic inversion 16 translocations (such as patient 2 in this report) not detected by standard cytogenetics or FISH and for rational design of probes for quantitative analysis by real-time PCR. PMID:14736823

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

PubMed Central

Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D.C.

2013-01-01

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments. PMID:23746509

Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

PubMed Central

2013-01-01

Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion.

PubMed

Smith, Matthew B; Karatekin, Erdem; Gohlke, Andrea; Mizuno, Hiroaki; Watanabe, Naoki; Vavylonis, Dimitrios

2011-10-05

Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values ∼0.5 μm(2)/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface.

PubMed

Imanaka, Hiroyuki; Yamadzumi, Daisuke; Yanagita, Keisuke; Ishida, Naoyuki; Nakanishi, Kazuhiro; Imamura, Koreyoshi

2016-03-01

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a "cushion" between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016. © 2016 American Institute of Chemical Engineers.

Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.

2007-06-01

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, andmore » YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA regulation.« less

Luminescence in the fluoride-containing phosphate-based glasses: A possible origin of their high resistance to nanosecond pulse laser-induced damage

PubMed Central

Wang, Pengfei; Lu, Min; Gao, Fei; Guo, Haitao; Xu, Yantao; Hou, Chaoqi; Zhou, Zhiwei; Peng, Bo

2015-01-01

Fusion power offers the prospect of an almost inexhaustible source of energy for future generations. It was reported that fusion fuel gains exceeding unity on the National Ignition Facility (NIF) were achieved, but so far great deal of scientific and engineering challenges have to be overcome for realizing fusion power generation. There is a bottleneck for color-separation gratings in NIF and other similar inertial confinement fusion (ICF) lasers. Here we show a series of high performance phosphate-based glasses that can transmit the third harmonic frequency (3ω) laser light with high efficiency meanwhile filter the fundamental (1ω) and the second harmonic frequency (2ω) laser lights through direct absorption, and especially they exhibit excellent damage threshold induced by nanosecond pulse laser compared with that of the fused silica used in NIF. Yellowish-orange fluorescence emits during the laser-material interaction process, and it can be tailored through regulating the glass structure. Study on its structural origin suggests that the fluorescence emission is a key factor that conduces to the high laser-induced damage resistance of these glasses. The results also indicated the feasibility of utilizing these high performance glasses in novel color separation optics, allowing novel design for the final optics assembly in ICF lasers. PMID:25716328

Luminescence in the fluoride-containing phosphate-based glasses: a possible origin of their high resistance to nanosecond pulse laser-induced damage.

PubMed

Wang, Pengfei; Lu, Min; Gao, Fei; Guo, Haitao; Xu, Yantao; Hou, Chaoqi; Zhou, Zhiwei; Peng, Bo

2015-02-26

Fusion power offers the prospect of an almost inexhaustible source of energy for future generations. It was reported that fusion fuel gains exceeding unity on the National Ignition Facility (NIF) were achieved, but so far great deal of scientific and engineering challenges have to be overcome for realizing fusion power generation. There is a bottleneck for color-separation gratings in NIF and other similar inertial confinement fusion (ICF) lasers. Here we show a series of high performance phosphate-based glasses that can transmit the third harmonic frequency (3ω) laser light with high efficiency meanwhile filter the fundamental (1ω) and the second harmonic frequency (2ω) laser lights through direct absorption, and especially they exhibit excellent damage threshold induced by nanosecond pulse laser compared with that of the fused silica used in NIF. Yellowish-orange fluorescence emits during the laser-material interaction process, and it can be tailored through regulating the glass structure. Study on its structural origin suggests that the fluorescence emission is a key factor that conduces to the high laser-induced damage resistance of these glasses. The results also indicated the feasibility of utilizing these high performance glasses in novel color separation optics, allowing novel design for the final optics assembly in ICF lasers.

Membrane fusion activity of vesicular stomatitis virus glycoprotein G is induced by low pH but not by heat or denaturant.

PubMed

Yao, Yi; Ghosh, Kakoli; Epand, Raquel F; Epand, Richard M; Ghosh, Hara P

2003-06-05

The fusogenic envelope glycoprotein G of the rhabdovirus vesicular stomatitis virus (VSV) induces membrane fusion at acidic pH. At acidic pH the G protein undergoes a major structural reorganization leading to the fusogenic conformation. However, unlike other viral fusion proteins, the low-pH-induced conformational change of VSV G is completely reversible. As well, the presence of an alpha-helical coiled-coil motif required for fusion by a number of viral and cellular fusion proteins was not predicted in VSV G protein by using a number of algorithms. Results of pH dependence of the thermal stability of G protein as determined by intrinsic Trp fluorescence and circular dichroism (CD) spectroscopy show that the G protein is equally stable at neutral or acidic pH. Destabilization of G structure at neutral pH with either heat or urea did not induce membrane fusion or conformational change(s) leading to membrane fusion. Taken together, these data suggest that the mechanism of VSV G-induced fusion is distinct from the fusion mechanism of fusion proteins that involve a coiled-coil motif.

Water secretion associated with exocytosis in endocrine cells revealed by micro forcemetry and evanescent wave microscopy.

PubMed Central

Tsuboi, Takashi; Kikuta, Toshiteru; Sakurai, Takashi; Terakawa, Susumu

2002-01-01

It has been a long belief that release of substances from the cell to the extracellular milieu by exocytosis is completed by diffusion of the substances from secretory vesicles through the fusion pore. Involvement of any mechanical force that may be superposed on the diffusion to enhance the releasing process has not been elucidated to date. We tackled this problem in cultured bovine chromaffin cells using direct and sensitive methods: the laser-trap forcemetry and the evanescent-wave fluorescence microscopy. With a laser beam, we trapped a micro bead in the vicinity of a cell (with 1 microm of separation) and observed movements of the bead optically. Electrical stimulation of the cell induced many of rapid and transient movements of the bead in a direction away from the cell surface. Upon the same stimulation, secretory vesicles stained with a fluorescent probe, acridine orange, and excited under the evanescent field illumination, showed a flash-like response: a transient increase in fluorescence intensity associated with a diffuse cloud of brightness, followed by a complete disappearance. These mechanical and fluorescence transients indicate a directional flow of substances. Blockers of the Cl(-) channel suppressed the rates of both responses in a characteristic way but not exocytotic fusion itself. Immunocytochemical studies revealed the presence of Cl(-) and K(+) channels on the vesicle membranes. These results suggest that the externalization of hormones or transmitters upon exocytosis of vesicles is augmented by secretion of water from the vesicle membrane through the widened fusion pore, possibly modulating the rate and reach of the hormone or transmitter release and facilitating transport of the signal molecules in intercellular spaces. PMID:12080110

Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

PubMed Central

Francin-Allami, Mathilde; Saumonneau, Amélie; Lavenant, Laurence; Bouder, Axelle; Sparkes, Imogen; Hawes, Chris; Popineau, Yves

2011-01-01

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP ‘core’. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions. PMID:21617248

CXCR2 inverse agonism detected by arrestin redistribution.

PubMed

Kredel, Simone; Wolff, Michael; Wiedenmann, Jörg; Moepps, Barbara; Nienhaus, G Ulrich; Gierschik, Peter; Kistler, Barbara; Heilker, Ralf

2009-10-01

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria-derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo-monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td-RFP611 (Arr-td-RFP611) or enhanced green fluorescent protein (EGFP; Arr-EGFP), were found to colocalize with internalized fluorescently labeled Gro-alpha a few minutes after Gro-alpha addition. Intriguingly, however, Arr-td-RFP611 and Arr-EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2-activating ligand. Under these conditions, Arr-td-RFP611 showed a largely homogeneous cytosolic distribution, whereas Arr-EGFP segregated, to a large degree, into granular spots. These observations indicate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr-EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC(50) value similar to that observed for Arr-EGFP redistribution. Thus, the redistribution assay, when based on Arr-EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr-td-RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism.

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

PubMed

Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

2013-08-01

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Ratiometric Two-Photon Fluorescent Probe for Tracking the Lysosomal ATP Level: Direct in cellulo Observation of Lysosomal Membrane Fusion Processes.

PubMed

Jun, Yong Woong; Wang, Taejun; Hwang, Sekyu; Kim, Dokyoung; Ma, Donghee; Kim, Ki Hean; Kim, Sungjee; Jung, Junyang; Ahn, Kyo Han

2018-06-05

Vesicles exchange its contents through membrane fusion processes-kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We disclose a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging technique, lysosomal membrane fusion process in cells has been directly observed along with the concentration of its content-lysosomal ATP. Results show that the kiss-and-run process between lysosomes proceeds through repeating transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling of the Human Alveolar Rhabdomyosarcoma Pax3-Foxo1 Chromosome Translocation in Mouse Myoblasts Using CRISPR-Cas9 Nuclease

PubMed Central

Lagutina, Irina V.; Valentine, Virginia; Picchione, Fabrizio; Harwood, Frank; Valentine, Marcus B.; Villarejo-Balcells, Barbara; Carvajal, Jaime J.; Grosveld, Gerard C.

2015-01-01

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice. PMID:25659124

Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

PubMed

Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

2017-07-25

Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.

A SNAP-Tagged Derivative of HIV-1—A Versatile Tool to Study Virus-Cell Interactions

PubMed Central

Eckhardt, Manon; Anders, Maria; Muranyi, Walter; Heilemann, Mike; Krijnse-Locker, Jacomine; Müller, Barbara

2011-01-01

Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIVSNAP, which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIVSNAP represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy. PMID:21799764

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells

NASA Astrophysics Data System (ADS)

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2011-12-01

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging αvβ3 integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging. Electronic supplementary information (ESI) available. See DOI: 10.1039/c1nr11132a

GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes.

PubMed

Pélissier, Hélène C; Peters, Winfried S; Collier, Ray; van Bel, Aart J E; Knoblauch, Michael

2008-11-01

Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.

GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

PubMed Central

Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

2008-01-01

Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195

Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry

PubMed Central

Brauchle, Michael; Hansen, Simon; Caussinus, Emmanuel; Lenard, Anna; Ochoa-Espinosa, Amanda; Scholz, Oliver; Sprecher, Simon G.; Plückthun, Andreas; Affolter, Markus

2014-01-01

ABSTRACT Protein–protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level. PMID:25416061

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.

PubMed

Villarreal, Seth; Lee, Sung Hoon; Wu, Ling-Gang

2017-09-04

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

PubMed Central

Hsieh, Ya-Ju; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

2014-01-01

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.

PubMed

Sehgal, Lalit; Budnar, Srikanth; Bhatt, Khyati; Sansare, Sneha; Mukhopadhaya, Amitabha; Kalraiya, Rajiv D; Dalal, Sorab N

2012-10-01

The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo.

Green fluorescent protein (GFP): is seeing believing and is that enough?

PubMed

Shorter, Susan A; Pettit, Marie W; Dyer, Paul D R; Coakley Youngs, Emma; Gorringe-Pattrick, Monique A M; El-Daher, Samer; Richardson, Simon

Intracellular compartmentalisation is a significant barrier to the successful nucleocytosolic delivery of biologics. The endocytic system has been shown to be responsible for compartmentalisation, providing an entry point, and trigger(s) for the activation of drug delivery systems. Consequently, many of the technologies used to understand endocytosis have found utility within the field of drug delivery. The use of fluorescent proteins as markers denoting compartmentalisation within the endocytic system has become commonplace. Several of the limitations associated with the use of green fluorescent protein (GFP) within the context of drug delivery have been explored here by asking a series of related questions: (1) Are molecules that regulate fusion to a specific compartment (i.e. Rab- or SNARE-GFP fusions) a good choice of marker for that compartment? (2) How reliable was GFP-marker overexpression when used to define a given endocytic compartment? (3) Can glutathione-s-transferase (GST) fused in frame with GFP (GST-GFP) act as a fluid phase endocytic probe? (4) Was GFP fluorescence a robust indicator of (GFP) protein integrity? This study concluded that there are many appropriate and useful applications for GFP; however, thought and an understanding of the biological and physicochemical character of these markers are required for the generation of meaningful data.

Imaging Erg and Jun transcription factor interaction in living cells using fluorescence resonance energy transfer analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Camuzeaux, Barbara; Spriet, Corentin; Heliot, Laurent

2005-07-15

Physical interactions between transcription factors play important roles in modulating gene expression. Previous in vitro studies have shown a transcriptional synergy between Erg protein, an Ets family member, and Jun/Fos heterodimer, members of the bZip family, which requires direct Erg-Jun protein interactions. Visualization of protein interactions in living cells is a new challenge in biology. For this purpose, we generated fusion proteins of Erg, Fos, and Jun with yellow and cyan fluorescent proteins, YFP and CFP, respectively. After transient expression in HeLa cells, interactions of the resulting fusion proteins were explored by fluorescence resonance energy transfer microscopy (FRET) in fixedmore » and living cells. FRET between YFP-Erg and CFP-Jun was monitored by using photobleaching FRET and fluorescence lifetime imaging microscopy. Both techniques revealed the occurrence of intermolecular FRET between YFP-Erg and CFP-Jun. This is stressed by loss of FRET with an YFP-Erg version carrying a point mutation in its ETS domain. These results provide evidence for the interaction of Erg and Jun proteins in living cells as a critical prerequisite of their transcriptional synergy, but also for the essential role of the Y371 residue, conserved in most Ets proteins, in this interaction.« less

Protein- protein interaction detection system using fluorescent protein microdomains

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

Kinase gene fusions in defined subsets of melanoma.

PubMed

Turner, Jacqueline; Couts, Kasey; Sheren, Jamie; Saichaemchan, Siriwimon; Ariyawutyakorn, Witthawat; Avolio, Izabela; Cabral, Ethan; Glogowska, Magdelena; Amato, Carol; Robinson, Steven; Hintzsche, Jennifer; Applegate, Allison; Seelenfreund, Eric; Gonzalez, Rita; Wells, Keith; Bagby, Stacey; Tentler, John; Tan, Aik-Choon; Wisell, Joshua; Varella-Garcia, Marileila; Robinson, William

2017-01-01

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break-apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10-BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Fusion Algorithm for GFP Image and Phase Contrast Image of Arabidopsis Cell Based on SFL-Contourlet Transform

PubMed Central

Feng, Peng; Wang, Jing; Wei, Biao; Mi, Deling

2013-01-01

A hybrid multiscale and multilevel image fusion algorithm for green fluorescent protein (GFP) image and phase contrast image of Arabidopsis cell is proposed in this paper. Combining intensity-hue-saturation (IHS) transform and sharp frequency localization Contourlet transform (SFL-CT), this algorithm uses different fusion strategies for different detailed subbands, which include neighborhood consistency measurement (NCM) that can adaptively find balance between color background and gray structure. Also two kinds of neighborhood classes based on empirical model are taken into consideration. Visual information fidelity (VIF) as an objective criterion is introduced to evaluate the fusion image. The experimental results of 117 groups of Arabidopsis cell image from John Innes Center show that the new algorithm cannot only make the details of original images well preserved but also improve the visibility of the fusion image, which shows the superiority of the novel method to traditional ones. PMID:23476716

Kinase Gene Fusions in Defined Subsets of Melanoma

PubMed Central

Turner, Jacqueline; Couts, Kasey; Sheren, Jamie; Saichaemchan, Siriwimon; Ariyawutyakorn, Witthawat; Avolio, Izabela; Cabral, Ethan; Glogowska, Magdelena; Amato, Carol; Robinson, Steven; Hintzsche, Jennifer; Applegate, Allison; Seelenfreund, Eric; Gonzalez, Rita; Wells, Keith; Bagby, Stacey; Tentler, John; Tan, Aik-Choon; Wisell, Joshua; Varella-Garcia, Marileila; Robinson, William

2017-01-01

Summary Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break-apart fluorescence in-situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by IHC or sequencing and one was found to be an ARMC10-BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought-and should be further explored particularly in melanomas lacking known driver mutations. PMID:27864876

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

PubMed

Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

Mechanism of Fusion Triggering by Human Parainfluenza Virus Type III

PubMed Central

Porotto, Matteo; Palmer, Samantha G.; Palermo, Laura M.; Moscona, Anne

2012-01-01

Parainfluenza viruses enter host cells by fusing the viral and target cell membranes via concerted action of their two envelope glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion protein (F). Receptor-bound HN triggers F to undergo conformational changes that render it fusion-competent. To address the role of receptor engagement and to elucidate how HN and F interact during the fusion process, we used bimolecular fluorescence complementation to follow the dynamics of human parainfluenza virus type 3 (HPIV3) HN/F pairs in living cells. We show that HN and F associate before receptor engagement. HN drives the formation of HN-F clusters at the site of fusion, and alterations in HN-F interaction determine the fusogenicity of the glycoprotein pair. An interactive site, at the HN dimer interface modulates HN fusion activation property, which is critical for infection of the natural host. This first evidence for the sequence of initial events that lead to viral entry may indicate a new paradigm for understanding Paramyxovirus infection. PMID:22110138

Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

PubMed

Moua, Pachai S; Gonzalez, Alfonso; Oshiro, Kristin T; Tam, Vivian; Li, Zhiguo Harry; Chang, Jennifer; Leung, Wilson; Yon, Amy; Thor, Der; Venkatram, Sri; Franz, Andreas H; Risser, Douglas D; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

2016-08-01

The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of Rheb in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

PubMed

Swer, Pynskhem Bok; Bhadoriya, Pooja; Saran, Shweta

2014-03-01

Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52 percent identity and 100 percent similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the beta-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis[W

PubMed Central

Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

2012-01-01

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

The bacterial secondary metabolite 2,4-diacetylphloroglucinol impairs mitochondrial function and affects calcium homeostasis in Neurospora crassa.

PubMed

Troppens, Danielle M; Chu, Meiling; Holcombe, Lucy J; Gleeson, Olive; O'Gara, Fergal; Read, Nick D; Morrissey, John P

2013-07-01

The bacterial secondary metabolite 2,4-diacetylphloroglucinol (DAPG) is of interest as an active ingredient of biological control strains of Pseudomonas fluorescens and as a potential lead pharmaceutical molecule because of its capacity to inhibit growth of diverse microbial and non-microbial cells. The mechanism by which this occurs is unknown and in this study the filamentous fungus Neurospora crassa was used as a model to investigate the effects of DAPG on a eukaryotic cell. Colony growth, conidial germination and cell fusion assays confirmed the inhibitory nature of DAPG towards N. crassa. A number of different fluorescent dyes and fluorescent protein reporters were used to assess the effects of DAPG treatment on mitochondrial and other cellular functions. DAPG treatment led to changes in mitochondrial morphology, and rapid loss of mitochondrial membrane potential. These effects are likely to be responsible for the toxicity of DAPG. It was also found that DAPG treatment caused extracellular calcium to be taken up by conidial germlings leading to a transient increase in cytosolic free Ca(2+) with a distinct concentration dependent Ca(2+) signature. Copyright © 2013 Elsevier Inc. All rights reserved.

Noninvasive imaging of protein-protein interactions in living animals

NASA Astrophysics Data System (ADS)

Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

2002-05-01

Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

PubMed

Arii, Yasuhiro; Yamaguchi, Hidenori; Fukuoka, Shin-Ichi

2007-10-01

The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.

Distinct roles for key karyogamy proteins during yeast nuclear fusion.

PubMed

Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D

2009-09-01

During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-06-01

Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

PubMed Central

Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

2016-01-01

Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Charalel, Joseph K; Viana, Matheus P; Garcia, Enrique J; Sing, Cierra N; Koenigsberg, Andrea; Swayne, Theresa C; Vevea, Jason D; Boldogh, Istvan R; Rafelski, Susanne M; Pon, Liza A

2016-03-01

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud. © 2016 Higuchi-Sanabria et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Recurrent LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes promote tumor cell motility in human osteosarcoma.

PubMed

Yang, Jilong; Annala, Matti; Ji, Ping; Wang, Guowen; Zheng, Hong; Codgell, David; Du, Xiaoling; Fang, Zhiwei; Sun, Baocun; Nykter, Matti; Chen, Kexin; Zhang, Wei

2014-10-10

The identification of fusion genes such as SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/TPM4-ALK and EWS-FLI1 in human sarcomas has provided important insight into the diagnosis and targeted therapy of sarcomas. No recurrent fusion has been reported in human osteosarcoma. Transcriptome sequencing was used to characterize the gene fusions and mutations in 11 human osteosarcomas. Nine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway. Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas. Expression of LRP1-SNRNP25 fusion gene promoted SAOS-2 osteosarcoma cell migration and invasion. Expression of KCNMB4-CCND3 fusion gene promoted SAOS-2 cell migration. Our study represents the first whole transcriptome analysis of untreated human osteosarcoma. Our discovery of two osteosarcoma specific fusion genes associated with osteosarcoma cellular motility highlights the heterogeneity of osteosarcoma and provides opportunities for new treatment modalities.

Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

PubMed

Zhao, Jing; Zhao, Jin-Yin; Chen, Zhi-Xia; Zhong, Wei; Li, Long-Yun; Liu, Li-Cheng; Hu, Xiao-Xu; Chen, Wei-Jun; Wang, Meng-Zhao

2016-12-20

Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

Development and Validation of a High-Content Bimolecular Fluorescence Complementation Assay for Small Molecule Inhibitors of HIV-1 Nef Dimerization

PubMed Central

Poe, Jerrod A.; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E.

2014-01-01

Nef is an HIV-1 accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus an mRFP reporter were expressed from a single vector, separated by picornavirus ‘2A’ linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type vs. dimerization-defective Nef were very clearly separated, with Z-factors consistently in the 0.6–0.7 range. A fully automated pilot screen of the NIH Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function. PMID:24282155

In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter.

PubMed

Luker, Gary D; Luker, Kathryn E; Sharma, Vijay; Pica, Christina M; Dahlheimer, Julie L; Ocheskey, Joe A; Fahrner, Timothy J; Milbrandt, Jeffrey; Piwnica-Worms, David

2002-01-01

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

PubMed

Beadling, Carol; Wald, Abigail I; Warrick, Andrea; Neff, Tanaya L; Zhong, Shan; Nikiforov, Yuri E; Corless, Christopher L; Nikiforova, Marina N

2016-03-01

Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

UGT-29 protein expression and localization during bacterial infection in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Wong, Rui-Rui; Lee, Song-Hua; Nathan, Sheila

2014-09-01

The nematode Caenorhabditis elegans is routinely used as an animal model to delineate complex molecular mechanisms involved in the host response to pathogen infection. Following up on an earlier study on host-pathogen interaction, we constructed a ugt-29::GFP transcriptional fusion transgenic worm strain to examine UGT-29 protein expression and localization upon bacterial infection. UGT-29 orthologs can be found in higher organisms including humans and is proposed as a member of the UDP-Glucoronosyl Transferase family of proteins which are involved in phase II detoxification of compounds detrimental to the host organism. Under uninfected conditions, UGT-29::GFP fusion protein was highly expressed in the C. elegans anterior pharynx and intestine, two major organs involved in detoxification. We further evaluated the localization of the enzyme in worms infected with the bacterial pathogen, Burkholderia pseudomallei. The infected ugt-29::GFP transgenic strain exhibited increased fluorescence in the pharynx and intestine with pronounced fluorescence also extending to body wall muscle. This transcriptional fusion GFP transgenic worm is a convenient and direct tool to provide information on UGT detoxification enzyme gene expression and could be a useful tool for a number of diverse applications.

Putative role of membranes in the HIV fusion inhibitor enfuvirtide mode of action at the molecular level.

PubMed Central

Veiga, Salomé; Henriques, Sónia; Santos, Nuno C; Castanho, Miguel

2004-01-01

Partition of the intrinsically fluorescent HIV fusion inhibitor enfuvirtide into lipidic membranes is relatively high (Delta G =6.6 kcal x mol(-1)) and modulated by cholesterol. A shallow position in the lipidic matrix makes it readily available for interaction with gp41. No conformational energetic barrier prevents enfuvirtide from being active in both aqueous solution and lipidic membranes. Lipidic membranes may play a key role in the enfuvirtide biochemical mode of action. PMID:14514352

Osteoclast fusion is initiated by a small subset of RANKL-stimulated monocyte progenitors, which can fuse to RANKL-unstimulated progenitors.

PubMed

Levaot, Noam; Ottolenghi, Aner; Mann, Mati; Guterman-Ram, Gali; Kam, Zvi; Geiger, Benjamin

2015-10-01

Osteoclasts are multinucleated, bone-resorbing cells formed via fusion of monocyte progenitors, a process triggered by prolonged stimulation with RANKL, the osteoclast master regulator cytokine. Monocyte fusion into osteoclasts has been shown to play a key role in bone remodeling and homeostasis; therefore, aberrant fusion may be involved in a variety of bone diseases. Indeed, research in the last decade has led to the discovery of genes regulating osteoclast fusion; yet the basic cellular regulatory mechanism underlying the fusion process is poorly understood. Here, we applied a novel approach for tracking the fusion processes, using live-cell imaging of RANKL-stimulated and non-stimulated progenitor monocytes differentially expressing dsRED or GFP, respectively. We show that osteoclast fusion is initiated by a small (~2.4%) subset of precursors, termed "fusion founders", capable of fusing either with other founders or with non-stimulated progenitors (fusion followers), which alone, are unable to initiate fusion. Careful examination indicates that the fusion between a founder and a follower cell consists of two distinct phases: an initial pairing of the two cells, typically lasting 5-35 min, during which the cells nevertheless maintain their initial morphology; and the fusion event itself. Interestingly, during the initial pre-fusion phase, a transfer of the fluorescent reporter proteins from nucleus to nucleus was noticed, suggesting crosstalk between the founder and follower progenitors via the cytoplasm that might directly affect the fusion process, as well as overall transcriptional regulation in the developing heterokaryon. Copyright © 2015 Elsevier Inc. All rights reserved.

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

PubMed

Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

2014-06-01

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

PubMed

Hebert, Benedict; Costantino, Santiago; Wiseman, Paul W

2005-05-01

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

[The Influence of New Medium with RGD on Cell Growth,Cell Fusion and Expression of Exogenous Gene].

PubMed

Wang, Pei-Pei; Wei, Da-Peng; Zhu, Tong-Bo

2018-03-01

To investigate the influence of a new culture medium added with RGD on cell growth,cell fusion and expression of exogenous gene. A new medium was prepared by adding different concentrations of RGD to ordinary culture medium. The optimum concentration of RGD was determined by observation of the growth of human pancreatic epithelial cell line HPDE6-C7. After determining the optimum concentration of RGD,different concentrations of cells HPDE6-C7 (5×10 4 ,10 5 ,5×10 5 mL －1 ) were inoculated in the two mediums. The morphology,adherence,growth and density of the cells were observed by inverted microscope; The ratio of clone formation and the positive rate of cloning were compared between the two cultures after fusion; The fluorescence intensity after the transfection of plasmid with green fluorescent protein ( GFP ) and the protein expression after transfection of plasmid with KRAS were observed to campare the expression of exogenous genes between the new medium with ordinary medium. Firstly,the optimal concentration of RGD was 10 ng/mL. Compared with the normal medium,the cultured cells with RGD had better morphology,adhesion and faster proliferation. In addition,both of the number and positive rate of clones formed in the new medium were significantly higher than that in the ordinary medium ( P <0.05);The fluorescence intensity after transfection of exogenous gene GFP in the new medium was significantly higher than that in normal medium ( P <0.05); Expression level of exogenous gene KRAS of the new medium was also significantly higher than that in normal medium. The new culture medium has highlighted advantages in cell growth,cell fusion and expression of exogenous genes. RGD peptide has widely prospect and potential value in the cell culture. Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).

Arabidopsis adaptor protein 1G is critical for pollen development.

PubMed

Feng, Chong; Wang, Jia-Gang; Liu, Hai-Hong; Li, Sha; Zhang, Yan

2017-09-01

Pollen development is a pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 (AP1G) mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development. © 2017 Institute of Botany, Chinese Academy of Sciences.

Dichotomy of Genetic Abnormalities in PEComas with Therapeutic Implications

PubMed Central

Agaram, Narasimhan P; Sung, Yun-Shao; Zhang, Lei; Chen, Chun-Liang; Chen, Hsiao-Wei; Singer, Samuel; Dickson, Mark A.; Berger, Michael F.; Antonescu, Cristina R

2014-01-01

Perivascular epithelioid cell neoplasms (PEComa) are a family of rare mesenchymal tumors with hybrid myo-melanocytic differentiation. Although most PEComas harbor loss of function TSC1/TSC2 mutations, a small subset were reported to carry TFE3 gene rearrangements. As no comprehensive genomic study has addressed the molecular classification of PEComa, we sought to investigate by multiple methodologies the incidence and spectrum of genetic abnormalities and their potential genotype-phenotype correlations in a large group of 38 PEComas. The tumors were located in soft tissue (11 cases) and visceral sites (27) including uterus, kidney, liver, lung and urinary bladder. Combined RNA sequencing and Fluorescence In Situ Hybridization (FISH) analysis identified 9 (23%) TFE3 gene rearranged tumors, with 3 cases showing a SFPQ/PSF-TFE3 fusion and one case a novel DVL2-TFE3 gene fusion. The TFE3-positive lesions showed a distinctive nested/alveolar morphology and were equally distributed between soft tissue and visceral sites. Additionally, novel RAD51B gene rearrangements were identified in 3 (8%) uterine PEComas, which showed a complex fusion pattern and were fused to RRAGB/OPHN1 genes in two cases. Other non-recurrent gene fusions, HTR4-ST3GAL1 and RASSF1-PDZRN3, were identified in 2 cases. Targeted exome sequencing using the IMPACT assay was used to address if the presence of gene fusions are mutually exclusive from TSC gene abnormalities. TSC2 mutations were identified in 80% of the TFE3 fusion-negative cases tested. Co-existent TP53 mutations were identified in 63% of the TSC2 mutated PEComas. Our results showed that TFE3-rearranged PEComas lacked co-existing TSC2 mutations, indicating alternative pathways of tumorigenesis. In summary, this comprehensive genetic analysis significantly expands our understanding of molecular alterations in PEComas and brings forth the genetic heterogeneity of these tumors. PMID:25651471

Direct transfer of viral and cellular proteins from varicella-zoster virus-infected non-neuronal cells to human axons.

PubMed

Grigoryan, Sergei; Yee, Michael B; Glick, Yair; Gerber, Doron; Kepten, Eldad; Garini, Yuval; Yang, In Hong; Kinchington, Paul R; Goldstein, Ronald S

2015-01-01

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.

Direct Transfer of Viral and Cellular Proteins from Varicella-Zoster Virus-Infected Non-Neuronal Cells to Human Axons

PubMed Central

Grigoryan, Sergei; Yee, Michael B; Glick, Yair; Gerber, Doron; Kepten, Eldad; Garini, Yuval; Yang, In Hong; Kinchington, Paul R.; Goldstein, Ronald S.

2015-01-01

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk. PMID:25973990

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family.

PubMed

Bahaji, Abdellatif; Ovecka, Miroslav; Bárány, Ivett; Risueño, María Carmen; Muñoz, Francisco José; Baroja-Fernández, Edurne; Montero, Manuel; Li, Jun; Hidalgo, Maite; Sesma, María Teresa; Ezquer, Ignacio; Testillano, Pilar S; Pozueta-Romero, Javier

2011-04-01

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.

Prevalence and clinicopathological characteristics of ALK fusion subtypes in lung adenocarcinomas from Chinese populations.

PubMed

Zheng, Difan; Wang, Rui; Zhang, Yang; Pan, Yunjian; Cheng, Xinghua; Cheng, Chao; Zheng, Shanbo; Li, Hang; Gong, Ranxia; Li, Yuan; Shen, Xuxia; Sun, Yihua; Chen, Haiquan

2016-04-01

We performed this retrospective study to have a comprehensive investigation of the clinicopathological characteristics of ALK fusion-positive lung adenocarcinoma in Chinese populations. We screened 1407 patients with primary lung adenocarcinoma from October 2007 to May 2013. Quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and fluorescence in situ hybridization were performed to detect ALK fusion genes, with validation of positive results using immunohistochemistry. Clinicopathological characteristics were collected to assess prognosis in ALK fusion-positive patients. Of 1407 patients with lung adenocarcinoma, there were 74 (5.3 %) ALK fusion-positive patients. Patients harboring ALK fusion were significantly younger (56.0 years vs. 59.8 years p = 0.002) and were more likely to have advanced stages (stage III or stage IV) (OR 1.761; 95 % CI 1.10-2.82, p = 0.017). Lepidic predominant adenocarcinoma was rarely found in ALK fusion patients (2.7 vs. 13.5 % p = 0.025), while IMA (invasive mucinous adenocarcinoma) predominant adenocarcinoma was more frequently found (21.6 vs. 5.0 % p < 0.001). ALK fusion was neither a risk factor nor protective factor in relapse-free survival and overall survival. Male, current smoker, and EML4-ALK variant 3 indicated poor prognosis among ALK fusion-positive lung adenocarcinomas. ALK fusion was detected in 5.3 % (74/1407) of the Chinese patients with lung adenocarcinoma. ALK fusion defines a molecular subset of lung adenocarcinoma with unique clinicopathological characteristics. Different ALK fusion variants determine distinct prognoses.

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag

PubMed Central

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant. PMID:26539718

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

PubMed

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

PubMed

Winters, Jennifer L; Davila, Jaime I; McDonald, Amber M; Nair, Asha A; Fadra, Numrah; Wehrs, Rebecca N; Thomas, Brittany C; Balcom, Jessica R; Jin, Long; Wu, Xianglin; Voss, Jesse S; Klee, Eric W; Oliver, Gavin R; Graham, Rondell P; Neff, Jadee L; Rumilla, Kandelaria M; Aypar, Umut; Kipp, Benjamin R; Jenkins, Robert B; Jen, Jin; Halling, Kevin C

2018-06-13

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Functional analysis of two genes coding for distinct cation diffusion facilitators of the ectomycorrhizal Zn-accumulating fungus Russula atropurpurea.

PubMed

Sácký, Jan; Leonhardt, Tereza; Kotrba, Pavel

2016-04-01

Russula atropurpurea can accumulate remarkably high concentrations of Zn in its sporocarps. We have previously demonstrated that 40 % of the intracellular Zn in this species is sequestered by MT-like RaZBP peptides. To see what other mechanisms for the handling of the accumulated Zn are available to R. atropurpurea, we searched its transcriptome for cDNAs coding for transporters of the cation diffusion facilitator (CDF) family. The transcriptome search enabled us to identify RaCDF1 and RaCDF2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of RaCDF1 and its translational fusion with green fluorescent protein (GFP) protected the yeasts against Zn and Co, but not Cd or Mn, toxicity and led to increased Zn accumulation in the cells. The GFP fluorescence, observed in the RaCDF1::GFP-expressing yeasts on tonoplasts, indicated that the RaCDF1-mediated Zn and Co tolerance was a result of vacuolar sequestration of the metals. The expression of RaCDF2 supported Zn, but not Mn, tolerance in the yeasts and reduced the cellular uptake of Zn, which is congruent with the proposed idea of the Zn-efflux function of RaCDF2, supported by the localization of GFP-derived fluorescence on the plasma membrane of the yeasts expressing functional RaCDF2::GFP. Contrarily, RaCDF2 increased the sensitivity to Co and Cd in the yeasts and significantly promoted Cd uptake, which suggested that it can act as a bidirectional metal transporter. The notion that RaCDF1 and RaCDF2 are genuine CDF transporters in R. atropurputrea was further reinforced by the fact that the RaCDF-associated metal tolerance and uptake phenotypes were lost upon the replacement of histidyl (in RaCDF1) and aspartyl (in RaCDF2), which are highly conserved in the second transmembrane domain and known to be essential for the function of CDF proteins.

Site-specific labeling of proteins by using biotin protein ligase conjugated with fluorophores.

PubMed

Sueda, Shinji; Yoneda, Sawako; Hayashi, Hideki

2011-06-14

Biotin protein ligase (BPL) mediates the covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP). This biotinylation in Sulfolobus tokodaii is unique in that BPL forms a tight complex with the product, biotinylated BCCP, and this property was exploited for fluorescent labeling of a membrane protein. Thus, the truncated form of BCCP (BCCPΔ100, 69 residues) was fused to either the N or C terminus of the bradykinin B2 receptor (B2R). The resulting fusion proteins, BCCPΔ100-B2R and B2R-BCCPΔ100, respectively, were separately expressed in mammalian HEK293 cells, and labeled with BPL conjugated with a fluorophore: either fluorescein, DyLight549 or green fluorescent protein. The fusion proteins were biotinylated and bound to BPL, thereby giving rise to strong fluorescence along the periphery of the cell. Some were capable of binding bradykinin and an antagonist. When stimulated with the former, the receptor translocated to the cytosol; this suggests that the labeled receptor retains its integrity in terms of ligand-binding and translocation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alignment and absolute wavelength calibration of imaging Bragg spectrometers.

PubMed

Bertschinger, G; Marchuk, O; Barnsley, R

2016-11-01

In the present and the next generation of fusion devices, imaging Bragg spectrometers are key diagnostics to measure plasma parameters in the hot core, especially ion temperature and plasma rotation. The latter quantities are routinely obtained using the Doppler-width and -shift of the emitted spectral lines, respectively. Line shift measurements require absolute accuracies Δλ/λ of about 10 ppm, where λ-is the observed wavelength. For ITER and the present fusion devices, spectral lines of He-and H-like argon, iron, and krypton as well as Ne-like tungsten are foreseen for the measurements. For these lines, Kα lines can be found, some in higher order, which fit into the narrow energy window of the spectrometers. For arbitrary wavelength settings, Kα lines are also used to measure the miscut of the spherical crystals; afterwards the spectrometers can be set according to the geometrical imaging properties using coordinate measurement machines. For the spectrometers measuring Lyα lines of H-like ions, fluorescence targets can provide in situ localized calibration lines on the spectra. The fluorescence targets are used best in transmission and are excited by the thermal x-ray radiation of the plasma. An analytic theory of fluorescence is worked out.

Development of a bioluminescence resonance energy transfer (BRET) for monitoring estrogen receptor alpha activation

NASA Astrophysics Data System (ADS)

Michelini, Elisa; Mirasoli, Mara; Karp, Matti; Virta, Marko; Roda, Aldo

2004-06-01

Estrogen receptor (ER) is a ligand-activated transcriptional factor, able to dimerize after activation and to bind specific DNA sequences (estrogen response elements), thus activating gene target transcription. Since ER homo- and hetero-dimerization (giving a-a and a-b isoforms) is a fundamental step for receptor activation, we developed an assay for detecting compounds that induce human ERa homo-dimerization based on bioluminescence resonance energy transfer (BRET). BRET is a non-radiative energy transfer, occurring between a luminescent donor and a fluorescent acceptor, that strictly depends on the closeness between the two proteins and can therefore be used for studying protein-protein interactions. We cloned ERa coding sequence in frame with either a variant of the green fluorescent protein (enhanced yellow fluorescent protein, EYFP) or Renilla luciferase (RLuc). Upon ERa homo-dimerization, BRET process takes place in the presence of the RLuc substrate coelenterazine resulting in EYFP emission at its characteristic wavelength. The ER alpha-Rluc and ER alpha-EYFP fusion proteins were cloned, then the occurrence of BRET in the presence of ER alpha activators was assayed both in vivo, within cells, and in vitro, with purified fusion proteins.

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

PubMed Central

Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

2014-01-01

Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. PMID:25044501

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

PubMed

Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

2014-10-01

Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

DOE PAGES

Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.; ...

2016-10-21

Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump,more » change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.« less

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

NASA Astrophysics Data System (ADS)

Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

2000-04-01

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.

Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump,more » change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.« less

Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells

PubMed Central

Karatekin, Erdem; Rothman, James E.

2013-01-01

Many biological processes rely on membrane fusion, therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE liposomes and target-membrane-associated t-SNARE-reconstituted planar, supported bilayers (SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorsecence microscopy. Unlike most previous attempts, fusion here is dependent on SNAP25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of (i) bilayers covered with a thin layer of poly(ethylene glycol) to control bilayer-bilayer and bilayer-substrate interactions, (ii) microfluidic flow channels which presents many advantages such as the removal of non-specifically bound liposomes by flow. The protocol takes 6–8 days to complete. Analysis can take up to two weeks. PMID:22517259

MicroRNA Detection by DNA-Mediated Liposome Fusion.

PubMed

Jumeaux, Coline; Wahlsten, Olov; Block, Stephan; Kim, Eunjung; Chandrawati, Rona; Howes, Philip D; Höök, Fredrik; Stevens, Molly M

2018-03-02

Membrane fusion is a process of fundamental importance in biological systems that involves highly selective recognition mechanisms for the trafficking of molecular and ionic cargos. Mimicking natural membrane fusion mechanisms for the purpose of biosensor development holds great potential for amplified detection because relatively few highly discriminating targets lead to fusion and an accompanied engagement of a large payload of signal-generating molecules. In this work, sequence-specific DNA-mediated liposome fusion is used for the highly selective detection of microRNA. The detection of miR-29a, a known flu biomarker, is demonstrated down to 18 nm within 30 min with high specificity by using a standard laboratory microplate reader. Furthermore, one order of magnitude improvement in the limit of detection is demonstrated by using a novel imaging technique combined with an intensity fluctuation analysis, which is coined two-color fluorescence correlation microscopy. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

PubMed

Lete, Marta G; Byrne, Richard D; Alonso, Alicia; Poccia, Dominic; Larijani, Banafshé

2017-01-15

Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P 3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P 3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes. © 2017. Published by The Company of Biologists Ltd.

A plant cell-based system that predicts aβ42 misfolding: potential as a drug discovery tool for Alzheimer's disease.

PubMed

Zhao, Tiehan; Zeng, Ying; Kermode, Allison R

2012-11-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid β (Aβ) peptides and the failure of mechanisms to clear toxic aggregates. The Aβ42 peptide is considered to be a causative factor that underlies the pathophysiology of AD, in part due to its propensity for misfolding and aggregation; the small oligomers that result represent toxic species. Thus agents that prevent Aβ42 misfolding/aggregation or, alternatively improve Aβ42 oligomer clearance, may have significant therapeutic value. We have developed the basis for a drug screening system based on transgenic plant cells that express Aβ42 fusion proteins to serve as the reliable indicators of the general conformational status of Aβ42. Within cells of transgenic tobacco and Nicotiana benthamiana, misfolding of Aβ42 causes the misfolding of a GFP fusion partner, and consequently there is a loss of fluorescence associated with the native GFP protein. In a similar fusion consisting of Aβ42 linked to hygromycin phosphotransferase II (Hpt II), a hygromycin-resistance marker, misfolding of Aβ42 leads to a misfolded Hpt II, and consequently the transgenic cells are unable to grow on media containing hygromycin. Importantly, substitution of the 'aggregation-prone' Aβ42 with a missense mutant of Aβ42 (F19S/L34F) that is not prone to misfolding/aggregation, 'rescues' both fusion partners. Several 'positive control' chemicals that represent inhibitors of Aβ42 aggregation, including curcumin, epigallocatechin-3-gallate (EGCG), and resveratrol show efficacy in preventing the Aβ42-fusion proteins from misfolding/aggregating in the transgenic plant cells. We discuss the potential of the two fusion protein systems to serve as the basis for an inexpensive, selective, and efficient screening system in which a plant cell can fluoresce or survive only in the presence of drug candidates that are able to prevent Aβ42 misfolding/aggregation. Copyright © 2012 Elsevier Inc. All rights reserved.

Novel BCOR-MAML3 and ZC3H7B-BCOR Gene Fusions in Undifferentiated Small Blue Round Cell Sarcomas.

PubMed

Specht, Katja; Zhang, Lei; Sung, Yun-Shao; Nucci, Marisa; Dry, Sarah; Vaiyapuri, Sumathi; Richter, Gunther H S; Fletcher, Christopher D M; Antonescu, Cristina R

2016-04-01

Small blue round cell tumors (SBRCTs) are a heterogenous group of tumors that are difficult to diagnose because of overlapping morphologic, immunohistochemical, and clinical features. About two-thirds of EWSR1-negative SBRCTs are associated with CIC-DUX4-related fusions, whereas another small subset shows BCOR-CCNB3 X-chromosomal paracentric inversion. Applying paired-end RNA sequencing to an SBRCT index case of a 44-year-old man, we identified a novel BCOR-MAML3 chimeric fusion, which was validated by reverse transcription polymerase chain reaction and fluorescence in situ hybridization techniques. We then screened a total of 75 SBRCTs lacking EWSR1, FUS, SYT, CIC, and BCOR-CCNB3 abnormalities for BCOR break-apart probes by fluorescence in situ hybridization to detect potential recurrent BCOR gene rearrangements outside the typical X-chromosomal inversion. Indeed, 8/75 (11%) SBRCTs showed distinct BCOR gene rearrangements, with 2 cases each showing either a BCOR-MAML3 or the alternative ZC3H7B-BCOR fusion, whereas no fusion partner was detected in the remaining 4 cases. Gene expression of the BCOR-MAML3-positive index case showed a distinct transcriptional profile with upregulation of HOX-gene signature, compared with classic Ewing's sarcoma or CIC-DUX4-positive SBRCTs. The clinicopathologic features of the SBRCTs with alternative BCOR rearrangements were also compared with a group of BCOR-CCNB3 inversion-positive cases, combining 11 from our files with a meta-analysis of 42 published cases. The BCOR-CCNB3-positive tumors occurred preferentially in children and in bone, in contrast to alternative BCOR-rearranged SBRCTs, which presented in young adults, with a variable anatomic distribution. Furthermore, BCOR-rearranged tumors often displayed spindle cell areas, either well defined in intersecting fascicles or blending with the round cell component, which appears distinct from most other fusion-positive SBRCTs and shares histologic overlap with poorly differentiated synovial sarcoma.

Chimera: a Bioconductor package for secondary analysis of fusion products.

PubMed

Beccuti, Marco; Carrara, Matteo; Cordero, Francesca; Lazzarato, Fulvio; Donatelli, Susanna; Nadalin, Francesca; Policriti, Alberto; Calogero, Raffaele A

2014-12-15

Chimera is a Bioconductor package that organizes, annotates, analyses and validates fusions reported by different fusion detection tools; current implementation can deal with output from bellerophontes, chimeraScan, deFuse, fusionCatcher, FusionFinder, FusionHunter, FusionMap, mapSplice, Rsubread, tophat-fusion and STAR. The core of Chimera is a fusion data structure that can store fusion events detected with any of the aforementioned tools. Fusions are then easily manipulated with standard R functions or through the set of functionalities specifically developed in Chimera with the aim of supporting the user in managing fusions and discriminating false-positive results. © The Author 2014. Published by Oxford University Press.

Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.

PubMed

Liu, Di; Guo, Hua; Zheng, Wenyun; Zhang, Na; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

2016-06-01

Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease.

Modulation of Membrane Protein Lateral Mobility by Polyphosphates and Polyamines

NASA Astrophysics Data System (ADS)

Schindler, Melvin; Koppel, Dennis E.; Sheetz, Michael P.

1980-03-01

The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of ``fluorescence redistribution after fusion.'' Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugawara, Taishi; Ito, Keisuke; Shiroishi, Mitsunori

2009-05-15

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these fivemore » bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-{beta}-D-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.« less

Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer

PubMed Central

Rogers, Toni-Maree; Arnau, Gisela Mir; Ryland, Georgina L.; Huang, Stephen; Lira, Maruja E.; Emmanuel, Yvette; Perez, Omar D.; Irwin, Darryl; Fellowes, Andrew P.; Wong, Stephen Q.; Fox, Stephen B.

2017-01-01

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86–96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof–of–principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting. PMID:28181564

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

PubMed

Corcoran, Jennifer A; Salsman, Jayme; de Antueno, Roberto; Touhami, Ahmed; Jericho, Manfred H; Clancy, Eileen K; Duncan, Roy

2006-10-20

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization

PubMed Central

Hensing, Thomas; Schrock, Alexa B.; Allen, Justin; Sanford, Eric; Gowen, Kyle; Kulkarni, Atul; He, Jie; Suh, James H.; Lipson, Doron; Elvin, Julia A.; Yelensky, Roman; Chalmers, Zachary; Chmielecki, Juliann; Peled, Nir; Klempner, Samuel J.; Firozvi, Kashif; Frampton, Garrett M.; Molina, Julian R.; Menon, Smitha; Brahmer, Julie R.; MacMahon, Heber; Nowak, Jan; Ou, Sai-Hong Ignatius; Zauderer, Marjorie; Ladanyi, Marc; Zakowski, Maureen; Fischbach, Neil; Ross, Jeffrey S.; Stephens, Phil J.; Miller, Vincent A.; Wakelee, Heather

2016-01-01

Introduction. For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. Materials and Methods. Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. Results. A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. Conclusion. Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. Implications for Practice: Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing. PMID:27245569

Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization.

PubMed

Ali, Siraj M; Hensing, Thomas; Schrock, Alexa B; Allen, Justin; Sanford, Eric; Gowen, Kyle; Kulkarni, Atul; He, Jie; Suh, James H; Lipson, Doron; Elvin, Julia A; Yelensky, Roman; Chalmers, Zachary; Chmielecki, Juliann; Peled, Nir; Klempner, Samuel J; Firozvi, Kashif; Frampton, Garrett M; Molina, Julian R; Menon, Smitha; Brahmer, Julie R; MacMahon, Heber; Nowak, Jan; Ou, Sai-Hong Ignatius; Zauderer, Marjorie; Ladanyi, Marc; Zakowski, Maureen; Fischbach, Neil; Ross, Jeffrey S; Stephens, Phil J; Miller, Vincent A; Wakelee, Heather; Ganesan, Shridar; Salgia, Ravi

2016-06-01

For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing. ©AlphaMed Press.

Fusion enhancement at near and sub-barrier energies in 19O + 12C

DOE PAGES

Singh, Varinderjit; Vadas, J.; Steinbach, T. K.; ...

2016-12-12

Measuring the fusion excitation function for an isotopic chain of projectile nuclei provides a stringent test of a microscopic description of fusion. We report the first measurement of the fusion excitation function at near-barrier energies for the 19O+ 12C system. The measured excitation function is compared with the fusion excitation function of 18O+ 12C. A significant enhancement in the fusion probability of 19O ions with a 12C target as compared to 18O ions is observed. As a result, the experimental cross-sections observed at near-barrier energies are compared with a state-of-the-art microscopic model.

Human-technology interaction for standoff IED detection

NASA Astrophysics Data System (ADS)

Zhang, Evan; Zou, Yiyang; Zachrich, Liping; Fulton, Jack

2011-03-01

IEDs kill our soldiers and innocent people every day. Lessons learned from Iraq and Afghanistan clearly indicated that IEDs cannot be detected/defeated by technology alone; human-technology interaction must be engaged. In most cases, eye is the best detector, brain is the best computer, and technologies are tools, they must be used by human being properly then can achieve full functionality. In this paper, a UV Raman/fluorescence, CCD and LWIR 3 sensor fusion system for standoff IED detection and a handheld fusion system for close range IED detection are developed and demonstrated. We must train solders using their eyes or CCD/LWIR cameras to do wide area search while on the move to find small suspected area first then use the spectrometer because the laser spot is too small, to scan a one-mile long and 2-meter wide road needs 185 days although our fusion system can detect the IED in 30m with 1s interrogating time. Even if the small suspected area (e.g., 0.5mx0.5m) is found, human eyes still cannot detect the IED, soldiers must use or interact with the technology - laser based spectrometer to scan the area then they are able to detect and identify the IED in 10 minutes not 185 days. Therefore, the human-technology interaction approach will be the best solution for IED detection.

The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

PubMed

Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

2015-04-21

A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

3D fluorescence anisotropy imaging using selective plane illumination microscopy.

PubMed

Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

2015-08-24

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Sepiella japonica.

PubMed

Huo, Liping; Bao, Miaomiao; Lv, Zhenming; Chi, Changfeng; Wang, Tianming; Liu, Huihui

2018-05-01

Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-κB (NF-κB). In this study a novel isoform of MyD88 in Sepiella japonica (SjMyD88) was cloned and functionally characterized (GenBank accession no. AQY56781.1). The complete cDNA sequence of SjMyD88 was 1912 bp and contained a 1017 bp open reading frame encoding 338 amino acid residues, which was similar to its mollusk orthologues in the length. BLASTp analysis suggested the deduced amino acids sequence of SjMyD88 shared high identity to the known MyD88, for instance, 64% identity with Octopus bimaculoides. Sequence analysis revealed two conserved domains, the N-terminal DD and the C-terminal TIR domain appeared in SjMyD88, which was consistent with MyD88 proteins from other species. The fusion expression of SjMyD88 and green fluorescent protein (EGFP) in HEK293 cells was conducted and cytoplasm localization was detected. Meanwhile, the TIR-pmCherry fusion protein showed red fluorescence and mainly distributed in the cytoplasm. After cotransfection MyD88-EGFP and TIR-pmCherry red obviously overlapped and changed to yellowish green. The results suggested that there was the interaction between homologous TIR-pmcherry and MyD88-EGFP. Tissues expression profiles analysis showed that SjMyD88 ubiquitously expressed in all tested tissues with the highest expression in the gills and livers except reproductive related tissue, and it was significantly induced in livers under LPS stress. These data provide insight into the roles of SjMyD88 in the TLR signaling pathway of S. japonica in response to pathogenic bacteria. Copyright © 2018. Published by Elsevier Ltd.

A measurement fusion method for nonlinear system identification using a cooperative learning algorithm.

PubMed

Xia, Youshen; Kamel, Mohamed S

2007-06-01

Identification of a general nonlinear noisy system viewed as an estimation of a predictor function is studied in this article. A measurement fusion method for the predictor function estimate is proposed. In the proposed scheme, observed data are first fused by using an optimal fusion technique, and then the optimal fused data are incorporated in a nonlinear function estimator based on a robust least squares support vector machine (LS-SVM). A cooperative learning algorithm is proposed to implement the proposed measurement fusion method. Compared with related identification methods, the proposed method can minimize both the approximation error and the noise error. The performance analysis shows that the proposed optimal measurement fusion function estimate has a smaller mean square error than the LS-SVM function estimate. Moreover, the proposed cooperative learning algorithm can converge globally to the optimal measurement fusion function estimate. Finally, the proposed measurement fusion method is applied to ARMA signal and spatial temporal signal modeling. Experimental results show that the proposed measurement fusion method can provide a more accurate model.

Application of semiconductor fluorescent nanocrystals as optical probes for rapid early viral detection

NASA Astrophysics Data System (ADS)

Bentzen, Elizabeth L.; House, Frances; Tomlinson, Ian D.; Rosenthal, Sandra J.; Crowe, James E.; Wright, David D.

2005-04-01

Fluorescence is a tool widely employed in biological assays. Fluorescent semiconducting nanocrystals, quantum dots (QDs), are beginning to find their way into the tool box of many biologist, chemist and biochemist. These quantum dots are an attractive alternative to the traditional organic dyes due to their broad excitation spectra, narrow emission spectra and photostability. Non-specific binding is a frequently encountered problem with fluorescent labeling in biological assays. In these studies various cell lines were examined for non-specific binding to quantum dots. Evidence suggests that non-specific binding is related to cell type and, may be significantly reduced by functionalizing quantum dots with polyethyleneglycol ligands (PEG). In addition quantum dots were used to detect and monitor the progession of the viral glycoproteins ,F (fusion) and G (attachment), from Respiratory Syncytial Virus (RSV) in HEp-2 cells. RSV is the most common cause of lower respiratory tract infection in children worldwide and the most common cause of hospitalization of infants in the US. Antiviral therapy is available for treatment of RSV but is only effective if given within the first 48 hours of infection. Existing test methods require a virus level of at least 1000-fold of the amount needed for infection of most children and require several days to weeks to obtain results. The use of quantum dots may provide an early, rapid method for detection and provide insight into the trafficking of viral proteins during the course of infection.

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis.

PubMed

Weber, K; Mock, U; Petrowitz, B; Bartsch, U; Fehse, B

2010-04-01

Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10(6) per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

RET fusion as a novel driver of medullary thyroid carcinoma.

PubMed

Grubbs, Elizabeth G; Ng, Patrick Kwok-Shing; Bui, Jacquelin; Busaidy, Naifa L; Chen, Ken; Lee, Jeffrey E; Lu, Xinyan; Lu, Hengyu; Meric-Bernstam, Funda; Mills, Gordon B; Palmer, Gary; Perrier, Nancy D; Scott, Kenneth L; Shaw, Kenna R; Waguespack, Steven G; Williams, Michelle D; Yelensky, Roman; Cote, Gilbert J

2015-03-01

Oncogenic RET tyrosine kinase gene fusions and activating mutations have recently been identified in lung cancers, prompting initiation of targeted therapy trials in this disease. Although RET point mutation has been identified as a driver of tumorigenesis in medullary thyroid carcinoma (MTC), no fusions have been described to date. We evaluated the role of RET fusion as an oncogenic driver in MTC. We describe a patient who died from aggressive sporadic MTC < 10 months after diagnosis. Her tumor was evaluated by means of next-generation sequencing, including an intronic capture strategy. A reciprocal translocation involving RET intron 12 was identified. The fusion was validated using a targeted break apart fluorescence in situ hybridization probe, and RNA sequencing confirmed the existence of an in-frame fusion transcript joining MYH13 exon 35 with RET exon 12. Ectopic expression of fusion product in a murine Ba/F3 cell reporter model established strong oncogenicity. Three tyrosine kinase inhibitors currently used to treat MTC in clinical practice blocked tumorigenic cell growth. This finding represents the report of a novel RET fusion, the first of its kind described in MTC. The finding of this potential novel oncogenic mechanism has clear implications for sporadic MTC, which in the majority of cases has no driver mutation identified. The presence of a RET fusion also provides a plausible target for RET tyrosine kinase inhibitor therapies.

Engineering and Functional Characterization of Fusion Genes Identifies Novel Oncogenic Drivers of Cancer. | Office of Cancer Genomics

Cancer.gov

Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK, and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in TCGA datasets.

Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

NASA Technical Reports Server (NTRS)

Love, J.; Scott, A. C.; Thompson, W. F.; Brown, C. S. (Principal Investigator)

2000-01-01

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.

Influence of incomplete fusion on complete fusion at energies above the Coulomb barrier

NASA Astrophysics Data System (ADS)

Shuaib, Mohd; Sharma, Vijay R.; Yadav, Abhishek; Sharma, Manoj Kumar; Singh, Pushpendra P.; Singh, Devendra P.; Kumar, R.; Singh, R. P.; Muralithar, S.; Singh, B. P.; Prasad, R.

2017-10-01

In the present work, excitation functions of several reaction residues in the system 19F+169Tm, populated via the complete and incomplete fusion processes, have been measured using off-line γ-ray spectroscopy. The analysis of excitation functions has been done within the framework of statistical model code pace4. The excitation functions of residues populated via xn and pxn channels are found to be in good agreement with those estimated by the theoretical model code, which confirms the production of these residues solely via complete fusion process. However, a significant enhancement has been observed in the cross-sections of residues involving α-emitting channels as compared to the theoretical predictions. The observed enhancement in the cross-sections has been attributed to the incomplete fusion processes. In order to have a better insight into the onset and strength of incomplete fusion, the incomplete fusion strength function has been deduced. At present, there is no theoretical model available which can satisfactorily explain the incomplete fusion reaction data at energies ≈4-6 MeV/nucleon. In the present work, the influence of incomplete fusion on complete fusion in the 19F+169Tm system has also been studied. The measured cross-section data may be important for the development of reactor technology as well. It has been found that the incomplete fusion strength function strongly depends on the α-Q value of the projectile, which is found to be in good agreement with the existing literature data. The analysis strongly supports the projectile-dependent mass-asymmetry systematics. In order to study the influence of Coulomb effect ({Z}{{P}}{Z}{{T}}) on incomplete fusion, the deduced strength function for the present work is compared with the nearby projectile-target combinations. The incomplete fusion strength function is found to increase linearly with {Z}{{P}}{Z}{{T}}, indicating a strong influence of Coulomb effect in the incomplete fusion reactions.

Systematic identification and analysis of frequent gene fusion events in metabolic pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, Christopher S.; Lerma-Ortiz, Claudia; Gerdes, Svetlana Y.

Here, gene fusions are the most powerful type of in silico-derived functional associations. However, many fusion compilations were made when <100 genomes were available, and algorithms for identifying fusions need updating to handle the current avalanche of sequenced genomes. The availability of a large fusion dataset would help probe functional associations and enable systematic analysis of where and why fusion events occur. As a result, here we present a systematic analysis of fusions in prokaryotes. We manually generated two training sets: (i) 121 fusions in the model organism Escherichia coli; (ii) 131 fusions found in B vitamin metabolism. These setsmore » were used to develop a fusion prediction algorithm that captured the training set fusions with only 7 % false negatives and 50 % false positives, a substantial improvement over existing approaches. This algorithm was then applied to identify 3.8 million potential fusions across 11,473 genomes. The results of the analysis are available in a searchable database. A functional analysis identified 3,000 reactions associated with frequent fusion events and revealed areas of metabolism where fusions are particularly prevalent. In conclusion, customary definitions of fusions were shown to be ambiguous, and a stricter one was proposed. Exploring the genes participating in fusion events showed that they most commonly encode transporters, regulators, and metabolic enzymes. The major rationales for fusions between metabolic genes appear to be overcoming pathway bottlenecks, avoiding toxicity, controlling competing pathways, and facilitating expression and assembly of protein complexes. Finally, our fusion dataset provides powerful clues to decipher the biological activities of domains of unknown function.« less

Systematic identification and analysis of frequent gene fusion events in metabolic pathways

DOE PAGES

Henry, Christopher S.; Lerma-Ortiz, Claudia; Gerdes, Svetlana Y.; ...

2016-06-24

Here, gene fusions are the most powerful type of in silico-derived functional associations. However, many fusion compilations were made when <100 genomes were available, and algorithms for identifying fusions need updating to handle the current avalanche of sequenced genomes. The availability of a large fusion dataset would help probe functional associations and enable systematic analysis of where and why fusion events occur. As a result, here we present a systematic analysis of fusions in prokaryotes. We manually generated two training sets: (i) 121 fusions in the model organism Escherichia coli; (ii) 131 fusions found in B vitamin metabolism. These setsmore » were used to develop a fusion prediction algorithm that captured the training set fusions with only 7 % false negatives and 50 % false positives, a substantial improvement over existing approaches. This algorithm was then applied to identify 3.8 million potential fusions across 11,473 genomes. The results of the analysis are available in a searchable database. A functional analysis identified 3,000 reactions associated with frequent fusion events and revealed areas of metabolism where fusions are particularly prevalent. In conclusion, customary definitions of fusions were shown to be ambiguous, and a stricter one was proposed. Exploring the genes participating in fusion events showed that they most commonly encode transporters, regulators, and metabolic enzymes. The major rationales for fusions between metabolic genes appear to be overcoming pathway bottlenecks, avoiding toxicity, controlling competing pathways, and facilitating expression and assembly of protein complexes. Finally, our fusion dataset provides powerful clues to decipher the biological activities of domains of unknown function.« less

XRF inductive bead fusion and PLC based control system

NASA Astrophysics Data System (ADS)

Zhu, Jin-hong; Wang, Ying-jie; Shi, Hong-xin; Chen, Qing-ling; Chen, Yu-xi

2009-03-01

In order to ensure high-quality X-ray fluorescence spectrometry (XRF) analysis, an inductive bead fusion machine was developed. The prototype consists of super-audio IGBT induction heating power supply, rotation and swing mechanisms, and programmable logic controller (PLC). The system can realize sequence control, mechanical movement control, output current and temperature control. Experimental results show that the power supply can operate at an ideal quasi-resonant state, in which the expected power output and the required temperature can be achieved for rapid heating and the uniform formation of glass beads respectively.

Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

PubMed

Kraft, Thomas E; Hresko, Richard C; Hruz, Paul W

2015-12-01

The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses. © 2015 The Protein Society.

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

PubMed Central

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y.; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality. PMID:24505408

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

PubMed

Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

2014-01-01

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

The pharmacological efficacy of the anti-IL17 scFv and sTNFR1 bispecific fusion protein in inflammation mouse stimulated by LPS.

PubMed

Yang, Yongbi; Zhang, Teng; Cao, Hongxue; Yu, Dan; Zhang, Tong; Zhao, Shaojuan; Jing, Xiaohui; Song, Liying; Liu, Yunye; Che, Ruixiang; Liu, Xin; Li, Deshan; Ren, Guiping

2017-08-01

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our study found that a bispecific fusion protein treatment can ameliorate the lung injury induced by LPS. However, the molecular mechanisms which bispecific fusion protein ameliorates acute lung injury remain unclear. In this study, we found that the bispecific fusion protein treatment inhibited the nuclear transcription of NF-κB in confocal laser scanning fluorescence microscopy, the bispecific fusion protein exert protective effects in the cell model of ALI induced by lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway and mediate inflammation. Moreover, the treatment of the bispecific fusion protein show its efficacy in animal models stimulated by LPS, the results of real-time PCR and ELISA demonstrate that bispecific fusion protein treatment effectively inhibited the over-expression of inflammatory cytokines(tumor necrosis factor α, interleukin 1β and interleukin 17). In addition, LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology, which was meliorated by bispecific fusion protein treatment. Collectively, these results demonstrate that bispecific fusion protein treatment ameliorates LPS-induced ALI through reducing inflammatory cytokines and lung inflammation, which may be associated with the decreased the nuclear transcription of NF-κB. The bispecific fusion protein may be useful as a novel therapy to treat ALI. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

PubMed

Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

2014-05-01

Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A plasmid collection for PCR-based gene targeting in the filamentous ascomycete Ashbya gossypii.

PubMed

Kaufmann, Andreas

2009-08-01

PCR-based gene targeting with heterologous markers is an efficient method to delete genes, generate gene fusions, and modulate gene expression. For the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, several plasmid collections are available covering a wide range of tags and markers. For several reasons, many of these cassettes cannot be used in the filamentous ascomycete Ashbya gossypii. This article describes the construction of 93 heterologous modules for C- and N-terminal tagging and promoter replacements in A. gossypii. The performance of 12 different fluorescent tags was evaluated by monitoring their brightness, detectability, and photostability when fused to the myosin light-chain protein Mlc2. Furthermore, the thiamine-repressible S. cerevisiae THI13 promoter was established to regulate gene expression in A. gossypii. This collection will help accelerate analysis of gene function in A. gossypii and in other ascomycetes where S. cerevisiae promoter elements are functional.

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

Localization of a bacterial group II intron-encoded protein in human cells.

PubMed

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

2015-08-05

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

Localization of a bacterial group II intron-encoded protein in human cells

PubMed Central

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

2015-01-01

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells

PubMed Central

Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

2014-01-01

We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation. PMID:24817874

Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells.

PubMed

Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

2014-01-01

We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.

[Application of polyclonal break-apart probes in the diagnosis of Xp11.2 translocation renal cell carcinoma].

PubMed

Chen, Xiancheng; Gan, Weidong; Ye, Qing; Yang, Jun; Guo, Hongqian; Li, Dongmei

2014-12-16

To explore the value of self-designed fluorescent in situ hybridization (FISH) polyclonal break-apart probes specific for TFE3 gene in the diagnosis of Xp11.2 translocation renal cell carcinoma. All tissue samples were collected from 2006 to 2013, including Xp11.2 translocation renal cell carcinoma (n = 10), renal clear cell carcinoma (n = 10) and renal papillary cell carcinoma (n = 10). FISH was conducted for paraffin-embedded tumor tissue sections with probes. The types of fluorescence were observed by fluorescent microscopy to determine the existence or non-existence of translocated TFE3 gene. All sections were successfully probed. The split red and green signals within a single nucleus were detected simultaneously in 9 cases of Xp11.2 translocation renal cell carcinoma as diagnosed by traditional pathological and immunohistochemical methods. And it was consistent with the initial diagnosis. Detection of fusion signal in 1/10 and negative FISH result did not conform to the initial diagnosis. The fluorescent types of renal clear cell carcinoma and renal papillary cell carcinoma were all fusion signals. FISH tests were negative for renal clear and papillary cell carcinomas. Xp11.2 translocation renal cell carcinomas diagnosed by traditional pathological and immunohistochemical methods are sometimes misdiagnosed. Detecting the translocation of TFE3 gene with FISH polyclonal break-apart probes is both accurate and reliable for diagnosing Xp11.2 translocation renal cell carcinoma.

Low Copy Numbers of DC-SIGN in Cell Membrane Microdomains: Implications for Structure and Function

PubMed Central

Liu, Ping; Wang, Xiang; Itano, Michelle S.; Neumann, Aaron K.; de Silva, Aravinda M.; Jacobson, Ken; Thompson, Nancy L.

2014-01-01

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1μm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (~50nm) pathogen, dengue virus, leading to infection of host cells. PMID:24313910

A new protein-protein interaction sensor based on tripartite split-GFP association.

PubMed

Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

2013-10-04

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association

PubMed Central

Cabantous, Stéphanie; Nguyen, Hau B.; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A.; Favre, Gilles; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2013-01-01

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence. PMID:24092409

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome of Turnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta▿

PubMed Central

Beauchemin, Chantal; Boutet, Nathalie; Laliberté, Jean-François

2007-01-01

The RNA genome of Turnip mosaic virus is covalently linked at its 5′ end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication. PMID:17079311

High-level production of human interleukin-10 fusions in tobacco cell suspension cultures

PubMed Central

Kaldis, Angelo; Ahmad, Adil; Reid, Alexandra; McGarvey, Brian; Brandle, Jim; Ma, Shengwu; Jevnikar, Anthony; Kohalmi, Susanne E; Menassa, Rima

2013-01-01

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER. PMID:23297698

Visualizing Herpesvirus Procapsids in Living Cells.

PubMed

Maier, Oana; Sollars, Patricia J; Pickard, Gary E; Smith, Gregory A

2016-11-15

A complete understanding of herpesvirus morphogenesis requires studies of capsid assembly dynamics in living cells. Although fluorescent tags fused to the VP26 and pUL25 capsid proteins are available, neither of these components is present on the initial capsid assembly, the procapsid. To make procapsids accessible to live-cell imaging, we made a series of recombinant pseudorabies viruses that encoded green fluorescent protein (GFP) fused in frame to the internal capsid scaffold and maturation protease. One recombinant, a GFP-VP24 fusion, maintained wild-type propagation kinetics in vitro and approximated wild-type virulence in vivo The fusion also proved to be well tolerated in herpes simplex virus. Viruses encoding GFP-VP24, along with a traditional capsid reporter fusion (pUL25/mCherry), demonstrated that GFP-VP24 was a reliable capsid marker and revealed that the protein remained capsid associated following entry into cells and upon nuclear docking. These dual-fluorescent viruses made possible the discrimination of procapsids during infection and monitoring of capsid shell maturation kinetics. The results demonstrate the feasibility of imaging herpesvirus procapsids and their morphogenesis in living cells and indicate that the encapsidation machinery does not substantially help coordinate capsid shell maturation. The family Herpesviridae consists of human and veterinary pathogens that cause a wide range of diseases in their respective hosts. These viruses share structurally related icosahedral capsids that encase the double-stranded DNA (dsDNA) viral genome. The dynamics of capsid assembly and maturation have been inaccessible to examination in living cells. This study has overcome this technical hurdle and provides new insights into this fundamental stage of herpesvirus infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Varinderjit; Vadas, J.; Steinbach, T. K.

Measuring the fusion excitation function for an isotopic chain of projectile nuclei provides a stringent test of a microscopic description of fusion. We report the first measurement of the fusion excitation function at near-barrier energies for the 19O+ 12C system. The measured excitation function is compared with the fusion excitation function of 18O+ 12C. A significant enhancement in the fusion probability of 19O ions with a 12C target as compared to 18O ions is observed. As a result, the experimental cross-sections observed at near-barrier energies are compared with a state-of-the-art microscopic model.

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

PubMed

Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

2003-08-01

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila.

PubMed

Köhler, R; Bubert, A; Goebel, W; Steinert, M; Hacker, J; Bubert, B

2000-01-01

The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.

ICG-enhanced imaging of arthritis with an integrated Optical Imaging/X-ray System

PubMed Central

Meier, Reinhard; Krug, Christian; Golovko, Daniel; Boddington, Sophie; Piontek, Guido; Rudelius, Martina; Sutton, Elizabeth J.; Baur-Melnyk, Andrea; Jones, Ella F.; Daldrup-Link, Heike E.

2010-01-01

Background Optical Imaging (OI) is a promising technique that is quick, inexpensive and, in combination with Indocyanine Green (ICG), an FDA-approved fluorescent dye, could provide early detection of rheumatoid arthritis. Objective The purpose of this study was to evaluate a combined X-ray/OI imaging system for ICG-enhanced detection of arthritic joints in a rat model of antigen induced arthritis. Methods Arthritis of the knee and ankle joints was induced in six Harlan rats with peptidoglycan polysaccharide polymers (PGPS). Three rats served as non-treated controls. Optical imaging of the knee and ankle joints was done with an integrated OI/X-ray system before and up to 24h post intravenous injection (p.i.) of 10mg/kg ICG. The fluorescence signal intensities of arthritic and normal joints were compared for significant differences using generalized estimation equation models. Specimen of knee and ankle joints were further processed and evaluated by histology. Results ICG provided a significant increase in fluorescence signal of arthritic joints compared to baseline values immediately after administration (p<0.05). The fluorescence signal of arthritic joints was significantly higher compared to the non-arthritic control joints at 1 - 720 min p.i. (p<0.05). Fusion of ICG-enhanced OI and X-rays allowed for anatomical co-registration of the inflamed tissue with the associated joint. H&E stains confirmed marked synovial inflammation of arthritic joints and absence of inflammation in control joints. Conclusion ICG-enhanced OI is a clinically applicable tool for detection of arthritic tissue. Using relatively high doses of ICG, a long term fluorescence enhancement of arthritic joints can be achieved. This may facilitate simultaneous evaluations of multiple joints in a clinical setting. Fusion of ICG-OI scans with X-ray imaging increases anatomical resolution. PMID:20506388

A Large Nonconserved Region of the Tethering Protein Leashin Is Involved in Regulating the Position, Movement, and Function of Woronin Bodies in Aspergillus oryzae

PubMed Central

Han, Pei; Jin, Feng Jie; Kitamoto, Katsuhiko

2014-01-01

The Woronin body is a Pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. Leashin (LAH) is a large Woronin body tethering protein that contains highly conserved N- and C-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. As the involvement of the nonconserved region in Woronin body function has not been investigated, here, we functionally characterized individual regions of the LAH protein of Aspergillus oryzae (AoLAH). In an Aolah disruptant, no Woronin bodies were tethered to the septum, and hyphae had a reduced ability to prevent excessive cytoplasmic loss upon hyphal wounding. Localization analysis revealed that the N-terminal region of AoLAH associated with Woronin bodies dependently on AoWSC, which is homologous to Neurospora crassa WSC (Woronin body sorting complex), and that the C-terminal region was localized to the septum. Elastic movement of Woronin bodies was observed when visualized with an AoLAH N-terminal-region–enhanced green fluorescent protein (EGFP) fusion protein. An N- and C-terminal fusion construct lacking the nonconserved middle region of AoLAH was sufficient for the tethering of Woronin bodies to the septum. However, Woronin bodies were located closer to the septum and exhibited impaired elastic movement. Moreover, expression of middle-region-deleted AoLAH in the Aolah disruptant did not restore the ability to prevent excessive cytoplasmic loss. These findings indicate that the nonconserved middle region of AoLAH has functional importance for regulating the position, movement, and function of Woronin bodies. PMID:24813188

A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors

PubMed Central

Corridon, Peter R.; Rhodes, George J.; Leonard, Ellen C.; Basile, David P.; Gattone, Vincent H.; Bacallao, Robert L.

2013-01-01

Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10–50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys. PMID:23467422

Fusion of small unilamellar vesicles induced by bovine serum albumin fragments.

PubMed

Garcia, L A; Schenkman, S; Araujo, P S; Chaimovich, H

1983-07-01

The limited pepsin proteolysis products of bovine serum albumin, fragment A (residues 307-586) and fragment B (residues 1-306), induced the fusion of small unilamellar vesicles of egg phosphatidyl choline at concentrations near 5 microM. Fusion was demonstrated and analyzed on the basis of: a) time-dependent changes in absorbance; b) dilution of the fluorescent label 2-(10-(1-pyrene)decanoyl) phosphatidyl choline, incorporated into a small percentage of the vesicles, as measured by the decrease in the excimer to monomer (E/M) ratio; c) increase of the average hydrodynamic radius of the liposomes, estimated by Sepharose 4B filtration, and d) the strict inverse relationship between the size of the liposomes and their E/M ratios. Albumin fragment B, like albumin, induced the formation of large aggregates in which rapid cooperative fusion produced vesicles having a large hydrodynamic radius. Fragment A did not produce large aggregates and the initial fusion products exhibited a hydrodynamic radius. Fragment A did not produce large aggregates and the initial fusion products exhibited a hydrodynamic radius smaller than those obtained with fragment B. Albumin and fragments A and B are fusogenic only at pH below 4.0. These data discussed in terms of a general model for a signal-dependent protein-induced membrane fusion.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport

PubMed Central

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-01-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. PMID:25657322

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

PubMed

Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

2017-07-01

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells.

PubMed

Li, Ke; Zhang, Zhi-Ping; Luo, Ming; Yu, Xiang; Han, Yu; Wei, Hong-Ping; Cui, Zong-Qiang; Zhang, Xian-En

2012-01-07

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging α(v)β(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.

Local protein dynamics during microvesicle exocytosis in neuroendocrine cells.

PubMed

Somasundaram, Agila; Taraska, Justin

2018-06-06

Calcium triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine transporter (VAChT) tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are pre-clustered at fusion sites and rapidly lost at fusion. The ATPase NSF is locally recruited at fusion. Interestingly, the endocytic BAR domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites, and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the over-expression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

Membrane Fusion Proteins as Nanomachines

NASA Astrophysics Data System (ADS)

Tamm, Lukas

2009-03-01

Membrane fusion is key to fertilization, virus infection, and neurotransmission. Specific proteins work like nanomachines to stitch together fluid, yet highly ordered lipid bilayers. The energy gained from large exothermic conformational changes of these proteins is utilized to fuse lipid bilayers that do not fuse spontaneously. Structural studies using x-ray crystallography and NMR spectroscopy have yielded detailed information about architecture and inner workings of these molecular machines. The question now is: how is mechanical energy gained from such protein transformations harnessed to transform membrane topology? To answer this question, we have determined that a boomerang-shaped structure of the influenza fusion peptide is critical to generate a high-energy binding intermediate in the target membrane and to return the ``boomerang'' to its place of release near the viral membrane for completion of the fusion cycle. In presynaptic exocytosis, receptor and acceptor SNAREs are zippered to form a helical bundle that is arrested shortly before the membrane. Ca binding to interlocked synaptotagmin releases the fusion block. Structural NMR and single molecule fluorescence data are combined to arrive at and further refine this picture.

Belief Function Based Decision Fusion for Decentralized Target Classification in Wireless Sensor Networks

PubMed Central

Zhang, Wenyu; Zhang, Zhenjiang

2015-01-01

Decision fusion in sensor networks enables sensors to improve classification accuracy while reducing the energy consumption and bandwidth demand for data transmission. In this paper, we focus on the decentralized multi-class classification fusion problem in wireless sensor networks (WSNs) and a new simple but effective decision fusion rule based on belief function theory is proposed. Unlike existing belief function based decision fusion schemes, the proposed approach is compatible with any type of classifier because the basic belief assignments (BBAs) of each sensor are constructed on the basis of the classifier’s training output confusion matrix and real-time observations. We also derive explicit global BBA in the fusion center under Dempster’s combinational rule, making the decision making operation in the fusion center greatly simplified. Also, sending the whole BBA structure to the fusion center is avoided. Experimental results demonstrate that the proposed fusion rule has better performance in fusion accuracy compared with the naïve Bayes rule and weighted majority voting rule. PMID:26295399

Respiratory Chain Complexes in Dynamic Mitochondria Display a Patchy Distribution in Life Cells

PubMed Central

Muster, Britta; Kohl, Wladislaw; Wittig, Ilka; Strecker, Valentina; Joos, Friederike; Haase, Winfried; Bereiter-Hahn, Jürgen; Busch, Karin

2010-01-01

Background Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. Methodology/Principal Findings The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. Conclusion/Significance Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion. PMID:20689601

Sister kinetochores are mechanically fused during meiosis I in yeast.

PubMed

Sarangapani, Krishna K; Duro, Eris; Deng, Yi; Alves, Flavia de Lima; Ye, Qiaozhen; Opoku, Kwaku N; Ceto, Steven; Rappsilber, Juri; Corbett, Kevin D; Biggins, Sue; Marston, Adèle L; Asbury, Charles L

2014-10-10

Production of healthy gametes requires a reductional meiosis I division in which replicated sister chromatids comigrate, rather than separate as in mitosis or meiosis II. Fusion of sister kinetochores during meiosis I may underlie sister chromatid comigration in diverse organisms, but direct evidence for such fusion has been lacking. We used laser trapping and quantitative fluorescence microscopy to study native kinetochore particles isolated from yeast. Meiosis I kinetochores formed stronger attachments and carried more microtubule-binding elements than kinetochores isolated from cells in mitosis or meiosis II. The meiosis I-specific monopolin complex was both necessary and sufficient to drive these modifications. Thus, kinetochore fusion directs sister chromatid comigration, a conserved feature of meiosis that is fundamental to Mendelian inheritance. Copyright © 2014, American Association for the Advancement of Science.

Engineering and Functional Characterization of Fusion Genes Identifies Novel Oncogenic Drivers of Cancer.

PubMed

Lu, Hengyu; Villafane, Nicole; Dogruluk, Turgut; Grzeskowiak, Caitlin L; Kong, Kathleen; Tsang, Yiu Huen; Zagorodna, Oksana; Pantazi, Angeliki; Yang, Lixing; Neill, Nicholas J; Kim, Young Won; Creighton, Chad J; Verhaak, Roel G; Mills, Gordon B; Park, Peter J; Kucherlapati, Raju; Scott, Kenneth L

2017-07-01

Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK , and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in The Cancer Genome Atlas datasets. In addition to confirming oncogenic activity of the known fusion oncogenes engineered by our construction strategy, we validated five novel fusion genes involving MET, NTRK2 , and BRAF kinases that exhibited potent transforming activity and conferred sensitivity to FDA-approved kinase inhibitors. Our fusion construction strategy also enabled domain-function studies of BRAF fusion genes. Our results confirmed other reports that the transforming activity of BRAF fusions results from truncation-mediated loss of inhibitory domains within the N-terminus of the BRAF protein. BRAF mutations residing within this inhibitory region may provide a means for BRAF activation in cancer, therefore we leveraged the modular design of our fusion gene construction methodology to screen N-terminal domain mutations discovered in tumors that are wild-type at the BRAF mutation hotspot, V600. We identified an oncogenic mutation, F247L, whose expression robustly activated the MAPK pathway and sensitized cells to BRAF and MEK inhibitors. When applied broadly, these tools will facilitate rapid fusion gene construction for subsequent functional characterization and translation into personalized treatment strategies. Cancer Res; 77(13); 3502-12. ©2017 AACR . ©2017 American Association for Cancer Research.

Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications.

PubMed

Mizukami, Shin; Hori, Yuichiro; Kikuchi, Kazuya

2014-01-21

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

Overexpression of neurofilament H disrupts normal cell structure and function

NASA Technical Reports Server (NTRS)

Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

2002-01-01

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.

PubMed

Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily; Potempa, Marc; Schiffer, Celia A; Janzen, William; Swanstrom, Ronald

2013-07-23

The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.

A C. trachomatis Cloning Vector and the Generation of C. trachomatis Strains Expressing Fluorescent Proteins under the Control of a C. trachomatis Promoter

PubMed Central

Agaisse, Hervé; Derré, Isabelle

2013-01-01

Here we describe a versatile cloning vector for conducting genetic experiments in C. trachomatis. We successfully expressed various fluorescent proteins (i.e. GFP, mCherry and CFP) from C. trachomatis regulatory elements (i.e. the promoter and terminator of the incDEFG operon) and showed that the transformed strains produced wild type amounts of infectious particles and recapitulated major features of the C. trachomatis developmental cycle. C. trachomatis strains expressing fluorescent proteins are valuable tools for studying the C. trachomatis developmental cycle. For instance, we show the feasibility of investigating the dynamics of inclusion fusion and interaction with host proteins and organelles by time-lapse video microscopy. PMID:23441233

A new human lung adenocarcinoma cell line harboring the EML4-ALK fusion gene.

PubMed

Isozaki, Hideko; Yasugi, Masayuki; Takigawa, Nagio; Hotta, Katsuyuki; Ichihara, Eiki; Taniguchi, Akihiko; Toyooka, Shinichi; Hashida, Shinsuke; Sendo, Toshiaki; Tanimoto, Mitsune; Kiura, Katsuyuki

2014-10-01

The echinoderm microtubule associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene was identified in patients with non-small cell lung cancer. To the best of our knowledge, there are only three cell lines harboring the EML4-ALK fusion gene, which have contributed to the development of therapeutic strategies. Therefore, we tried to establish a new lung cancer cell line harboring EML4-ALK. A 61-year-old Japanese female presented with chest discomfort. She was diagnosed with left lung adenocarcinoma with T4N3M1 Stage IV. Although she was treated with chemotherapy, her disease progressed with massive pleural effusion. Because the EML4-ALK rearrangement was found in a biopsied specimen using fluorescence in situ hybridization, she was treated with crizotinib. She did well for 3 months. Tumor cells were obtained from the malignant pleural effusion before treatment with crizotinib. Cells continued to proliferate substantially for several weeks. The cell line was designated ABC-11. The EML4-ALK fusion protein and genes were identified in ABC-11 cells using fluorescence in situ hybridization and immunohistochemistry, respectively. ABC-11 cells were sensitive to crizotinib and next-generation ALK inhibitors (ceritinib and AP26113), as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Phosphorylated ALK protein and its downstream signaling were suppressed by treatment with crizotinib in western blotting. Furthermore, we could transplant ABC-11 cells subcutaneously into BALB/c nu/nu mice. We successfully established a new lung adenocarcinoma cell line harboring the EML4-ALK fusion gene. This cell line could contribute to future research of EML4-ALK-positive lung cancer both in vivo and in vitro. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Integrating the genomic architecture of human nucleolar organizer regions with the biophysical properties of nucleoli.

PubMed

Mangan, Hazel; Gailín, Michael Ó; McStay, Brian

2017-12-01

Nucleoli are the sites of ribosome biogenesis and the largest membraneless subnuclear structures. They are intimately linked with growth and proliferation control and function as sensors of cellular stress. Nucleoli form around arrays of ribosomal gene (rDNA) repeats also called nucleolar organizer regions (NORs). In humans, NORs are located on the short arms of all five human acrocentric chromosomes. Multiple NORs contribute to the formation of large heterochromatin-surrounded nucleoli observed in most human cells. Here we will review recent findings about their genomic architecture. The dynamic nature of nucleoli began to be appreciated with the advent of photodynamic experiments using fluorescent protein fusions. We review more recent data on nucleoli in Xenopus germinal vesicles (GVs) which has revealed a liquid droplet-like behavior that facilitates nucleolar fusion. Further analysis in both XenopusGVs and Drosophila embryos indicates that the internal organization of nucleoli is generated by a combination of liquid-liquid phase separation and active processes involving rDNA. We will attempt to integrate these recent findings with the genomic architecture of human NORs to advance our understanding of how nucleoli form and respond to stress in human cells. © 2017 Federation of European Biochemical Societies.

Repressing a Repressor

PubMed Central

Silverstone, Aron L.; Jung, Hou-Sung; Dill, Alyssa; Kawaide, Hiroshi; Kamiya, Yuji; Sun, Tai-ping

2001-01-01

RGA (for repressor of ga1-3) and SPINDLY (SPY) are likely repressors of gibberellin (GA) signaling in Arabidopsis because the recessive rga and spy mutations partially suppressed the phenotype of the GA-deficient mutant ga1-3. We found that neither rga nor spy altered the GA levels in the wild-type or the ga1-3 background. However, expression of the GA biosynthetic gene GA4 was reduced 26% by the rga mutation, suggesting that partial derepression of the GA response pathway by rga resulted in the feedback inhibition of GA4 expression. The green fluorescent protein (GFP)–RGA fusion protein was localized to nuclei in transgenic Arabidopsis. This result supports the predicted function of RGA as a transcriptional regulator based on sequence analysis. Confocal microscopy and immunoblot analyses demonstrated that the levels of both the GFP-RGA fusion protein and endogenous RGA were reduced rapidly by GA treatment. Therefore, the GA signal appears to derepress the GA signaling pathway by degrading the repressor protein RGA. The effect of rga on GA4 gene expression and the effect of GA on RGA protein level allow us to identify part of the mechanism by which GA homeostasis is achieved. PMID:11449051

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Stephanie B.; Harding, Eric C.; Knapp, Patrick F.

The burning core of an inertial confinement fusion (ICF) plasma produces bright x-rays at stagnation that can directly diagnose core conditions essential for comparison to simulations and understanding fusion yields. These x-rays also backlight the surrounding shell of warm, dense matter, whose properties are critical to understanding the efficacy of the inertial confinement and global morphology. In this work, we show that the absorption and fluorescence spectra of mid-Z impurities or dopants in the warm dense shell can reveal the optical depth, temperature, and density of the shell and help constrain models of warm, dense matter. This is illustrated bymore » the example of a high-resolution spectrum collected from an ICF plasma with a beryllium shell containing native iron impurities. Lastly, analysis of the iron K-edge provides model-independent diagnostics of the shell density (2.3 × 10 24 e/cm 3) and temperature (10 eV), while a 12-eV red shift in Kβ and 5-eV blue shift in the K-edge discriminate among models of warm dense matter: Both shifts are well described by a self-consistent field model based on density functional theory but are not fully consistent with isolated-atom models using ad-hoc density effects.« less

A novel fusion of HNRNPA1-ALK in inflammatory myofibroblastic tumor of urinary bladder.

PubMed

Inamura, Kentaro; Kobayashi, Maki; Nagano, Hiroko; Sugiura, Yoshiya; Ogawa, Masahiro; Masuda, Hitoshi; Yonese, Junji; Ishikawa, Yuichi

2017-11-01

Here, we report an inflammatory myofibroblastic tumor (IMT) of the urinary bladder with a novel HNRNPA1-ALK fusion. To the best of our knowledge, this is the first case of a tumor with HNRNPA1-ALK fusion. A 42-year-old Japanese man underwent total cystectomy because of an invasive urinary bladder tumor. Grossly, the tumor had invaded the peribladder fat tissue. Histologically, it comprised spindle neoplastic cells with intermingled inflammatory cells. Immunohistochemically, it was positive for ALK, SMA, desmin, cytokeratin, and vimentin, consistent with the immunohistochemical characteristics of IMTs. Fluorescence in situ hybridization demonstrated an ALK split, and the presence of HNRNPA1-ALK was revealed by RNA sequencing. We identified a novel transcript fusion of exon 2 of HNRNPA1 and exon 18 of ALK, resulting in ALK protein overexpression. These findings provide useful information on the biology and tumorigenesis of IMTs, thus facilitating the development of molecular-targeted therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo

PubMed Central

Bittner, Mary A.; Lawrence, Daniel A.

2017-01-01

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species. PMID:28882880

Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor.

PubMed

Charest-Morin, Xavier; Lodge, Robert; Marceau, François

2018-05-01

To support bradykinin (BK) B 2 receptor (B 2 R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B 2 Rs (immunoblots, epifluorescence microscopy). APEX2-(NG) 15 -MK is a bona fide agonist of the rat, but not of the human B 2 R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3',5,5'-tetramethylbenzidine (luminescence or colourimetric B 2 R detection, cell well plate format). APEX2-(NG) 15 -MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B 2 R in the concentration range of 50-600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B 2 R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B 2 R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

Enhanced metastatic capacity of breast cancer cells after interaction and hybrid formation with mesenchymal stroma/stem cells (MSC).

PubMed

Melzer, Catharina; von der Ohe, Juliane; Hass, Ralf

2018-01-05

Fusion of breast cancer cells with tumor-associated populations of the microenvironment including mesenchymal stroma/stem-like cells (MSC) represents a rare event in cell communication whereby the metastatic capacity of those hybrid cells remains unclear. Functional changes were investigated in vitro and in vivo following spontaneous fusion and hybrid cell formation between primary human MSC and human MDA-MB-231 breast cancer cells. Thus, lentiviral eGFP-labeled MSC and breast cancer cells labeled with mcherry resulted in dual-fluorescing hybrid cells after co-culture. Double FACS sorting and single cell cloning revealed two different aneuploid male hybrid populations (MDA-hyb1 and MDA-hyb2) with different STR profiles, pronounced telomerase activities, and enhanced proliferative capacities as compared to the parental cells. Microarray-based mRNA profiling demonstrated marked regulation of genes involved in epithelial-mesenchymal transition and increased expression of metastasis-associated genes including S100A4. In vivo studies following subcutaneous injection of the breast cancer and the two hybrid populations substantiated the in vitro findings by a significantly elevated tumor growth of the hybrid cells. Moreover, both hybrid populations developed various distant organ metastases in a much shorter period of time than the parental breast cancer cells. Together, these data demonstrate spontaneous development of new tumor cell populations exhibiting different parental properties after close interaction and subsequent fusion of MSC with breast cancer cells. This formation of tumor hybrids contributes to continuously increasing tumor heterogeneity and elevated metastatic capacities.

Comprehensive analysis of the MYB-NFIB gene fusion in salivary adenoid cystic carcinoma: Incidence, variability, and clinicopathologic significance.

PubMed

Mitani, Yoshitsugu; Li, Jie; Rao, Pulivarthi H; Zhao, Yi-Jue; Bell, Diana; Lippman, Scott M; Weber, Randal S; Caulin, Carlos; El-Naggar, Adel K

2010-10-01

The objectives of this study were to determine the incidence of the MYB-NFIB fusion in salivary adenoid cystic carcinoma (ACC), to establish the clinicopathologic significance of the fusion, and to analyze the expression of MYB in ACCs in the context of the MYB-NFIB fusion. We did an extensive analysis involving 123 cancers of the salivary gland, including primary and metastatic ACCs, and non-ACC salivary carcinomas. MYB-NFIB fusions were identified by reverse transcriptase-PCR (RT-PCR) and sequencing of the RT-PCR products, and confirmed by fluorescence in situ hybridization. MYB RNA expression was determined by quantitative RT-PCR and protein expression was analyzed by immunohistochemistry. The MYB-NFIB fusion was detected in 28% primary and 35% metastatic ACCs, but not in any of the non-ACC salivary carcinomas analyzed. Different exons in both the MYB and NFIB genes were involved in the fusions, resulting in expression of multiple chimeric variants. Notably, MYB was overexpressed in the vast majority of the ACCs, although MYB expression was significantly higher in tumors carrying the MYB-NFIB fusion. The presence of the MYB-NFIB fusion was significantly associated (P = 0.03) with patients older than 50 years of age. No correlation with other clinicopathologic markers, factors, and survival was found. We conclude that the MYB-NFIB fusion characterizes a subset of ACCs and contributes to MYB overexpression. Additional mechanisms may be involved in MYB overexpression in ACCs lacking the MYB-NFIB fusion. These findings suggest that MYB may be a specific novel target for tumor intervention in patients with ACC. ©2010 AACR.

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging.

PubMed

Li, Tengfei; Bourgeois, Jean-Pierre; Celli, Susanna; Glacial, Fabienne; Le Sourd, Anne-Marie; Mecheri, Salah; Weksler, Babette; Romero, Ignacio; Couraud, Pierre-Olivier; Rougeon, François; Lafaye, Pierre

2012-10-01

Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.

Improving the photostability of bright monomeric orange and red fluorescent proteins.

PubMed

Shaner, Nathan C; Lin, Michael Z; McKeown, Michael R; Steinbach, Paul A; Hazelwood, Kristin L; Davidson, Michael W; Tsien, Roger Y

2008-06-01

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dinh, Phat X.; Panda, Debasis; Das, Phani B.

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutationmore » occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.« less

Method for screening inhibitors of the toxicity of Bacillus anthracis

DOEpatents

Cirino, Nick M.; Jackson, Paul J.; Lehnert, Bruce E.

2001-01-01

The protective antigen (PA) of Bacillus anthracis is integral to the mechanism of anthrax poisoning. The cloning, expression and purification of a 32 kDa B. anthracis PA fragment (PA32) is described. This fragment has also been expressed as a fusion construct to stabilized green fluorescent protein (EGFP-PA32). Both proteins were capable of binding to specific cell surface receptors as determined by fluorescent microscopy and a flow cytometric assay. To confirm binding specificity in the flow cytometric assay, non-fluorescent PA83 or PA32 was used to competitively inhibit fluorescent EGFP-PA32 binding to cell receptors. This assay can be employed as a rapid screen for compounds which disrupts binding of PA to cells. Additionally, the high intracellular expression levels and ease of purification make this recombinant protein an attractive vaccine candidate or therapeutic treatment for anthrax poisoning.

Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

PubMed Central

Theis, Thomas; Mishra, Bibhudatta; von der Ohe, Maren; Loers, Gabriele; Prondzynski, Maksymilian; Pless, Ole; Blackshear, Perry J.; Schachner, Melitta; Kleene, Ralf

2013-01-01

Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system. PMID:23329829

To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

PubMed Central

Sznajder, Anna

2014-01-01

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure. PMID:24907326

To be or not to be a poly(3-hydroxybutyrate) (PHB) depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, highly active PHB depolymerases with no detectable role in mobilization of accumulated PHB.

PubMed

Sznajder, Anna; Jendrossek, Dieter

2014-08-01

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Automated Segmentation of Light-Sheet Fluorescent Imaging to Characterize Experimental Doxorubicin-Induced Cardiac Injury and Repair.

PubMed

Packard, René R Sevag; Baek, Kyung In; Beebe, Tyler; Jen, Nelson; Ding, Yichen; Shi, Feng; Fei, Peng; Kang, Bong Jin; Chen, Po-Heng; Gau, Jonathan; Chen, Michael; Tang, Jonathan Y; Shih, Yu-Huan; Ding, Yonghe; Li, Debiao; Xu, Xiaolei; Hsiai, Tzung K

2017-08-17

This study sought to develop an automated segmentation approach based on histogram analysis of raw axial images acquired by light-sheet fluorescent imaging (LSFI) to establish rapid reconstruction of the 3-D zebrafish cardiac architecture in response to doxorubicin-induced injury and repair. Input images underwent a 4-step automated image segmentation process consisting of stationary noise removal, histogram equalization, adaptive thresholding, and image fusion followed by 3-D reconstruction. We applied this method to 3-month old zebrafish injected intraperitoneally with doxorubicin followed by LSFI at 3, 30, and 60 days post-injection. We observed an initial decrease in myocardial and endocardial cavity volumes at day 3, followed by ventricular remodeling at day 30, and recovery at day 60 (P < 0.05, n = 7-19). Doxorubicin-injected fish developed ventricular diastolic dysfunction and worsening global cardiac function evidenced by elevated E/A ratios and myocardial performance indexes quantified by pulsed-wave Doppler ultrasound at day 30, followed by normalization at day 60 (P < 0.05, n = 9-20). Treatment with the γ-secretase inhibitor, DAPT, to inhibit cleavage and release of Notch Intracellular Domain (NICD) blocked cardiac architectural regeneration and restoration of ventricular function at day 60 (P < 0.05, n = 6-14). Our approach provides a high-throughput model with translational implications for drug discovery and genetic modifiers of chemotherapy-induced cardiomyopathy.

NUTM1 Gene Fusions Characterize a Subset of Undifferentiated Soft Tissue and Visceral Tumors.

PubMed

Dickson, Brendan C; Sung, Yun-Shao; Rosenblum, Marc K; Reuter, Victor E; Harb, Mohammed; Wunder, Jay S; Swanson, David; Antonescu, Cristina R

2018-05-01

NUT midline carcinoma is an aggressive tumor that occurs mainly in the head and neck and, less frequently, the mediastinum and lung. Following identification of an index case of a NUTM1 fusion positive undifferentiated soft tissue tumor, we interrogated additional cases of primary undifferentiated soft tissue and visceral tumors for NUTM1 abnormalities. Targeted next-generation sequencing was performed on RNA extracted from formalin-fixed paraffin-embedded tissue, and results validated by fluorescence in situ hybridization using custom bacterial artificial chromosome probes. Six patients were identified: mean age of 42 years (range, 3 to 71 y); equal sex distribution; and, tumors involved the extremity soft tissues (N=2), kidney (N=2), stomach, and brain. On systemic work-up at presentation all patients lacked a distant primary tumor. Morphologically, the tumors were heterogenous, with undifferentiated round-epithelioid-rhabdoid cells arranged in solid sheets, nests, and cords. Mitotic activity was generally brisk. Four cases expressed pancytokeratin, but in only 2 cases was this diffuse. Next-generation sequencing demonstrated the following fusions: BRD4-NUTM1 (3 cases), BRD3-NUTM1, MXD1-NUTM1, and BCORL1-NUTM1. Independent testing by fluorescence in situ hybridization confirmed the presence of NUTM1 and partner gene rearrangement. This study establishes that NUT-associated tumors transgress the midline and account for a subset of primitive neoplasms occurring in soft tissue and viscera. Tumors harboring NUTM1 gene fusions are presumably underrecognized, and the extent to which they account for undifferentiated mesenchymal, neuroendocrine, and/or epithelial neoplasms is unclear. Moreover, the relationship, if any, between NUT-associated tumors in soft tissue and/or viscera, and conventional NUT carcinoma, remains to be elucidated.

Characterization of the functional requirements of West Nile virus membrane fusion.

PubMed

Moesker, Bastiaan; Rodenhuis-Zybert, Izabela A; Meijerhof, Tjarko; Wilschut, Jan; Smit, Jolanda M

2010-02-01

Flaviviruses infect their host cells by a membrane fusion reaction. In this study, we performed a functional analysis of the membrane fusion properties of West Nile virus (WNV) with liposomal target membranes. Membrane fusion was monitored continuously using a lipid mixing assay involving the fluorophore, pyrene. Fusion of WNV with liposomes occurred on the timescale of seconds and was strictly dependent on mildly acidic pH. Optimal fusion kinetics were observed at pH 6.3, the threshold for fusion being pH 6.9. Preincubation of the virus alone at pH 6.3 resulted in a rapid loss of fusion capacity. WNV fusion activity is strongly promoted by the presence of cholesterol in the target membrane. Furthermore, we provide direct evidence that cleavage of prM to M is a requirement for fusion activity of WNV.

TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate.

PubMed

Guo, Charles C; Dancer, Jane Y; Wang, Yan; Aparicio, Ana; Navone, Nora M; Troncoso, Patricia; Czerniak, Bogdan A

2011-01-01

Recent studies have shown that most prostate cancers carry the TMPRSS2-ERG gene fusion. Here we evaluated the TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate (n = 12) in comparison with small cell carcinoma of the urinary bladder (n = 12) and lung (n = 11). Fluorescence in situ hybridization demonstrated rearrangement of the ERG gene in 8 cases of prostatic small cell carcinoma (67%), and the rearrangement was associated with deletion of the 5' ERG gene in 7 cases, but rearrangement of the ERG gene was not present in any small cell carcinoma of the urinary bladder or lung. Next we evaluated the TMPRSS2-ERG gene fusion in nude mouse xenografts that were derived from 2 prostatic small cell carcinomas carrying the TMPRSS2-ERG gene fusion. Two transcripts encoded by the TMPRSS2-ERG gene fusion were detected by reverse transcriptase polymerase chain reaction, and DNA sequencing demonstrated that the 2 transcripts were composed of fusions of exon 1 of the TMPRSS2 gene to exon 4 or 5 of the ERG gene. Our study demonstrates the specific presence of TMPRSS2-ERG gene fusion in prostatic small cell carcinoma, which may be helpful in distinguishing small cell carcinoma of prostatic origin from nonprostatic origins. The high prevalence of the TMPRSS2-ERG gene fusion in prostatic small cell carcinoma as well as adenocarcinoma implies that small cell carcinoma may share a common pathogenic pathway with adenocarcinoma in the prostate. Copyright © 2011 Elsevier Inc. All rights reserved.

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

PubMed Central

Pfeiffer, Daniel

2012-01-01

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with “phasin 2 motifs.” To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules. PMID:22923598

Localization of poly(3-hydroxybutyrate) (PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16.

PubMed

Pfeiffer, Daniel; Jendrossek, Dieter

2012-11-01

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.

Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene

PubMed Central

Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.

2010-01-01

Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2. Although fluorescence was observed, there were discrepancies between in vivo imaging and ex vivo imaging as well as between nuclear imaging and fluorescent imaging. Conclusion These studies showed that the SSTR2-EGFP fusion construct can be used for in vivo nuclear and optical imaging of gene transfer. PMID:20720053

Fluorescence and absorption spectroscopy for warm dense matter studies and ICF plasma diagnostics

NASA Astrophysics Data System (ADS)

Hansen, Stephanie

2017-10-01

The burning core of an inertial confinement fusion (ICF) plasma at stagnation is surrounded by a shell of warm, dense matter whose properties are difficult both to model (due to a complex interplay of thermal, degeneracy, and strong coupling effects) and to diagnose (due to low emissivity and high opacity). We demonstrate a promising technique to study the warm dense shells of ICF plasmas based on the fluorescence emission of dopants or impurities in the shell material. This emission, which is driven by x-rays produced in the hot core, exhibits signature changes in response to compression and heating. High-resolution measurements of absorption and fluorescence features can refine our understanding of the electronic structure of material under high compression, improve our models of density-driven phenomena such as ionization potential depression and plasma polarization shifts, and help diagnose shell density, temperature, mass distribution, and residual motion in ICF plasmas at stagnation. Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA-0003525. This work was supported by the U.S. Department of Energy, Office of Science Early Career Research Program, Office of Fusion Energy Sciences under FWP-14-017426.

Role of partial linear momentum transfer on incomplete fusion reaction

NASA Astrophysics Data System (ADS)

Ali, Sabir; Ahmad, Tauseef; Kumar, Kamal; Gull, Muntazir; Rizvi, I. A.; Agarwal, Avinash; Ghugre, S. S.; Sinha, A. K.; Chaubey, A. K.

2018-04-01

Measurements of forward recoil range distributions (FRRDs) of the evaporation residues, populated in the 20Ne+51V reaction at E_{lab}≈ 145 MeV, have been carried out using the offline characteristic γ-ray detection method. The observation does corroborate the presence of complete fusion (CF) process in the population of p xn channel residues and both complete as well as incomplete fusion (ICF) processes in the population of α emitting channel residues. The FRRDs of p xn channel residues comprise single peak only, whereas α emitting channel residues have multiple peaks in their FRRDs. CF cross section data were used to extract the fusion functions. Extracted fusion functions were found to be suppressed with respect to the universal fusion function which is used as a uniform standard reference. The observed contribution arising from the ICF process in the population of α emitting channel residues is explained in terms of breakup fusion model.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

NUTM2A-CIC fusion small round cell sarcoma: a genetically distinct variant of CIC-rearranged sarcoma.

PubMed

Sugita, Shintaro; Arai, Yasuhito; Aoyama, Tomoyuki; Asanuma, Hiroko; Mukai, Wakako; Hama, Natsuko; Emori, Makoto; Shibata, Tatsuhiro; Hasegawa, Tadashi

2017-07-01

CIC-rearranged sarcoma is a new entity of undifferentiated small round cell sarcoma characterized by chimeric fusions with CIC rearrangement. We report a NUTM2A-CIC fusion sarcoma in a 43-year-old woman who died of rapidly progressive disease. Histologic analysis revealed multinodular proliferation of small round tumor cells with mild nuclear pleomorphism. The sclerotic fibrous septa separated the tumor into multiple nodules. Immunohistochemistry showed that the tumor cells were diffusely positive for vimentin, focally positive for cytokeratin, and negative for CD99 and NKX2.2. Tumor cells were also negative for ETV4, which was recently identified as a specific marker for CIC-rearranged sarcoma. High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded clinical sample unveiled a novel NUTM2A-CIC fusion between NUTM2A exon 7 and CIC exon 12, and fluorescence in situ hybridization identified CIC and NUTM2A split signals. This case shared several clinicopathological findings with previously reported CIC-rearranged cases. We recognized the tumor as a genetically distinct variant of CIC-rearranged sarcomas with a novel NUTM2A-CIC fusion. Copyright © 2017. Published by Elsevier Inc.

Design, synthesis, and spectroscopic properties of extended and fused pyrrolo-dC and pyrrolo-C analogs.

PubMed

Noé, Mary S; Ríos, Andro C; Tor, Yitzhak

2012-06-15

The syntheses of four fluorescent nucleoside analogs, related to pyrrolo-C (PyC) and pyrrolo-dC (PydC) through the conjugation or fusion of a thiophene moiety, are described. A thorough photophysical analysis of the nucleosides, in comparison to PyC, is reported.

[Molecular genetics in chronic myeloid leukemia with variant Ph translocation].

PubMed

Wu, Wei; Li, Jian-yong; Zhu, Yu; Qiu, Hai-rong; Pan, Jin-lan; Xu, Wei; Chen, Li-juan; Shen, Yun-feng; Xue, Yong-quan

2007-08-01

To explore the value of fluorescence in situ hybridization (FISH) and multiplex fluorescence in situ hybridization (M-FISH) techniques in the detection of genetic changes in chronic myeloid leukemia (CML) with variant Philadelphia translocation (vPh). Cytogenetic preparations from 10 CML patients with vPh confirmed by R banding were assayed with dual color dual fusion FISH technique. If only one fusion signal was detected in interphase cells, metaphase cells were observed to determine if there were derivative chromosome 9[der (9)] deletions. Meanwhile, the same cytogenetic preparations were assayed with M-FISH technique. Of the 10 CML patients with vPh, 5 were detected with der (9) deletions by FISH technique. M-FISH technique revealed that besides the chromosome 22, chromosomes 1, 3, 5, 6, 8, 10, 11 and 17 were also involved in the vPh. M-FISH technique also detected the abnormalities which were not found with conventional cytogenetics (CC), including two never reported abnormalities. The combination of CC, FISH and M-FISH technique could refine the genetic diagnosis of CML with vPh.

Distinct modes of perimembrane TRP channel turnover revealed by TIR-FRAP.

PubMed

Ghosh, Debapriya; Segal, Andrei; Voets, Thomas

2014-11-19

Transient Receptor Potential (TRP) channels form a broadly expressed and functionally diverse family of cation channels involved in various (patho)physiological processes. Whereas the mechanisms that control opening of TRP channels have been extensively studied, little is known about the transport processes of TRP channels to and within the plasma membrane. Here we used Total Internal Reflection--Fluorescence Recovery after Photobleaching (TIR-FRAP) to selectively visualize and bleach the fluorescently labeled TRP channels TRPV2 and TRPM4 in close proximity of the glass-plasma membrane interface, allowing detailed analysis of their perimembrane dynamics. We show that recovery of TRPM4 occurs via 200-nm diameter transport vesicles, and demonstrate the full fusion of such vesicles with the plasma membrane. In contrast, TRPV2 recovery proceeded mainly via lateral diffusion from non-bleached areas of the plasma membrane. Analysis of the two-dimensional channel diffusion kinetics yielded 2D diffusion coefficients ranging between 0.1 and 0.3 μm(2)/s, suggesting that these TRP channels move relatively unrestricted within the plasma membrane. These data demonstrate distinct modes of TRP channel turnover at the plasma membrane and illustrate the usefulness of TIR-FRAP to monitor these processes with high resolution.

Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

PubMed Central

Ai, Hui-wang; Olenych, Scott G; Wong, Peter; Davidson, Michael W; Campbell, Robert E

2008-01-01

Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants. PMID:18325109

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

PubMed

Roubalova, Lenka; Vosahlikova, Miroslava; Brejchova, Jana; Sykora, Jan; Rudajev, Vladimir; Svoboda, Petr

2015-01-01

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins--towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation.

PubMed

Daskalova, Sasha M; Radder, Josiah E; Cichacz, Zbigniew A; Olsen, Sam H; Tsaprailis, George; Mason, Hugh; Lopez, Linda C

2010-08-24

Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform. Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.

The polyomavirus BK agnoprotein co-localizes with lipid droplets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unterstab, Gunhild; Gosert, Rainer; Leuenberger, David

Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures. We found that agnoprotein co-localizes with lipid droplets (LD) in primary human renal tubular epithelial cells as well as in other cells supporting BKV replication in vitro (UTA, Vero cells). Using agnoprotein-enhanced green fluorescent protein (EGFP) fusion constructs, we demonstrate that agnoprotein aa 20-42 are required for targeting LD, whereas aa 1-20 or aa 42-66more » were not. Agnoprotein aa 22-40 are predicted to form an amphipathic helix, and mutations A25D and F39E, disrupting its hydrophobic domain, prevented LD targeting. However, changing the phosphorylation site serine-11 to alanine or aspartic acid did not alter LD co-localization. Our findings provide new clues to unravel agnoprotein function.« less

Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae.

PubMed

Lunprom, Siriporn; Pongcharoen, Pongsanat; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi; Akiyama, Koichi

2015-01-01

Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

PubMed

Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

2002-08-01

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

PubMed

Puri, Nidhi; Manoharlal, Raman; Sharma, Monika; Sanglard, Dominique; Prasad, Rajendra

2011-01-07

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded. Copyright © 2010 Elsevier Inc. All rights reserved.

Xp11.2 translocation renal cell carcinoma with TFE3 gene fusion: A case report.

PubMed

Pan, Xiang; Quan, Jing; Zhao, Liwen; Li, Wenhua; Wei, Benlin; Yang, Shangqi; Lai, Yongqing

2018-01-01

Xp11.2 translocation renal cell carcinoma (RCC) with transcription factor E3 (TFE3) gene fusion is a rare tumor, and the prognosis of this tumor is poorer compared with that of other subtypes of RCC. The patient presented herein was a 70-year-old man who presented with a solid mass sized ~8.2×6.1 cm in the right kidney and underwent radical right nephrectomy. Following pathological and immunohistochemical (IHC) examination and fluorescent in situ hybridization (FISH), the patient was diagnosed with Xp11.2 translocation RCC with TFE3 gene fusion. These tumors are more commonly encountered in children rather than in adults, and adult Xp11.2 translocation RCC is associated with a poorer prognosis compared with its pediatric counterpart. IHC assay and FISH are important diagnostic methods. However, there is currently no established effective treatment for Xp11.2 RCC.

Xp11.2 translocation renal cell carcinoma with TFE3 gene fusion: A case report

PubMed Central

Pan, Xiang; Quan, Jing; Zhao, Liwen; Li, Wenhua; Wei, Benlin; Yang, Shangqi; Lai, Yongqing

2018-01-01

Xp11.2 translocation renal cell carcinoma (RCC) with transcription factor E3 (TFE3) gene fusion is a rare tumor, and the prognosis of this tumor is poorer compared with that of other subtypes of RCC. The patient presented herein was a 70-year-old man who presented with a solid mass sized ~8.2×6.1 cm in the right kidney and underwent radical right nephrectomy. Following pathological and immunohistochemical (IHC) examination and fluorescent in situ hybridization (FISH), the patient was diagnosed with Xp11.2 translocation RCC with TFE3 gene fusion. These tumors are more commonly encountered in children rather than in adults, and adult Xp11.2 translocation RCC is associated with a poorer prognosis compared with its pediatric counterpart. IHC assay and FISH are important diagnostic methods. However, there is currently no established effective treatment for Xp11.2 RCC. PMID:29399348

Inducible model for β-six-mediated site-specific recombination in mammalian cells

PubMed Central

Servert, Pilar; Garcia-Castro, Javier; Díaz, Vicente; Lucas, Daniel; Gonzalez, Manuel A.; Martínez-A, Carlos; Bernad, Antonio

2006-01-01

The prokaryotic β recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in chromatin-integrated substrates. Here, we demonstrate that an enhanced green fluorescent protein (EGFP)-fused version of β recombinase (β-EGFP) is fully active, retaining most specific activity. It is used to develop a recombination-dependent activatable gene expression (RAGE) system based on the androgen receptor (AR) ligand-binding domain (LBD). Two hybrid molecules, a direct fusion of the LBD-AR to the C-terminus of β recombinase (β-AR) and a triple fusion of β-EGFP to the same ligand-binding domain (β-EGFP-AR), were engineered and their subcellular behavior, stability and catalytic activity were evaluated. Both chimeric β recombinase proteins showed in vivo inducible recombinogenic activity dependent on addition of an androgen receptor agonist, although the β-AR fusion protein demonstrated more accurate ligand-dependent translocation from cytoplasm to nucleus. PMID:16394020

Determination of major elements by wavelength-dispersive X-ray fluorescence spectrometry and trace elements by inductively coupled plasma mass spectrometry in igneous rocks from the same fused sample (110 mg)

NASA Astrophysics Data System (ADS)

Amosova, Alena A.; Panteeva, Svetlana V.; Chubarov, Victor M.; Finkelshtein, Alexandr L.

2016-08-01

The fusion technique is proposed for simultaneous determination of 35 elements from the same sample. Only 110 mg of rock sample was used to obtain fused glasses for quantitative determination of 10 major elements by wavelength dispersive X-ray fluorescence analysis, 16 rare earth elements and some other trace elements by inductively coupled plasma mass spectrometry analysis. Fusion was performed with 1.1 g of lithium metaborate and LiBr solution as the releasing agent in platinum crucible in electric furnace at 1100 °C. The certified reference materials of ultramafic, mafic, intermediate and felsic igneous rocks have been applied to obtain the calibration curves for rock-forming oxides (Na2O, MgO, Al2O3, SiO2, P2O5, K2O, CaO, TiO2, MnO, Fe2O3) and some trace elements (Ba, Sr, Zr) determination by X-ray fluorescence analysis. The repeatability does not exceed the allowable standard deviation for a wide range of concentrations. In the most cases the relative standard deviation was less than 5%. Obtained glasses were utilized for the further determination of rare earth (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) and some other (Ba, Sr, Zr, Rb, Cs, Y, Nb, Hf, Ta, Th and U) trace elements by inductively coupled plasma mass spectrometry analysis with the same certified reference materials employed. The results could mostly be accepted as satisfactory. The proposed procedure essentially reduces the expenses in comparison with separate sample preparation for inductively coupled plasma mass spectrometry and X-ray fluorescence analysis.

Live cell imaging of in vitro human trophoblast syncytialization.

PubMed

Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

2014-06-01

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.

PubMed

Powell, Anne E; Anderson, Eric C; Davies, Paige S; Silk, Alain D; Pelz, Carl; Impey, Soren; Wong, Melissa H

2011-02-15

The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells. ©2011 AACR.

[Mutations of EGFR gene and EML4-ALK fusion gene in superficial lymph node of non-small cell lung cancer].

PubMed

Wei, Lili; Li, Xingzhou; Yu, Zhonghe

2015-07-14

To explore the mutation status of epidermal growth factor receptor (EGFR) fusion gene and microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene in superficial lymph nodes of non-small cell lung cancer (NSCLC). The technique of fluorescent quantitative polymerase chain reaction (FQ-PCR) was employed for detecting the mutation rate of EGFR gene and EML4-ALK fusion gene for 40 cases of superficial lymph node tissue of NSCLC inpatients at General Military Hospital of Beijing PLA Command from February 2013 to November 2014. And then the correlations were analyzed between EMIA-ALK fusion gene and EGFR gene with clinical features and the clinical efficacies of targeted therapy. The mutation rate of EGFR gene was 35% (14/40) and 50% (10/20) in non-smokers and 46.7% (14/30) in adenocarcinoma patients. The mutation distribution was as follows: exon 18 (n = 1), exon 19 (n =8) and exon 21 (n =5). The mutation rate of EML4-ALK fusion gene was 2. 5% (1/40). EGFR gene mutation was predominantly present in non-smokers (P < 0. 05) and adenocarcinoma (P <0. 01) while no significant difference existed between gender, age or stage (P >0. 05). Those on a targeted therapy had a disease control rate of 93. 3%. Both EGFR gene and EMI4-ALK fusion gene may be detected in superficial lymph nodes of NSCLC patients. The mutation rate of EGFR gene is high in adenocarcinoma and non-smokers while EML4-ALK fusion gene has a low mutation rate.

In vitro capacitation and acrosome reaction in sperm of the phyllostomid bat Artibeus jamaicensis.

PubMed

Álvarez-Guerrero, Alma; González-Díaz, Francisco; Medrano, Alfredo; Moreno-Mendoza, Norma

2016-04-01

Sperm capacitation occurs during the passage of sperm through the female reproductive tract. Once the sperm binds to the pellucid zone, the acrosome reaction to enable penetration of the oocyte is completed. In this study, sperm of Artibeus jamaicensis bat was used to evaluate both capacitation status and the acrosome reaction under in vitro conditions, incubating sperm at 32 and 37°C with and without progesterone. Sperm was incubated at different times to assess sperm cells' functionality in terms of capacitation and acrosome reaction, using the chlortetracycline staining, lectin fluoresceinisocyanate conjugate-Pisum sativum agglutinin (FITC-PSA), and transmission electron microscopy. Sperm cells that presented uniform fluorescence throughout the head and mid-piece were classified as non-capacitated. Subsequently, sperm cells, which were observed with fluorescence only in the anterior portion of the head and mid-piece, were classified as capacitated. Sperm cells with no fluorescence in the head, but fluorescence in the mid-piece, were categorized as sperm cells that have carried out the acrosome reaction. During the acrosome reaction, sperm cells showed changes in their morphology, so it was not possible to distinguish the plasma and acrosomal membranes. Around the entire head, it was not possible to distinguish the fusion points between these membranes that made it possible for the acrosomal reaction to take place and thus to release the enzymes necessary to penetrate the pellucid zone. In conclusion, under appropriate in vitro conditions and by supplementing the culture medium with progesterone, A. jamaicensis bat sperm cells are able to be capacitated in a period from 6 to 8 h and to carry out the acrosome reaction.

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

PubMed

James, Declan J; Kowalchyk, Judith; Daily, Neil; Petrie, Matt; Martin, Thomas F J

2009-10-13

Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.

Strong expression of the neuronal transcription factor FOXP2 is linked to an increased risk of early PSA recurrence in ERG fusion-negative cancers.

PubMed

Stumm, Laura; Burkhardt, Lia; Steurer, Stefan; Simon, Ronald; Adam, Meike; Becker, Andreas; Sauter, Guido; Minner, Sarah; Schlomm, Thorsten; Sirma, Hüseyin; Michl, Uwe

2013-07-01

Transcription factors of the forkhead box P (FOXP1-4) family have been implicated in various human cancer types before. The relevance and role of neuronal transcription factor FOXP2 in prostate cancer is unknown. A tissue microarray containing samples from more than 11 000 prostate cancers from radical prostatectomy specimens with clinical follow-up data was analysed for FOXP2 expression by immunohistochemistry. FOXP2 data were also compared with pre-existing ERG fusion (by fluorescence in situ hybridisation and immunohistochemistry) and cell proliferation (Ki67 labelling index) data. There was a moderate to strong FOXP2 protein expression in basal and secretory cells of normal prostatic glands. As compared with normal cells, FOXP2 expression was lost or reduced in 25% of cancers. Strong FOXP2 expression was linked to advanced tumour stage, high Gleason score, presence of lymph node metastases and early tumour recurrence (p<0.0001; each) in ERG fusion-negative, but not in ERG fusion-positive cancers. High FOXP2 expression was linked to high Ki67 labelling index (p<0.0001) in all cancers irrespective of ERG fusion status. These data demonstrate that similar high FOXP2 protein levels as in normal prostate epithelium exert a 'paradoxical' oncogenic role in 'non fusion-type' prostate cancer. It may be speculated that interaction of FOXP2 with members of pathways that are specifically activated in 'non fusion-type' cancers may be responsible for this phenomenon.

HIP1-ALK, a novel fusion protein identified in lung adenocarcinoma.

PubMed

Hong, Mineui; Kim, Ryong Nam; Song, Ji-Young; Choi, So-Jung; Oh, Ensel; Lira, Maruja E; Mao, Mao; Takeuchi, Kengo; Han, Joungho; Kim, Jhingook; Choi, Yoon-La

2014-03-01

The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3. In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene's transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing. The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame. The novel fusion gene and its protein HIP1-ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.

Role of Abl kinase and the Wave2 signaling complex in HIV-1 entry at a post-hemifusion step.

PubMed

Harmon, Brooke; Campbell, Nancy; Ratner, Lee

2010-06-17

Entry of human immunodeficiency virus type 1 (HIV-1) commences with binding of the envelope glycoprotein (Env) to the receptor CD4, and one of two coreceptors, CXCR4 or CCR5. Env-mediated signaling through coreceptor results in Galphaq-mediated Rac activation and actin cytoskeleton rearrangements necessary for fusion. Guanine nucleotide exchange factors (GEFs) activate Rac and regulate its downstream protein effectors. In this study we show that Env-induced Rac activation is mediated by the Rac GEF Tiam-1, which associates with the adaptor protein IRSp53 to link Rac to the Wave2 complex. Rac and the tyrosine kinase Abl then activate the Wave2 complex and promote Arp2/3-dependent actin polymerization. Env-mediated cell-cell fusion, virus-cell fusion and HIV-1 infection are dependent on Tiam-1, Abl, IRSp53, Wave2, and Arp3 as shown by attenuation of fusion and infection in cells expressing siRNA targeted to these signaling components. HIV-1 Env-dependent cell-cell fusion, virus-cell fusion and infection were also inhibited by Abl kinase inhibitors, imatinib, nilotinib, and dasatinib. Treatment of cells with Abl kinase inhibitors did not affect cell viability or surface expression of CD4 and CCR5. Similar results with inhibitors and siRNAs were obtained when Env-dependent cell-cell fusion, virus-cell fusion or infection was measured, and when cell lines or primary cells were the target. Using membrane curving agents and fluorescence microscopy, we showed that inhibition of Abl kinase activity arrests fusion at the hemifusion (lipid mixing) step, suggesting a role for Abl-mediated actin remodeling in pore formation and expansion. These results suggest a potential utility of Abl kinase inhibitors to treat HIV-1 infected patients.

Green fluorescent protein as a reporter of gene expression and protein localization.

PubMed

Kain, S R; Adams, M; Kondepudi, A; Yang, T T; Ward, W W; Kitts, P

1995-10-01

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.

Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence.

PubMed

Leroch, Michaela; Mernke, Dennis; Koppenhoefer, Dieter; Schneider, Prisca; Mosbach, Andreas; Doehlemann, Gunther; Hahn, Matthias

2011-05-01

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.

A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.

PubMed

Jeong, Hee-Jin; Itayama, Shuya; Ueda, Hiroshi

2015-03-26

An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

A Signal-On Fluorosensor Based on Quench-Release Principle for Sensitive Detection of Antibiotic Rapamycin

PubMed Central

Jeong, Hee-Jin; Itayama, Shuya; Ueda, Hiroshi

2015-01-01

An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose “Q’-body”, which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q’-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q’-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin. PMID:25822756

TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

2012-06-01

Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

StarD7 behaves as a fusogenic protein in model and cell membrane bilayers.

PubMed

Angeletti, Sofía; Sanchez, Julieta M; Chamley, Larry W; Genti-Raimondi, Susana; Perillo, María A

2012-03-01

StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid-membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC-DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC-DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid-water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein. Copyright © 2011 Elsevier B.V. All rights reserved.

Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain.

PubMed

Leontovyc, Ivan; Habart, David; Loukotova, Sarka; Kosinova, Lucie; Kriz, Jan; Saudek, Frantisek; Koblas, Tomas

2017-01-01

Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP+ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29-71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

PubMed

Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

2016-02-12

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts*

PubMed Central

Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

2016-01-01

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. PMID:26670608

Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hernaez, Bruno; Escribano, Jose M.; Alonso, Covadonga

2006-06-20

Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to themore » wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations.« less

LucY: A Versatile New Fluorescent Reporter Protein

PubMed Central

Auldridge, Michele E.; Franz, Laura P.; Bingman, Craig A.; Yennamalli, Ragothaman M.; Phillips, George N.; Mead, David; Steinmetz, Eric J.

2015-01-01

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions. PMID:25906065

LucY: A Versatile New Fluorescent Reporter Protein.

PubMed

Auldridge, Michele E; Cao, Hongnan; Sen, Saurabh; Franz, Laura P; Bingman, Craig A; Yennamalli, Ragothaman M; Phillips, George N; Mead, David; Steinmetz, Eric J

2015-01-01

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.

LucY: A versatile new fluorescent reporter protein

DOE PAGES

Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; ...

2015-04-23

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrastmore » to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.« less

Pedicle screw fixation for isthmic spondylolisthesis: does posterior lumbar interbody fusion improve outcome over posterolateral fusion?

PubMed

La Rosa, Giovanni; Conti, Alfredo; Cacciola, Fabio; Cardali, Salvatore; La Torre, Domenico; Gambadauro, Nicola Maria; Tomasello, Francesco

2003-09-01

Posterolateral fusion involving instrumentation-assisted segmental fixation represents a valid procedure in the treatment of lumbar instability. In cases of anterior column failure, such as in isthmic spondylolisthesis, supplemental posterior lumbar interbody fusion (PLIF) may improve the fusion rate and endurance of the construct. Posterior lumbar interbody fusion is, however, a more demanding procedure and increases costs and risks of the intervention. The advantages of this technique must, therefore, be weighed against those of a simple posterior lumbar fusion. Thirty-five consecutive patients underwent pedicle screw fixation for isthmic spondylolisthesis. In 18 patients posterior lumbar fusion was performed, and in 17 patients PLIF was added. Clinical, economic, functional, and radiographic data were assessed to determine differences in clinical and functional results and biomechanical properties. At 2-year follow-up examination, the correction of subluxation, disc height, and foraminal area were maintained in the group in which a PLIF procedure was performed, but not in the posterolateral fusion-only group (p < 0.05). Nevertheless, no statistical intergroup differences were demonstrated in terms of neurological improvement (p = 1), economic (p = 0.43), or functional (p = 0.95) outcome, nor in terms of fusion rate (p = 0.49). The authors' findings support the view that an interbody fusion confers superior mechanical strength to the spinal construct; when posterolateral fusion is the sole intervention, progressive loss of the extreme correction can be expected. Such mechanical insufficiency, however, did not influence clinical outcome.

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

PubMed

Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

2013-05-28

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Effect of Surgical Approach on Pulmonary Function in Adolescent Idiopathic Scoliosis Patients: A Systemic Review and Meta-analysis.

PubMed

Lee, Andy C H; Feger, Mark A; Singla, Anuj; Abel, Mark F

2016-11-15

Systemic review and meta-analysis. To analyze the effect of spinal fusion and instrumentation for adolescent idiopathic scoliosis (AIS) on absolute pulmonary function test (PFTs). Pulmonary function is correlated with severity of deformity in AIS patients and studies that have analyzed the effect of spinal fusion and instrumentation on PFTs for AIS have reported inconsistent results. There is a need to analyze the effect of spinal fusion on PFTs with stratification by surgical approach. Our analysis included 22 studies. Cohen's d effect sizes were calculated for absolute PFT outcome measures with 95% confidence intervals (CI). Meta-analyses were performed at each postoperative time frame for six homogeneous surgical approaches: (i) combined anterior release and posterior fusion with instrumentation; (ii) combined video assisted anterior release and posterior fusion with instrumentation without thoracoplasty; (iii) posterior fusion with instrumentation without thoracoplasty; (iv) anterior fusion with instrumentation and without thoracoplasty; (v) video assisted anterior fusion with instrumentation without thoracoplasty; and (vi) any scoliosis surgery with additional thoracoplasty. Anterior spinal fusion with instrumentation, any scoliosis surgery with concomitant thoracoplasty, or video-assisted anterior fusion with instrumentation for AIS had similar absolute PFTs at their 2 year postoperative follow up compared with their preoperative PFTs (effect sizes ranging from -0.2-0.2 with all CI crossing "0"). Posterior spinal fusion with instrumentation (with or without an anterior release) demonstrated small to moderate increases in PFTs 2 years postoperatively (effect sizes ranging from 0.35-0.65 with all CI not crossing "0"). Anterior fusion with instrumentation, regardless of the approach, and any scoliosis surgery with concomitant thoracoplasty do not lead to significant change in pulmonary functions 2 year after surgery. Posterior spinal fusion with instrumentation (with or without an anterior release) resulted in small to moderate increases in PFTs. N/A.

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

PubMed

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

PubMed

Xu, Kai; Chan, Yee-Peng; Bradel-Tretheway, Birgit; Akyol-Ataman, Zeynep; Zhu, Yongqun; Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z Hong; Broder, Christopher C; Aguilar, Hector C; Nikolov, Dimitar B

2015-12-01

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

PubMed

Wang, Honglin; Sun, Yue; Chang, Jianhong; Zheng, Fangfang; Pei, Haixia; Yi, Yanjun; Chang, Caren; Dong, Chun-Hai

2016-07-01

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.

PubMed

Müller, Andreas; Neukam, Martin; Ivanova, Anna; Sönmez, Anke; Münster, Carla; Kretschmar, Susanne; Kalaidzidis, Yannis; Kurth, Thomas; Verbavatz, Jean-Marc; Solimena, Michele

2017-02-02

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Spatially resolved x-ray fluorescence spectroscopy of beryllium capsule implosions at the NIF

NASA Astrophysics Data System (ADS)

MacDonald, M. J.; Bishel, D. T.; Saunders, A. M.; Scott, H. A.; Kyrala, G.; Kline, J.; MacLaren, S.; Thorn, D. B.; Yi, S. A.; Zylstra, A. B.; Falcone, R. W.; Doeppner, T.

2017-10-01

Beryllium ablators used in indirectly driven inertial confinement fusion implosions are doped with copper to prevent preheat of the cryogenic hydrogen fuel. Here, we present analysis of spatially resolved copper K- α fluorescence spectra from the beryllium ablator layer. It has been shown that K- α fluorescence spectroscopy can be used to measure plasma conditions of partially ionized dopants in high energy density systems. In these experiments, K-shell vacancies in the copper dopant are created by the hotspot emission at stagnation, resulting in K-shell fluorescence at bang time. Spatially resolved copper K- α emission spectra are compared to atomic kinetics and radiation code simulations to infer density and temperature profiles. This work was supported by the US DOE under Grant No. DE-NA0001859, under the auspices of the US DOE by Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and by Los Alamos National Laboratory under contract DE-AC52-06NA52396.

Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

PubMed

Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

2016-02-01

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice. © 2015 The Protein Society.

Chromophore Protonation State Controls Photoswitching of the Fluoroprotein asFP595

PubMed Central

Schäfer, Lars V.; Groenhof, Gerrit; Boggio-Pasqua, Martial; Robb, Michael A.; Grubmüller, Helmut

2008-01-01

Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins. PMID:18369426

Protein-specific localization of a rhodamine-based calcium-sensor in living cells.

PubMed

Best, Marcel; Porth, Isabel; Hauke, Sebastian; Braun, Felix; Herten, Dirk-Peter; Wombacher, Richard

2016-06-28

A small synthetic calcium sensor that can be site-specifically coupled to proteins in living cells by utilizing the bio-orthogonal HaloTag labeling strategy is presented. We synthesized an iodo-derivatized BAPTA chelator with a tetramethyl rhodamine fluorophore that allows further modification by Sonogashira cross-coupling. The presented calcium sensitive dye shows a 200-fold increase in fluorescence upon calcium binding. The derivatization with an aliphatic linker bearing a terminal haloalkane-function by Sonogashira cross-coupling allows the localization of the calcium sensor to Halo fusion proteins which we successfully demonstrate in in vitro and in vivo experiments. The herein reported highly sensitive tetramethyl rhodamine based calcium indicator, which can be selectively localized to proteins, is a powerful tool to determine changes in calcium levels inside living cells with spatiotemporal resolution.

Low-Level Laser Effect on Proliferation, Migration, and Antiapoptosis of Mesenchymal Stem Cells.

PubMed

Yin, Kan; Zhu, Rongjia; Wang, Shihua; Zhao, Robert Chunhua

2017-05-15

Mesenchymal stem cells (MSCs) have been proved to be an important element in cell-based therapy. Photobiomodulation used extremely low-level lasers (LLLs) to affect the behavior of cells. The effect mechanism of LLLs on MSCs from human remained to be discovered. In this study, cell viability was assessed using MTS assays and cell cycle was evaluated by fluorescence-activated cell sorting (FACS). The influence of LLLs on mitochondrial biogenesis (fission or fusion) and function (ATP, reactive oxygen species [ROS], nitric oxide [NO]) was evaluated by transmission electron microscope, FACS, quantitative real time polymerase chain reaction (q-PCR), and immunocytochemistry. Cell migration and cytoskeleton alteration (actin and tubulin) were evaluated using transwell assay, immunocytochemistry, enzyme-linked immunosorbent assay, and western blotting. Cell apoptosis was evaluated using FACS, immunocytochemistry, and western blotting. We investigated that certain influence of LLLs on MSCs in vitro 6 or 24 h after 1 h of LLL irradiation. The mechanism of the effects included proliferation rate increase mediated by increased S phase proportion; mitochondrial biogenesis and function alteration mediated by fusion (Mfn1, Mfn2, and Opa-1) and fission (Fis1, Drp-1, and MTP18)-related proteins, NRF1, TFAM, PGC-1a, and upregulated intracellular ROS and NO concentration; migration acceleration through the ERK1/2 and FAK pathway and upregulation of growth factors such as HGF and PDGF; and resistance to apoptosis with increased Bcl-2 and decreased Bax, or through tunneling nanotube formation between LLL-treated MSCs and 5-fluorouracil-induced apoptotic MSCs. These observations suggested that LLLs enhanced stem cell survival and therapeutic function, which could appear to be an innovative pretreatment in the application of MSCs.

Bridging the gap: functional healing of embryonic small intestine ex vivo

PubMed Central

Coletta, Riccardo; Roberts, Neil A.; Oltrabella, Francesca; Khalil, Basem A.; Morabito, Antonino

2015-01-01

Abstract The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2–3 mm tubes, which were placed in pairs, separated by a small gap, on semi‐permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum‐free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin+ neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies. ©2015. The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd PMID:26234729

Designing Image Operators for MRI-PET Image Fusion of the Brain

NASA Astrophysics Data System (ADS)

Márquez, Jorge; Gastélum, Alfonso; Padilla, Miguel A.

2006-09-01

Our goal is to obtain images combining in a useful and precise way the information from 3D volumes of medical imaging sets. We address two modalities combining anatomy (Magnetic Resonance Imaging or MRI) and functional information (Positron Emission Tomography or PET). Commercial imaging software offers image fusion tools based on fixed blending or color-channel combination of two modalities, and color Look-Up Tables (LUTs), without considering the anatomical and functional character of the image features. We used a sensible approach for image fusion taking advantage mainly from the HSL (Hue, Saturation and Luminosity) color space, in order to enhance the fusion results. We further tested operators for gradient and contour extraction to enhance anatomical details, plus other spatial-domain filters for functional features corresponding to wide point-spread-function responses in PET images. A set of image-fusion operators was formulated and tested on PET and MRI acquisitions.

General software design for multisensor data fusion

NASA Astrophysics Data System (ADS)

Zhang, Junliang; Zhao, Yuming

1999-03-01

In this paper a general method of software design for multisensor data fusion is discussed in detail, which adopts object-oriented technology under UNIX operation system. The software for multisensor data fusion is divided into six functional modules: data collection, database management, GIS, target display and alarming data simulation etc. Furthermore, the primary function, the components and some realization methods of each modular is given. The interfaces among these functional modular relations are discussed. The data exchange among each functional modular is performed by interprocess communication IPC, including message queue, semaphore and shared memory. Thus, each functional modular is executed independently, which reduces the dependence among functional modules and helps software programing and testing. This software for multisensor data fusion is designed as hierarchical structure by the inheritance character of classes. Each functional modular is abstracted and encapsulated through class structure, which avoids software redundancy and enhances readability.

Microscopie de fluorescence de protéines autofluorescentes uniques pour la biologie cellulaire

NASA Astrophysics Data System (ADS)

Cognet, Laurent; Coussen, Françoise; Choquet, Daniel; Lounis, Brahim

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells. To cite this article: L. Cognet et al., C. R. Physique 3 (2002) 645-656.

The ultimate picture-the combination of live cell superresolution microscopy and single molecule tracking yields highest spatio-temporal resolution.

PubMed

Dersch, Simon; Graumann, Peter L

2018-06-01

We are witnessing a breathtaking development in light (fluorescence) microscopy, where structures can be resolved down to the size of a ribosome within cells. This has already yielded surprising insight into the subcellular structure of cells, including the smallest cells, bacteria. Moreover, it has become possible to visualize and track single fluorescent protein fusions in real time, and quantify molecule numbers within individual cells. Combined, super resolution and single molecule tracking are pushing the limits of our understanding of the spatio-temporal organization even of the smallest cells to an unprecedented depth. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transformation with green fluorescent protein of Trichoderma harzianum 1051, a strain with biocontrol activity against Crinipellis perniciosa, the agent of witches'-broom disease of cocoa.

PubMed

Inglis, Peter W.; Queiroz, Paulo R.; Valadares-Inglis, M. Cléria

1999-04-01

A plasmid vector for fungal expression of an enhanced, red-shifted variant of the Aequoria victoriae green fluorescent protein was constructed by fusion of the EGFP gene to the highly expressed Aspergillus nidulans gpd promoter and the A. nidulans trpC terminator. This construction was introduced by cotransformation, using benomyl selection, into Trichoderma harzianum strain 1051, a strain being evaluated for the biological control of witches'-broom disease of cocoa caused by Crinipellis perniciosa. Epifluorescence microscopy was used to monitor germination and attachment of stable transformant conidia on the surface of C. perniciosa hyphae.

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

PubMed

Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

2016-05-25

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.

Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes.

PubMed

Suzuki, Akiko; Endo, Takeshi

2002-02-06

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.

Janus dendrimersomes coassembled from fluorinated, hydrogenated, and hybrid Janus dendrimers as models for cell fusion and fission.

PubMed

Xiao, Qi; Sherman, Samuel E; Wilner, Samantha E; Zhou, Xuhao; Dazen, Cody; Baumgart, Tobias; Reed, Ellen H; Hammer, Daniel A; Shinoda, Wataru; Klein, Michael L; Percec, Virgil

2017-08-22

A three-component system of Janus dendrimers (JDs) including hydrogenated, fluorinated, and hybrid hydrogenated-fluorinated JDs are reported to coassemble by film hydration at specific ratios into an unprecedented class of supramolecular Janus particles (JPs) denoted Janus dendrimersomes (JDSs). They consist of a dumbbell-shaped structure composed of an onion-like hydrogenated vesicle and an onion-like fluorinated vesicle tethered together. The synthesis of dye-tagged analogs of each JD component enabled characterization of JDS architectures with confocal fluorescence microscopy. Additionally, a simple injection method was used to prepare submicron JDSs, which were imaged with cryogenic transmission electron microscopy (cryo-TEM). As reported previously, different ratios of the same three-component system yielded a variety of structures including homogenous onion-like vesicles, core-shell structures, and completely self-sorted hydrogenated and fluorinated vesicles. Taken together with the JDSs reported herein, a self-sorting pathway is revealed as a function of the relative concentration of the hybrid JD, which may serve to stabilize the interface between hydrogenated and fluorinated bilayers. The fission-like pathway suggests the possibility of fusion and fission processes in biological systems that do not require the assistance of proteins but instead may result from alterations in the ratios of membrane composition.

Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

PubMed

Chura-Chambi, Rosa Maria; Arcuri, Helen Andrade; Lino, Felipe; Versati, Natan; Palma, Mario Sergio; Favaro, Denize C; Morganti, Ligia

2017-05-01

Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Activity of Fusion Prophenoloxidase-GFP and Its Potential Applications for Innate Immunity Study

PubMed Central

Yang, Bing; Lu, Anrui; Peng, Qin; Ling, Qing-Zhi; Ling, Erjun

2013-01-01

Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu2+ was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo. PMID:23717543

Fusion cross sections for reactions involving medium and heavy nucleus-nucleus systems

NASA Astrophysics Data System (ADS)

Atta, Debasis; Basu, D. N.

2014-12-01

Existing data on near-barrier fusion excitation functions of medium and heavy nucleus-nucleus systems have been analyzed by using a simple diffused-barrier formula derived assuming the Gaussian shape of the barrier-height distributions. The fusion cross section is obtained by folding the Gaussian barrier distribution with the classical expression for the fusion cross section for a fixed barrier. The energy dependence of the fusion cross section, thus obtained, provides good description to the existing data on near-barrier fusion and capture excitation functions for medium and heavy nucleus-nucleus systems. The theoretical values for the parameters of the barrier distribution are estimated which can be used for fusion or capture cross-section predictions that are especially important for planning experiments for synthesizing new superheavy elements.

Towards fully automated Identification of Vesicle-Membrane Fusion Events in TIRF Microscopy

NASA Astrophysics Data System (ADS)

Vallotton, Pascal; James, David E.; Hughes, William E.

2007-11-01

Total Internal Reflection Fluorescence Microscopy (TIRFM) is imposing itself as the tool of choice for studying biological activity in close proximity to the plasma membrane. For example, the exquisite selectivity of TIRFM allows monitoring the diffusion of GFP-phogrin vesicles and their recruitment to the plasma membrane in pancreatic β-cells. We present a novel computer vision system for automatically identifying the elusive fusion events of GFP-phogrin vesicles with the plasma membrane. Our method is based on robust object tracking and matched filtering. It should accelerate the quantification of TIRFM data and allow the extraction of more biological information from image data to support research in diabetes and obesity.

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Outcome of transforaminal lumbar interbody fusion in spondylolisthesis-A clinico-radiological correlation.

PubMed

Balasubramanian, Vijay Anand; Douraiswami, Balaji; Subramani, Suresh

2018-06-01

Lumbar spondylolisthesis is a common cause of morbidity in middle aged individuals. Spinal fusion with instrumentation has become the gold standard for lumbar segmental instability. Studies which correlate the improvement in radiology postoperatively with functional outcome show contrasting reports. This study is aimed at finding the correlation between clinical and radiological outcomes after surgery with transforaminal lumbar interbody fusion. A retrospective study in 35 patients who underwent transforaminal lumbar interbody fusion in a period of 1 year was done. Preoperative pain (VAS Score), functional ability (ODI), radiological parameters (slip angle, slip grade, disc height, foraminal height, lumbar lordosis) were compared with postoperative recordings at the last followup. Functional improvement (Macnab's criteria) and fusion (Lee's fusion criteria) were assessed. Statistical analysis was done with student's paired t -test and Pearson's correlation coefficient. VAS score, ODI improved from 8 to 2 and 70 to 15 respectively. Slip angle improved from 23°to 5° on an average. 80% patients showed fusion and 85% showed good clinical outcome at 1 year followup. Analyzing with Pearson correlation coefficient showed no significant relation between pain scores and radiological parameters. But there was statistically significant relation between radiological fusion and the final clinical outcome. TLIF produces spinal fusion in most individuals. Strong spinal fusion is essential for good clinical outcome in spondylolisthesis patients who undergo TLIF. Reduction in slip is not necessary for all patients with listhesis.

Revisions to the JDL data fusion model

NASA Astrophysics Data System (ADS)

Steinberg, Alan N.; Bowman, Christopher L.; White, Franklin E.

1999-03-01

The Data Fusion Model maintained by the Joint Directors of Laboratories (JDL) Data Fusion Group is the most widely-used method for categorizing data fusion-related functions. This paper discusses the current effort to revise the expand this model to facilitate the cost-effective development, acquisition, integration and operation of multi- sensor/multi-source systems. Data fusion involves combining information - in the broadest sense - to estimate or predict the state of some aspect of the universe. These may be represented in terms of attributive and relational states. If the job is to estimate the state of a people, it can be useful to include consideration of informational and perceptual states in addition to the physical state. Developing cost-effective multi-source information systems requires a method for specifying data fusion processing and control functions, interfaces, and associate databases. The lack of common engineering standards for data fusion systems has been a major impediment to integration and re-use of available technology: current developments do not lend themselves to objective evaluation, comparison or re-use. This paper reports on proposed revisions and expansions of the JDL Data FUsion model to remedy some of these deficiencies. This involves broadening the functional model and related taxonomy beyond the original military focus, and integrating the Data Fusion Tree Architecture model for system description, design and development.

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules.

PubMed

Kasai, Kazuo; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

2008-07-01

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic beta cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.

Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus.

PubMed

Kim, So Yeon; Gitai, Zemer; Kinkhabwala, Anika; Shapiro, Lucy; Moerner, W E

2006-07-18

The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.

Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells.

PubMed

Eggenberger, Kai; Mink, Christian; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter

2011-01-03

The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.

MagIC, a genetically encoded fluorescent indicator for monitoring cellular Mg2+ using a non-Förster resonance energy transfer ratiometric imaging approach

NASA Astrophysics Data System (ADS)

Koldenkova, Vadim Pérez; Matsuda, Tomoki; Nagai, Takeharu

2015-10-01

Intracellular Mg roles are commensurate with its abundance in the cell cytoplasm. However, little is known about Mg subcellular dynamics, primarily due to the lack of suitable Mg-selective tools to monitor this ion in intracellular compartments. To cope with this lack, we developed a Mg-sensitive indicator-MagIC (indicator for Magnesium Imaging in Cell) -composed of a functionalized yellow fluorescent protein (FP) variant fused to a red-emitting FP serving as a reference, thus allowing ratiometric imaging of Mg. MagIC expressed in mammalian cells is homogeneously distributed between the cytosol and nucleus but its fusion with appropriate targeting sequences redirects it to mitochondria or the endoplasmic reticulum. MagIC shows little interference by intracellular Ca [Kd(Mg2+)=5.1 mM Kd(Ca2+)=4.8 mM] and its kinetic properties (k=84 s-1) approach those of indicator dyes. With MagIC, as reported previously, we also observed a cytosolic Mg increase provoked by application of 50 mM MgCl2 in the medium. This effect is, however, mimicked by 75 mM KCl or 150 mM D-sorbitol addition, indicating that it is a response to the associated hyperosmotic shock and not to Mg itself. Our results confirm the functionality of MagIC as a useful tool for the long-awaited possibility of prolonged and organelle-specific monitoring of cellular Mg.

Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2β in the human osteosarcoma cell line U2OS.

PubMed

Ben-Shoshan, Shirley Oren; Simon, Amos J; Jacob-Hirsch, Jasmine; Shaklai, Sigal; Paz-Yaacov, Nurit; Amariglio, Ninette; Rechavi, Gideon; Trakhtenbrot, Luba

2014-01-28

Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2β plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2β in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein. By knocking down the expression of LAP2β, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2β knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2β knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2β knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression. Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2β reduced expression. Our study implies on a novel role of LAP2β in the maintenance of cell ploidy status. LAP2β depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.