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Sample records for cell-free endosome fusion

  1. Zn2+ depletion blocks endosome fusion.

    PubMed Central

    Aballay, A; Sarrouf, M N; Colombo, M I; Stahl, P D; Mayorga, L S

    1995-01-01

    Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca2+ concentrations, i.e. < 30 nM. Moreover, fusion is not affected when intravesicular Ca2+ is depleted by preincubation of vesicles with calcium ionophores (5 microM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca2+. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid], a relatively specific Ca2+ chelator, inhibits fusion. BAPTA binds other metals besides Ca2+. We present evidence that BAPTA inhibition is due not to Ca2+ chelation but to Zn2+ depletion. TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca2+, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn2+. Zn(2+)-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process. Images Figure 1 PMID:8554539

  2. Fusion of Enveloped Viruses in Endosomes.

    PubMed

    White, Judith M; Whittaker, Gary R

    2016-06-01

    Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion. PMID:26935856

  3. Rab5-mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani.

    PubMed

    Singh, Sudha B; Tandon, Ruchi; Krishnamurthy, Ganga; Vikram, Rajagopal; Sharma, Nimisha; Basu, Sandip K; Mukhopadhyay, Amitabha

    2003-11-01

    To understand the trafficking of endocytosed hemoglobin (Hb) in Leishmania, we investigated the characteristics of in vitro fusion between endosomes containing biotinylated Hb (BHb) and avidin-horseradish peroxidase (AHRP). We showed that early endosome fusion in Leishmania is temperature and cytosol dependent and is inhibited by ATP depletion, ATPgammaS, GTPgammaS and N-ethylmaleimide treatment. The Rab5 homolog from Leishmania donovani, LdRab5, was cloned and expressed. Our results showed that homotypic fusion between the early endosomes in Leishmania is Rab5 dependent. Early endosomes containing BHb fused efficiently with late endosomes in a process regulated by Rab7, whereas no fusion between early and late endosomes was detected using fluid phase markers. Pre-treatment of early endosomes containing BHb with monoclonal antibody specific for the C-terminus of the Hb receptor (HbR) or the addition of the C-terminal cytoplasmic fragment of the HbR specifically inhibited the fusion with late endosomes, suggesting that signal(s) mediated through the HbR cytoplasmic tail promotes the fusion of early endosomes containing Hb with late endosomes. PMID:14592970

  4. Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system.

    PubMed Central

    Gruenberg, J E; Howell, K E

    1986-01-01

    We have used defined subcellular fractions to reconstitute in a cell-free system vesicle fusions occurring in the endocytic pathway. The endosomal fractions were prepared by immuno-isolation using as antigen an epitope located on a foreign protein, the transmembrane glycoprotein G (G-protein) of vesicular stomatitis virus. The G-protein was first implanted in the cell plasma membrane and subsequently endocytosed for 15 to 30 min at 37 degrees C. The endosomal fractions were immuno-isolated on a solid support using as antigen the cytoplasmic domain of the G-protein in combination with a specific monoclonal antibody. For comparative studies the plasma membrane was immuno-isolated from cells in the absence of G internalization with a monoclonal antibody against the exoplasmic domain of the G-protein. The immuno-isolated endosomal vesicles contained 70% of horseradish peroxidase internalized in the endosome fluid phase, exhibited an acidic luminal pH as shown by acridine orange fluorescence and differed in their protein composition from the immuno-isolated plasma membrane fraction. The fusion of endocytic vesicles originating from different stages of the pathway was studied in a cell-free assay using both a bio-chemical and a morphological detection system. These well defined endosomal vesicles were immuno-isolated with the G-protein on the solid support and provided the recipient compartment of the fusion (acceptor). They were mixed with a post-nuclear supernatant containing endosomes loaded with exogenous lactoperoxidase (donor) at 37 degrees C. Fusion delivered the donor peroxidase to the lumen of acceptor vesicles permitting fusion-specific iodination of the G-protein itself. The fusion of vesicles required ATP and was detected only with an endosomal fraction prepared after internalization of the G-protein for 15 min at 37 degrees C but not with a plasma membrane or with an endosomal fraction prepared after 30 min G-protein internalization. Images Fig. 1. Fig. 2

  5. Cell-to-Cell Transfer of HIV-1 via Virological Synapses Leads to Endosomal Virion Maturation that Activates Viral Membrane Fusion

    PubMed Central

    Dale, Benjamin M.; McNerney, Gregory P.; Thompson, Deanna L.; Hubner, Wolfgang; de los Reyes, Kevin; Chuang, Frank Y.S.; Huser, Thomas; Chen, Benjamin K.

    2012-01-01

    SUMMARY HIV-1 can infect T cells by cell-free virus or by direct virion transfer between cells through cell contact-induced structures called virological synapses (VS). During VS-mediated infection, virions accumulate within target cell endosomes. We show that after crossing the VS, the transferred virus undergoes both maturation and viral membrane fusion. Following VS transfer, viral membrane fusion occurs with delayed kinetics and transferred virions display reduced sensitivity to patient antisera compared to mature, cell-free virus. Furthermore, particle fusion requires that the transferred virions undergo proteolytic maturation within acceptor cell endosomes, which occurs over several hours. Rapid, live cell confocal microscopy demonstrated that viral fusion can occur in compartments that have moved away from the VS. Thus, HIV particle maturation activates viral fusion in target CD4+ T cell endosomes following transfer across the VS and may represent a pathway by which HIV evades antibody neutralization. PMID:22177560

  6. Mucolipin-3 Regulates Luminal Calcium, Acidification, and Membrane Fusion in the Endosomal Pathway*

    PubMed Central

    Lelouvier, Benjamin; Puertollano, Rosa

    2011-01-01

    Mucolipin-3 (MCOLN3) is a pH-regulated Ca2+ channel that localizes to the endosomal pathway. Gain-of-function mutation in MCOLN3 causes the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction, and coat color dilution. The Va phenotype results from a punctual mutation (A419P) in the pore region of MCOLN3 that locks the channel in an open conformation causing massive entry of Ca2+ inside cells and inducing cell death by apoptosis. Overexpression of wild-type MCOLN3 produces severe alterations of the endosomal pathway, including enlargement and clustering of endosomes, delayed EGF receptor degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. To understand better the physiological role of MCOLN3, we inhibited MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3. Remarkably, we found that impairment of MCOLN3 activity caused a significant accumulation of luminal Ca2+ in endosomes. This accumulation led to severe defects in endosomal acidification as well as to increased endosomal fusion. Our findings reveal a prominent role for MCOLN3 in regulating Ca2+ homeostasis at the endosomal pathway and confirm the importance of luminal Ca2+ for proper acidification and membrane fusion. PMID:21245134

  7. Cell-free fusion of bacteria-containing phagosomes with endocytic compartments

    PubMed Central

    Becken, Ulrike; Jeschke, Andreas; Veltman, Katharina; Haas, Albert

    2010-01-01

    Uptake of microorganisms by professional phagocytic cells leads to formation of a new subcellular compartment, the phagosome, which matures by sequential fusion with early and late endocytic compartments, resulting in oxidative and nonoxidative killing of the enclosed microbe. Few tools are available to study membrane fusion between phagocytic and late endocytic compartments in general and with pathogen-containing phagosomes in particular. We have developed and applied a fluorescence microscopy assay to study fusion of microbe-containing phagosomes with different-aged endocytic compartments in vitro. This revealed that fusion of phagosomes containing nonpathogenic Escherichia coli with lysosomes requires Rab7 and SNARE proteins but not organelle acidification. In vitro fusion experiments with phagosomes containing pathogenic Salmonella enterica serovar Typhimurium indicated that reduced fusion of these phagosomes with early and late endocytic compartments was independent of endosome and cytosol sources and, hence, a consequence of altered phagosome quality. PMID:21071675

  8. Cell-free fusion of bacteria-containing phagosomes with endocytic compartments.

    PubMed

    Becken, Ulrike; Jeschke, Andreas; Veltman, Katharina; Haas, Albert

    2010-11-30

    Uptake of microorganisms by professional phagocytic cells leads to formation of a new subcellular compartment, the phagosome, which matures by sequential fusion with early and late endocytic compartments, resulting in oxidative and nonoxidative killing of the enclosed microbe. Few tools are available to study membrane fusion between phagocytic and late endocytic compartments in general and with pathogen-containing phagosomes in particular. We have developed and applied a fluorescence microscopy assay to study fusion of microbe-containing phagosomes with different-aged endocytic compartments in vitro. This revealed that fusion of phagosomes containing nonpathogenic Escherichia coli with lysosomes requires Rab7 and SNARE proteins but not organelle acidification. In vitro fusion experiments with phagosomes containing pathogenic Salmonella enterica serovar Typhimurium indicated that reduced fusion of these phagosomes with early and late endocytic compartments was independent of endosome and cytosol sources and, hence, a consequence of altered phagosome quality. PMID:21071675

  9. VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

    PubMed Central

    Marshall, Misty R.; Pattu, Varsha; Halimani, Mahantappa; Maier-Peuschel, Monika; Müller, Martha-Lena; Becherer, Ute; Hong, Wanjin; Hoth, Markus; Tschernig, Thomas

    2015-01-01

    Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated. PMID:26124288

  10. Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes

    PubMed Central

    Cox, Reagan G.; Mainou, Bernardo A.; Johnson, Monika; Hastings, Andrew K.; Schuster, Jennifer E.; Dermody, Terence S.; Williams, John V.

    2015-01-01

    Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments. PMID:26629703

  11. Cell-Penetrating Peptide Induces Leaky Fusion of Liposomes Containing Late Endosome-Specific Anionic Lipid

    PubMed Central

    Yang, Sung-Tae; Zaitseva, Elena; Chernomordik, Leonid V.; Melikov, Kamran

    2010-01-01

    Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them. PMID:20959093

  12. IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion

    PubMed Central

    Chin, Christopher R.; Savidis, George; Brass, Abraham L.; Melikyan, Gregory B.

    2014-01-01

    Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes. PMID:24699674

  13. Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes

    PubMed Central

    Wartosch, Lena; Günesdogan, Ufuk; Graham, Stephen C; Luzio, J Paul

    2015-01-01

    The mammalian homotypic fusion and vacuole protein sorting (HOPS) complex is comprised of six subunits: VPS11, VPS16, VPS18, VPS39, VPS41 and the Sec1/Munc18 (SM) family member VPS33A. Human HOPS has been predicted to be a tethering complex required for fusion of intracellular compartments with lysosomes, but it remains unclear whether all HOPS subunits are required. We showed that the whole HOPS complex is required for fusion of endosomes with lysosomes by monitoring the delivery of endocytosed fluorescent dextran to lysosomes in cells depleted of individual HOPS proteins. We used the crystal structure of the VPS16/VPS33A complex to design VPS16 and VPS33A mutants that no longer bind each other and showed that, unlike the wild-type proteins, these mutants no longer rescue lysosome fusion with endosomes or autophagosomes in cells depleted of the endogenous proteins. There was no effect of depleting either VIPAR or VPS33B, paralogs of VPS16 and VPS33A, on fusion of lysosomes with either endosomes or autophagosomes and immunoprecipitation showed that they form a complex distinct from HOPS. Our data demonstrate the necessity of recruiting the SM protein VPS33A to HOPS via its interaction with VPS16 and that HOPS proteins, but not VIPAR or VPS33B, are essential for fusion of endosomes or autophagosomes with lysosomes. PMID:25783203

  14. Inhibition of endosomal fusion activity of influenza virus by Rheum tanguticum (da-huang)

    PubMed Central

    Lin, Ta-Jen; Lin, Chwan-Fwu; Chiu, Cheng-Hsun; Lee, Ming-Chung; Horng, Jim-Tong

    2016-01-01

    Rhubarb (Rheum tanguticum; da-huang in Chinese medicine) is a herbal medicine that has been used widely for managing fever and removing toxicity. In this study, we investigated how rhubarb inhibits influenza virus during the early stage of the infectious cycle using different functional assays. A non-toxic ethanolic extract of rhubarb (Rex) inhibited several H1N1 subtypes of influenza A viruses in Madin–Darby canine kidney cells, including strains that are clinically resistant to oseltamivir. Time course analysis of Rex addition showed that viral entry was one of the steps that was inhibited by Rex. We also confirmed that Rex effectively inhibited viral attachment and penetration into the host cells. The inhibition of red blood cell haemolysis and cell–cell fusion by Rex suggests that Rex may block haemagglutinin-mediated fusion (virus–endosome fusion) during the fusion/uncoating step. Rex has the capacity to inhibit influenza viruses by blocking viral endocytosis. Thus, rhubarb might provide an alternative therapeutic approach when resistant viruses become more prevalent. PMID:27302738

  15. Inhibition of endosomal fusion activity of influenza virus by Rheum tanguticum (da-huang).

    PubMed

    Lin, Ta-Jen; Lin, Chwan-Fwu; Chiu, Cheng-Hsun; Lee, Ming-Chung; Horng, Jim-Tong

    2016-01-01

    Rhubarb (Rheum tanguticum; da-huang in Chinese medicine) is a herbal medicine that has been used widely for managing fever and removing toxicity. In this study, we investigated how rhubarb inhibits influenza virus during the early stage of the infectious cycle using different functional assays. A non-toxic ethanolic extract of rhubarb (Rex) inhibited several H1N1 subtypes of influenza A viruses in Madin-Darby canine kidney cells, including strains that are clinically resistant to oseltamivir. Time course analysis of Rex addition showed that viral entry was one of the steps that was inhibited by Rex. We also confirmed that Rex effectively inhibited viral attachment and penetration into the host cells. The inhibition of red blood cell haemolysis and cell-cell fusion by Rex suggests that Rex may block haemagglutinin-mediated fusion (virus-endosome fusion) during the fusion/uncoating step. Rex has the capacity to inhibit influenza viruses by blocking viral endocytosis. Thus, rhubarb might provide an alternative therapeutic approach when resistant viruses become more prevalent. PMID:27302738

  16. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    PubMed

    Hernáez, Bruno; Guerra, Milagros; Salas, María L; Andrés, Germán

    2016-04-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  17. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    PubMed Central

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  18. Vanadate from air pollutant inhibits hrs-dependent endosome fusion and augments responsiveness to toll-like receptors.

    PubMed

    Zelnikar, Mojca; Benčina, Mojca; Jerala, Roman; Manček-Keber, Mateja

    2014-01-01

    There is a well-established association between exposure to air pollutants and pulmonary injuries. For example, metals found in ROFA (residual oil fly ash) increase susceptibility of mice as well as humans to microbial infections. In our research, we have found that vanadate substantially increased the response of several Toll-like receptors (TLRs) to stimulation with their ligands. Although vanadate caused generation of reactive oxygen species (ROS), the addition of ROS scavenger N-acetyl cysteine (NAC) had no effect on augmented lipopolysaccharide (LPS) stimulation. We further showed that vanadate inhibits endosome fusion. This effect was determined by measuring the size of endosomes, NF-κB activity and TLR4 degradation in Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) overexpressed cells. Moreover, we identified the role of Hrs phosphorylation in these processes. Based on our findings, we can conclude that vanadate potentiates TLR4 activity by increasing Hrs phosphorylation status, reducing the size of Hrs/TLR4-positive endosomes and impacting TLR4 degradation, thus contributing to the detrimental effects of air pollutants on human health. PMID:24901993

  19. A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

    PubMed

    Tian, Xuejun; Gala, Upasana; Zhang, Yongping; Shang, Weina; Nagarkar Jaiswal, Sonal; di Ronza, Alberto; Jaiswal, Manish; Yamamoto, Shinya; Sandoval, Hector; Duraine, Lita; Sardiello, Marco; Sillitoe, Roy V; Venkatachalam, Kartik; Fan, Hengyu; Bellen, Hugo J; Tong, Chao

    2015-03-01

    Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis. PMID:25811491

  20. A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

    PubMed Central

    Zhang, Yongping; Shang, Weina; Nagarkar Jaiswal, Sonal; di Ronza, Alberto; Jaiswal, Manish; Yamamoto, Shinya; Sandoval, Hector; Duraine, Lita; Sardiello, Marco; Sillitoe, Roy V.; Venkatachalam, Kartik; Fan, Hengyu; Bellen, Hugo J.; Tong, Chao

    2015-01-01

    Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis. PMID:25811491

  1. Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast.

    PubMed

    Hart, P D; Young, M R

    1991-10-01

    The weak base ammonium chloride has been previously reported to inhibit lysosomal movements and phagosome-lysosome (Ph-L) fusion in cultured mouse macrophages (M phi), thus reducing delivery, to an intraphagosomal infection, of endocytosed solutes that have concentrated in secondary lysosomes. We have now addressed the question, whether NH4Cl might affect any direct interaction (if it exists) between such infection phagosomes and earlier, nonlysosomal compartments of the endocytic pathway, i.e., solute-containing endosomes. The phagosomes studied were formed after ingestion of the mouse pathogen Mycobacterium microti and the nonpathogenic yeast Saccharomyces cerevisiae; and the endosomes were formed after nonreceptor-mediated endocytosis of electronopaque and fluorescent soluble markers. By electron microscopy, survey of the cell profiles of M phi that had been treated with 10 mM NH4Cl so that Ph-L fusion was prevented, and that displayed many ferritin-labeled endosomes, revealed numerous examples of the fusion of electronlucent endosomes, revealed numerous examples of the fusion of electronlucent vesicles with phagosomes, whether containing M. microti bacilli or S. cerevisiae yeasts. Fusion was recognized by transfer of label and by morphological evidence of fusion in progress. The fusing vesicles were classed as endosomes, not NH4Cl-lysosomes, by their appearance and provenance, and because lysosome participation was excluded by the concurrent, NH4Cl-caused block of Ph-L fusion and associated lysosomal stasis. No evidence of such phagosome-endosome (Ph-E) fusion was observed in profiles from M phi treated with chloroquine, nor in those from normal, untreated M phi. NH4Cl-treated living M phi that had ingested yeasts at 37 degrees C, followed by endocytosis of lucifer yellow at 17 degrees C (to accumulate labeled endosomes and postpone label passing to lysosomes), were then restored to 37 degrees C. Fluorescence microscopy showed that as many as half of the yeast

  2. Numb regulates vesicular docking for homotypic fusion of early endosomes via membrane recruitment of Mon1b.

    PubMed

    Shao, Ximing; Liu, Yi; Yu, Qian; Ding, Zhihao; Qian, Wenyu; Zhang, Lei; Zhang, Jianchao; Jiang, Nan; Gui, Linfei; Xu, Zhiheng; Hong, Yang; Ma, Yifan; Wei, Yanjie; Liu, Xiaoqing; Jiang, Changan; Zhu, Minyan; Li, Hongchang; Li, Huashun

    2016-05-01

    Numb is an endocytic protein that plays crucial roles in diverse cellular processes such as asymmetric cell division, cell migration and differentiation. However, the molecular mechanism by which Numb regulates endocytic trafficking is poorly understood. Here, we demonstrate that Numb is a docking regulator for homotypic fusion of early endosomes (EEs). Numb depletion causes clustered but unfused EEs, which can be rescued by overexpressing cytosolic Numb 65 and Numb 71 but not plasma membrane-attached Numb 66 or Numb 72. Time-lapse analysis reveals that paired vesicles tend to tether but not fuse with each other in the absence of Numb. We further show that Numb binds to another docking regulator, Mon1b, and is required for the recruitment of cytosolic Mon1b to the EE membrane. Consistent with this, deletion of Mon1b causes similar defects in EE fusion. Our study thus identifies a novel mechanism by which Numb regulates endocytic sorting by mediating EE fusion. PMID:26987402

  3. Activation of the Nipah Virus Fusion Protein in MDCK Cells Is Mediated by Cathepsin B within the Endosome-Recycling Compartment

    PubMed Central

    Diederich, Sandra; Sauerhering, Lucie; Weis, Michael; Altmeppen, Hermann; Schaschke, Norbert; Reinheckel, Thomas; Erbar, Stephanie

    2012-01-01

    Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV. PMID:22278224

  4. The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

    PubMed

    Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

    2016-09-01

    Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. PMID:27588602

  5. Endosomal Interactions during Root Hair Growth.

    PubMed

    von Wangenheim, Daniel; Rosero, Amparo; Komis, George; Šamajová, Olga; Ovečka, Miroslav; Voigt, Boris; Šamaj, Jozef

    2015-01-01

    The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes-termed herein as dancing-endosomes-which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth. PMID:26858728

  6. Recycling endosomes

    PubMed Central

    Goldenring, James R

    2015-01-01

    The endosomal membrane recycling system represents a dynamic conduit for sorting and re-exporting internalized membrane constituents. The recycling system is composed of multiple tubulovesicular recycling pathways that likely confer distinct trafficking pathways for individual cargoes. In addition, elements of the recycling system are responsible for assembly and maintenance of apical membrane specializations including primary cilia and apical microvilli. The existence of multiple intersecting and diverging recycling tracks likely accounts for specificity in plasma membrane recycling trafficking. PMID:26022676

  7. Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

    PubMed

    Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

    2016-03-01

    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion. PMID:26667208

  8. Membrane Tethering Complexes in the Endosomal System

    PubMed Central

    Spang, Anne

    2016-01-01

    Vesicles that are generated by endocytic events at the plasma membrane are destined to early endosomes. A prerequisite for proper fusion is the tethering of two membrane entities. Tethering of vesicles to early endosomes is mediated by the class C core vacuole/endosome tethering (CORVET) complex, while fusion of late endosomes with lysosomes depends on the homotypic fusion and vacuole protein sorting (HOPS) complex. Recycling through the trans-Golgi network (TGN) and to the plasma membrane is facilitated by the Golgi associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes, respectively. However, there are other tethering functions in the endosomal system as there are multiple pathways through which proteins can be delivered from endosomes to either the TGN or the plasma membrane. Furthermore, proteins that may be part of novel tethering complexes have been recently identified. Thus, it is likely that more tethering factors exist. In this review, I will provide an overview of different tethering complexes of the endosomal system and discuss how they may provide specificity in membrane traffic. PMID:27243003

  9. Endosomal Interactions during Root Hair Growth

    PubMed Central

    von Wangenheim, Daniel; Rosero, Amparo; Komis, George; Šamajová, Olga; Ovečka, Miroslav; Voigt, Boris; Šamaj, Jozef

    2016-01-01

    The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes—termed herein as dancing-endosomes—which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth. PMID:26858728

  10. Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.

    PubMed

    Zhang, Xu; Lian, Jiazhang; Kai, Lei; Huang, Lei; Cen, Peilin; Xu, Zhinan

    2014-02-01

    Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹⁴ cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve. PMID:24411442

  11. ER–endosome contact sites: molecular compositions and functions

    PubMed Central

    Raiborg, Camilla; Wenzel, Eva M; Stenmark, Harald

    2015-01-01

    Recent studies have revealed the existence of numerous contact sites between the endoplasmic reticulum (ER) and endosomes in mammalian cells. Such contacts increase during endosome maturation and play key roles in cholesterol transfer, endosome positioning, receptor dephosphorylation, and endosome fission. At least 7 distinct contact sites between the ER and endosomes have been identified to date, which have diverse molecular compositions. Common to these contact sites is that they impose a close apposition between the ER and endosome membranes, which excludes membrane fusion while allowing the flow of molecular signals between the two membranes, in the form of enzymatic modifications, or ion, lipid, or protein transfer. Thus, ER–endosome contact sites ensure coordination of molecular activities between the two compartments while keeping their general compositions intact. Here, we review the molecular architectures and cellular functions of known ER–endosome contact sites and discuss their implications for human health. PMID:26041457

  12. The structure and function of presynaptic endosomes

    SciTech Connect

    Jähne, Sebastian; Rizzoli, Silvio O.; Helm, Martin S.

    2015-07-15

    The function of endosomes and of endosome-like structures in the presynaptic compartment is still controversial. This is in part due to the absence of a consensus on definitions and markers for these compartments. Synaptic endosomes are sometimes seen as stable organelles, permanently present in the synapse. Alternatively, they are seen as short-lived intermediates in synaptic vesicle recycling, arising from the endocytosis of large vesicles from the plasma membrane, or from homotypic fusion of small vesicles. In addition, the potential function of the endosome is largely unknown in the synapse. Some groups have proposed that the endosome is involved in the sorting of synaptic vesicle proteins, albeit others have produced data that deny this possibility. In this review, we present the existing evidence for synaptic endosomes, we discuss their potential functions, and we highlight frequent technical pitfalls in the analysis of this elusive compartment. We also sketch a roadmap to definitely determine the role of synaptic endosomes for the synaptic vesicle cycle. Finally, we propose a common definition of synaptic endosome-like structures.

  13. Selective degradation of insulin within rat liver endosomes

    SciTech Connect

    Doherty, J.J. II; Kay, D.G.; Lai, W.H.; Posner, B.I.; Bergeron, J.J. )

    1990-01-01

    To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor.

  14. Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

    PubMed

    Liutkus, Mantas; Fraser, Samuel A; Caron, Karine; Stigers, Dannon J; Easton, Christopher J

    2016-05-17

    Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis. PMID:26918308

  15. Late endosomes: sorting and partitioning in multivesicular bodies.

    PubMed

    Piper, R C; Luzio, J P

    2001-09-01

    Late endosomes, which have the morphological characteristics of multivesicular bodies, have received relatively little attention in comparison with early endosomes and lysosomes. Recent work in mammalian and yeast cells has given insights into their structure and function, including the generation of their multivesicular morphology. Lipid partitioning to create microdomains enriched in specific lipids is observed in late endosomes, with some lumenal vesicles enriched in lysobisphosphatidic acid and others in phosphatidylinositol 3-phosphate. Sorting of membrane proteins into the lumenal vesicles may occur because of the properties of their trans-membrane domains, or as a result of tagging with ubiquitin. Yeast class E Vps proteins and their mammalian orthologs are the best candidates to make up the protein machinery that controls inward budding, a process that starts in early endosomes. Late endosomes are able to undergo homotypic fusion events and also heterotypic fusion with lysosomes, a process that delivers endocytosed macromolecules for proteolytic degradation. PMID:11555415

  16. A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

    SciTech Connect

    Hirota, Yuko; Kuronita, Toshio; Fujita, Hideaki; Tanaka, Yoshitaka

    2007-12-07

    Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.

  17. Molecular assemblies and membrane domains in multivesicular endosome dynamics

    SciTech Connect

    Falguieres, Thomas; Luyet, Pierre-Philippe; Gruenberg, Jean

    2009-05-15

    Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo 'back-fusion' with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.

  18. Molecular assemblies and membrane domains in multivesicular endosome dynamics.

    PubMed

    Falguières, Thomas; Luyet, Pierre-Philippe; Gruenberg, Jean

    2009-05-15

    Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo "back-fusion" with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic. PMID:19133258

  19. Clathrin regenerates synaptic vesicles from endosomes

    PubMed Central

    Watanabe, Shigeki; Trimbuch, Thorsten; Camacho-Pérez, Marcial; Rost, Benjamin R.; Brokowski, Bettina; Söhl-Kielczynski, Berit; Felies, Annegret; Davis, M. Wayne; Rosenmund, Christian; Jorgensen, Erik M.

    2014-01-01

    Summary Ultrafast endocytosis can retrieve a single large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation. The endosome is resolved into coated vesicles after 3 seconds, which in turn become small-diameter synaptic vesicles 5-6 seconds after stimulation. We disrupted clathrin function using RNAi and found that clathrin is not required for ultrafast endocytosis but is required to generate synaptic vesicles from the endosome. Ultrafast endocytosis fails when actin polymerization is disrupted, or when neurons are stimulated at room temperature instead of physiological temperature. In the absence of ultrafast endocytosis, synaptic vesicles are retrieved directly from the plasma membrane by clathrin-mediated endocytosis. These results explain in large part discrepancies among published experiments concerning the role of clathrin in synaptic vesicle endocytosis. PMID:25296249

  20. Endosomal escape: a bottleneck in intracellular delivery.

    PubMed

    Shete, Harshad K; Prabhu, Rashmi H; Patravale, Vandana B

    2014-01-01

    With advances in therapeutic science, apart from drugs, newer bioactive moieties like oligonucleotides, proteins, peptides, enzymes and antibodies are constantly being introduced for the betterment of therapeutic efficacy. These moieties have intracellular components of the cells like cytoplasm and nucleus as one of their pharmacological sites for exhibiting therapeutic activity. Despite their promising efficacy, their intracellular bioavailability has been critically hampered leading to failure in the treatment of numerous diseases and disorders. The endosomal uptake pathway is known to be a rate-limiting barrier for such systems. Bioactive molecules get trapped in the endosomal vesicles and degraded in the lysosomal compartment, necessitating the need for effective strategies that facilitate the endosomal escape and enhance the cytosolic bioavailability of bioactives. Microbes like viruses and bacteria have developed their innate mechanistic tactics to translocate their genome and toxins by efficiently penetrating the host cell membrane. Understanding this mechanism and exploring it further for intracellular delivery has opened new avenues to surmount the endosomal barrier. These strategies include membrane fusion, pore formation and proton sponge effects. On the other hand, progress in designing a novel smart polymeric carrier system that triggers endosomal escape by undergoing modulations in the intracellular milieu has further led to an improvement in intracellular delivery. These comprise pH, enzyme and temperature-induced modulators, synthetic cationic lipids and photo-induced physical disruption. Each of the aforementioned strategies has its own unique mechanism to escape the endosome. This review recapitulates the numerous strategies designed to surmount the bottleneck of endosomal escape and thereby achieve successful intracellular uptake of bioactives. PMID:24730275

  1. Chasing Ebola through the Endosomal Labyrinth

    PubMed Central

    2016-01-01

    ABSTRACT During virus entry, the surface glycoprotein of Ebola virus (EBOV) undergoes a complex set of transformations within the endosomal network. Tools to study EBOV entry have been limited to static immunofluorescence or biochemical and functional analysis. In a recent article in mBio, Spence et al. reported a novel, live-cell-imaging method that tracks this transformational journey of EBOV in real time [J. S. Spence, T. B. Krause, E. Mittler, R. K. Jangra, and K. Chandran, mBio 7(1):e01857-15, 2016, http://dx.doi.org/10.1128/mBio.01857-15]. The assay validates known mechanisms of EBOV entry and sheds light on some novel intricacies. Direct evidence supports the hypothesis that fusion is a rare event that starts in maturing early endosomes, is completed in late endosomes, and occurs entirely in Niemann-Pick C1 (NPC1)-positive (NPC1+) compartments. The study demonstrated that lipid mixing and productive fusion are temporally decoupled, with different energetic barriers and a protease-dependent step between the two events. Analysis of the mechanism of action of an important class of EBOV neutralizing antibodies, such as KZ52 and ZMapp, provides direct evidence that these antibodies act by inhibiting the membrane fusion. PMID:27006455

  2. Endosome-to-cytosol transport of viral nucleocapsids.

    PubMed

    Le Blanc, Isabelle; Luyet, Pierre-Philippe; Pons, Véronique; Ferguson, Charles; Emans, Neil; Petiot, Anne; Mayran, Nathalie; Demaurex, Nicolas; Fauré, Julien; Sadoul, Rémy; Parton, Robert G; Gruenberg, J

    2005-07-01

    During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns3P) signalling via the PtdIns3P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns3P and their effectors. PMID:15951806

  3. Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes

    PubMed Central

    Li, Xinran; Garrity, Abigail G; Xu, Haoxing

    2013-01-01

    Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca2+. Small GTPases act as master regulators and molecular switches in a GTP-dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane-bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment-specific manner to mediate transport and signalling. Finally, Ca2+, a locally acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca2+ signalling. Importantly, impaired phosphoinositide and Ca2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of lysosome storage diseases. PMID:23878375

  4. Drosophila Strip serves as a platform for early endosome organization during axon elongation

    PubMed Central

    Sakuma, Chisako; Kawauchi, Takeshi; Haraguchi, Shuka; Shikanai, Mima; Yamaguchi, Yoshifumi; Gelfand, Vladimir I.; Luo, Liqun; Miura, Masayuki; Chihara, Takahiro

    2014-01-01

    Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. Here we show that Drosophila Strip is a key regulator for neuronal morphogenesis. strip knockdown disturbs the early endosome clustering and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that plays important roles in neuronal morphogenesis. PMID:25312435

  5. The intact structural form of LLO in endosomes cannot protect against listeriosis.

    PubMed

    Rodriguez-Del Rio, Estela; Frande-Cabanes, Elisabet; Tobes, Raquel; Pareja, Eduardo; Lecea-Cuello, M Jesús; Ruiz-Sáez, Marta; Carrasco-Marín, Eugenio; Alvarez-Dominguez, Carmen

    2011-01-01

    LLO is the major immuno-dominant antigen in listeriosis and is also required for protective immunity. Two forms of LLO can be observed in endosomal membranes, a LLO intact form and a Ctsd-processed LLO(1-491) form. Endosomes obtained from resting macrophages contained only LLO intact forms, while endosomes obtained from IFN-activated macrophages contained both forms. Both types of endosomes elicited LLO(90-91)/CD8(+) and LLO(189-201)/CD4(+) specific immune responses. However, only endosomes containing the Ctsd-processed LLO(1-491) form showed significant CD4(+) and CD8(+) T cell responses similar to LM infected bone marrow derived macrophages and characteristic of protective Listeria immunity. Moreover, endosomes with intact LLO could not confer protection as vaccine carriers against murine listeriosis. While endosomes with Ctsd-processed LLO(1-491) form showed a moderate ability, slightly lower than high efficiency vaccine vectors as MØ infected with LM. These studies argue that all cell-free membrane vesicles might serve as valid vaccine carriers against infectious agents. Exclusively those cell-free vesicles MIIC competent for LLO processing are protective vaccines vectors since they recruit significant numbers of mature dendritic cells to the vaccination sites and contain a LLO(1-491) form that might be accessible for MHC class I and class II antigen presentation. PMID:22003433

  6. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

    PubMed

    Sriram, V; Krishnan, K S; Mayor, Satyajit

    2003-05-12

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function. PMID:12743107

  7. Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex

    SciTech Connect

    Reenstra, W.W.; Bae, H.R.; Verkman, A.S. Univ. of California, San Francisco ); Sabolic, I. Harvard Medical School, Charlestown, MA )

    1992-01-14

    Regulation of Cl conductance by protein kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by ({gamma}-{sup 32}P)ATP. These results suggest that, in a cell-free system, protein kinase A increases Cl conductance in endosomes from kidney proximal tubule by a phosphorylation mechanism. The labeled protein has a size similar to that of the 64-kDa putative kidney Cl channel reported by Landry et al. but is much smaller than the {approximately}170-kDa cystic fibrosis transmembrane conductance regulatory protein.

  8. A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo.

    PubMed

    Drengk, Anja; Fritsch, Jürgen; Schmauch, Christian; Rühling, Harald; Maniak, Markus

    2003-10-14

    The endocytic pathway depends on the actin cytoskeleton. Actin contributes to internalization at the plasma membrane and to subsequent trafficking steps like propulsion through the cytoplasm, fusion of phagosomes with early endosomes, and transport from early to late endosomes. In vitro studies with mammalian endosomes and yeast vacuoles implicate actin in membrane fusion. Here, we investigate the function of the actin coat that surrounds late endosomes in Dictyostelium. Latrunculin treatment leads to aggregation of these endosomes into grape-like clusters and completely blocks progression of endocytic marker. In addition, the cells round up and stop moving. Because this drug treatment perturbs all actin assemblies in the cell simultaneously, we used a novel targeting approach to specifically study the function of the cytoskeleton in one subcellular location. To this end, we constructed a hybrid protein targeting cofilin, an actin depolymerizing protein, to late endosomes. As a consequence, the endosomal compartments lost their actin coats and aggregated, but these cells remained morphologically normal, and the kinetics of endocytic marker trafficking were unaltered. Therefore, the actin coat prevents the clustering of endosomes, which could be one safeguard mechanism precluding their docking and fusion. PMID:14561408

  9. Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer.

    PubMed

    Acuña, Rodrigo; Bignon, Eduardo A; Mancini, Roberta; Lozach, Pierre-Yves; Tischler, Nicole D

    2015-11-01

    The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells. PMID:26310672

  10. Integrin endosomal signalling suppresses anoikis.

    PubMed

    Alanko, Jonna; Mai, Anja; Jacquemet, Guillaume; Schauer, Kristine; Kaukonen, Riina; Saari, Markku; Goud, Bruno; Ivaska, Johanna

    2015-11-01

    Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage independence and metastasis. PMID:26436690

  11. Endosomal Transportation via Ubiquitination

    PubMed Central

    Piper, Robert C.; Lehner, Paul J.

    2011-01-01

    Cell survival, growth, differentiation, and homeostasis all rely on exquisite control over the abundance of particular cell surface membrane proteins. Cell surface proteins must respond appropriately to environmental as well as intracellular cues, often undergoing regulated internalization and lysosomal degradation. In addition, cell surface proteins can sustain damage and must be recognized and removed. A unifying mechanism has now emerged for the trafficking of damaged and downregulated proteins to the lysosome by their attachment to ubiquitin, which serves as a sorting signal for clathrin-mediated internalization and sorting into the lumen of late endosomes. Major questions remain as to how this broad system is governed, how it is adapted to meet the needs of particular cell surface proteins, and whether Ub serves as more than a one-way ticket to the lysosome for degradation. Here we highlight recent insights into these questions and the challenges that remain. PMID:21955996

  12. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion

    PubMed Central

    Liu, Kai; Jian, Youli; Sun, Xiaojuan; Yang, Chengkui; Gao, Zhiyang; Zhang, Zhili; Liu, Xuezhao; Li, Yang; Xu, Jing; Jing, Yudong; Mitani, Shohei; He, Sudan

    2016-01-01

    Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion. PMID:26783301

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

    SciTech Connect

    Tatsumi, Akinori; Shoji, Jun-ya; Kikuma, Takashi; Arioka, Manabu; Kitamoto, Katsuhiko

    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 the 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.

  14. Recycling endosome membrane incorporation into the leading edge regulates lamellipodia formation and macrophage migration.

    PubMed

    Veale, Kelly J; Offenhäuser, Carolin; Whittaker, Shane P; Estrella, Ruby P; Murray, Rachael Z

    2010-10-01

    In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R-SNARE VAMP3 on the recycling endosome partnering with the surface Q-SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3-mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3-mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration. PMID:20604897

  15. Integrin endosomal signalling suppresses anoikis

    PubMed Central

    Alanko, Jonna; Mai, Anja; Jacquemet, Guillaume; Schauer, Kristine; Kaukonen, Riina; Saari, Markku; Goud, Bruno; Ivaska, Johanna

    2016-01-01

    Integrin containing focal adhesions (FAs) transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localises with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 (EEA1) and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage-independence and metastasis. Integrins are heterodimeric cell surface adhesion receptors functioning as integrators of the extra-cellular matrix (ECM) driven cues, the cellular cytoskeleton and the cellular signalling apparatus 1.Upon adhesion, integrins trigger the formation of plasma-membrane proximal large mechanosensing and signal-transmitting protein clusters depicted as “adhesomes” 2, 3. In addition, integrins undergo constant endocytic traffic to facilitate focal adhesion turnover, cell migration, invasion and cytokinesis 4. For other receptor systems it is well established that endocytic membrane traffic regulates bioavailability of cell-surface molecules and therefore the intensity and/or specificity of receptor-initiated signals 5, 6. Although active integrins and their ligands have been detected in endosomes 7–9 and increased integrin recycling to the plasma membrane contributes

  16. Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses

    PubMed Central

    He, Jing; Johnson, Jennifer L.; Monfregola, Jlenia; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Napolitano, Gennaro; Zhang, Jinzhong; Catz, Sergio D.

    2016-01-01

    The molecular mechanisms that regulate late endosomal maturation and function are not completely elucidated, and direct evidence of a calcium sensor is lacking. Here we identify a novel mechanism of late endosomal maturation that involves a new molecular interaction between the tethering factor Munc13-4, syntaxin 7, and VAMP8. Munc13-4 binding to syntaxin 7 was significantly increased by calcium. Colocalization of Munc13-4 and syntaxin 7 at late endosomes was demonstrated by high-resolution and live-cell microscopy. Munc13-4–deficient cells show increased numbers of significantly enlarged late endosomes, a phenotype that was mimicked by the fusion inhibitor chloroquine in wild-type cells and rescued by expression of Munc13-4 but not by a syntaxin 7–binding–deficient mutant. Late endosomes from Munc13-4-KO neutrophils show decreased degradative capacity. Munc13-4–knockout neutrophils show impaired endosomal-initiated, TLR9-dependent signaling and deficient TLR9-specific CD11b up-regulation. Thus we present a novel mechanism of late endosomal maturation and propose that Munc13-4 regulates the late endocytic machinery and late endosomal–associated innate immune cellular functions. PMID:26680738

  17. Recycling endosome tubule morphogenesis from sorting endosomes requires the kinesin motor KIF13A

    PubMed Central

    Delevoye, Cédric; Miserey-Lenkei, Stéphanie; Montagnac, Guillaume; Gilles-Marsens, Floriane; Paul-Gilloteaux, Perrine; Giordano, Francesca; Waharte, François; Marks, Michael S.; Goud, Bruno; Raposo, Graça

    2014-01-01

    Summary Early endosomes consist of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. The tubular transport intermediates that constitute recycling endosomes function in cell polarity, migration and cytokinesis. Endosomal tubulation and fission require both actin and intact microtubules, but while factors that stabilize recycling endosomal tubules have been identified, those required for tubule generation from vacuolar sorting endosomes remain unknown. We show that the microtubule motor KIF13A associates with recycling endosome tubules and controls their morphogenesis. Interfering with KIF13A function impairs the formation of endosomal tubules from sorting endosomes with consequent defects in endosome homeostasis and cargo recycling. Moreover, KIF13A interacts and cooperates with RAB11 to generate endosomal tubules. Our data illustrate how a microtubule motor couples early endosome morphogenesis to its motility and function. PMID:24462287

  18. Methods for analyzing the role of phospholipase A2 enzymes in endosome membrane tubule formation

    PubMed Central

    Kalkofen, Danielle N.; de Figueiredo, Paul; Brown, William J.

    2016-01-01

    Cargo export from mammalian endosomal compartments often involves membrane tubules, into which soluble and membrane-bound cargos are segregated for subsequent intracellular transport. These membrane tubules are highly dynamic and their formation is mediated by a variety of endosome-associated proteins. However, little is known about how these membrane tubules are temporally or spatially regulated, so other tubule-associated proteins are likely to be discovered and analyzed. Therefore, methods to examine the biogenesis and regulation of endosome membrane tubules will prove to be valuable for cell biologists. In this chapter, we describe methods for studying this process using both cell-free, in vitro reconstitution assays, and in vivo image analysis tools. PMID:26360034

  19. Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects.

    PubMed

    Cataldo, Anne M; Mathews, Paul M; Boiteau, Anne Boyer; Hassinger, Linda C; Peterhoff, Corrinne M; Jiang, Ying; Mullaney, Kerry; Neve, Rachael L; Gruenberg, Jean; Nixon, Ralph A

    2008-08-01

    Endocytic dysfunction is an early pathological change in Alzheimer's disease (AD) and Down's syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains. PMID:18535180

  20. Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity.

    PubMed

    van der Kant, Rik; Jonker, Caspar T H; Wijdeven, Ruud H; Bakker, Jeroen; Janssen, Lennert; Klumperman, Judith; Neefjes, Jacques

    2015-12-18

    Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway. PMID:26463206

  1. γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways.

    PubMed

    Inoue, Hiroki; Matsuzaki, Yuka; Tanaka, Ayaka; Hosoi, Kaori; Ichimura, Kaoru; Arasaki, Kohei; Wakana, Yuichi; Asano, Kenichi; Tanaka, Masato; Okuzaki, Daisuke; Yamamoto, Akitsugu; Tani, Katsuko; Tagaya, Mitsuo

    2015-08-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways. PMID:26101353

  2. Endocytosis and Endosomal Trafficking in Plants.

    PubMed

    Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

    2016-04-29

    Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants. PMID:27128466

  3. Fusion

    NASA Astrophysics Data System (ADS)

    Herman, Robin

    1990-10-01

    The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

  4. Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L.

    PubMed

    van der Kant, Rik; Fish, Alexander; Janssen, Lennert; Janssen, Hans; Krom, Sabine; Ho, Nataschja; Brummelkamp, Thijn; Carette, Jan; Rocha, Nuno; Neefjes, Jacques

    2013-08-01

    Late endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire cargo from early endosomes, phagosomes and autophagosome. Defects in lysosomal dynamics cause severe neurodegenerative and developmental diseases, such as Niemann-Pick type C disease and ARC syndrome, yet little is known about the regulation of late endosomal fusion in a mammalian system. Mammalian endosomes destined for fusion need to be transported over very long distances before they tether to initiate contact. Here, we describe that lysosomal tethering and transport are combined processes co-regulated by one multi-protein complex: RAB7-RILP-ORP1L. We show that RILP directly and concomitantly binds the tethering HOPS complex and the p150(Glued) subunit of the dynein motor. ORP1L then functions as a cholesterol-sensing switch controlling RILP-HOPS-p150(Glued) interactions. We show that RILP and ORP1L control Ebola virus infection, a process dependent on late endosomal fusion. By combining recruitment and regulation of both the dynein motor and HOPS complex into a single multiprotein complex, the RAB7-RILP-ORP1L complex efficiently couples and regulates the timing of microtubule minus-end transport and fusion, two major events in endosomal biology. PMID:23729732

  5. Vicenistatin induces early endosome-derived vacuole formation in mammalian cells.

    PubMed

    Nishiyama, Yuko; Ohmichi, Tomohiro; Kazami, Sayaka; Iwasaki, Hiroki; Mano, Kousuke; Nagumo, Yoko; Kudo, Fumitaka; Ichikawa, Sosaku; Iwabuchi, Yoshiharu; Kanoh, Naoki; Eguchi, Tadashi; Osada, Hiroyuki; Usui, Takeo

    2016-05-01

    Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity. PMID:27104762

  6. Endosomal vesicles as vehicles for viral genomes

    PubMed Central

    Nour, Adel M.; Modis, Yorgo

    2014-01-01

    The endocytic pathway is the principal cell entry pathway for large cargo and pathogens. Among the wide variety of specialized lipid structures within endosomes, the intraluminal vesicles formed in early endosomes and transferred to late endosomal compartments are emerging as critical effectors of viral infection and immune recognition. Various viruses deliver their genomes into these intraluminal vesicles, which serve as vehicles to transport the genome to the nuclear periphery for replication. When secreted as exosomes, intraluminal vesicles containing viral genomes can infect permissive cells, or activate immune responses in myeloid cells. We therefore propose that endosomal intraluminal vesicles and exosomes are key effectors of viral pathogenesis. PMID:24746011

  7. Convergence of Non-clathrin- and Clathrin-derived Endosomes Involves Arf6 Inactivation and Changes in Phosphoinositides

    PubMed Central

    Naslavsky, Naava; Weigert, Roberto; Donaldson, Julie G.

    2003-01-01

    The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor α subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the “classical” early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP2-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways. PMID:12589044

  8. Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

    SciTech Connect

    Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S. . E-mail: lmayorga@fcm.uncu.edu.ar

    2005-04-01

    The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

  9. Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

    PubMed Central

    Schluter, Cayetana; Lam, Karen K.Y.; Brumm, Jochen; Wu, Bella W.; Saunders, Matthew; Stevens, Tom H.

    2008-01-01

    Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. PMID:18216282

  10. Rab GTPase regulation of retromer-mediated cargo export during endosome maturation

    PubMed Central

    Liu, Ting-Ting; Gomez, Timothy S.; Sackey, Bridget K.; Billadeau, Daniel D.; Burd, Christopher G.

    2012-01-01

    The retromer complex, composed of sorting nexin subunits and a Vps26/Vps29/Vps35 trimer, mediates sorting of retrograde cargo from the endosome to the trans-Golgi network. The retromer trimer subcomplex is an effector of Rab7 (Ypt7 in yeast). Whereas endosome targeting of human retromer has been shown to require Rab7-GTP, targeting of yeast retromer to the endosome is independent of Ypt7-GTP and requires the Vps5 and Vps17 retromer sorting nexin subunits. An evolutionarily conserved amino acid segment within Vps35 is required for Ypt7/Rab7 recognition in vivo by both yeast and human retromer, establishing that Rab recognition is a conserved feature of this subunit. Recognition of Ypt7 by retromer is required for its function in retrograde sorting, and in yeast cells lacking the guanine nucleotide exchange factor for Ypt7, retrograde cargo accumulates in endosomes that are decorated with retromer, revealing an additional role for Rab recognition at the cargo export stage of the retromer functional cycle. In addition, yeast retromer trimer antagonizes Ypt7-regulated organelle tethering and fusion of endosomes/vacuoles via recognition of Ypt7. Thus retromer has dual roles in retrograde cargo export and in controlling the fusion dynamics of the late endovacuolar system. PMID:22593205

  11. Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.

    PubMed

    Tornieri, Karine; Zlatic, Stephanie A; Mullin, Ariana P; Werner, Erica; Harrison, Robert; L'hernault, Steven W; Faundez, Victor

    2013-12-20

    Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion. PMID:23918659

  12. Ferlins Show Tissue-Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans-Golgi/Recycling Ferlins.

    PubMed

    Redpath, Gregory M I; Sophocleous, Reece A; Turnbull, Lynne; Whitchurch, Cynthia B; Cooper, Sandra T

    2016-03-01

    Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+) -regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+) -dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+) -triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations. PMID:26707827

  13. Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels.

    PubMed

    Mazuel, François; Espinosa, Ana; Luciani, Nathalie; Reffay, Myriam; Le Borgne, Rémi; Motte, Laurence; Desboeufs, Karine; Michel, Aude; Pellegrino, Teresa; Lalatonne, Yoann; Wilhelm, Claire

    2016-08-23

    Quantitative studies of the long-term fate of iron oxide nanoparticles inside cells, a prerequisite for regenerative medicine applications, are hampered by the lack of suitable biological tissue models and analytical methods. Here, we propose stem-cell spheroids as a tissue model to track intracellular magnetic nanoparticle transformations during long-term tissue maturation. We show that global spheroid magnetism can serve as a fingerprint of the degradation process, and we evidence a near-complete nanoparticle degradation over a month of tissue maturation, as confirmed by electron microscopy. Remarkably, the same massive degradation was measured at the endosome level by single-endosome nanomagnetophoretic tracking in cell-free endosomal extract. Interestingly, this spectacular nanoparticle breakdown barely affected iron homeostasis: only the genes coding for ferritin light chain (iron loading) and ferroportin (iron export) were up-regulated 2-fold by the degradation process. Besides, the magnetic and tissular tools developed here allow screening of the biostability of magnetic nanomaterials, as demonstrated with iron oxide nanocubes and nanodimers. Hence, stem-cell spheroids and purified endosomes are suitable models needed to monitor nanoparticle degradation in conjunction with magnetic, chemical, and biological characterizations at the cellular scale, quantitatively, in the long term, in situ, and in real time. PMID:27419260

  14. Actin-dependent propulsion of endosomes and lysosomes byrecruitment of n-wasp

    SciTech Connect

    Taunton J; Rowning BA; Coughlin ML; Wu M; Moon RT; Mitchison TJ; Larabell CA

    2000-02-07

    We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation,dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA),and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP, The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.

  15. ER-endosome contact sites in endosome positioning and protrusion outgrowth.

    PubMed

    Raiborg, Camilla; Wenzel, Eva M; Pedersen, Nina M; Stenmark, Harald

    2016-04-15

    The endoplasmic reticulum (ER) makes abundant contacts with endosomes, and the numbers of contact sites increase as endosomes mature. It is already clear that such contact sites have diverse compositions and functions, but in this mini-review we will focus on two particular types of ER-endosome contact sites that regulate endosome positioning. Formation of ER-endosome contact sites that contain the cholesterol-binding protein oxysterol-binding protein-related protein 1L (ORP1L) is coordinated with loss of the minus-end-directed microtubule motor Dynein from endosomes. Conversely, formation of ER-endosome contact sites that contain the Kinesin-1-binding protein Protrudin results in transfer of the plus-end-directed microtubule motor Kinesin-1 from ER to endosomes. We discuss the possibility that formation of these two types of contact sites is coordinated as a 'gear-shift' mechanism for endosome motility, and we review evidence that Kinesin-1-mediated motility of late endosomes (LEs) to the cell periphery promotes outgrowth of neurites and other protrusions. PMID:27068952

  16. Lysobisphosphatidic acid controls endosomal cholesterol levels.

    PubMed

    Chevallier, Julien; Chamoun, Zeina; Jiang, Guowei; Prestwich, Glenn; Sakai, Naomi; Matile, Stefan; Parton, Robert G; Gruenberg, Jean

    2008-10-10

    Most cell types acquire cholesterol by endocytosis of circulating low density lipoprotein, but little is known about the mechanisms of intra-endosomal cholesterol transport and about the primary cause of its aberrant accumulation in the cholesterol storage disorder Niemann-Pick type C (NPC). Here we report that lysobisphosphatidic acid (LBPA), an unconventional phospholipid that is only detected in late endosomes, regulates endosomal cholesterol levels under the control of Alix/AlP1, which is an LBPA-interacting protein involved in sorting into multivesicular endosomes. We find that Alix down-expression decreases both LBPA levels and the lumenal vesicle content of late endosomes. Cellular cholesterol levels are also decreased, presumably because the storage capacity of endosomes is affected and thus cholesterol clearance accelerated. Both lumenal membranes and cholesterol can be restored in Alix knockdown cells by exogenously added LBPA. Conversely, we also find that LBPA becomes limiting upon pathological cholesterol accumulation in NPC cells, because the addition of exogenous LBPA, but not of LBPA isoforms or analogues, partially reverts the NPC phenotype. We conclude that LBPA controls the cholesterol capacity of endosomes. PMID:18644787

  17. Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

    PubMed Central

    Barbero, Pierre; Bittova, Lenka; Pfeffer, Suzanne R.

    2002-01-01

    Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion. PMID:11827983

  18. Lipid Sorting and Multivesicular Endosome Biogenesis

    PubMed Central

    Bissig, Christin

    2013-01-01

    Intracellular organelles, including endosomes, show differences not only in protein but also in lipid composition. It is becoming clear from the work of many laboratories that the mechanisms necessary to achieve such lipid segregation can operate at very different levels, including the membrane biophysical properties, the interactions with other lipids and proteins, and the turnover rates or distribution of metabolic enzymes. In turn, lipids can directly influence the organelle membrane properties by changing biophysical parameters and by recruiting partner effector proteins involved in protein sorting and membrane dynamics. In this review, we will discuss how lipids are sorted in endosomal membranes and how they impact on endosome functions. PMID:24086044

  19. Modulation of Endosomal Escape of IRQ-PEGylated Nano-carrier

    NASA Astrophysics Data System (ADS)

    Mudhakir, Diky; Akita, Hidetaka; Harashima, Hideyoshi

    2011-12-01

    The novel IRQ peptide is one of cell penetrating peptides (CPPs) that has ability to induce endosomal escape. It has been demonstrated that IRQ ligand had ability to facilitate an escape of liposomes encapsulating siRNA from the endosomes presumably by fusion-independent mechanism [1,2]. In the present study, we attempted to modulate the intracellular trafficking of IRQ-modified nano-carrier in term of escaping process by changing the lipid composition. The peptide was attached to the terminal end of maleimide group of polyethylene glycol-modified liposomes (IRQ-PEG-Lip). The liposomes were composed of DOTAP, DOPE and cholesterol and it was labeled by water soluble sulpho-rhodamine B (Sr-B). The escape of PEG-coated liposomes was then observed by confocal laser scanning microscope after the endosomes were stained with Lysosensor. The results exhibited that IRQ-PEG-Lip was escaped from endosomal compartment after 1 h transfection when 40% of DOPE was incorporated into the nanostructure comparing to that of PEG-Lip. These results are consistent with the previous results that the IRQ facilitates endosomal escape via independent-mechanism. However, IRQ-PEG-Lip were then completely co-localized in the acidic compartment when density of DOPE was reduced approximately 20%. These results indicated that the utilizing of DOPE is important for the escape process even in the presence of hydrophilic PEG polymer. In conclusion, the regulation of endosomal escape ability of the PEGylated-IRQ nano-carrier was induced by fusion-independent manner as well as fusogenic lipid.

  20. Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9

    PubMed Central

    Shideler, Tess; Nickerson, Daniel P.; Merz, Alexey J.; Odorizzi, Greg

    2015-01-01

    Vps9 and Muk1 are guanine nucleotide exchange factors (GEFs) in Saccharomyces cerevisiae that regulate membrane trafficking in the endolysosomal pathway by activating Rab5 GTPases. We show that Vps9 is the primary Rab5 GEF required for biogenesis of late endosomal multivesicular bodies (MVBs). However, only Vps9 (but not Muk1) is required for the formation of aberrant class E compartments that arise upon dysfunction of endosomal sorting complexes required for transport (ESCRTs). ESCRT dysfunction causes ubiquitinated transmembrane proteins to accumulate at endosomes, and we demonstrate that endosomal recruitment of Vps9 is promoted by its ubiquitin-binding CUE domain. Muk1 lacks ubiquitin-binding motifs, but its fusion to the Vps9 CUE domain allows Muk1 to rescue endosome morphology, cargo trafficking, and cellular stress-tolerance phenotypes that result from loss of Vps9 function. These results indicate that ubiquitin binding by the CUE domain promotes Vps9 function in endolysosomal membrane trafficking via promotion of localization. PMID:25673804

  1. Linkage of azurophil granule secretion in neutrophils to chloride ion transport and endosomal transcytosis.

    PubMed Central

    Fittschen, C; Henson, P M

    1994-01-01

    Neutrophils contain at least two types of secretory granules. The present work links the secretion of the (lysosomal type) azurophil granules, but not that of specific granules, to endosomal transport mechanisms. (a) Selective stimulation of azurophil granule secretion by the Na-ionophore Monensin, or nonselective stimulation by FMLP after cytochalasin B pretreatment elicited marked pinocytic activity in parallel with azurophil granule release, whereas FMLP alone, selective for specific granules, elicited little fluid pinocytosis. (b) Pinosomes thus formed fused with azurophil granules, suggesting that exocytosis of azurophil granules might occur via endosomal organelles. This hypothesis was tested by determining the effect on the endosomal pathway(s) of two treatments that selectively prevent the release of azurophil granule contents without interfering with specific granule secretion, namely replacement of Cl- with gluconate- or the addition of zinc. Replacement of Cl- was found to impair the pinocytosis process itself, whereas ZnSO4 appeared to prevent the fusion between endosomes and azurophil granules. These data support the concept that the (lysosomal type) azurophil granules, but not the specific granules, are secreted through the endosomal pathway. Images PMID:8282794

  2. Studying lipids involved in the endosomal pathway.

    PubMed

    Bissig, Christin; Johnson, Shem; Gruenberg, Jean

    2012-01-01

    Endosomes along the degradation pathway exhibit a multivesicular appearance and differ in their lipid compositions. Association of proteins to specific membrane lipids and presumably also lipid-lipid interactions contribute to the formation of functional membrane platforms that regulate endosome biogenesis and function. This chapter provides a brief review of the functions of endosomal lipids in the degradation pathway, a discussion of techniques that allow studying lipid-based mechanisms and a selection of step-by-step protocols for in vivo and in vitro methods commonly used to study lipid roles in endocytosis. The techniques described here have been used to elucidate the function of the late endosomal lipid lysobisphosphatidic acid and allow the monitoring of lipid distribution, levels and dynamics, as well as the characterization of lipid-binding partners. PMID:22325596

  3. Lipid compartmentalization in the endosome system.

    PubMed

    Hullin-Matsuda, Françoise; Taguchi, Tomohiko; Greimel, Peter; Kobayashi, Toshihide

    2014-07-01

    Lipids play an essential role in the structure of the endosomal membranes as well as in their dynamic rearrangement during the transport of internalized cargoes along the endocytic pathway. In this review, we discuss the function of endosomal lipids mainly in mammalian cells, focusing on two well-known components of the lipid rafts, sphingomyelin and cholesterol, as well as on three anionic phospholipids, phosphatidylserine, polyphosphoinositides and the atypical phospholipid, bis(monoacylglycero)phosphate/lysobisphosphatidic acid. We detail the structure, metabolism, distribution and role of these lipids in the endosome system as well as their importance in pathological conditions where modification of the endosomal membrane flow can lead to various diseases such as lipid-storage diseases, myopathies and neuropathies. PMID:24747366

  4. Viral membrane fusion

    PubMed Central

    Harrison, Stephen C.

    2015-01-01

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. PMID:25866377

  5. ER contact sites direct late endosome transport.

    PubMed

    Wijdeven, Ruud H; Jongsma, Marlieke L M; Neefjes, Jacques; Berlin, Ilana

    2015-12-01

    Endosomes shuttle select cargoes between cellular compartments and, in doing so, maintain intracellular homeostasis and enable interactions with the extracellular space. Directionality of endosomal transport critically impinges on cargo fate, as retrograde (microtubule minus-end directed) traffic delivers vesicle contents to the lysosome for proteolysis, while the opposing anterograde (plus-end directed) movement promotes recycling and secretion. Intriguingly, the endoplasmic reticulum (ER) is emerging as a key player in spatiotemporal control of late endosome and lysosome transport, through the establishment of physical contacts with these organelles. Earlier studies have described how minus-end-directed motor proteins become discharged from vesicles engaged at such contact sites. Now, Raiborg et al. implicate ER-mediated interactions, induced by protrudin, in loading plus-end-directed motor kinesin-1 onto endosomes, thereby stimulating their transport toward the cell's periphery. In this review, we recast the prevailing concepts on bidirectional late endosome transport and discuss the emerging paradigm of inter-compartmental regulation from the ER-endosome interface viewpoint. PMID:26440125

  6. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    SciTech Connect

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  7. The Legionella pneumophila Effector Protein, LegC7, Alters Yeast Endosomal Trafficking

    PubMed Central

    O’Brien, Kevin M.; Lindsay, Elizabeth L.; Starai, Vincent J.

    2015-01-01

    The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome. PMID:25643265

  8. Regulation of Early Endosomal Entry by the Drosophila Tumor Suppressors Rabenosyn and Vps45

    PubMed Central

    Morrison, Holly A.; Dionne, Heather; Rusten, Tor Erik; Brech, Andreas; Fisher, William W.; Pfeiffer, Barret D.; Celniker, Susan E.; Stenmark, Harald

    2008-01-01

    The small GTPase Rab5 has emerged as an important regulator of animal development, and it is essential for endocytic trafficking. However, the mechanisms that link Rab5 activation to cargo entry into early endosomes remain unclear. We show here that Drosophila Rabenosyn (Rbsn) is a Rab5 effector that bridges an interaction between Rab5 and the Sec1/Munc18-family protein Vps45, and we further identify the syntaxin Avalanche (Avl) as a target for Vps45 activity. Rbsn and Vps45, like Avl and Rab5, are specifically localized to early endosomes and are required for endocytosis. Ultrastructural analysis of rbsn, Vps45, avl, and Rab5 null mutant cells, which show identical defects, demonstrates that all four proteins are required for vesicle fusion to form early endosomes. These defects lead to loss of epithelial polarity in mutant tissues, which overproliferate to form neoplastic tumors. This work represents the first characterization of a Rab5 effector as a tumor suppressor, and it provides in vivo evidence for a Rbsn–Vps45 complex on early endosomes that links Rab5 to the SNARE fusion machinery. PMID:18685079

  9. Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes

    PubMed Central

    Tornieri, Karine; Zlatic, Stephanie A.; Mullin, Ariana P.; Werner, Erica; Harrison, Robert; L'Hernault, Steven W.; Faundez, Victor

    2013-01-01

    Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome–lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion. PMID:23918659

  10. Structural and functional analysis of endosomal compartments in epithelial cells.

    PubMed

    Bay, Andres E Perez; Schreiner, Ryan; Rodriguez-Boulan, Enrique

    2015-01-01

    Epithelial cells display segregated early endosomal compartments, termed apical sorting endosomes and basolateral sorting endosomes, that converge into a common late endosomal-lysosomal degradative compartment and common recycling endosomes (CREs). Unlike recycling endosomes of nonpolarized cells, CREs have the ability to sort apical and basolateral plasma membrane proteins into distinct apical and basolateral recycling routes, utilizing mechanisms similar to those employed by the trans Golgi network in the biosynthetic pathway. The apical recycling route includes an additional compartment, the apical recycling endosomes, consisting of multiple vesicles bundled around the basal body. Recent evidence indicates that, in addition to their role in internalizing ligands and recycling their receptors back to the cell surface, endosomal compartments act as intermediate stations in the biosynthetic routes to the plasma membrane. Here we review methods employed by our laboratory to study the endosomal compartments of epithelial cells and their multiple trafficking roles. PMID:26360040

  11. Endotoxin Priming of Neutrophils Requires Endocytosis and NADPH Oxidase-dependent Endosomal Reactive Oxygen Species*

    PubMed Central

    Lamb, Fred S.; Hook, Jessica S.; Hilkin, Brieanna M.; Huber, Jody N.; Volk, A. Paige Davis; Moreland, Jessica G.

    2012-01-01

    NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91phox, p40phox, p67phox, and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin. PMID:22235113

  12. Cell-free DNA: Comparison of Technologies.

    PubMed

    Dar, Pe'er; Shani, Hagit; Evans, Mark I

    2016-06-01

    Cell-free fetal DNA screening for Down syndrome has gained rapid acceptance over the past few years with increasing market penetration. Three main laboratory methodologies are currently used: a massive parallel shotgun sequencing (MPSS), a targeted massive parallel sequencing (t-MPS) and a single nucleotide polymorphism (SNP) based approach. Although each of these technologies has its own advantages and disadvantages, the performance of all was shown to be comparable and superior to that of traditional first-trimester screening for the detection of trisomy 21 in a routine prenatal population. Differences in performance were predominantly shown for chromosomal anomalies other than trisomy 21. Understanding the limitations and benefits of each technology is essential for proper counseling to patients. These technologies, as well as few investigational technologies described in this review, carry a great potential beyond screening for the common aneuploidies. PMID:27235906

  13. Signal processing by the endosomal system.

    PubMed

    Villaseñor, Roberto; Kalaidzidis, Yannis; Zerial, Marino

    2016-04-01

    Cells need to decode chemical or physical signals from their environment in order to make decisions on their fate. In the case of signalling receptors, ligand binding triggers a cascade of chemical reactions but also the internalization of the activated receptors in the endocytic pathway. Here, we highlight recent studies revealing a new role of the endosomal network in signal processing. The diversity of entry pathways and endosomal compartments is exploited to regulate the kinetics of receptor trafficking, and interactions with specific signalling adaptors and effectors. By governing the spatio-temporal distribution of signalling molecules, the endosomal system functions analogously to a digital-analogue computer that regulates the specificity and robustness of the signalling response. PMID:26921695

  14. Hepatic Endosome Protein Profiling in Apolipoprotein E Deficient Mice Expressing Apolipoprotein B48 but not B100

    PubMed Central

    Chen, AnShu; Guo, ZhongMao; Zhou, LiChun; Yang, Hong

    2011-01-01

    Liver cells absorb apolipoprotein (Apo) B48-carrying lipoproteins in ApoE’s absence, albeit not as efficiently as the ApoE-mediated process. Our objective was to identify differentially expressed hepatic endosome proteins in mice expressing ApoB48 but lacking ApoE and ApoB100 expression (ApoE−/−/B48/48). We purified early and late endosomes from ApoE−/−/B48/48 and wild-type mouse’s livers. In ApoE−/−/B48/48 mouse’s hepatic endosomes, proteomic analysis revealed elevated protein levels of major urinary protein 6 (MUP), calreticulin, protein disulfide isomerases (PDI) A1, and A3. These proteins are capable of interacting with lipids/lipoproteins and triggering receptor-mediated endocytosis. In addition, hepatic endosomes from ApoE−/− /B48/48 mice exhibited significantly reduced protein levels of haptoglobin, hemopexin, late endosome/lysosome interacting protein, cell division control protein 2 homolog, γ-soluble Nethylmaleimide- sensitive factor attachment protein, vacuolar ATP synthase catalytic subunit A1, dipeptidyl peptidases II, cathepsin B, D, H, and Z. These proteins participate in plasma heme clearance, receptor-mediated signaling, membrane fusion, endosomal/lysosomal acidification, and protein degradation. The significance of increasing endosomal MUP, calreticulin and PDIs in ApoE−/−/B48/48 mouse liver cells is not clear; however, reducing endosomal/ lysosomal membrane proteins and hydrolases might be, at least partially, responsible for the retarded clearance of plasma ApoB-carrying lipoproteins in ApoE−/−/B48/48 mice. PMID:21837265

  15. Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy

    PubMed Central

    Lim, Ji Hyae; Kim, Min Hyoung; Han, You Jung; Lee, Da Eun; Park, So Yeon; Han, Jung Yeol; Kim, Moon Young; Ryu, Hyun Mee

    2013-01-01

    Background Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA) with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy. Methodology/Principal Findings A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC) curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both). The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001) than in SA women with normal karyotype (r = 0.465, P = 0.002) and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037). The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852–0.945) and 0.939 (95% CI, 0.903–0.975), respectively. Conclusions Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA with fetal

  16. Fluorescent In Situ Folding Control for Rapid Optimization of Cell-Free Membrane Protein Synthesis

    PubMed Central

    Müller-Lucks, Annika; Bock, Sinja; Wu, Binghua; Beitz, Eric

    2012-01-01

    Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality. PMID:22848743

  17. Translation in cell-free systems

    SciTech Connect

    Jagus, R.

    1987-01-01

    The simplest, unambiguous identification of a particular mRNA is the identification of its protein product. This can be established by translation of the mRNA of interest in a cell-free protein-synthesizing system. Messenger RNA protein product identification is important in the isolation of a particular mRNA species for cDNA cloning and in the identification of positive cDNA clones. The two high-activity translation systems in common use are those prepared from rabbit reticulocytes and from wheat germ. Both systems are easy to prepare, and both are available commercially. Each has advantages and disadvantages over the other and a choice between the two will depend on the type of mRNAs to be translated, the prejudices of experience, and availability. The main disadvantage of the reticulocyte system is that it requires removal of endogenous mRNA. However, this is a relatively simple procedure. The wheat germ system does not require removal of endogenous mRNA and may translate weakly initiating mRNAs more efficiently. However, ionic optima for translation in the wheat germ system are more sensitive to the nature and concentration of mRNA and may need to be determined for each template. The biggest problem with the use of the wheat germ system is its tendency to produce incomplete translation products due to premature termination.

  18. Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium

    PubMed Central

    Wavre-Shapton, Silène T.; Meschede, Ingrid P.; Seabra, Miguel C.; Futter, Clare E.

    2014-01-01

    ABSTRACT Defects in phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) are associated with aging and retinal disease. The daily burst of rod outer segment (ROS) phagocytosis by the RPE provides a unique opportunity to analyse phagosome processing in vivo. In mouse retinae, phagosomes containing stacked rhodopsin-rich discs were identified by immuno-electron microscopy. Early apical phagosomes stained with antibodies against both cytoplasmic and intradiscal domains of rhodopsin. During phagosome maturation, a remarkably synchronised loss of the cytoplasmic epitope coincided with movement to the cell body and preceded phagosome–lysosome fusion and disc degradation. Loss of the intradiscal rhodopsin epitope and disc digestion occurred upon fusion with cathepsin-D-positive lysosomes. The same sequential stages of phagosome maturation were identified in cultured RPE and macrophages challenged with isolated POS. Loss of the cytoplasmic rhodopsin epitope was insensitive to pH but sensitive to protease inhibition and coincided with the interaction of phagosomes with endosomes. Thus, during pre-lysosomal maturation of ROS-containing phagosomes, limited rhodopsin processing occurs upon interaction with endosomes. This potentially provides a sensitive readout of phagosome–endosome interactions that is applicable to multiple phagocytes. PMID:25074813

  19. Plant RNA processing: soybean pre-mRNA in a pea cell-free extract

    SciTech Connect

    Schuler, M.A.; Hanley, B.A.

    1987-05-01

    Using a pea cell-free extract they have demonstrated the splicing of an SP6 fusion transcript containing an intron derived from the soybean seed storage protein ..beta..-subunit gene. Intron 115 from the conglycinin gene was cloned into a SP6 vector and transcribed using standard recombinant DNA techniques. Incubation of radioactively labeled fusion transcripts in the cell-free system produced a number of products which were identified by primer extension and S1 nuclease analysis. All the products are linear RNA molecules. Lariat intermediates, similar to those found in the yeast and HeLa cell RNA processing systems, have not been detected. The linear RNA products detected in their plant in vitro processing system have various portions of the intron removed which suggests that alternative splice sites are used in processing of this plant intron due to activation of cryptic splice sites or creation of splice sites in the fusion construction. The kinetics of the reactions and parameters of the extract are similar to those determined for the HeLa cell system. Sucrose gradient analysis has demonstrated that the plant RNA products sedimented in a 30S particle, similar in size to that found for the spliceosome of the HeLa cell system.

  20. Saposin C-LBPA interaction in late-endosomes/lysosomes.

    PubMed

    Chu, Zhengtao; Witte, David P; Qi, Xiaoyang

    2005-02-15

    Acidic phospholipids and saposins associations are involved in the degradation process of glycosphingolipids/sphingolipids in late endosomes/lysosomes. In this report, we showed the colocalization of saposin C and lysobisphosphatidic acid (LBPA) in human fibroblasts by using cytoimmunofluorescence analysis. This colocalization pattern was not seen with other saposins. Large numbers of saposins A, B, and D illustrated the staining patterns that differ from LBPA. In addition, ingested anti-LBPA antibody altered the location of saposin C in human wild-type fibroblasts. In vitro assays demonstrated that saposin C at nM concentrations induced membrane fusion of LBPA containing phospholipid vesicles. Under the same condition, other saposins had no fusion induction on these vesicles. These results suggested a specific interaction between saposin C and LBPA. Total saposin-deficient fibroblasts showed a massive accumulation of multivesicular bodies (MVBs) by electron microscopic analysis. No significant increase of MVBs was found in saposins A and B deficient cells. Interestingly, the accumulated MVBs were significantly reduced by loading saposin C alone into the total saposin-deficient cells. Therefore, we propose that saposin C-LBPA interaction plays a role in the regulation of MVB formation in cells. PMID:15652344

  1. Sip1, a Conserved AP-1 Accessory Protein, Is Important for Golgi/Endosome Trafficking in Fission Yeast

    PubMed Central

    Yu, Yang; Kita, Ayako; Udo, Masako; Katayama, Yuta; Shintani, Mami; Park, Kwihwa; Hagihara, Kanako; Umeda, Nanae; Sugiura, Reiko

    2012-01-01

    We had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4+/sip1+, which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1-deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl− and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking. PMID:23028933

  2. Nuclear envelope-associated endosomes deliver surface proteins to the nucleus.

    PubMed

    Chaumet, Alexandre; Wright, Graham D; Seet, Sze Hwee; Tham, Keit Min; Gounko, Natalia V; Bard, Frederic

    2015-01-01

    Endocytosis directs molecular cargo along three main routes: recycling to the cell surface, transport to the Golgi apparatus or degradation in endolysosomes. Pseudomonas exotoxin A (PE) is a bacterial protein that typically traffics to the Golgi and then the endoplasmic reticulum before translocating to the cytosol. Here we show that a substantial fraction of internalized PE is also located in nuclear envelope-associated endosomes (NAE), which display limited mobility, exhibit a propensity to undergo fusion and readily discharge their contents into the nuclear envelope. Electron microscopy and protein trapping in the nucleus indicate that NAE mediate PE transfer into the nucleoplasm. RNAi screening further revealed that NAE-mediated transfer depends on the nuclear envelope proteins SUN1 and SUN2, as well as the Sec61 translocon complex. These data reveal a novel endosomal route from the cell surface to the nucleoplasm that facilitates the accumulation of extracellular and cell surface proteins in the nucleus. PMID:26356418

  3. Neurotrophin signaling endosomes: biogenesis, regulation, and functions.

    PubMed

    Yamashita, Naoya; Kuruvilla, Rejji

    2016-08-01

    In the nervous system, communication between neurons and their post-synaptic target cells is critical for the formation, refinement and maintenance of functional neuronal connections. Diffusible signals secreted by target tissues, exemplified by the family of neurotrophins, impinge on nerve terminals to influence diverse developmental events including neuronal survival and axonal growth. Key mechanisms of action of target-derived neurotrophins include the cell biological processes of endocytosis and retrograde trafficking of their Trk receptors from growth cones to cell bodies. In this review, we summarize the molecular mechanisms underlying this endosome-mediated signaling, focusing on the instructive role of neurotrophin signaling itself in directing its own trafficking. Recent studies have linked impaired neurotrophin trafficking to neurodevelopmental disorders, highlighting the relevance of neurotrophin endosomes in human health. PMID:27327126

  4. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    PubMed Central

    2011-01-01

    Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments. PMID:22145853

  5. Probing cell-free gene expression noise in femtoliter volumes

    SciTech Connect

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta R; Collier, Pat; Simpson, Michael L

    2013-01-01

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell relevant 20 fL volumes (between the volumes of E. coli and S. cerevisiae), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely at this volume, and we analyze gene expression noise. Noise analysis reveals signatures of translational bursting while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  6. Viral membrane fusion

    SciTech Connect

    Harrison, Stephen C.

    2015-05-15

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

  7. Plant endosomal NHX antiporters: Activity and function.

    PubMed

    Qiu, Quan-Sheng

    2016-05-01

    The Arabidopsis NHX antiporter family contains eight members that are divided into three subclasses: vacuolar, endosomal, and plasma membrane. While the plasma membrane and vacuolar NHXs have been studied extensively, the activity and function of the endosomal NHXs are beginning to be discovered. AtNHX5 and AtNHX6 are endosomal Na(+),K(+)/H(+) antiporters that share high sequence similarity. They are localized in the Golgi, trans-Golgi network (TGN), and prevacuolear compartment (PVC). Studies have shown that AtNHX5 and AtNHX6 mediate K(+) and Na(+) transport, and regulate cellular pH homeostasis. Sequence alignment has found that AtNHX5 and AtNHX6 contain four conserved acidic amino acid residues in transmembrane domains that align with yeast and human NHXs. Three of these conserved acidic residues are critical for K(+) transport and seedling growth in Arabidopsis. Moreover, studies have shown that the precursors of the seed storage proteins are missorted to the apoplast in the nhx5 nhx6 knockout mutant, suggesting that AtNHX5 and AtNHX6 regulate protein transport into the vacuole. Further analysis found that AtNHX5 and AtNHX6 regulated the binding of VSR to its cargoes. Taken together, AtNHX5 and AtNHX6 play an important role in cellular ion and pH homeostasis, and are essential for protein transport into the vacuole. PMID:26890367

  8. Altered Endosome Biogenesis in Prostate Cancer has Biomarker Potential

    PubMed Central

    Johnson, Ian R D; Parkinson-Lawrence, Emma J; Shandala, Tetyana; Weigert, Roberto; Butler, Lisa M; Brooks, Doug A

    2016-01-01

    Prostate cancer is the second most common form of cancer in males, affecting one in eight men by the time they reach the age of 70. Current diagnostic tests for prostate cancer have significant problems with both false negatives and false positives, necessitating the search for new molecular markers. A recent investigation of endosomal and lysosomal proteins revealed that the critical process of endosomal biogenesis might be altered in prostate cancer. Here, a panel of endosomal markers was evaluated in prostate cancer and non-malignant cells and a significant increase in gene and protein expression was found for early, but not late endosomal proteins. There was also a differential distribution of early endosomes, and altered endosomal traffic and signalling of the transferrin receptors (TFRC and TFR2) in prostate cancer cells. These findings support the concept that endosome biogenesis and function is altered in prostate cancer. Microarray analysis of a clinical cohort confirmed the altered endosomal gene expression observed in cultured prostate cancer cells. Furthermore, in prostate cancer patient tissue specimens, the early endosomal marker and adaptor protein APPL1 showed consistently altered basement membrane histology in the vicinity of tumours and concentrated staining within tumour masses. These novel observations on altered early endosome biogenesis provide a new avenue for prostate cancer biomarker investigation and suggest new methods for the early diagnosis and accurate prognosis of prostate cancer. PMID:25080433

  9. Macroendocytosis and endosome processing in snake motor boutons.

    PubMed

    Teng, Haibing; Lin, Michael Y; Wilkinson, Robert S

    2007-07-01

    We have examined the processing of endosomes formed by macroendocytosis (ME), or bulk membrane retrieval, in active motor terminal boutons at the snake nerve-muscle synapse. Endocytic probes were imaged at light (FM1-43) and electron (horseradish peroxidase (HRP)) levels over stimulus frequencies representing low, intermediate and high levels of use. Endosomes formed rapidly (1-2 s) at all frequencies, concomitant with clathrin-mediated vesicular endocytosis (CME). Endosomes dissipated rapidly into vesicles (approximately 10 s). The dissipation rate was not influenced by activity. Many endosomes split into clusters of 2-20 smaller endosomes of varying size. Vesicles budded from these smaller endosomes, from large endosomes that had not undergone fission into smaller ones, and from precursor membrane infoldings that had not yet internalized. In snake, exocytosed vesicular membrane is not competent for reuse until after a delay (> 3 min). We found that time required for endosome processing is not responsible for this delay. Endosome processing might, however, limit availability of some vesicles for release at very high levels of use. Generally, endosome processing paralleled that of vesicles internalized directly from the plasma membrane via CME, regardless of stimulus frequency. There was no evidence for differential recruitment of ME versus CME depending upon level of use. PMID:17478535

  10. Effects of Endosomal Photodamage on Membrane Recycling and Endocytosis

    PubMed Central

    Kessel, David; Santiago, Ann Marie; Andrzejak, Michelle

    2011-01-01

    The flux of receptor-independent endocytosis can be estimated by addition of wortmannin to cell cultures. Membrane influx is unaffected but traffic out of late endosomes is impaired, resulting in a substantial enlargement of these organelles. Using the 1c1c7 murine hepatoma, we investigated the effect of endosomal photodamage on this endocytic pathway. We previously reported that photodamage catalyzed by the lysosomal photosensitizer NPe6 prevented wortmannin-induced endosomal swelling, indicating an earlier block in the process. In this study, we show that endosomal photodamage, initiated by photodamage from an asymmetrically-substituted porphine or a phthalocyanine, also prevents wortmannin-induced endosomal swelling, even when the PDT dose is insufficient to cause endosomal disruption. As the PDT dose is increased, endosomal breakage occurs, as does apoptosis and cell death. Very high PDT doses result in necrosis. We propose that photodamage to endosomes results in alterations in the endosomal structure such that influx of new material is inhibited and receptor-independent endocytosis is prevented. In an additional series of studies, we found that the swollen late endosomes induced by wortmannin are unable to retain previously accumulated fluorescent probes or photosensitizers. PMID:21208213

  11. Overview of Production of Protein Using Cell-free Systems

    PubMed Central

    Gao, Fei Philip

    2014-01-01

    One of the most important steps in protein research is production of the target protein. Cell based systems are mature tools that have long been used to express recombinant proteins by manipulation of the expression organisms. However, it is often challenging to find suitable cell systems that allow for rapid screening of conditions and constructs to produce properly folded, functional proteins in a cost effective manner. As a result, cell-free protein production emerged as an attractive alternative to cell-based protein expression methods because of its advantages including speed, simplicity, and adaptability to various formats. Efforts have been made in recent years to overcome a few major obstacles that had been preventing the system from being more widely used. These advances have led to the revitalization of cell-free expression systems to meet the increasing demands for protein production, and many research institutions and companies have developed unique and innovative cell-free systems. This poster will present the history and development of the cell-free method, and the updated techniques of various cell-free systems. Examples will be presented to demonstrate that the cell-free system can be a true alternative to cell based protein expression systems and offers a powerful technology for accelerating the production of recombinant protein.

  12. Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications.

    PubMed

    Rani, Sweta; Ryan, Aideen E; Griffin, Matthew D; Ritter, Thomas

    2015-05-01

    Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy. PMID:25868399

  13. Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications

    PubMed Central

    Rani, Sweta; Ryan, Aideen E; Griffin, Matthew D; Ritter, Thomas

    2015-01-01

    Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy. PMID:25868399

  14. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport.

    PubMed

    Kobayashi, T; Beuchat, M H; Lindsay, M; Frias, S; Palmiter, R D; Sakuraba, H; Parton, R G; Gruenberg, J

    1999-06-01

    The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC. PMID:10559883

  15. Cathepsin W Is Required for Escape of Influenza A Virus from Late Endosomes

    PubMed Central

    Edinger, Thomas O.; Pohl, Marie O.; Yángüez, Emilio

    2015-01-01

    ABSTRACT Human cathepsin W (CtsW) is a cysteine protease, which was identified in a genome-wide RNA interference (RNAi) screen to be required for influenza A virus (IAV) replication. In this study, we show that reducing the levels of expression of CtsW reduces viral titers for different subtypes of IAV, and we map the target step of CtsW requirement to viral entry. Using a set of small interfering RNAs (siRNAs) targeting CtsW, we demonstrate that knockdown of CtsW results in a decrease of IAV nucleoprotein accumulation in the nuclei of infected cells at 3 h postinfection. Assays specific for the individual stages of IAV entry further show that attachment, internalization, and early endosomal trafficking are not affected by CtsW knockdown. However, we detected impaired escape of viral particles from late endosomes in CtsW knockdown cells. Moreover, fusion analysis with a dual-labeled influenza virus revealed a significant reduction in fusion events, with no detectable impact on endosomal pH, suggesting that CtsW is required at the stage of viral fusion. The defect in IAV entry upon CtsW knockdown could be rescued by ectopic expression of wild-type CtsW but not by the expression of a catalytically inactive mutant of CtsW, suggesting that the proteolytic activity of CtsW is required for successful entry of IAV. Our results establish CtsW as an important host factor for entry of IAV into target cells and suggest that CtsW could be a promising target for the development of future antiviral drugs. PMID:26060270

  16. Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1.

    PubMed

    Wang, Han; Shi, Yi; Song, Jian; Qi, Jianxun; Lu, Guangwen; Yan, Jinghua; Gao, George F

    2016-01-14

    Filoviruses, including Ebola and Marburg, cause fatal hemorrhagic fever in humans and primates. Understanding how these viruses enter host cells could help to develop effective therapeutics. An endosomal protein, Niemann-Pick C1 (NPC1), has been identified as a necessary entry receptor for this process, and priming of the viral glycoprotein (GP) to a fusion-competent state is a prerequisite for NPC1 binding. Here, we have determined the crystal structure of the primed GP (GPcl) of Ebola virus bound to domain C of NPC1 (NPC1-C) at a resolution of 2.3 Å. NPC1-C utilizes two protruding loops to engage a hydrophobic cavity on head of GPcl. Upon enzymatic cleavage and NPC1-C binding, conformational change in the GPcl further affects the state of the internal fusion loop, triggering membrane fusion. Our data therefore provide structural insights into filovirus entry in the late endosome and the molecular basis for design of therapeutic inhibitors of viral entry. PMID:26771495

  17. Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape.

    PubMed

    Kim, Ji-Sun; Choi, Dong-Ki; Shin, Ju-Yeon; Shin, Seung-Min; Park, Seong-Wook; Cho, Hyun-Soo; Kim, Yong-Sung

    2016-08-10

    Endosomal escape after endocytosis is a critical step for protein-based agents to exhibit their effects in the cytosol of cells. However, antibodies internalized into cells by endocytosis cannot reach the cytosol due to their inability to escape from endosomes. Here, we report a unique endosomal escape mechanism of the IgG-format TMab4 antibody, which can reach the cytosol of living cells after internalization. Dissociation of TMab4 from its cell surface receptor heparan sulfate proteoglycan by activated heparanase in acidified early endosomes and then local structural changes of the endosomal escape motif of TMab4 in response to the acidified endosomal pH were critical for the formation of membrane pores through which TMab4 escaped into the cytosol. Identification of structural determinants of endosomal escape led us to generate a TMab4 variant with ~3-fold improved endosomal escape efficiency. Our finding of the endosomal escape mechanism of the cytosol-penetrating antibody and its improvement will establish a platform technology that enables a full-length IgG antibody to directly target cytosolic proteins. PMID:27264553

  18. Isolation of highly purified, functional endosomes from toad urinary bladder.

    PubMed Central

    Hammond, T G; Morré, D J; Harris, H W; Zeidel, M L

    1993-01-01

    Endosomes are difficult to isolate as they share size and density properties with much more abundant cellular organelles such as mitochondria. In cultured cell lines the tandem use of charge-dependent isolation techniques and differential centrifugation is necessary to isolate endosomes. Endosomal populations of the toad urinary bladder are of special interest because they are thought to contain a water channel. Understanding of the molecular structure of the water channel has been constrained, as there is currently no practical method to isolate functional water-channel-containing vesicles. This study reports the tandem use of charge-dependent techniques and centrifugation to isolate populations of endosomes from the toad urinary bladder. To purify water-channel-containing vesicles aqueous two-phase partition was utilized to fractionate a preparation partially purified by differential centrifugation. Populations of endosomes were analysed by small-particle flow cytometry techniques. A 5-fold enrichment in endosomes, achieved with aqueous two-phase partition, allowed us to identify two populations of endosomes of diverse size in a toad bladder endosomal fraction. Preenrichment also improved the efficiency of flow cytometry sorting, allowing isolation of the two endosomal populations in sufficient quantities for secondary analysis. A population of larger endosomes had very high water permeability, indicating the presence of water channels. The two populations had different SDS/PAGE fingerprints. Electron micrographs of the flow-sorted material shows a uniform population of membrane vesicles devoid of mitochondria and other identifiable cellular organelles. Hence, aqueous two-phase partition and flow cytometry allow identification of two populations of endosomes in the toad urinary bladder which have diverse structural and functional properties. Isolation of functional water-channel-containing vesicles allows co-localization of water-channel function with candidate

  19. Amiodarone impairs trafficking through late endosomes inducing a Niemann-Pick C-like phenotype.

    PubMed

    Piccoli, Elena; Nadai, Matteo; Caretta, Carla Mucignat; Bergonzini, Valeria; Del Vecchio, Claudia; Ha, Huy Riem; Bigler, Laurent; Dal Zoppo, Daniele; Faggin, Elisabetta; Pettenazzo, Andrea; Orlando, Rocco; Salata, Cristiano; Calistri, Arianna; Palù, Giorgio; Baritussio, Aldo

    2011-11-01

    Patients treated with amiodarone accumulate lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate, in airway secretions and develop in different tissues vacuoles and inclusion bodies thought to originate from endosomes. To clarify the origin of these changes, we studied in vitro the effects of amiodarone on endosomal activities like transferrin recycling, Shiga toxin processing, ESCRT-dependent lentivirus budding, fluid phase endocytosis, proteolysis and exosome secretion. Furthermore, since the accumulation of LBPA might point to a broader disturbance in lipid homeostasis, we studied the effect of amiodarone on the distribution of LBPA, unesterified cholesterol, sphingomyelin and glycosphyngolipids. Amiodarone analogues were also studied, including the recently developed derivative dronedarone. We found that amiodarone does not affect early endosomal activities, like transferrin recycling, Shiga toxin processing and lentivirus budding. Amiodarone, instead, interferes with late compartments of the endocytic pathway, blocking the progression of fluid phase endocytosis and causing fusion of organelles, collapse of lumenal structures, accumulation of undegraded substrates and amassing of different types of lipids. Not all late endocytic compartments are affected, since exosome secretion is spared. These changes recall the Niemann-Pick type-C phenotype (NPC), but originate by a different mechanism, since, differently from NPC, they are not alleviated by cholesterol removal. Studies with analogues indicate that basic pKa and high water-solubility at acidic pH are crucial requirements for the interference with late endosomes/lysosomes and that, in this respect, dronedarone is at least as potent as amiodarone. These findings may have relevance in fields unrelated to rhythm control. PMID:21878321

  20. IL4/PGE{sub 2} induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

    SciTech Connect

    Wainszelbaum, Marisa J.; Proctor, Brandon M.; Pontow, Suzanne E.; Stahl, Philip D. . E-mail: pstahl@cellbiology.wustl.edu; Barbieri, M. Alejandro

    2006-07-15

    The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E{sub 2} (PGE{sub 2}) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.

  1. The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

    NASA Astrophysics Data System (ADS)

    Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

    2014-07-01

    Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

  2. Ion-induced fluorescence imaging of endosomes

    NASA Astrophysics Data System (ADS)

    Norarat, R.; Marjomäki, V.; Chen, X.; Zhaohong, M.; Minqin, R.; Chen, C.-B.; Bettiol, A. A.; Whitlow, H. J.; Watt, F.

    2013-07-01

    Imaging laboratories at Jyväskylä and Singapore are collaborating on the development of fluorescence imaging of cytoplasmic endosomes using a combination of proton induced fluorescence (PIF) with direct Scanning Transmission Ion Microscopy (direct-STIM) for sub-cellular structural imaging. A549 lung carcinoma cells were cultivated and stained for epidermal growth factor receptor (EGFR) and receptor α2β1 integrin. In this paper, we demonstrate that cells can be imaged at sub-150 nm resolution using the PIF technique. In addition, the same target cell was imaged at 50 and 25 nm resolution by using proton and He-STIM, respectively. The combination of both techniques offer a powerful tool to improve fluorescence imaging beyond optical diffraction limits.

  3. Entry of Bluetongue Virus Capsid Requires the Late Endosome-specific Lipid Lysobisphosphatidic Acid.

    PubMed

    Patel, Avnish; Mohl, Bjorn-Patrick; Roy, Polly

    2016-06-01

    The entry of viruses into host cells is one of the key processes of infection. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show, utilizing an in vitro liposome penetration assay and cell biology, that bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosome-specific lipid lysobisphosphatidic acid for productive membrane penetration and viral entry. Further, we provide preliminary evidence that lipid lysobisphosphatidic acid facilitates pore expansion during membrane penetration, suggesting a mechanism for lipid factor requirement of BTV. This finding indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry. PMID:27036941

  4. Entry of Bluetongue Virus Capsid Requires the Late Endosome-specific Lipid Lysobisphosphatidic Acid*

    PubMed Central

    Patel, Avnish; Mohl, Bjorn-Patrick; Roy, Polly

    2016-01-01

    The entry of viruses into host cells is one of the key processes of infection. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show, utilizing an in vitro liposome penetration assay and cell biology, that bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosome-specific lipid lysobisphosphatidic acid for productive membrane penetration and viral entry. Further, we provide preliminary evidence that lipid lysobisphosphatidic acid facilitates pore expansion during membrane penetration, suggesting a mechanism for lipid factor requirement of BTV. This finding indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry. PMID:27036941

  5. Sealable femtoliter chamber arrays for cell-free biology

    DOE PAGESBeta

    Retterer, Scott T.; Fowlkes, Jason Davidson; Collier, Charles Patrick; Simpson, Michael L.; Norred, Sarah Elizabeth; Caveney, Patrick M.; Boreyko, Jonathan B.

    2015-03-11

    Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g. global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) devicemore » contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Lastly, we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques to characterize CFPS gene circuits and their interactions with the cell-free environment.« less

  6. Sealable femtoliter chamber arrays for cell-free biology

    SciTech Connect

    Retterer, Scott T.; Fowlkes, Jason Davidson; Collier, Charles Patrick; Simpson, Michael L.; Norred, Sarah Elizabeth; Caveney, Patrick M.; Boreyko, Jonathan B.

    2015-03-11

    Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g. global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Lastly, we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques to characterize CFPS gene circuits and their interactions with the cell-free environment.

  7. Sealable Femtoliter Chamber Arrays for Cell-free Biology

    PubMed Central

    Norred, Sarah Elizabeth; Caveney, Patrick M.; Retterer, Scott T.; Boreyko, Jonathan B.; Fowlkes, Jason D.; Collier, Charles Patrick; Simpson, Michael L.

    2015-01-01

    Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment. PMID:25867144

  8. Cell-free preparation of functional and triggerable giant proteoliposomes.

    PubMed

    Liu, Yan-Jun; Hansen, Gregory P R; Venancio-Marques, Anna; Baigl, Damien

    2013-11-25

    Heat, we leak: We express a membrane protein outside well-defined giant liposomes obtained by gravity-transferred sucrose-in-oil droplets into a cell-free, reconstituted expression system. We show that the presence of the liposome is necessary during expression for efficient protein insertion into the membrane and that temperature can trigger the resulting membrane function. PMID:24115581

  9. Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

    PubMed Central

    McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

    2012-01-01

    Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

  10. Annexin A8 Regulates Late Endosome Organization and Function

    PubMed Central

    Goebeler, Verena; Poeter, Michaela; Zeuschner, Dagmar; Gerke, Volker

    2008-01-01

    Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility. PMID:18923148

  11. Importance of the N-Terminal Domain of the Qb-SNARE Vti1p for Different Membrane Transport Steps in the Yeast Endosomal System

    PubMed Central

    Gossing, Michael; Chidambaram, Subbulakshmi; Fischer von Mollard, Gabriele

    2013-01-01

    SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) on transport vesicles and target membranes are crucial for vesicle targeting and fusion. They form SNARE complexes, which contain four α-helical SNARE motifs contributed by three or four different SNAREs. Most SNAREs function only in a single transport step. The yeast SNARE Vti1p participates in four distinct SNARE complexes in transport from the trans Golgi network to late endosomes, in transport to the vacuole, in retrograde transport from endosomes to the trans Golgi network and in retrograde transport within the Golgi. So far, all vti1 mutants investigated had mutations within the SNARE motif. Little is known about the function of the N-terminal domain of Vti1p, which forms a three helix bundle called Habc domain. Here we generated a temperature-sensitive mutant of this domain to study the effects on different transport steps. The secondary structure of wild type and vti1-3 Habc domain was analyzed by circular dichroism spectroscopy. The amino acid exchanges identified in the temperature-sensitive vti1-3 mutant caused unfolding of the Habc domain. Transport pathways were investigated by immunoprecipitation of newly synthesized proteins after pulse-chase labeling and by fluorescence microscopy of a GFP-tagged protein cycling between plasma membrane, early endosomes and Golgi. In vti1-3 cells transport to the late endosome and assembly of the late endosomal SNARE complex was blocked at 37°C. Retrograde transport to the trans Golgi network was affected while fusion with the vacuole was possible but slower. Steady state levels of SNARE complexes mediating these steps were less affected than that of the late endosomal SNARE complex. As different transport steps were affected our data demonstrate the importance of a folded Vti1p Habc domain for transport. PMID:23776654

  12. VARP Is Recruited on to Endosomes by Direct Interaction with Retromer, Where Together They Function in Export to the Cell Surface

    PubMed Central

    Hesketh, Geoffrey G.; Pérez-Dorado, Inmaculada; Jackson, Lauren P.; Wartosch, Lena; Schäfer, Ingmar B.; Gray, Sally R.; McCoy, Airlie J.; Zeldin, Oliver B.; Garman, Elspeth F.; Harbour, Michael E.; Evans, Philip R.; Seaman, Matthew N.J.; Luzio, J. Paul; Owen, David J.

    2014-01-01

    Summary VARP is a Rab32/38 effector that also binds to the endosomal/lysosomal R-SNARE VAMP7. VARP binding regulates VAMP7 participation in SNARE complex formation and can therefore influence VAMP7-mediated membrane fusion events. Mutant versions of VARP that cannot bind Rab32:GTP, designed on the basis of the VARP ankyrin repeat/Rab32:GTP complex structure described here, unexpectedly retain endosomal localization, showing that VARP recruitment is not dependent on Rab32 binding. We show that recruitment of VARP to the endosomal membrane is mediated by its direct interaction with VPS29, a subunit of the retromer complex, which is involved in trafficking from endosomes to the TGN and the cell surface. Transport of GLUT1 from endosomes to the cell surface requires VARP, VPS29, and VAMP7 and depends on the direct interaction between VPS29 and VARP. Finally, we propose that endocytic cycling of VAMP7 depends on its interaction with VARP and, consequently, also on retromer. PMID:24856514

  13. Endocytosis and early endosome motility in filamentous fungi

    PubMed Central

    Steinberg, Gero

    2014-01-01

    Hyphal growth of filamentous fungi requires microtubule-based long-distance motility of early endosomes. Since the discovery of this process in Ustilago maydis, our understanding of its molecular basis and biological function has greatly advanced. Studies in U. maydis and Aspergillus nidulans reveal a complex interplay of the motor proteins kinesin-3 and dynein, which co-operate to support bi-directional motion of early endosomes. Genetic screening has shed light on the molecular mechanisms underpinning motor regulation, revealing Hook protein as general motor adapters on early endosomes. Recently, fascinating insight into unexpected roles for endosome motility has emerged. This includes septin filament formation and cellular distribution of the machinery for protein translation. PMID:24835422

  14. Live cell imaging of endosomal trafficking in fungi.

    PubMed

    Baumann, Sebastian; Takeshita, Norio; Grün, Nathalie; Fischer, Reinhard; Feldbrügge, Michael

    2015-01-01

    Endosomes are multipurpose membranous carriers important for endocytosis and secretion. During membrane trafficking, endosomes transport lipids, proteins, and even RNAs. In highly polarized cells such as fungal hyphae, they shuttle bidirectionally along microtubules mediated by molecular motors like kinesins and dynein. For in vivo studies of these highly dynamic protein/membrane complexes, advanced fluorescence microscopy is instrumental. In this chapter, we describe live cell imaging of endosomes in two distantly related fungal model systems, the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans. We provide insights into live cell imaging of dynamic endosomal proteins and RNA, dual-color detection for colocalization studies, as well as fluorescence recovery after photobleaching (FRAP) for quantification and photo-activated localization microscopy (PALM) for super-resolution. These methods described in two well-studied fungal model systems are applicable to a broad range of other organisms. PMID:25702128

  15. Endosomes: Emerging Platforms for Integrin-Mediated FAK Signalling.

    PubMed

    Alanko, Jonna; Ivaska, Johanna

    2016-06-01

    Integrins are vital cell adhesion receptors with the ability to transmit extracellular matrix (ECM) cues to intracellular signalling pathways. ECM-integrin signalling regulates various cellular functions such as cell survival and movement. Integrin signalling has been considered to occur exclusively from adhesion sites at the plasma membrane (PM). However, recent data demonstrates integrin signalling also from endosomes. Integrin-mediated focal adhesion kinase (FAK) signalling is strongly dependent on integrin endocytosis, and endosomal FAK signalling facilitates cancer metastasis by supporting anchorage-independent growth and anoikis resistance. Here we discuss the possible mechanisms and functions of endosomal FAK signalling compared with its previously known roles in other cellular locations and discuss the potential of endosomal FAK as novel target for future cancer therapies. PMID:26944773

  16. Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.

    PubMed

    Calder, Lesley J; Rosenthal, Peter B

    2016-09-01

    The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy. PMID:27501535

  17. Vps1 in the late endosome-to-vacuole traffic.

    PubMed

    Hayden, Jacob; Williams, Michelle; Granich, Ann; Ahn, Hyoeun; Tenay, Brandon; Lukehart, Joshua; Highfill, Chad; Dobard, Sarah; Kim, Kyoungtae

    2013-03-01

    Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 delta cells accumulated FM4-64 to a greater extent than wild-type (WT))cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1's implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole. PMID:23385815

  18. Cell-free protein synthesis: applications come of age.

    PubMed

    Carlson, Erik D; Gan, Rui; Hodgman, C Eric; Jewett, Michael C

    2012-01-01

    Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications. PMID:22008973

  19. Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

    SciTech Connect

    Blanchette, C D; Woo, Y; Thomas, C; Shen, N; Sulchek, T A; Hiddessen, A L

    2008-12-22

    Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification process to demonstrate

  20. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes. PMID:26237676

  1. “Late” Macroendosomes and Acidic Endosomes in Vertebrate Motor Nerve Terminals

    PubMed Central

    Stewart, Richard S.; Teng, Haibing; Wilkinson, Robert S.

    2014-01-01

    Activity at the vertebrate nerve—muscle synapse creates large macroendosomes (MEs) via bulk membrane infolding. Visualized with the endocytic probe FM1-43, most (94%) of the ~25 MEs/terminal created by brief (30-Hz, 18-second) stimulation dissipate rapidly (~1 minute) into vesicles. Others, however, remain for hours. Here we study these “ late” MEs by using 4D live imaging over a period of ~1 hour after stimulation. We find that some (51/398 or 13%) disappear spontaneously via exocytosis, releasing their contents into the extracellular milieu. Others (at least 15/1,960 or 1%) fuse or closely associate with a second class of endosomes that take up acidophilic dyes (acidic endosomes [AEs]). AEs are plentiful (~47/terminal) and exist independent of stimulation. Unlike MEs, which exhibit Brownian motion, AEs exhibit directed motion (average, 83 nm/sec) on microtubules within and among terminal boutons. AEs populate the axon as well, where movement is predominantly retrograde. They share biochemical and immunohistochemical markers (e.g., lysosomal-associated membrane protein [LAMP-1]) with lysosomes. Fusion/association of MEs with AEs suggests a sorting/degradation pathway in nerve terminals wherein the role of AEs is similar to that of lysosomes. Based on our data, we propose that MEs serve as sorting endosomes. Thus their contents, which include plasma membrane proteins, vesicle proteins, and extracellular levels of Ca2+, can be targeted either toward the reformation and budding of synaptic vesicles, toward secretion via exocytosis, or toward a degradation process that utilizes AEs either for lysis within the terminal or for transport toward the cell body. PMID:22740045

  2. High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format

    PubMed Central

    Quast, Robert B.; Sonnabend, Andrei; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

    2016-01-01

    Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2. PMID:27456041

  3. Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery.

    PubMed

    Qian, Ziqing; LaRochelle, Jonathan R; Jiang, Bisheng; Lian, Wenlong; Hard, Ryan L; Selner, Nicholas G; Luechapanichkul, Rinrada; Barrios, Amy M; Pei, Dehua

    2014-06-24

    Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4-12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape. PMID:24896852

  4. Endosomal sorting of VAMP3 is regulated by PI4K2A.

    PubMed

    Jović, Marko; Kean, Michelle J; Dubankova, Anna; Boura, Evzen; Gingras, Anne-Claude; Brill, Julie A; Balla, Tamas

    2014-09-01

    Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells. PMID:25002402

  5. Carbon monoxide impairs mitochondria-dependent endosomal maturation and antigen presentation in dendritic cells.

    PubMed

    Riquelme, Sebastián A; Pogu, Julien; Anegon, Ignacio; Bueno, Susan M; Kalergis, Alexis M

    2015-12-01

    Heme-oxygenase 1 (HO-1) prevents T cell-mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome-to-lysosome fusion, and antigen processing, dampening the production of peptide-MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4(+) T-cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8(+) T cell-dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T-cell priming by blocking an unknown mitochondria-dependent antigen-processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen-containing endosomes. In conclusion, CO produced by HO-1 impairs antigen-dependent inflammation by regulating DC immunogenicity by a mitochondria-dependent mechanism. PMID:26461179

  6. Multivesicular Bodies Mature from the Trans-Golgi Network/Early Endosome in Arabidopsis[W

    PubMed Central

    Scheuring, David; Viotti, Corrado; Krüger, Falco; Künzl, Fabian; Sturm, Silke; Bubeck, Julia; Hillmer, Stefan; Frigerio, Lorenzo; Robinson, David G.; Pimpl, Peter; Schumacher, Karin

    2011-01-01

    The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference–mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole. PMID:21934143

  7. Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes*

    PubMed Central

    Chen, Xi; Khambu, Bilon; Zhang, Hao; Gao, Wentao; Li, Min; Chen, Xiaoyun; Yoshimori, Tamotsu; Yin, Xiao-Ming

    2014-01-01

    Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62. PMID:24619419

  8. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.

    PubMed

    Lönn, Peter; Kacsinta, Apollo D; Cui, Xian-Shu; Hamil, Alexander S; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F

    2016-01-01

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells. PMID:27604151

  9. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics

    PubMed Central

    Lönn, Peter; Kacsinta, Apollo D.; Cui, Xian-Shu; Hamil, Alexander S.; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F.

    2016-01-01

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells. PMID:27604151

  10. Alix regulates cortical actin and the spatial distribution of endosomes.

    PubMed

    Cabezas, Alicia; Bache, Kristi G; Brech, Andreas; Stenmark, Harald

    2005-06-15

    Alix/AIP1 is a proline-rich protein that has been implicated in apoptosis, endocytic membrane trafficking and viral budding. To further elucidate the functions of Alix, we used RNA interference to specifically suppress its expression. Depletion of Alix caused a striking redistribution of early endosomes from a peripheral to a perinuclear location. The redistribution of endosomes did not affect transferrin recycling or degradation of endocytosed epidermal growth factor receptors, although the uptake of transferrin was mildly reduced when Alix was downregulated. Quantitative immunoelectron microscopy showed that multivesicular endosomes of Alix-depleted cells contained normal amounts of CD63, whereas their levels of lysobisphosphatidic acid were reduced. Alix depletion also caused an accumulation of unusual actin structures that contained clathrin and cortactin, a protein that couples membrane dynamics to the cortical actin cytoskeleton. Our results suggest that Alix functions in the actin-dependent intracellular positioning of endosomes, but that it is not essential for endocytic recycling or for trafficking of membrane proteins between early and late endosomes in non-polarised cells. PMID:15914539

  11. Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

    PubMed Central

    Mari, Muriel; Griffith, Janice; Reggiori, Fulvio

    2014-01-01

    Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations. PMID:25046212

  12. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    NASA Astrophysics Data System (ADS)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  13. Rab11-endosomes contribute to mitotic spindle orientation

    PubMed Central

    Hehnly, Heidi; Doxsey, Stephen

    2014-01-01

    During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 and endosomes in mitosis. Here we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind γ-TuRC components and associate with tubulin in vitro. Rab11-depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively-active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization and generates robust spindles. This suggests a fundamental role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes co-contribute to these processes through retrograde transport to poles by dynein. PMID:24561039

  14. Rab11 endosomes contribute to mitotic spindle organization and orientation.

    PubMed

    Hehnly, Heidi; Doxsey, Stephen

    2014-03-10

    During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. Here, we show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. We found that mitotic recycling endosomes bind γ-TuRC components and associate with tubulin in vitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores γ-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. We propose that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein. PMID:24561039

  15. Lipid peroxidation causes endosomal antigen release for cross-presentation.

    PubMed

    Dingjan, Ilse; Verboogen, Daniëlle Rj; Paardekooper, Laurent M; Revelo, Natalia H; Sittig, Simone P; Visser, Linda J; Mollard, Gabriele Fischer von; Henriet, Stefanie Sv; Figdor, Carl G; Ter Beest, Martin; van den Bogaart, Geert

    2016-01-01

    Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer. PMID:26907999

  16. Lipid peroxidation causes endosomal antigen release for cross-presentation

    PubMed Central

    Dingjan, Ilse; Verboogen, Daniëlle RJ; Paardekooper, Laurent M; Revelo, Natalia H; Sittig, Simone P; Visser, Linda J; Mollard, Gabriele Fischer von; Henriet, Stefanie SV; Figdor, Carl G; ter Beest, Martin; van den Bogaart, Geert

    2016-01-01

    Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer. PMID:26907999

  17. Cell-Free Protein Expression under Macromolecular Crowding Conditions

    PubMed Central

    Ge, Xumeng; Luo, Dan; Xu, Jianfeng

    2011-01-01

    Background Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process. Methodology/Principal Findings Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments. Conclusions/Significance Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE. PMID:22174874

  18. Cell-free DNA screening and sex chromosome aneuploidies.

    PubMed

    Mennuti, Michael T; Chandrasekaran, Suchitra; Khalek, Nahla; Dugoff, Lorraine

    2015-10-01

    Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed. PMID:26088741

  19. #36: Prenatal aneuploidy screening using cell-free DNA.

    PubMed

    2015-06-01

    Recent advances in technology have created exciting opportunities to expand and improve genetic testing options that are available to women during pregnancy. However, the novelty and complexity of these technologies, combined with the commercial interest to implement these tests rapidly into routine clinical care, have created challenges for physicians and patients and potentially will lead to misunderstanding, misuse, and unintended consequences. The purpose of this document was to aid clinicians in their day-to-day practice of counseling patients regarding prenatal aneuploidy testing options with cell-free DNA screening, which includes how it compares to current testing methods, potential benefits and harms, and its limitations and caveats. PMID:25813012

  20. Cell-free expression of functional receptor tyrosine kinases

    PubMed Central

    He, Wei; Scharadin, Tiffany M.; Saldana, Matthew; Gellner, Candice; Hoang-Phou, Steven; Takanishi, Christina; Hura, Gregory L.; Tainer, John A; Carraway III, Kermit L.; Henderson, Paul T.; Coleman, Matthew A.

    2015-01-01

    Receptor tyrosine kinases (RTKs) play critical roles in physiological and pathological processes, and are important anticancer drug targets. In vitro mechanistic and drug discovery studies of full-length RTKs require protein that is both fully functional and free from contaminating proteins. Here we describe a rapid cell-free and detergent-free co-translation method for producing full-length and functional ERBB2 and EGFR receptor tyrosine kinases supported by water-soluble apolipoprotein A-I based nanolipoprotein particles. PMID:26274523

  1. Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome.

    PubMed Central

    Webb, G C; Zhang, J; Garlow, S J; Wesp, A; Riezman, H; Jones, E W

    1997-01-01

    Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole. PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids. A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins. Several of these proteins are thought to function in the vacuolar/lysosomal pathway. Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function. Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome. Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function. pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome. Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway. Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions. We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome. Images PMID:9168472

  2. Herpesvirus Entry into Host Cells Mediated by Endosomal Low pH.

    PubMed

    Nicola, Anthony V

    2016-09-01

    Herpesviral pathogenesis stems from infection of multiple cell types including the site of latency and cells that support lytic replication. Herpesviruses utilize distinct cellular pathways, including low pH endocytic pathways, to enter different pathophysiologically relevant target cells. This review details the impact of the mildly acidic milieu of endosomes on the entry of herpesviruses, with particular emphasis on herpes simplex virus 1 (HSV-1). Epithelial cells, the portal of primary HSV-1 infection, support entry via low pH endocytosis mechanisms. Mildly acidic pH triggers reversible conformational changes in the HSV-1 class III fusion protein glycoprotein B (gB). In vitro treatment of herpes simplex virions with a similar pH range inactivates infectivity, likely by prematurely activating the viral entry machinery in the absence of a target membrane. How a given herpesvirus mediates both low pH and pH-independent entry events is a key unresolved question. PMID:27126894

  3. Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

    PubMed

    Pasqual, Giulia; Rojek, Jillian M; Masin, Mark; Chatton, Jean-Yves; Kunz, Stefan

    2011-09-01

    The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor. PMID:21931550

  4. Old World Arenaviruses Enter the Host Cell via the Multivesicular Body and Depend on the Endosomal Sorting Complex Required for Transport

    PubMed Central

    Pasqual, Giulia; Rojek, Jillian M.; Masin, Mark; Chatton, Jean-Yves; Kunz, Stefan

    2011-01-01

    The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor. PMID:21931550

  5. Cell-free production of transducible transcription factors for nuclear reprogramming.

    PubMed

    Yang, William C; Patel, Kedar G; Lee, Jieun; Ghebremariam, Yohannes T; Wong, H Edward; Cooke, John P; Swartz, James R

    2009-12-15

    Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target. PMID:19718703

  6. Regulation of liver metabolism by the endosomal GTPase Rab5.

    PubMed

    Zeigerer, Anja; Bogorad, Roman L; Sharma, Kirti; Gilleron, Jerome; Seifert, Sarah; Sales, Susanne; Berndt, Nikolaus; Bulik, Sascha; Marsico, Giovanni; D'Souza, Rochelle C J; Lakshmanaperumal, Naharajan; Meganathan, Kesavan; Natarajan, Karthick; Sachinidis, Agapios; Dahl, Andreas; Holzhütter, Hermann-Georg; Shevchenko, Andrej; Mann, Matthias; Koteliansky, Victor; Zerial, Marino

    2015-05-12

    The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases. PMID:25937276

  7. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

    PubMed Central

    Zheng, Yi-Min; Melikyan, Gregory B.; Liu, Shan-Lu; Cohen, Fredric S.

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

  8. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

    PubMed

    Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min; Melikyan, Gregory B; Liu, Shan-Lu; Cohen, Fredric S

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

  9. Cell-free metabolic engineering: Biomanufacturing beyond the cell

    SciTech Connect

    Dudley, QM; Karim, AS; Jewett, MC

    2014-10-15

    Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L-1 h(-1), reaction scales of >100 L, and new directions in protein purification, spatial organization, and enzyme stability. In the coming years, CFME will offer exciting opportunities to: (i) debug and optimize biosynthetic pathways; (ii) carry out design-build-test iterations without re-engineering organisms; and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility.

  10. Cell-free metabolic engineering: biomanufacturing beyond the cell.

    PubMed

    Dudley, Quentin M; Karim, Ashty S; Jewett, Michael C

    2015-01-01

    Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L(-1) h(-1) , reaction scales of >100 L, and new directions in protein purification, spatial organization, and enzyme stability. In the coming years, CFME will offer exciting opportunities to: (i) debug and optimize biosynthetic pathways; (ii) carry out design-build-test iterations without re-engineering organisms; and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility. PMID:25319678

  11. Cell-Free Metabolic Engineering: Biomanufacturing beyond the cell

    PubMed Central

    Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C.

    2014-01-01

    Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L−1 hr−1, reaction scales of >100L, and new directions in protein purification, spatial organization and enzyme stability. In the coming years, CFME will offer exciting opportunities to (i) debug and optimize biosynthetic pathways, (ii) carry out design-build-test iterations without re-engineering organisms, and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility. PMID:25319678

  12. Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

    PubMed Central

    Heard, William; Sklenář, Jan; Tomé, Daniel F. A.; Robatzek, Silke; Jones, Alexandra M. E.

    2015-01-01

    The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein

  13. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    NASA Astrophysics Data System (ADS)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  14. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    SciTech Connect

    Belfort, Georges; Grimaldi, Joseph J.

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

  15. Spinal fusion

    MedlinePlus

    ... Anterior spinal fusion; Spine surgery - spinal fusion; Low back pain - fusion; Herniated disk - fusion ... If you had chronic back pain before surgery, you will likely still have some pain afterward. Spinal fusion is unlikely to take away all your pain ...

  16. Cell-free NADPH oxidase activation assays: "in vitro veritas".

    PubMed

    Pick, Edgar

    2014-01-01

    The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

  17. Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

    PubMed Central

    2015-01-01

    To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

  18. Energizing eukaryotic cell-free protein synthesis with glucose metabolism.

    PubMed

    Anderson, Mark J; Stark, Jessica C; Hodgman, C Eric; Jewett, Michael C

    2015-07-01

    Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms. PMID:26054976

  19. Use of cell free DNA in breast oncology.

    PubMed

    Canzoniero, Jenna VanLiere; Park, Ben Ho

    2016-04-01

    Cell free DNA (cfDNA) are short fragments of nucleic acids present in circulation outside of cells. In patients with cancer, some portion of cfDNA is derived from tumor cells, termed circulating tumor DNA (ctDNA), and contains the same mutations and genetic changes as the cancer. The development of new, more effective methods to detect these changes has led to increased interest in developing ctDNA as a biomarker for cancer. Here we will review current literature on the use of ctDNA, with an emphasis on breast cancer, for cancer detection, prognosis, monitoring response to therapy, and tracking the rise of new mutant subclones. PMID:27012505

  20. Strategies for Implementing Cell-Free DNA Testing.

    PubMed

    Cuckle, Howard

    2016-06-01

    Maternal plasma cell-free (cf) DNA testing has higher discriminatory power for aneuploidy than any conventional multi-marker screening test. Several strategies have been suggested for introducing it into clinical practice. Secondary cfDNA, restricted only to women with positive conventional screening test, is generally cost saving and minimizes the need for invasive prenatal diagnosis but leads to a small loss in detection. Primary cfDNA, replacing conventional screening or retaining the nuchal translucency scan, is not currently cost-effective for third-party payers. Contingent cfDNA, testing about 20% of women with the highest risks based on a conventional test, is the preferred approach. PMID:27235907

  1. Endosomal system genetics and autism spectrum disorders: A literature review.

    PubMed

    Patak, Jameson; Zhang-James, Yanli; Faraone, Stephen V

    2016-06-01

    Autism spectrum disorders (ASDs) are a group of debilitating neurodevelopmental disorders thought to have genetic etiology, due to their high heritability. The endosomal system has become increasingly implicated in ASD pathophysiology. In an attempt to summarize the association between endosomal system genes and ASDs we performed a systematic review of the literature. We searched PubMed for relevant articles. Simons Foundation Autism Research Initiative (SFARI) gene database was used to exclude articles regarding genes with less than minimal evidence for association with ASDs. Our search retained 55 articles reviewed in two categories: genes that regulate and genes that are regulated by the endosomal system. Our review shows that the endosomal system is a novel pathway implicated in ASDs as well as other neuropsychiatric disorders. It plays a central role in aspects of cellular physiology on which neurons and glial cells are particularly reliant, due to their unique metabolic and functional demands. The system shows potential for biomarkers and pharmacological intervention and thus more research into this pathway is warranted. PMID:27048963

  2. Self-assembling dual component nanoparticles with endosomal escape capability.

    PubMed

    Wong, Adelene S M; Mann, Sarah K; Czuba, Ewa; Sahut, Audrey; Liu, Haiyin; Suekama, Tiffany C; Bickerton, Tayla; Johnston, Angus P R; Such, Georgina K

    2015-04-21

    This study reports a novel nanoparticle system with simple and modular one-step assembly, which can respond intelligently to biologically relevant variations in pH. Importantly, these particles also show the ability to induce escape from the endosomal/lysosomal compartments of the cell, which is integral to the design of efficient polymeric delivery systems. The nanoparticles were formed by the nanoprecipitation of pH-responsive poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) and poly(2-(diethylamino)ethyl methacrylate)-b-poly(ethylene glycol) (PDEAEMA-b-PEG). Rhodamine B octadecyl ester perchlorate was successfully encapsulated within the hydrophobic core of the nanoparticle upon nanoprecipitation into PBS at pH 8. These particles disassembled when the pH was reduced below 6.8 at 37 °C. Cellular experiments showed the successful uptake of the nanoparticles into the endosomal/lysosomal compartments of 3T3 fibroblast cells. The ability to induce escape from the endosomes was demonstrated by the use of calcein, a membrane-impermeable fluorophore. The modular nature of these particles combined with promising endosomal escape capabilities make these dual component PDEAEMA nanoparticles useful for drug and gene delivery applications. PMID:25731820

  3. Cooperation of phosphoinositides and BAR domain proteins in endosomal tubulation.

    PubMed

    Shinozaki-Narikawa, Naeko; Kodama, Tatsuhiko; Shibasaki, Yoshikazu

    2006-11-01

    Phosphorylated derivatives of phosphatidylinositol (PtdIns) regulate many intracellular events, including vesicular trafficking and actin remodeling, by recruiting proteins to their sites of function. PtdIns(4,5)-bisphosphate [PI(4,5)P2] and related phosphoinositides are mainly synthesized by type I PtdIns-4-phosphate 5-kinases (PIP5Ks). We found that PIP5K induces endosomal tubules in COS-7 cells. ADP-ribosylation factor (ARF) 6 has been shown to act upstream of PIP5K and regulate endocytic transport and tubulation. ARF GAP with coiled-coil, ankyrin repeat, and pleckstrin homology domains 1 (ACAP1) has guanosine triphosphatase-activating protein (GAP) activity for ARF6. While there were few tubules induced by the expression of ACAP1 alone, numerous endosomal tubules were induced by coexpression of PIP5K and ACAP1. ACAP1 has a pleckstrin homology (PH) domain known to bind phosphoinositide and a Bin/amphiphysin/Rvs (BAR) domain that has been reported to detect membrane curvature. Truncated and point mutations in the ACAP1 BAR and PH domains revealed that both BAR and PH domains are required for tubulation. These results suggest that two ARF6 downstream molecules, PIP5K and ACAP1, function together in endosomal tubulation and that phosphoinositide levels may regulate endosomal dynamics. PMID:17010122

  4. Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion.

    PubMed

    Dolat, Lee; Spiliotis, Elias T

    2016-08-29

    Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate-dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes. PMID:27551056

  5. Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

    PubMed

    Aceti, David J; Bingman, Craig A; Wrobel, Russell L; Frederick, Ronnie O; Makino, Shin-Ichi; Nichols, Karl W; Sahu, Sarata C; Bergeman, Lai F; Blommel, Paul G; Cornilescu, Claudia C; Gromek, Katarzyna A; Seder, Kory D; Hwang, Soyoon; Primm, John G; Sabat, Grzegorz; Vojtik, Frank C; Volkman, Brian F; Zolnai, Zsolt; Phillips, George N; Markley, John L; Fox, Brian G

    2015-06-01

    Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed. PMID:25854603

  6. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

    SciTech Connect

    Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

    2010-01-20

    The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

  7. Overview of Cell-Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

    PubMed Central

    Chong, Shaorong

    2014-01-01

    During early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owning to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate and wheat germ extracts to recent insect and human cell extracts to defined systems reconstituted from purified recombinant components. Though each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This article provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology. PMID:25271714

  8. Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications.

    PubMed

    Chong, Shaorong

    2014-01-01

    During the early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from "traditional" E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology. PMID:25271714

  9. Visualization of endosome dynamics in living nerve terminals with four-dimensional fluorescence imaging.

    PubMed

    Stewart, Richard S; Kiss, Ilona M; Wilkinson, Robert S

    2014-01-01

    Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections. PMID:24799002

  10. Cell-free circulating tumor DNA in cancer.

    PubMed

    Qin, Zhen; Ljubimov, Vladimir A; Zhou, Cuiqi; Tong, Yunguang; Liang, Jimin

    2016-01-01

    Cancer is a common cause of death worldwide. Despite significant advances in cancer treatments, the morbidity and mortality are still enormous. Tumor heterogeneity, especially intratumoral heterogeneity, is a significant reason underlying difficulties in tumor treatment and failure of a number of current therapeutic modalities, even of molecularly targeted therapies. The development of a virtually noninvasive "liquid biopsy" from the blood has been attempted to characterize tumor heterogeneity. This review focuses on cell-free circulating tumor DNA (ctDNA) in the bloodstream as a versatile biomarker. ctDNA analysis is an evolving field with many new methods being developed and optimized to be able to successfully extract and analyze ctDNA, which has vast clinical applications. ctDNA has the potential to accurately genotype the tumor and identify personalized genetic and epigenetic alterations of the entire tumor. In addition, ctDNA has the potential to accurately monitor tumor burden and treatment response, while also being able to monitor minimal residual disease, reducing the need for harmful adjuvant chemotherapy and allowing more rapid detection of relapse. There are still many challenges that need to be overcome prior to this biomarker getting wide adoption in the clinical world, including optimization, standardization, and large multicenter trials. PMID:27056366

  11. Controls to validate plasma samples for cell free DNA quantification.

    PubMed

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund; Brandslund, Ivan; Jakobsen, Anders

    2015-06-15

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In conclusion we suggest a new method to improve the accuracy of cfDNA measurements easily incorporated in the current technology. PMID:25896958

  12. Cell-free protein synthesis and assembly on a biochip

    NASA Astrophysics Data System (ADS)

    Heyman, Yael; Buxboim, Amnon; Wolf, Sharon G.; Daube, Shirley S.; Bar-Ziv, Roy H.

    2012-06-01

    Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.

  13. Cell-free protein synthesis in microfluidic array devices.

    PubMed

    Mei, Qian; Fredrickson, Carl K; Simon, Andrew; Khnouf, Ruba; Fan, Z Hugh

    2007-01-01

    We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield. PMID:17924644

  14. Cell-free Circulating miRNA Biomarkers in Cancer

    PubMed Central

    Mo, Meng-Hsuan; Chen, Liang; Fu, Yebo; Wang, Wendy; Fu, Sidney W.

    2012-01-01

    Considerable attention and an enormous amount of resources have been dedicated to cancer biomarker discovery and validation. However, there are still a limited number of useful biomarkers available for clinical use. An ideal biomarker should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. Commonly used circulating biomarkers are proteins in serum, most of which require labor-intensive analysis hindered by low sensitivity in early tumor detection. Since the deregulation of microRNA (miRNA) is associated with cancer development and progression, profiling of circulating miRNAs has been used in a number of studies to identify novel minimally invasive miRNA biomarkers. In this review, we discuss the origin of the circulating cell-free miRNAs and their carriers in blood. We summarize the clinical use and function of potentially promising miRNA biomarkers in a variety of different cancers, along with their downstream target genes in tumor initiation and development. Additionally, we analyze some technical challenges in applying miRNA biomarkers to clinical practice. PMID:23074383

  15. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    PubMed

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. PMID:21371942

  16. Probiotic Properties of Lyophilized Cell Free Extract of Lactobacillus casei

    PubMed Central

    Saadatzadeh, Afrooz; Fazeli, Mohamma Reza; Jamalifar, Hossein; Dinarvand, Rassoul

    2013-01-01

    Background In recent years there have been considerable interests in the use of probiotic live cells for nutritional and therapeutic purposes. This strategy can be concomitant with some limitations such as survival of live cell during the GI-transit and their effective delivery to target tissues upon ingestion. Several attempts have been made to overcome these limitations such as their microencapsulation, spray-drying and lyophilization. Objectives In this study extract of cultured probiotics without cells was evaluated for its antimicrobial effects, antioxidant activity, and its stability. Materials and Methods In this work the potential of lyophilized-cell-free-probiotic-extract (LPE) as a suitable alternative strategy for the preparation of probiotic-products was investigated. The main aim of this study was to find out the antibacterial and antioxidant activity of LPE and also its stability. LPE was obtained by centrifugation and subsequent lyophilization of the collected supernatant from culture media of Lactobacillus casei. An enzymatic reagent-kit was used for detection of its content of lactic acid. Antibacterial test was performed using agar cup-plat-method, the DPPH scavenging -assay was used to determine its antioxidant activity and during a storage course, LPE was under a long-term stability study. Results Results showed that, LPE had more antipathogenic effects, antioxidant activity, and stability during storage-time when compared to fresh probiotic-extract. Conclusions Employing the LPE as a new approach, gives novel concept of probiotic-products in food and medical marketing. PMID:24624202

  17. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    PubMed Central

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J.; Kaur, Baljeet; Sarwar, Naveed; Seckl, Michael J.; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA) (from 9% to 53% of total cfDNA) in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis. PMID:26981554

  18. Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

    PubMed Central

    Nour, Adel M.; Li, Yue; Wolenski, Joseph; Modis, Yorgo

    2013-01-01

    Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion. PMID:24039574

  19. Separation and characterization of late endosomal membrane domains.

    PubMed

    Kobayashi, Toshihide; Beuchat, Marie-Hélène; Chevallier, Julien; Makino, Asami; Mayran, Nathalie; Escola, Jean-Michel; Lebrand, Cecile; Cosson, Pierre; Kobayashi, Tetsuyuki; Gruenberg, Jean

    2002-08-30

    Very little is known about the biophysical properties and the lipid or protein composition of membrane domains presumably present in endocytic and biosynthetic organelles. Here we analyzed the membrane composition of late endosomes by suborganellar fractionation in the absence of detergent. We found that the internal membranes of this multivesicular organelle can be separated from the limiting membrane and that each membrane population exhibited a defined composition. Our data also indicated that internal membranes may consist of at least two populations, containing primarily phosphatidylcholine or lysobisphosphatidic acid as major phospholipid, arguing for the existence of significant microheterogeneity within late endosomal membranes. We also found that lysobisphosphatidic acid exhibited unique pH-dependent fusogenic properties, and we speculated that this lipid is an ideal candidate to regulate the dynamic properties of this internal membrane mosaic. PMID:12065580

  20. ATP stimulates pannexin 1 internalization to endosomal compartments.

    PubMed

    Boyce, Andrew K J; Kim, Michelle S; Wicki-Stordeur, Leigh E; Swayne, Leigh Anne

    2015-09-15

    The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp(74)). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease. PMID:26195825

  1. MT1-MMP: Endosomal delivery drives breast cancer metastasis.

    PubMed

    Linder, Stefan

    2015-10-26

    The membrane-tethered membrane type 1-matrix metalloproteinase (MT1-MMP) mediates proteolysis-based invasive tumor growth. In this issue, Marchesin et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506002) describe a tug-of-war mechanism regulating dynein and kinesin motors to drive endosome tubulation and MT1-MMP delivery to the surface of cancer cells, identifying a crucial regulatory axis for tumor metastasis. PMID:26504163

  2. MT1-MMP: Endosomal delivery drives breast cancer metastasis

    PubMed Central

    2015-01-01

    The membrane-tethered membrane type 1–matrix metalloproteinase (MT1-MMP) mediates proteolysis-based invasive tumor growth. In this issue, Marchesin et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506002) describe a tug-of-war mechanism regulating dynein and kinesin motors to drive endosome tubulation and MT1-MMP delivery to the surface of cancer cells, identifying a crucial regulatory axis for tumor metastasis. PMID:26504163

  3. Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

    PubMed Central

    Hicke, L; Zanolari, B; Pypaert, M; Rohrer, J; Riezman, H

    1997-01-01

    Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes. Images PMID:9017592

  4. Inhibition of betanodavirus infection by inhibitors of endosomal acidification.

    PubMed

    Adachi, K; Ichinose, T; Takizawa, N; Watanabe, K; Kitazato, K; Kobayashi, N

    2007-01-01

    Betanodaviruses, members of the family Nodaviridae, have small positive-stranded bipartite RNA genomes and are the causal agent of viral nervous necrosis (VNN) in many species of marine farmed fish. In the aquaculture industry, outbreaks of betanodavirus infection and spread in larval and juvenile fish result in devastating damage and heavy economic loss. Although an urgent need exists to develop drugs that inhibit betanodavirus infection, there have been no reports about anti-betanodavirus drugs. Recently, it was reported that betanodaviruses were detected in the endosomes of infected cells, suggesting that betanodaviruses enter fish cells by endocytosis. This finding prompted us to examine whether blocking this endosomal pathway could provide a target for antiviral drug development. In this study, we examined the inhibitory effect of several lysosomotropic agents against betanodavirus infection in fish E-11 cells. The presence of 1 mM NH4Cl or 1 microM chloroquine in the medium inhibited the entry of betanodaviruses into cells and inhibited viral infection. The lysosomotropic agents bafilomycin A1 and monensin also inhibited virus-induced cytopathology and virus production. Our data demonstrate that inhibitors of endosomal acidification are candidates as antiviral agents against betanodavirus. PMID:17891330

  5. Cytoplasmic Delivery of Liposomes into MCF-7 Breast Cancer Cells Mediated by Cell-Specific Phage Fusion Coat Protein

    PubMed Central

    Wang, Tao; Yang, Shenghong; Petrenko, Valery A; Torchilin, Vladimir P

    2010-01-01

    Earlier, we have shown that doxorubicin-loaded liposomes (Doxil) modified with a chimeric phage fusion coat protein specific towards MCF-7 breast cancer cells identified from a phage landscape library demonstrated a significantly enhanced association with target cells and an increased cytotoxicity. Based on some structural similarities between the N-terminus of the phage potein and known fusogenic peptides, we hypothesized that, in addition to the specific targeting, the phage protein may possess endosome-escaping potential and an increased cytotoxicity of drug-loaded phage protein-targeted liposomes may be explained by an advantageous combination of both, cell targeting and endosomal escape of drug-loaded nanocarrier. The use of the fluorescence resonance energy transfer (FRET) technique allowed us to clearly demonstrate the pH-dependent membrane fusion activity of the phage protein. Endosomal escape and cytosolic delivery of phage-liposomes was visualized with fluorescence microscopy. Endosome acidification inhibition by bafilomycin A 1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, confirming its endosomal escape. Our results demonstrated an endosome-escaping property of the phage protein and provided an insight on mechanism of the enhanced cytotoxicity of phage-Doxil. PMID:20438086

  6. Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA

    PubMed Central

    Devaney, Stephanie A.; Palomaki, Glenn E.; Scott, Joan A.; Bianchi, Diana W.

    2015-01-01

    Context Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. Objective To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Data Sources Systematic review and meta-analysis with search of PubMed (January 1, 1997–April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Study Selection and Data Extraction Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. Results From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%–96.1%) and specificity, 98.6% (95% CI, 98.1%–99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989–0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979–0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993–0.998) for real-time quantitative PCR (RTQ-PCR) (P=.02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965–0.998) (<7 weeks); AUC, 0.994 (95% CI, 0.987–0.997) (7–12 weeks); AUC, 0.992 (95% CI, 0.983–0.996) (13

  7. Stochastic Fusion Simulations and Experiments Suggest Passive and Active Roles of Hemagglutinin during Membrane Fusion

    PubMed Central

    Lee, Donald W.; Thapar, Vikram; Clancy, Paulette; Daniel, Susan

    2014-01-01

    Influenza enters the host cell cytoplasm by fusing the viral and host membrane together. Fusion is mediated by hemagglutinin (HA) trimers that undergo conformational change when acidified in the endosome. It is currently debated how many HA trimers, w, and how many conformationally changed HA trimers, q, are minimally required for fusion. Conclusions vary because there are three common approaches for determining w and q from fusion data. One approach correlates the fusion rate with the fraction of fusogenic HA trimers and leads to the conclusion that one HA trimer is required for fusion. A second approach correlates the fusion rate with the total concentration of fusogenic HA trimers and indicates that more than one HA trimer is required. A third approach applies statistical models to fusion rate data obtained at a single HA density to establish w or q and suggests that more than one HA trimer is required. In this work, all three approaches are investigated through stochastic fusion simulations and experiments to elucidate the roles of HA and its ability to bend the target membrane during fusion. We find that the apparent discrepancies among the results from the various approaches may be resolved if nonfusogenic HA participates in fusion through interactions with a fusogenic HA. Our results, based on H3 and H1 serotypes, suggest that three adjacent HA trimers and one conformationally changed HA trimer are minimally required to induce membrane fusion (w = 3 and q = 1). PMID:24559987

  8. Ceramide formation mediated by acid sphingomyelinase facilitates endosomal escape of caliciviruses.

    PubMed

    Shivanna, Vinay; Kim, Yunjeong; Chang, Kyeong-Ok

    2015-09-01

    Our recent results demonstrated that bile acids facilitate virus escape from the endosomes into the cytoplasm for successful replication of porcine enteric calicivirus (PEC). We report a novel finding that bile acids can be substituted by cold treatment for endosomal escape and virus replication. This endosomal escape by cold treatment or bile acids is associated with ceramide formation by acid sphingomyelinase (ASM). ASM catalyzes hydrolysis of sphingomyelin into ceramide, which is known to destabilize lipid bilayer. Treatment of LLC-PK cells with bile acids or cold led to ceramide formation, and small molecule antagonists or siRNA of ASM blocked ceramide formation in the endosomes and significantly reduced PEC replication. Inhibition of ASM resulted in the retention of PEC, feline calicivirus or murine norovirus in the endosomes in correlation with reduced viral replication. These results suggest the importance of viral escape from the endosomes for the replication of various caliciviruses. PMID:25985440

  9. Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system.

    PubMed

    Salehi, Amin S M; Smith, Mark Thomas; Bennett, Anthony M; Williams, Jacob B; Pitt, William G; Bundy, Bradley C

    2016-02-01

    Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production. PMID:26380966

  10. Genome aberrations in canine mammary carcinomas and their detection in cell-free plasma DNA.

    PubMed

    Beck, Julia; Hennecke, Silvia; Bornemann-Kolatzki, Kirsten; Urnovitz, Howard B; Neumann, Stephan; Ströbel, Philipp; Kaup, Franz-Josef; Brenig, Bertram; Schütz, Ekkehard

    2013-01-01

    Mammary tumors are the most frequent cancers in female dogs exhibiting a variety of histopathological differences. There is lack of knowledge about the genomes of these common dog tumors. Five tumors of three different histological subtypes were evaluated. Massive parallel sequencing (MPS) was performed in comparison to the respective somatic genome of each animal. Copy number and structural aberrations were validated using droplet digital PCR (ddPCR). Using mate-pair sequencing chromosomal aneuploidies were found in two tumors, frequent smaller deletions were found in one, inter-chromosomal fusions in one other, whereas one tumor was almost normal. These aberrations affect several known cancer associated genes such as cMYC, and KIT. One common deletion of the proximal end of CFA27, harboring the tumor suppressor gene PFDN5 was detected in four tumors. Using ddPCR, this deletion was validated and detected in 50% of tumors (N = 20). Breakpoint specific dPCRs were established for four tumors and tumor specific cell-free DNA (cfDNA) was detected in the plasma. In one animal tumor-specific cfDNA was found >1 year after surgery, attributable to a lung metastasis. Paired-end sequencing proved that copy-number imbalances of the tumor are reflected by the cfDNA. This report on chromosomal instability of canine mammary cancers reveals similarities to human breast cancers as well as special canine alterations. This animal model provides a framework for using MPS for screening for individual cancer biomarkers with cost effective confirmation and monitoring using ddPCR. The possibility exists that ddPCR can be expanded to screening for common cancer related variants. PMID:24098698

  11. Genome Aberrations in Canine Mammary Carcinomas and Their Detection in Cell-Free Plasma DNA

    PubMed Central

    Beck, Julia; Hennecke, Silvia; Bornemann-Kolatzki, Kirsten; Urnovitz, Howard B.; Neumann, Stephan; Ströbel, Philipp; Kaup, Franz-Josef; Brenig, Bertram; Schütz, Ekkehard

    2013-01-01

    Mammary tumors are the most frequent cancers in female dogs exhibiting a variety of histopathological differences. There is lack of knowledge about the genomes of these common dog tumors. Five tumors of three different histological subtypes were evaluated. Massive parallel sequencing (MPS) was performed in comparison to the respective somatic genome of each animal. Copy number and structural aberrations were validated using droplet digital PCR (ddPCR). Using mate-pair sequencing chromosomal aneuploidies were found in two tumors, frequent smaller deletions were found in one, inter-chromosomal fusions in one other, whereas one tumor was almost normal. These aberrations affect several known cancer associated genes such as cMYC, and KIT. One common deletion of the proximal end of CFA27, harboring the tumor suppressor gene PFDN5 was detected in four tumors. Using ddPCR, this deletion was validated and detected in 50% of tumors (N = 20). Breakpoint specific dPCRs were established for four tumors and tumor specific cell-free DNA (cfDNA) was detected in the plasma. In one animal tumor-specific cfDNA was found >1 year after surgery, attributable to a lung metastasis. Paired-end sequencing proved that copy-number imbalances of the tumor are reflected by the cfDNA. This report on chromosomal instability of canine mammary cancers reveals similarities to human breast cancers as well as special canine alterations. This animal model provides a framework for using MPS for screening for individual cancer biomarkers with cost effective confirmation and monitoring using ddPCR. The possibility exists that ddPCR can be expanded to screening for common cancer related variants. PMID:24098698

  12. Studying the regulation of endosomal cAMP production in GPCR signaling

    PubMed Central

    Gidon, Alexandre; Feinstein, Timothy N.; Xiao, Kunhong; Vilardaga, Jean-Pierre

    2016-01-01

    We describe methods based on live cell fluorescent microscopy and mass spectrometry to characterize the mechanism of endosomal cAMP production and its regulation using the parathyroid hormone (PTH) type 1 receptor as a prime example. These methods permit to measure rapid changes of cAMP levels in response to PTH, kinetics of endosomal ligand–receptor interaction, pH changes associated with receptor trafficking, and to identify the endosomal receptor interactome. PMID:26928541

  13. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

    PubMed Central

    Burr, Ansen; Grant, Wilmon F.; Canfield, Elizabeth T.; Sorensen, Michelle L.; Moita, Luis F.; Lewinsohn, David M.

    2016-01-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  14. Human Na(+)/H(+) exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria.

    PubMed

    Brett, Christopher L; Wei, Ying; Donowitz, Mark; Rao, Rajini

    2002-05-01

    Since the discovery of the first intracellular Na(+)/H(+) exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na(+) into the vacuole, regulates intravesicular pH, and contributes to vacuolar biogenesis. In contrast, the human homolog of Nhx1, Na(+)/H(+) exchanger isoform 6 (NHE6), has been reported to localize to mitochondria when transiently expressed as a fusion with green fluorescent protein. This result warrants reevaluation because it conflicts with predictions from phylogenetic analyses. Here we demonstrate that when epitope-tagged NHE6 is transiently expressed in cultured mammalian cells, it does not colocalize with mitochondrial markers. It also does not colocalize with markers of the lysosome, late endosome, trans-Golgi network, or Golgi cisternae. Rather, NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane. These results suggest that, like its homologs in yeast and plants, NHE6 is an endosomal Na(+)/H(+) exchanger that may regulate intravesicular pH and volume and contribute to lysosomal biogenesis. PMID:11940519

  15. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells.

    PubMed

    Harriff, Melanie J; Karamooz, Elham; Burr, Ansen; Grant, Wilmon F; Canfield, Elizabeth T; Sorensen, Michelle L; Moita, Luis F; Lewinsohn, David M

    2016-03-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  16. Quantitative analysis of cell-free DNA in ovarian cancer

    PubMed Central

    SHAO, XUEFENG; He, YAN; JI, MIN; CHEN, XIAOFANG; QI, JING; SHI, WEI; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ovarian tumor cases, and 19 healthy, non-cancerous ovaries. bDNA techniques were used to detect serum cf-DNA concentrations. All data were analyzed using SPSS version 18.0. The cf-DNA levels were significantly increased in the ovarian cancer group compared with those of the benign ovarian tumor group and healthy ovarian group (P<0.01). Furthermore, cf-DNA levels were significantly increased in stage III and IV ovarian cancer compared with those of stages I and II (P<0.01). In addition, cf-DNA levels were significantly increased on the first day post-surgery (P<0.01), and subsequently demonstrated a gradual decrease. In the ovarian cancer group, the area under the receiver operating characteristic curve of cf-DNA and the sensitivity were 0.917 and 88.9%, respectively, which was higher than those of cancer antigen 125 (0.724, 75%) and human epididymis protein 4 (0.743, 80.6%). There was a correlation between the levels of serum cf-DNA and the occurrence and development of ovarian cancer in the patients evaluated. bDNA techniques possessed higher sensitivity and specificity than other methods for the detection of serum cf-DNA in patients exhibiting ovarian cancer, and bDNA techniques are more useful for detecting cf-DNA than other factors. Thus, the present study demonstrated the potential value for the use of bDNA as an adjuvant diagnostic method for ovarian cancer. PMID:26788153

  17. Expression optimization and synthetic gene networks in cell-free systems

    PubMed Central

    Karig, David K.; Iyer, Sukanya; Simpson, Michael L.; Doktycz, Mitchel J.

    2012-01-01

    Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion. PMID:22180537

  18. Localization of lysobisphosphatidic acid-rich membrane domains in late endosomes.

    PubMed

    Kobayashi, T; Startchev, K; Whitney, A J; Gruenber, J

    2001-03-01

    Late endosomes accumulate internal membranes within the lumen of the organelle. These internal membranes are enriched in the late endosome specific phospholipid, lysobisphosphatidic acid (LBPA). The organization of LBPA-rich membrane domains is not well characterized. Using an LBPA-specific monoclonal antibody (6C4), we show that these membrane domains are not accessible from the cytoplasm. Using fluorescence correlation spectroscopy, we also show that 6C4 only binds sonicated, but not intact, late endosomes, presumably reflecting the release of internal membranes upon endosome rupture. PMID:11347897

  19. Discovery of a vezatin-like protein for dynein-mediated early endosome transport

    PubMed Central

    Yao, Xuanli; Arst, Herbert N.; Wang, Xiangfeng; Xiang, Xin

    2015-01-01

    Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo. PMID:26378255

  20. Theoretical considerations on the role of membrane potential in the regulation of endosomal pH.

    PubMed Central

    Rybak, S L; Lanni, F; Murphy, R F

    1997-01-01

    Na+,K(+)-ATPase has been observed to partially inhibit acidification of early endosomes by increasing membrane potential, whereas chloride channels have been observed to enhance acidification in endosomes and lysosomes. However, little theoretical analysis of the ways in which different pumps and channels may interact has been carried out. We therefore developed quantitative models of endosomal pH regulation based on thermodynamic considerations. We conclude that 1) both size and shape of endosomes will influence steady-state endosomal pH whenever membrane potential due to the pH gradient limits proton pumping, 2) steady-state pH values similar to those observed in early endosomes of living cells can occur in endosomes containing just H(+)-ATPases and Na+,K(+)-ATPases when low endosomal buffering capacities are present, and 3) inclusion of active chloride channels results in predicted pH values well below those observed in vivo. The results support the separation of endocytic compartments into two classes, those (such as early endosomes) whose acidification is limited by attainment of a certain membrane potential, and those (such as lysosomes) whose acidification is limited by the attainment of a certain pH. The theoretical framework and conclusions described are potentially applicable to other membrane-enclosed compartments that are acidified, such as elements of the Golgi apparatus. PMID:9251786

  1. Modifying Risk of Aneuploidy with a Positive Cell-Free Fetal DNA Result.

    PubMed

    Long, A Ashleigh; Abuhamad, Alfred Z; Warsof, Steven L

    2016-06-01

    Noninvasive genomic assessments of the fetus while in utero have been made possible by the analysis of cell-free fetal DNA fragments from the serum of pregnant women, as part of a noninvasive prenatal testing screening strategy. Between 7% and 10% of total cell-free DNA in the maternal blood comes from placental trophoblasts, allowing for identification of the DNA associated with the fetal component of the placenta. Using simple venipuncture in the outpatient setting, this cell-free, extracellular fetal DNA can be isolated in the maternal serum from a single blood draw as early as the seventh week of gestation. PMID:27235910

  2. Role of the Small GTPase Rho3 in Golgi/Endosome Trafficking through Functional Interaction with Adaptin in Fission Yeast

    PubMed Central

    Kita, Ayako; Li, Cuifang; Yu, Yang; Umeda, Nanae; Doi, Akira; Yasuda, Mitsuko; Ishiwata, Shunji; Taga, Atsushi; Horiuchi, Yoshitaka; Sugiura, Reiko

    2011-01-01

    Background We had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Methodology/Principal Findings In the present study, we isolated rho3+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl− sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl−, and valproic acid. Green fluorescent protein (GFP)-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. Conclusions/Significance Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence of a direct link

  3. Translocation and clustering of endosomes and lysosomes depends on microtubules.

    PubMed

    Matteoni, R; Kreis, T E

    1987-09-01

    Indirect immunofluorescence labeling of normal rat kidney (NRK) cells with antibodies recognizing a lysosomal glycoprotein (LGP 120; Lewis, V., S.A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) reveals that lysosomes accumulate in the region around the microtubule-organizing center (MTOC). This clustering of lysosomes depends on microtubules. When the interphase microtubules are depolymerized by treatment of the cells with nocodazole or during mitosis, the lysosomes disperse throughout the cytoplasm. Lysosomes recluster rapidly (within 30-60 min) in the region of the centrosomes either upon removal of the drug, or, in telophase, when repolymerization of interphase microtubules has occurred. During this translocation process the lysosomes can be found aligned along centrosomal microtubules. Endosomes and lysosomes can be visualized by incubating living cells with acridine orange. We have analyzed the movement of these labeled endocytic organelles in vivo by video-enhanced fluorescence microscopy. Translocation of endosomes and lysosomes occurs along linear tracks (up to 10 microns long) by discontinuous saltations (with velocities of up to 2.5 microns/s). Organelles move bidirectionally with respect to the MTOC. This movement ceases when microtubules are depolymerized by treatment of the cells with nocodazole. After nocodazole washout and microtubule repolymerization, the translocation and reclustering of fluorescent organelles predominantly occurs in a unidirectional manner towards the area of the MTOC. Organelle movement remains unaffected when cells are treated with cytochalasin D, or when the collapse of intermediate filaments is induced by microinjected monoclonal antivimentin antibodies. It can be concluded that translocation of endosomes and lysosomes occurs along microtubules and is independent of the intermediate filament and microfilament networks. PMID:3308906

  4. Phosphoinositides and the regulation of tubular-based endosomal sorting.

    PubMed

    Cullen, Peter J

    2011-08-01

    From the pioneering work of Mabel and Lowell Hokin in the 1950s, the biology of this specific isomer of hexahydroxycyclohexane and its phosphorylated derivatives, in the form of inositol phosphates and phosphoinositides, has expanded to fill virtually every corner of cell biology, whole-organism physiology and development. In the present paper, I give a personal view of the role played by phosphoinositides in regulating the function of the endosomal network, and, in so doing, highlight some of the basic properties through which phosphoinositides regulate cell function. PMID:21787311

  5. Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.

    PubMed

    Wong, S H; Zhang, T; Xu, Y; Subramaniam, V N; Griffiths, G; Hong, W

    1998-06-01

    Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant alpha-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway. PMID:9614193

  6. LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function.

    PubMed

    Sepulveda, Fernando E; Burgess, Agathe; Heiligenstein, Xavier; Goudin, Nicolas; Ménager, Mickaël M; Romao, Maryse; Côte, Marjorie; Mahlaoui, Nizar; Fischer, Alain; Raposo, Graça; Ménasché, Gaël; de Saint Basile, Geneviève

    2015-02-01

    Chediak-Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome-like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling-late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal-sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13-4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin-containing vesicles into secretory cytotoxic granules. PMID:25425525

  7. EFFECT OF DIPHTHERIA TOXIN T-DOMAIN ON ENDOSOMAL pH.

    PubMed

    Labyntsev, A J; Korotkevych, N V; Kolybo, D V; Komisarenko, S V

    2015-01-01

    A key step in the mode of cytotoxic action of diphtheria toxin (DT) is the transfer of its catalytic domain (Cd) from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td), but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demonstrated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol. PMID:26547959

  8. Characterization of the Cell-Free Layer in a Microvessel by Computer Simulation

    NASA Astrophysics Data System (ADS)

    Jee, Sol Keun; Freund, Jonathon; Moser, Robert

    2006-11-01

    The cell-free layer between the erythrocyte-rich core of a micro-vessel and the vessel wall is a significant component of the hydrodynamics of the microcirculation. To investigate the mechanics of the cell-free layer, we simulate a two-dimensional periodic blood flow in a microvessel containing numerous erythrocytes, modeled as capsules with elastic shell membranes using the boundary integral method. Cell-cell interactions are mediated with an interaction potential which represents aggregation forces. Our model successfully recreates in-vivo hemodynamic properties such as blunt velocity profile and Fahraeus effect. The cell-free layer has a thickness of order one erythrocyte radius which is consistent with experimental results. To investigate the mechanics of the cell-free layer a number of numerical experiments were conducted, in which the effects of aggregation forces, and lubrication forces are investigated, by varying the aggregation potential, introducing artificial body forces and changing boundary condition.

  9. Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins.

    PubMed

    Nozawa, Akira; Tozawa, Yuzuru

    2014-01-01

    Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane proteins is the difficulty in producing sufficient quantities of functional protein for analysis. Here, we describe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modified with components necessary to produce functional protein. In this way we have developed modifications to a wheat germ cell-free system for the production of functional membrane proteins. Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins. These modified cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide variety of membrane proteins. PMID:24136528

  10. UNC93B1 mediates differential trafficking of endosomal TLRs

    PubMed Central

    Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

    2013-01-01

    UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.00291.001 PMID:23426999

  11. G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

    PubMed Central

    Marchese, Adriano; Paing, May M.; Temple, Brenda R.S.; Trejo, JoAnn

    2010-01-01

    The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs. PMID:17995450

  12. UNC93B1 mediates differential trafficking of endosomal TLRs.

    PubMed

    Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

    2013-01-01

    UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases.DOI:http://dx.doi.org/10.7554/eLife.00291.001. PMID:23426999

  13. Proteolytic processing of epidermal growth factor within endosomes

    SciTech Connect

    Gorman, R.M.; Savage C.R. Jr.; Poretz, R.D.; Schaudies, R.P.

    1986-05-01

    The authors have reported previously that EGF enters 3 biochemically distinct non-lysosomal intracellular compartments prior to detection within lysosomes. Earlier studies have demonstrated that EGF is processes by sequential removal of 1, 4 and 1 aminoacyl residues at the C-terminus. The final form, which lacks the 6 residues, accumulates in secondary lysosomes. After subcellular fractionation of fibroblasts exposed to /sup 125/I-EGF, ligand is detected with 3 non-lysosomal endocytic compartments and is fully processed prior to entrance into secondary lysosome. Following internalization, EGF enters an early endosomal compartment (E/sub 1/). The composition of the ligand (60%, -1 form; 40%, native form) represents an enhancement of the -1 form relative to that on the plasma membrane following the 90 min, 0/sup 0/ binding period. The proportion of different EGF forms in E/sub 1/ remains constant through the 2 min pulse and chase periods up to 30 min. However, in the ultimate endosomal compartment, E/sub 4/, the proportion of the -6 form increases from 25% at 15 min to greater than 75% in 30 min, with a concomitant decrease of the -1 and -5 forms. Secondary lysosomes contain an EGF composition similar to that found in E/sub 4/ at 30 min. Accordingly, it appears that EGF is processed to the -6 form following passage through E/sub 1/ and during its tenure in E/sub 4/.

  14. Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes

    PubMed Central

    Edagwa, Benson J.; Guo, Dongwei; Puligujja, Pavan; Chen, Han; McMillan, JoEllyn; Liu, Xinming; Gendelman, Howard E.; Narayanasamy, Prabagaran

    2014-01-01

    Eradication of Mycobacterium tuberculosis (MTB) infection requires daily administration of combinations of rifampin (RIF), isoniazid [isonicotinylhydrazine (INH)], pyrazinamide, and ethambutol, among other drug therapies. To facilitate and optimize MTB therapeutic selections, a mononuclear phagocyte (MP; monocyte, macrophage, and dendritic cell)-targeted drug delivery strategy was developed. Long-acting nanoformulations of RIF and an INH derivative, pentenyl-INH (INHP), were prepared, and their physicochemical properties were evaluated. This included the evaluation of MP particle uptake and retention, cell viability, and antimicrobial efficacy. Drug levels reached 6 μg/106 cells in human monocyte-derived macrophages (MDMs) for nanoparticle treatments compared with 0.1 μg/106 cells for native drugs. High RIF and INHP levels were retained in MDM for >15 d following nanoparticle loading. Rapid loss of native drugs was observed in cells and culture fluids within 24 h. Antimicrobial activities were determined against Mycobacterium smegmatis (M. smegmatis). Coadministration of nanoformulated RIF and INHP provided a 6-fold increase in therapeutic efficacy compared with equivalent concentrations of native drugs. Notably, nanoformulated RIF and INHP were found to be localized in recycling and late MDM endosomal compartments. These were the same compartments that contained the pathogen. Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.—Edagwa, B. J., Guo, D., Puligujja, P., Chen, H., McMillan, J., Liu, X., Gendelman, H. E., Narayanasamy, P. Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes. PMID:25122556

  15. EHD1 functions in endosomal recycling and confers salt tolerance.

    PubMed

    Bar, Maya; Leibman, Meirav; Schuster, Silvia; Pitzhadza, Hilla; Avni, Adi

    2013-01-01

    Endocytosis is a crucial process in all eukaryotic organisms including plants. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. Knock-down of EHD1 was shown to have a delayed recycling phenotype in mammalians. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD1 that are required for its activity have not been characterized. In this work we demonstrate that knock-down of EHD1 causes a delayed recycling phenotype and reduces Brefeldin A sensitivity in Arabidopsis seedlings. The EH domain of EHD1 was found to be crucial for the localization of EHD1 to endosomal structures. Mutant EHD1 lacking the EH domain did not localize to endosomal structures and showed a phenotype similar to that of EHD1 knock-down seedlings. Mutants lacking the coiled-coil domain, however, showed a phenotype similar to wild-type or EHD1 overexpression seedlings. Salinity stress is a major problem in current agriculture. Microarray data demonstrated that salinity stress enhances the expression of EHD1, and this was confirmed by semi quantitative RT-PCR. We demonstrate herein that transgenic plants over expressing EHD1 possess enhanced tolerance to salt stress, a property which also requires an intact EH domain. PMID:23342166

  16. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

    PubMed Central

    Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

    2015-01-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. PMID:26478227

  17. Cell-free protein synthesis systems derived from cultured mammalian cells.

    PubMed

    Brödel, Andreas K; Wüstenhagen, Doreen A; Kubick, Stefan

    2015-01-01

    We present a technology for the production of target proteins using novel cell-free systems derived from cultured human K562 cells and Chinese hamster ovary (CHO) cells. The protocol includes the cultivation of cells, the preparation of translationally active lysates, and the cell-free synthesis of desired proteins. An efficient expression vector based on the internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV) was constructed for both systems. The coupled batch-based platforms enable the synthesis of a broad range of target proteins such as cytosolic proteins, secreted proteins, membrane proteins embedded into endogenous microsomes, and glycoproteins. The glycosylation of erythropoietin demonstrates the successful performance of posttranslational modifications in the novel cell-free systems. Protein yields of approximately 20 μg/ml (K562-based cell-free system) and 50 μg/ml (CHO-based cell-free system) of active firefly luciferase are obtained in the coupled transcription-translation systems within 3 h. As a result, both cell-free protein synthesis systems serve as powerful tools for high-throughput proteomics. PMID:25502197

  18. Regulation of endosomal motility and degradation by amyotrophic lateral sclerosis 2/alsin

    PubMed Central

    Lai, Chen; Xie, Chengsong; Shim, Hoon; Chandran, Jayanth; Howell, Brian W; Cai, Huaibin

    2009-01-01

    Dysfunction of alsin, particularly its putative Rab5 guanine-nucleotide-exchange factor activity, has been linked to one form of juvenile onset recessive familial amyotrophic lateral sclerosis (ALS2). Multiple lines of alsin knockout (ALS2-/-) mice have been generated to model this disease. However, it remains elusive whether the Rab5-dependent endocytosis is altered in ALS2-/- neurons. To directly examine the Rab5-mediated endosomal trafficking in ALS2-/- neurons, we introduced green fluorescent protein (GFP)-tagged Rab5 into cultured hippocampal neurons to monitor the morphology and motility of Rab5-associated early endosomes. Here we report that Rab5-mediated endocytosis was severely altered in ALS2-/-neurons. Excessive accumulation of Rab5-positive vesicles was observed in ALS2-/- neurons, which correlated with a significant reduction in endosomal motility and augmentation in endosomal conversion to lysosomes. Consequently, a significant increase in endosome/lysosome-dependent degradation of internalized glutamate receptors was observed in ALS2-/- neurons. These phenotypes closely resembled the endosomal trafficking abnormalities induced by a constitutively active form of Rab5 in wild-type neurons. Therefore, our findings reveal a negatively regulatory mechanism of alsin in Rab5-mediated endosomal trafficking, suggesting that enhanced endosomal degradation in ALS2-/- neurons may underlie the pathogenesis of motor neuron degeneration in ALS2 and related motor neuron diseases. PMID:19630956

  19. Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges

    PubMed Central

    Erazo-Oliveras, Alfredo; Muthukrishnan, Nandhini; Baker, Ryan; Wang, Ting-Yi; Pellois, Jean-Philippe

    2012-01-01

    Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this review, the evidence for CPP-mediated endosomal escape is discussed. In addition, several strategies that have been utilized to enhance the endosomal escape of CPP-cargos are described. The recent development of branched systems that display multiple copies of a CPP is presented. The use of viral or synthetic peptides that can disrupt the endosomal membrane upon activation by the low pH of endosomes is also discussed. Finally, we survey how CPPs labeled with chromophores can be used in combination with light to stimulate endosomal lysis. The mechanisms and challenges associated with these intracellular delivery methodologies are discussed. PMID:24223492

  20. Touché! STARD3 and STARD3NL tether the ER to endosomes.

    PubMed

    Wilhelm, Léa P; Tomasetto, Catherine; Alpy, Fabien

    2016-04-15

    Membrane contact sites (MCSs) are subcellular regions where the membranes of distinct organelles come into close apposition. These specialized areas of the cell, which are involved in inter-organelle metabolite exchange, are scaffolded by specific complexes. STARD3 [StAR (steroidogenic acute regulatory protein)-related lipid transfer domain-3] and its close paralogue STARD3NL (STARD3 N-terminal like) are involved in the formation of contacts between late-endosomes and the endoplasmic reticulum (ER). The lipid transfer protein (LTP) STARD3 and STARD3NL, which are both anchored on the limiting membrane of late endosomes (LEs), interact with ER-anchored VAP [VAMP (vesicle-associated membrane protein)-associated protein] (VAP-A and VAP-B) proteins. This direct interaction allows ER-endosome contact formation. STARD3 or STARD3NL-mediated ER-endosome contacts, which affect endosome dynamics, are believed to be involved in cholesterol transport. PMID:27068960

  1. Plasma Cell-Free DNA in Paediatric Lymphomas

    PubMed Central

    Mussolin, Lara; Burnelli, Roberta; Pillon, Marta; Carraro, Elisa; Farruggia, Piero; Todesco, Alessandra; Mascarin, Maurizio; Rosolen, Angelo

    2013-01-01

    Background: Extracellular circulating DNA (cfDNA) can be found in small amounts in plasma of healthy individuals. Increased levels of cfDNA have been reported in patients with cancer of breast, cervix, colon, liver and it was shown that cfDNA can originate from both tumour and non-tumour cells. Objectives: Levels of cfDNA of a large series of children with lymphoma were evaluated and analyzed in relation with clinical characteristics. Methods: plasma cfDNA levels obtained at diagnosis in 201 paediatric lymphoma patients [43 Hodgkin lymphomas (HL), 45 anaplastic large cell lymphomas (ALCL), 88 Burkitt lymphomas (BL), 17 lymphoblastic (LBL), 8 diffuse large B cell lymphoma (DLBCL)] and 15 healthy individuals were determined using a quantitative PCR assay for POLR2 gene and, in addition, for NPM-ALK fusion gene in ALCL patients. Wilcoxon rank sum test was used to compare plasma levels among different patient subgroups and controls and to analyze relationship between levels of cfDNA and clinical characteristics. Results: Levels of cfDNA in lymphoma patients were significantly higher compared with controls (p<0.0001). CfDNA was associated with median age (p=0.01) in HL, and with stage in ALCL (p=0.01). In HL patients high cfDNA levels were correlated with poor prognosis (p=0.03). In ALCL we found that most of the cfDNA (77%) was non-tumor DNA. Conclusion: level of plasma cfDNA might constitute an important non-invasive tool at diagnosis in lymphoma patients' management; in particular in patients with HL, cfDNA seems to be a promising prognostic biomarker. PMID:23678368

  2. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    PubMed

    Brödel, Andreas K; Sonnabend, Andrei; Roberts, Lisa O; Stech, Marlitt; Wüstenhagen, Doreen A; Kubick, Stefan

    2013-01-01

    Internal ribosome entry site (IRES) elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems. PMID

  3. Different dimerisation mode for TLR4 upon endosomal acidification?

    PubMed Central

    Gangloff, Monique

    2012-01-01

    TLR4 is unique among pathogen-recognition receptors in that it initiates different pathways in different cellular locations. Binding of a bridging factor, Mal, allows recruitment of an adapter protein, MyD88, at the plasma membrane, which leads to the production of proinflammatory cytokines. Upon internalization, TLR4 uses a different bridging factor, TRAM, to activate a MyD88-independent pathway that results in type I interferon expression. Interestingly, both Mal and TRAM are localised initially at the plasma membrane. In this Opinion, I suggest a possible mechanism by which endosomal acidification triggers the differential adaptor usage of TLR4. I discuss the evidence of the pH sensitivity of TLR4 and propose a new dimerisation mode for TLR4 based on the crystal structure of the related receptor TLR3 bound to its ligand, double-stranded RNA. PMID:22196451

  4. Different dimerisation mode for TLR4 upon endosomal acidification?

    PubMed

    Gangloff, Monique

    2012-03-01

    TLR4 is unique among pathogen-recognition receptors in that it initiates different pathways in different cellular locations. Binding of a bridging factor, Mal, allows recruitment of an adapter protein, MyD88, at the plasma membrane, which leads to the production of proinflammatory cytokines. Upon internalization, TLR4 uses a different bridging factor, TRAM, to activate a MyD88-independent pathway that results in type I interferon expression. Interestingly, both Mal and TRAM are localised initially at the plasma membrane. In this Opinion, I suggest a possible mechanism by which endosomal acidification triggers the differential adaptor usage of TLR4. I discuss the evidence of the pH sensitivity of TLR4 and propose a new dimerisation mode for TLR4 based on the crystal structure of the related receptor TLR3 bound to its ligand, double-stranded RNA. PMID:22196451

  5. Loss of the Sec1/Munc18-family proteins VPS-33.2 and VPS-33.1 bypasses a block in endosome maturation in Caenorhabditis elegans

    PubMed Central

    Solinger, Jachen A.; Spang, Anne

    2014-01-01

    The end of the life of a transport vesicle requires a complex series of tethering, docking, and fusion events. Tethering complexes play a crucial role in the recognition of membrane entities and bringing them into close opposition, thereby coordinating and controlling cellular trafficking events. Here we provide a comprehensive RNA interference analysis of the CORVET and HOPS tethering complexes in metazoans. Knockdown of CORVET components promoted RAB-7 recruitment to subapical membranes, whereas in HOPS knockdowns, RAB-5 was found also on membrane structures close to the cell center, indicating the RAB conversion might be impaired in the absence of these tethering complexes. Unlike in yeast, metazoans have two VPS33 homologues, which are Sec1/Munc18 (SM)-family proteins involved in the regulation of membrane fusion. We assume that in wild type, each tethering complex contains a specific SM protein but that they may be able to substitute for each other in case of absence of the other. Of importance, knockdown of both SM proteins allowed bypass of the endosome maturation block in sand-1 mutants. We propose a model in which the SM proteins in tethering complexes are required for coordinated flux of material through the endosomal system. PMID:25273556

  6. Relationships between Cell-Free DNA and Serum Analytes in First and Second Trimesters of Pregnancy

    PubMed Central

    Vora, Neeta L.; Johnson, Kirby L.; Lambert-Messerlian, Geralyn; Tighiouart, Hocine; Peter, Inga; Urato, Adam C.; Bianchi, Diana W.

    2010-01-01

    Objective Circulating cell-free DNA and maternal serum analytes are indicators of fetal and placental condition. Little is known about the relationship of these noninvasive markers to each other, particularly in the first trimester. Our goal was to assess the relationship between first and second trimester cell-free DNA levels and maternal serum screening markers. Methods First and second trimester residual maternal serum samples from 50 women were obtained. First trimester (pregnancy-associated plasma protein A [PAPP-A] and β-hCG), and second trimester serum analytes (β-hCG, alpha-fetoprotein [AFP], unconjugated estriol and inhibin A) had been measured at the time of sample receipt. All fetuses were male, as confirmed by birth records. Cell-free DNA was extracted and measured by real-time quantitative polymerase chain reaction (PCR) amplification using glyceraldehyde phosphate dehydrogenase (GAPDH) and DYS1 as markers of total DNA and fetal DNA, respectively. Determination of linear associations between first and second trimester serum markers and cell-free DNA levels using Pearson correlations was performed. Results Statistically significant correlations between first trimester PAPP-A multiples of the median (MoMs) and both total (r=0.36, p=0.016) and fetal (r= 0.41, p=0.006) DNA in the first trimester were observed. There were no significant correlations between first trimester serum hCG or any second trimester serum marker with DNA levels. Conclusions Correlation between serum PAPP-A and first trimester circulating cell-free fetal and total DNA levels is a novel finding. PAPP-A is a glycoprotein of placental origin, and its correlation to cell-free fetal DNA in maternal serum suggests a common tissue origin, through apoptosis of placental cells. However, since PAPP-A and cell-free DNA were only marginally correlated and cell-free DNA can be reliably detected in the first trimester, the addition of cell-free DNA to serum screening strategies may be helpful in

  7. Expression Optimization and Inducible Negative Feedback in Cell-Free Systems

    SciTech Connect

    Karig, David K; Iyer, Sukanya; Simpson, Michael L; Doktycz, Mitchel John

    2012-01-01

    Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells. Cell-free approaches, on the other hand, offer simpler and more flexible contexts, but few synthetic systems based on cell-free protein expression have been constructed. Here, we evaluate cell-free regulatory systems based on T7 promoter driven expression, and we demonstrate negative feedback, an essential motif in many natural and engineered systems. First, we characterize variants of TetR and LacI repressible T7 promoters in a cell-free context and examine sequence elements that determine expression efficiency. Then, we explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in E. coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom up fashion.

  8. Rapid cell-free forward engineering of novel genetic ring oscillators

    PubMed Central

    Niederholtmeyer, Henrike; Sun, Zachary Z; Hori, Yutaka; Yeung, Enoch; Verpoorte, Amanda; Murray, Richard M; Maerkl, Sebastian J

    2015-01-01

    While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the ‘repressilator’, a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior. DOI: http://dx.doi.org/10.7554/eLife.09771.001 PMID:26430766

  9. Content of intrinsic disorder influences the outcome of cell-free protein synthesis

    PubMed Central

    Tokmakov, Alexander A.; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

    2015-01-01

    Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis. PMID:26359642

  10. Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination.

    PubMed

    Boland, Coilín; Li, Dianfan; Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-12-01

    Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved. PMID:25012698