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Sample records for organelle morphology examination

  1. Motile sperm organelle morphology examination (MSOME) and sperm head vacuoles: state of the art in 2013.

    PubMed

    Perdrix, Anne; Rives, Nathalie

    2013-01-01

    BACKGROUND Approximately 10 years after the first publication introducing the motile sperm organelle morphology examination (MSOME), many questions remained about sperm vacuoles: frequency, size, localization, mode of occurrence, biological significance and impact on male fertility potential. Many studies have tried to characterize sperm vacuoles, to determine the sperm abnormalities possibly associated with vacuoles, to test the diagnostic value of MSOME for male infertility or to question the benefits of intracytoplasmic morphologically selected sperm injection (IMSI). METHODS We searched PubMed for articles in the English language published in 2001-2012 regarding human sperm head vacuoles, MSOME and IMSI. RESULTS A bibliographic analysis revealed consensus for the following findings: (i) sperm vacuoles appeared frequently, often multiple and preferentially anterior; (ii) sperm vacuoles and sperm chromatin immaturity have been associated, particularly in the case of large vacuoles; (iii) teratozoospermia was a preferred indication of MSOME and IMSI. CONCLUSION The high-magnification system appears to be a powerful method to improve our understanding of human spermatozoa. However, its clinical use remains unclear in the fields of male infertility diagnosis and assisted reproduction techniques (ARTs). PMID:23825157

  2. Novel computer vision algorithm for the reliable analysis of organelle morphology in whole cell 3D images--A pilot study for the quantitative evaluation of mitochondrial fragmentation in amyotrophic lateral sclerosis.

    PubMed

    Lautenschläger, Janin; Lautenschläger, Christian; Tadic, Vedrana; Süße, Herbert; Ortmann, Wolfgang; Denzler, Joachim; Stallmach, Andreas; Witte, Otto W; Grosskreutz, Julian

    2015-11-01

    The function of intact organelles, whether mitochondria, Golgi apparatus or endoplasmic reticulum (ER), relies on their proper morphological organization. It is recognized that disturbances of organelle morphology are early events in disease manifestation, but reliable and quantitative detection of organelle morphology is difficult and time-consuming. Here we present a novel computer vision algorithm for the assessment of organelle morphology in whole cell 3D images. The algorithm allows the numerical and quantitative description of organelle structures, including total number and length of segments, cell and nucleus area/volume as well as novel texture parameters like lacunarity and fractal dimension. Applying the algorithm we performed a pilot study in cultured motor neurons from transgenic G93A hSOD1 mice, a model of human familial amyotrophic lateral sclerosis. In the presence of the mutated SOD1 and upon excitotoxic treatment with kainate we demonstrate a clear fragmentation of the mitochondrial network, with an increase in the number of mitochondrial segments and a reduction in the length of mitochondria. Histogram analyses show a reduced number of tubular mitochondria and an increased number of small mitochondrial segments. The computer vision algorithm for the evaluation of organelle morphology allows an objective assessment of disease-related organelle phenotypes with greatly reduced examiner bias and will aid the evaluation of novel therapeutic strategies on a cellular level. PMID:26440825

  3. Structure-Guided Mutations in the Terminal Organelle Protein MG491 Cause Major Motility and Morphologic Alterations on Mycoplasma genitalium.

    PubMed

    Martinelli, Luca; García-Morales, Luis; Querol, Enrique; Piñol, Jaume; Fita, Ignacio; Calisto, Bárbara M

    2016-04-01

    The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle. PMID:27082435

  4. Structure-Guided Mutations in the Terminal Organelle Protein MG491 Cause Major Motility and Morphologic Alterations on Mycoplasma genitalium

    PubMed Central

    Querol, Enrique; Piñol, Jaume; Fita, Ignacio; Calisto, Bárbara M.

    2016-01-01

    The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle. PMID:27082435

  5. Phenomenology based multiscale models as tools to understand cell membrane and organelle morphologies

    PubMed Central

    Ramakrishnan, N.; Radhakrishnan, Ravi

    2016-01-01

    An intriguing question in cell biology is “how do cells regulate their shape?” It is commonly believed that the observed cellular morphologies are a result of the complex interaction among the lipid molecules (constituting the cell membrane), and with a number of other macromolecules, such as proteins. It is also believed that the common biophysical processes essential for the functioning of a cell also play an important role in cellular morphogenesis. At the cellular scale—where typical dimensions are in the order of micrometers—the effects arising from the molecular scale can either be modeled as equilibrium or non-equilibrium processes. In this chapter, we discuss the dynamically triangulated Monte Carlo technique to model and simulate membrane morphologies at the cellular scale, which in turn can be used to investigate several questions related to shape regulation in cells. In particular, we focus on two specific problems within the framework of isotropic and anisotropic elasticity theories: namely, (i) the origin of complex, physiologically relevant, membrane shapes due to the interaction of the membrane with curvature remodeling proteins, and (ii) the genesis of steady state cellular shapes due to the action of non-equilibrium forces that are generated by the fission and fusion of transport vesicles and by the binding and unbinding of proteins from the parent membrane. PMID:27087801

  6. Droplet organelles?

    PubMed

    Courchaine, Edward M; Lu, Alice; Neugebauer, Karla M

    2016-08-01

    Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology. PMID:27357569

  7. The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology.

    PubMed

    Papatheodorou, Panagiotis; Domańska, Grazyna; Oxle, Marius; Mathieu, Johannes; Selchow, Olaf; Kenny, Brendan; Rassow, Joachim

    2006-04-01

    Enteropathogenic Escherichia coli (EPEC) is a human intestinal pathogen and a major cause of diarrhoea, particularly among infants in developing countries. EPEC target the Map and EspF multifunctional effector proteins to host mitochondria - organelles that play crucial roles in regulating cellular processes such as programmed cell death (apoptosis). While both molecules interfere with the organelles ability to maintain a membrane potential, EspF plays the predominant role and is responsible for triggering cell death. To learn more about the Map-mitochondria interaction, we studied Map localization to mitochondria with purified mitochondria (from mammalian and yeast cells) and within intact yeast. This revealed that (i) Map targeting is dependent on the predicted N-terminal mitochondrial targeting sequence, (ii) the N-terminal 44 residues are sufficient to target proteins to mitochondria and (iii) Map import involves the mitochondrial outer membrane translocase (Tom22 and Tom40), the mitochondrial membrane potential, and the matrix chaperone, mtHsp70. These results are consistent with Map import into the mitochondria matrix via the classical import mechanism. As all known, Map-associated phenotypes in mammalian cells are independent of mitochondrial targeting, this may indicate that import serves as a mechanism to remove Map from the cytoplasm thereby regulating cytoplasmic function. Intriguingly, Map, but not EspF, alters mitochondrial morphology with deletion analysis revealing important roles for residues 101-152. Changes in mitochondrial morphology have been linked to alterations in the ability of these organelles to regulate cellular processes providing a possible additional role for Map import into mitochondria. PMID:16548893

  8. Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology1

    PubMed Central

    Teardo, Enrico; Carraretto, Luca; De Bortoli, Sara; Costa, Alex; Behera, Smrutisanjita; Wagner, Richard; Lo Schiavo, Fiorella; Szabo, Ildiko

    2015-01-01

    Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view. PMID:25367859

  9. High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

    PubMed

    Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

    2015-06-16

    Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states. PMID:26005782

  10. Fluorescent Proteins in Cellular Organelles: Serious Pitfalls and Some Solutions

    PubMed Central

    Costantini, Lindsey M.

    2013-01-01

    Fluorescent proteins (FPs) have been powerful tools for cell biologists for over 15 years. The large variety of FPs available rarely comes with an instruction manual or a warning label. The potential pitfalls of the use of FPs in cellular organelles represent a significant concern for investigators. FPs generally did not evolve in the often distinctive physicochemical environments of subcellular organelles. In organelles, FPs can misfold, go dark, and even distort organelle morphology. In this minireview, we describe the issues associated with FPs in organelles and provide solutions to enable investigators to better exploit FP technology in cells. PMID:23971632

  11. Rapid method for the examination of platelet morphology.

    PubMed

    Yamashiro, S; Bast, T; Basrur, P K

    1983-05-01

    A simple, rapid method is described for preparing platelets of domestic animals for light and electron microscopic examinations. The method involves the isolation of the platelet rich plasma (PRP) by centrifugation of the whole blood for five minutes followed by recentrifugation of PRP in a Wintrobe tube at 4500 rpm for 20 minutes to separate the blood components into four distinct layers composed exclusively of plasma, leucocytes, platelets and erythrocytes. A short fixation (20 minutes) by replacing the plasma layer with glutaraldehyde before breaking the bottom of the tube with a glass cutter allows the cellular contents to be gently pushed out of the tube in the form of a cylindrical semisolid pellet, with a wooden applicator stick. A further three hour fixation of the pellet in fresh glutaraldehyde before cutting out the middle layer for routine processing for light and electron microscopy provides pure preparation of platelets with a minimum of morphological distortion. PMID:6878891

  12. Organelle morphogenesis by active membrane remodeling

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, N.; Ipsen, John H.; Rao, Madan; Kumar, P. B. Sunil

    Intracellular organelles are subject to a steady flux of lipids and proteins through active, energy consuming transport processes. Active fission and fusion are promoted by GTPases, e.g., Arf-Coatamer and the Rab-Snare complexes, which both sense and generate local membrane curvature. Here we investigate through Dynamical Triangulation Monte Carlo simulations, the role that these active processes play in determining the morphology and compositional segregation in closed membranes. Our results suggest that the ramified morphologies of organelles observed in-vivo are a consequence of driven nonequilibrium processes rather than equilibrium forces.

  13. Examining the Underlying Dimensions of Morphological Awareness and Vocabulary Knowledge

    ERIC Educational Resources Information Center

    Spencer, Mercedes; Muse, Andrea; Wagner, Richard K.; Foorman, Barbara; Petscher, Yaacov; Schatschneider, Christopher; Tighe, Elizabeth L.; Bishop, M. Denise

    2015-01-01

    We report results from two studies on the underlying dimensions of morphological awareness and vocabulary knowledge in elementary-aged children. In Study 1, 99 fourth-grade students were given multiple measures of morphological awareness and vocabulary. A single factor accounted for individual differences in all morphology and vocabulary…

  14. Morphological and ultrastructural examination of senescence in Morchella elata.

    PubMed

    He, Peixin; Cai, Yingli; Liu, Sumeng; Han, Li; Huang, Lina; Liu, Wei

    2015-11-01

    In recent years, the artificial cultivation of Morchella mushrooms that belong to Elata Clade, including Morchella elata, has been developed rapidly in China. However, the prominent problem of spawn aging has been frustrating the morel farming. In this paper, aging in M. elata was achieved from 12 to 17 subcultures and lifespan of 1536-2256 h by successive subculturing. The lifespan can be roughly divided into juvenile phase and senescent phase with respect to the mycelia linear growth rate. After a certain period of rapid growth with almost constant rate at the juvenile phase, the isolate entered the senescent phase characterized by slow down of mycelia growth, producing pigments ahead of time and final death of the apical hyphae. The period of the senescent phase was definitely 240-288 h; while that of the juvenile phase was diverse relying on different isolates. Moreover, microscopic study showed that angles between the leading and primary hyphae increased constantly with aging. In senesced hyphal cells of M. elata, the typical characteristics of autophagy (enlargement of vacuoles and existence of organelles sequestrated lysosomes) and apoptosis (condensation of the cytoplasm and nuclear and plasmolysis) were observed. In addition, in the final stage of senescence, the apical hyphae collapsed with the plasma membrane and all the cellular organelles disrupted, indicated a necrotic mode of cell death. Taken together, these data revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The characterization and molecular mechanism of autophagy, apoptosis and necrosis need further study and the systematic study of morel aging will be beneficial for the healthy development of morel farming. PMID:26281756

  15. Podosomes: Multipurpose organelles?

    PubMed

    Veillat, Veronique; Spuul, Pirjo; Daubon, Thomas; Egaña, Isabel; Kramer, Ijsbrand; Génot, Elisabeth

    2015-08-01

    Thirty years of research have accumulated ample evidence that podosome clusters qualify as genuine cellular organelles that are being found in more and more cell types. A podosome is a dynamic actin-based and membrane-bound microdomain and the organelle consists in an interconnected network of such basic units, forming a cytoskeletal superstructure linked to the plasma membrane. At this strategic location, podosomes are privileged sites of interactions with the pericellular environment that regulates their formation, density, lifetime, distribution, architecture and functioning. Actin polymerization is the driving force behind most podosome characteristics. In contrast to classical organelles, podosomes are not vital at the cell level but rather serve diverse and often intricate functions of which adhesion, matrix degradation and substrate sensing are the most established. These capabilities involve specific molecules, depend on podosome organization and may vary according to the cell type in which they form. Podosome-associated diseases manifest by loss or gain of podosome functions and include genetic diseases affecting podosome components and various cancers where tumor cells ectopically express podosome equivalents (invadopodia). PMID:26028292

  16. Correlative video-light-electron microscopy of mobile organelles.

    PubMed

    Beznoussenko, Galina V; Mironov, Alexander A

    2015-01-01

    Correlative microscopy is a method when for the analysis of the very same cell or tissue area, several different methods of light microscopy (LM) and then electron microscopy (EM) are used consecutively. The combination of LM and EM allows researchers to study phenomena at a global scale and then to look for unique or rare events for their subsequent EM examination. Unfortunately, the observation of living cells under EM is still impossible. LM provides the possibility to examine quickly many live cells, whereas EM provides the high level of resolution. On the other side, the final goal of any morphological analysis of a biological sample, whether it is an organism, organ, tissue, cell, organelle, or molecule, is to get an averaged three-dimensional model of the structure examined and to determine the chemical composition of it. This chapter describes the methodology of imaging with the help of CVLEM. The guidelines presented herein enable researchers to analyze structure of organelles and to obtain the three-dimensional model of the structure examined, and in particular rare events captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by EM. PMID:25702127

  17. Morphological Examination of the Obturator Notch and Canal in Cervidae

    PubMed Central

    TAE, Hyun-Jin; PARK, Byung-Yong; KIM, In-Shik; AHN, Dongchoon

    2014-01-01

    ABSTRACT The purpose of this study was to investigate gross findings of the obturator notch (ON) and obturator canal (OC) in Cervidae. A total of 183 pelvic girdles from 26 species of deer were examined, and the obturator canal (OC) was classified into 4 types based on the degree of separation from the obturator foramen (OF). The deep ON was observed primarily in the subfamily Capreolinae (telemetacarpal deer). The small bony OC was frequently observed in Hydropotes inermis, Mazama gouazoubira and Ozotoceros bezoarticus. A canal without a tubercle or bony bridge structure was mainly observed in the subfamily Cervinae (plesiometacarpal deer). These results suggest that the deep ONs or the OCs separated by bony structures are more common in telemetacarpal rather than plesiometacarpal deer. PMID:24430657

  18. [Morphologic examination of the urothelium after irradiation with laser energy].

    PubMed

    Hofmann, R; Hartung, R; Geissdörfer, K; Ascherl, R; Erhardt, W; Schmidt-Kloiber, H; Reichel, E; Schöffmann, H

    1987-01-01

    The energy of a Nd-YAG laser (1,064 nm wave length, 8 ns pulse duration) was used to irradiate the urothelium of the ureter or bladder and kidney parenchyma in pigs. Single pulse energy was 50-120 mJ with a 20-Hz repetition rate. The horizontal laser beam was reflected 90 degrees down by a 100% mirror and with a specially designed apparatus focussed on the surface of the tissue. Laser light from a quartz glass fiber was also focussed directly onto the tissue. Urothelium and kidney parenchyma were irradiated in 7 pigs. Tissue samples were examined histologically and raster electron microscopically 2, 4, 8 and 12 days after irradiation. No macroscopic lesion could be found. Maximum energy caused a small cone of 40 micron depth. No thermic effects or necrosis resulted, so that no harm is to be expected with unintentional irradiation. PMID:2896407

  19. Proteomics of Saccharomyces cerevisiae Organelles*

    PubMed Central

    Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan

    2010-01-01

    Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. PMID:19955081

  20. Diverse Bacterial Microcompartment Organelles

    PubMed Central

    Chowdhury, Chiranjit; Sinha, Sharmistha; Chun, Sunny; Yeates, Todd O.

    2014-01-01

    SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles. PMID:25184561

  1. The Plant Organelles Database 3 (PODB3) update 2014: integrating electron micrographs and new options for plant organelle research.

    PubMed

    Mano, Shoji; Nakamura, Takanori; Kondo, Maki; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nagatani, Akira; Nishimura, Mikio

    2014-01-01

    The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli. In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions of data from all plant researchers to enhance the utility and comprehensiveness of PODB3. PMID:24092884

  2. Cell Biology of Prokaryotic Organelles

    PubMed Central

    Murat, Dorothee; Byrne, Meghan; Komeili, Arash

    2010-01-01

    Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-bounded and lipid-bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance. PMID:20739411

  3. Compartmentalization and Organelle Formation in Bacteria

    PubMed Central

    Cornejo, Elias; Abreu, Nicole; Komeili, Arash

    2015-01-01

    A number of bacterial species rely on compartmentalization to gain specific functionalities that will provide them with a selective advantage. Here, we will highlight several of these modes of bacterial compartmentalization with an eye towards describing the mechanisms of their formation and their evolutionary origins. Spore formation in Bacillus subtilis, outer membrane biogenesis in Gram-negative bacteria and protein diffusion barriers of Caulobacter crescentus will be used to demonstrate the physical, chemical and compositional remodeling events that lead to compartmentalization. In addition, magnetosomes and carboxysomes will serve as models to examine the interplay between cytoskeletal systems and the subcellular positioning of organelles. PMID:24440431

  4. Cytoplasmic dynein is a minus end-directed motor for membranous organelles

    SciTech Connect

    Schroer, T.A.; Steuer, E.R.; Sheetz, M.P.

    1989-03-24

    The role of cytoplasmic dynein in microtubule-based organelle transport was examined using a reconstituted assay developed from chick embryo fibroblasts. Factors present in a high-speed cytosol caused the movement of purified organelles on microtubules predominantly in the minus end direction. Inactivation of cytoplasmic dynein in the high-speed cytosol by vanadate-mediated UV photocleavage inhibited minus end-directed organelle motility by over 90%. Addition of purified cytoplasmic dynein to the inactive cytosol restored minus end-directed organelle motility, although purified cytoplasmic dynein by itself did not support organelle movement. We propose that cytoplasmic dynein is the motor for minus end-directed organelle movement, but that additional cytosolic factors are also required to produce organelle motility.

  5. The function of genomes in bioenergetic organelles.

    PubMed Central

    Allen, John F

    2003-01-01

    Mitochondria and chloroplasts are energy-transducing organelles of the cytoplasm of eukaryotic cells. They originated as bacterial symbionts whose host cells acquired respiration from the precursor of the mitochondrion, and oxygenic photosynthesis from the precursor of the chloroplast. The host cells also acquired genetic information from their symbionts, eventually incorporating much of it into their own genomes. Genes of the eukaryotic cell nucleus now encode most mitochondrial and chloroplast proteins. Genes are copied and moved between cellular compartments with relative ease, and there is no obvious obstacle to successful import of any protein precursor from the cytosol. So why are any genes at all retained in cytoplasmic organelles? One proposal is that these small but functional genomes provide a location for genes that is close to, and in the same compartment as, their gene products. This co-location facilitates rapid and direct regulatory coupling. Redox control of synthesis de novo is put forward as the common property of those proteins that must be encoded and synthesized within mitochondria and chloroplasts. This testable hypothesis is termed CORR, for co-location for redox regulation. Principles, predictions and consequences of CORR are examined in the context of competing hypotheses and current evidence. PMID:12594916

  6. Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes

    PubMed Central

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles. PMID:17375895

  7. Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

    NASA Astrophysics Data System (ADS)

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

  8. Mechanisms of Polarized Organelle Distribution in Neurons

    PubMed Central

    Britt, Dylan J.; Farías, Ginny G.; Guardia, Carlos M.; Bonifacino, Juan S.

    2016-01-01

    Neurons are highly polarized cells exhibiting axonal and somatodendritic domains with distinct complements of cytoplasmic organelles. Although some organelles are widely distributed throughout the neuronal cytoplasm, others are segregated to either the axonal or somatodendritic domains. Recent findings show that organelle segregation is largely established at a pre-axonal exclusion zone (PAEZ) within the axon hillock. Polarized sorting of cytoplasmic organelles at the PAEZ is proposed to depend mainly on their selective association with different microtubule motors and, in turn, with distinct microtubule arrays. Somatodendritic organelles that escape sorting at the PAEZ can be subsequently retrieved at the axon initial segment (AIS) by a microtubule- and/or actin-based mechanism. Dynamic sorting along the PAEZ-AIS continuum can thus explain the polarized distribution of cytoplasmic organelles between the axonal and somatodendritic domains. PMID:27065809

  9. Organelle size control - increasing vacuole content activates SNAREs to augment organelle volume through homotypic fusion.

    PubMed

    Desfougères, Yann; Neumann, Heinz; Mayer, Andreas

    2016-07-15

    Cells control the size of their compartments relative to cell volume, but there is also size control within each organelle. Yeast vacuoles neither burst nor do they collapse into a ruffled morphology, indicating that the volume of the organellar envelope is adjusted to the amount of content. It is poorly understood how this adjustment is achieved. We show that the accumulating content of yeast vacuoles activates fusion of other vacuoles, thus increasing the volume-to-surface ratio. Synthesis of the dominant compound stored inside vacuoles, polyphosphate, stimulates binding of the chaperone Sec18/NSF to vacuolar SNAREs, which activates them and triggers fusion. SNAREs can only be activated by lumenal, not cytosolic, polyphosphate (polyP). Control of lumenal polyP over SNARE activation in the cytosol requires the cytosolic cyclin-dependent kinase Pho80-Pho85 and the R-SNARE Nyv1. These results suggest that cells can adapt the volume of vacuoles to their content through feedback from the vacuole lumen to the SNAREs on the cytosolic surface of the organelle. PMID:27252384

  10. Preparation of Mealybugs (Hemiptera: Pseudococcidae) for Genetic Characterization and Morphological Examination.

    PubMed

    Bahder, B W; Bollinger, M L; Sudarshana, M R; Zalom, F G

    2015-01-01

    Mealybugs (Hemiptera: Pseudococcidae) are economically significant agricultural pests on many different crops. Because of their small size and lack of easily visible characters for identification, determination of their taxonomic status is difficult and requires technical competency to prepare a slide-mounted specimen. The standard mounting technique does not allow for analysis of the genome of the specimen. Conversely, preparatory techniques for genetic analysis of mealybugs cause either loss of the entire individual or physical damage that can make morphology-based identification difficult. This study describes a simple protocol that does not impact physical integrity of the specimen for fixation and microscopic examination yet enables simultaneous DNA extraction for DNA-based identification of four mealybug species. All species prepared yielded high quality slide mounts, identified as Planococcus citri Risso, Pseudococcus viburni Signoret, Rhizoecus kondonis Kuwana, or Rhizoecus californicus Ferris. DNA extracted in this manner had higher purity and yield in the final eluate than in samples extracted using standard methods. DNA extracted was successfully amplified by polymerase chain reaction using primers for the cytochrome oxidase I gene and subsequently sequenced for all specimens. This protocol is likely to be applicable to other Hemiptera taxa that are preserved by slide mounting, allowing for both the preparation of a high-quality voucher specimen for morphological identification and simultaneous analysis of DNA for the same specimen. The methods used are technically less challenging than current standard procedures. PMID:26198869

  11. Single-prolonged stress induce different change in the cell organelle of the hippocampal cells: A study of ultrastructure.

    PubMed

    Wan, JunLai; Liu, Dongjuan; Zhang, Jie; Shi, Yuxiu; Han, Fang

    2016-01-01

    MRI studies have revealed structural and functional changes in the hippocampus of post-traumatic stress disorder (PTSD) patients. Previous studies conducted by us in a PTSD animal model found that single prolonged stress (SPS) induced abnormal morphological changes in hippocampal cells. The effects of SPS on cellular organelles of the hippocampal neurons remain unknown; however, these changes have been involved in SPS-induced abnormal hippocampal function. The aim of the present study is to examine ultrastructural changes in cellular organelles, including the lysosomes, mitochondria (Mit), Golgi apparatus, and endoplasmic reticulum (ER), following SPS exposure using transmission electron microscopy, enzyme histochemistry, and enzyme cytochemistry. First, morphological changes of the hippocampal cells and ultrastructural changes in cellular organelles, including lysosomes, ER, and Mit-induced by SPS were observed. Results from histo- and cytochemistry demonstrated that the Mit marker enzyme, cytochrome c oxidase (COX), and the lysosomal enzyme acid phosphatase, (ACP), increased following exposure to SPS. SPS induced COX release from Mit and led to a wider distribution of ACP in round lysosomes, NLY, and the Golgi. In addition, we found that SPS increased the presence of autophagosomes and induced changes in the autophagy-related protein, Beclin. These results indicated the differential effects of SPS on cellular organelles, that is, a positive effect on lysosomes as well as a negative effect on the Mit and ER. Increased lysosomal function may serve as protection against SPS-induced cell damage. Structural changes in the Mit and ER may be involved in SPS-induced disorders of energy metabolism and protein synthesis and export. PMID:26589383

  12. GOBASE: an organelle genome database

    PubMed Central

    O’Brien, Emmet A.; Zhang, Yue; Wang, Eric; Marie, Veronique; Badejoko, Wole; Lang, B. Franz; Burger, Gertraud

    2009-01-01

    The organelle genome database GOBASE, now in its 21st release (June 2008), contains all published mitochondrion-encoded sequences (∼913 000) and chloroplast-encoded sequences (∼250 000) from a wide range of eukaryotic taxa. For all sequences, information on related genes, exons, introns, gene products and taxonomy is available, as well as selected genome maps and RNA secondary structures. Recent major enhancements to database functionality include: (i) addition of an interface for RNA editing data, with substitutions, insertions and deletions displayed using multiple alignments; (ii) addition of medically relevant information, such as haplotypes, SNPs and associated disease states, to human mitochondrial sequence data; (iii) addition of fully reannotated genome sequences for Escherichia coli and Nostoc sp., for reference and comparison; and (iv) a number of interface enhancements, such as the availability of both genomic and gene-coding sequence downloads, and a more sophisticated literature reference search functionality with links to PubMed where available. Future projects include the transfer of GOBASE features to NCBI/GenBank, allowing long-term preservation of accumulated expert information. The GOBASE database can be found at http://gobase.bcm.umontreal.ca/. Queries about custom and large-scale data retrievals should be addressed to gobase@bch.umontreal.ca. PMID:18953030

  13. Laser Surgery: Organelles to Organs

    NASA Astrophysics Data System (ADS)

    Berns, Michael W. D.

    1998-03-01

    Understanding the physical mechanisms of light interaction with biological molecules and structure has resulted in the application of photons to a wide variety of biological and medical problems ranging from subcellular manipulation/surgery to the successful diagnosis and treatment of human disease. Mechanisms such as the generation and transfer of heat, light-driven chemistry (photochemistry), high peak power acoustic-mechanical effects, high photon-energy induced bond breaking, and optical induced forces through momentum transfer, are being utilized in single cells at the microscopic (submicron and micron) level as well as the macroscopic level in tissue and organs. At the subcellular level, focused laser microbeams (laser scissors and tweezers) are being used to cut and move chromosomes to study genetic function as well as to clone and sequence genes. The same laser technology is being used to manipulate a variety of cell organelles such as mitochondria, cell membranes, nucleoli, and mitochondria in order to study their functions in cell physiology. At the tissue level, lasers are being used to diagnose and treat malignancy in combination with light-activated drugs, to ablate cornea and other hard and soft tissue through ultraviolet photoablation, to selectively ablate structures within the skin under controlled heating/cooling conditions, and to differentiate normal from abnormal tissue using a variety of fluorescence detection and light scattering techniques.

  14. Endosymbiotic theory for organelle origins.

    PubMed

    Zimorski, Verena; Ku, Chuan; Martin, William F; Gould, Sven B

    2014-12-01

    Endosymbiotic theory goes back over 100 years. It explains the similarity of chloroplasts and mitochondria to free-living prokaryotes by suggesting that the organelles arose from prokaryotes through (endo)symbiosis. Gene trees provide important evidence in favour of symbiotic theory at a coarse-grained level, but the finer we get into the details of branches in trees containing dozens or hundreds of taxa, the more equivocal evidence for endosymbiotic events sometimes becomes. It seems that either the interpretation of some endosymbiotic events are wrong, or something is wrong with the interpretations of some gene trees having many leaves. There is a need for evidence that is independent of gene trees and that can help outline the course of symbiosis in eukaryote evolution. Protein import is the strongest evidence we have for the single origin of chloroplasts and mitochondria. It is probably also the strongest evidence we have to sort out the number and nature of secondary endosymbiotic events that have occurred in evolution involving the red plastid lineage. If we relax our interpretation of individual gene trees, endosymbiotic theory can tell us a lot. PMID:25306530

  15. Examination of changes in the morphology of lignocellulosic fibers treated with e-beam irradiation

    NASA Astrophysics Data System (ADS)

    Gryczka, Urszula; Migdal, Wojciech; Chmielewska, Dagmara; Antoniak, Magdalena; Kaszuwara, Waldemar; Jastrzebska, Agnieszka; Olszyna, Andrzej

    2014-01-01

    Ionizing radiation was applied as a substrate pretreatment method in the process of bioethanol production. The aim of the presented work was to determine the changes in the morphology of willow plant fibers caused by the interaction of a high energy electron beam with lignocellulosic biomass. The microstructure was examined with a scanning electron microscope and X-ray computer microtomography. Additionally, sorption analysis was carried out in order to determine specific surface area and porosity. The analysis carried out after the treatment of lignocellulose with an electron beam indicated destruction of cell walls, observed as a decrease in the smoothness and an increase in the roughness of the surface of the fibers. The changes in surface texture and fiber integrity affected the specific surface area and porosity of the tested samples. The specific surface area, the total volume of pores and the average pore diameter were calculated based on the isotherms of nitrogen sorption. The increase in the specific surface area was observed to occur simultaneously with the increase in the average diameter of pores.

  16. Light and Electron Microscopy Methods for Examination of Cochlear Morphology in Mouse Models of Deafness.

    PubMed

    Parker, Andrew; Chessum, Lauren; Mburu, Philomena; Sanderson, Jeremy; Bowl, Michael R

    2016-01-01

    Mice are an invaluable model organism for the study of auditory function. Even though there are differences in size and frequency response, the anatomy and physiology of the mouse and human ear are remarkably similar. In addition, the tools available for genetic manipulation in the mouse have enabled the generation of models carrying mutations in orthologous human deafness-causing genes, helping to validate these lesions and assess their functional consequence. Reciprocally, novel gene mutations discovered to cause auditory deficits in the mouse highlight potential new loci for human hearing loss, and expand our basic knowledge of the mechanisms and pathways important for the function of the mammalian ear. Microscopy and imaging are invaluable techniques that allow detailed characterization of cochlear pathologies associated with particular gene mutations. However, the highly organized, delicate, and intricate structures responsible for transduction of sound waves into nerve impulses are encapsulated in one of the hardest bones in the body - the temporal bone. This makes sample preparation without damage to the soft tissue, be it from dissection or processing, somewhat challenging. Fortunately, there are numerous methods for achieving high-quality images of the mouse cochlea. Reported in this article are a selection of sample preparation and imaging techniques that can be used routinely to assess cochlear morphology. Several protocols are also described for immunodetection of proteins in the cochlea. In addition, the advantages and disadvantages between different imaging platforms and their suitability for different types of microscopic examination are highlighted. © 2016 by John Wiley & Sons, Inc. PMID:27584554

  17. Cooperative protein transport in cellular organelles

    NASA Astrophysics Data System (ADS)

    Dmitrieff, S.; Sens, P.

    2011-04-01

    Compartmentalization into biochemically distinct organelles constantly exchanging material is one of the hallmarks of eukaryotic cells. In the most naive picture of interorganelle transport driven by concentration gradients, concentration differences between organelles should relax. We determine the conditions under which cooperative transport, i.e., based on molecular recognition, allows for the existence and maintenance of distinct organelle identities. Cooperative transport is also shown to control the flux of material transiting through a compartmentalized system, dramatically increasing the transit time under high incoming flux. By including chemical processing of the transported species, we show that this property provides a strong functional advantage to a system responsible for protein maturation and sorting.

  18. Organelle interactions and possible degradation pathways visualized in high-pressure frozen algal cells.

    PubMed

    Aichinger, N; Lütz-Meindl, U

    2005-08-01

    Summary Organelle interactions, although essential for both anabolic and catabolic pathways in plant cells have not been examined in detail so far. In the present study the structure of different organelle-organelle, organelle-vesicle and organelle-membrane interactions were investigated in growing and nongrowing cells of the green alga Micrasterias denticulata by use of high pressure freeze fixation and energy filtering transmission electron microscopy. It became clear that contacts between mitochondria always occur by formation of a cone-shaped protuberance of one of the mitochondria which penetrates into its fusion partner. In the same way, structural interactions between mitochondria and mucilage vesicles and between microbodies and mucilage vesicles are achieved. Lytic compartments contact mitochondria or mucilage vesicles again by forming protuberances and by extending their contents into the respective compartment. Detached portions of mitochondria are found inside lytic compartments as a consequence of such interactions. Mitochondria found in contact with the plasma membrane reveal structural disintegration. Our study shows that interactions of organelles and vesicles are frequent events in Micrasterias cells of different ages. The interactive contacts between lytic compartments and organelles or vesicles suggest a degradation pathway different from autophagy processes described in the literature. Both the interactions between vesicles and organelles and the degradation pathways occur independently from cytoskeleton function as demonstrated by use of cytochalasin D and the microtubule inhibitor amiprophos-methyl. PMID:16159344

  19. Dynein is the motor for retrograde axonal transport of organelles

    SciTech Connect

    Schnapp, B.J.; Reese, T.S.

    1989-03-01

    Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport.

  20. Membraneless organelles: Phasing in and out

    NASA Astrophysics Data System (ADS)

    Shorter, James

    2016-06-01

    The low-complexity-protein, liquid phases of membraneless organelles have now been established to selectively partition biomolecules. The specialized microenvironment that they provide differs chemically from the surrounding medium and enables specific nucleic-acid remodelling reactions.

  1. The bacterial magnetosome: a unique prokaryotic organelle.

    PubMed

    Lower, Brian H; Bazylinski, Dennis A

    2013-01-01

    The bacterial magnetosome is a unique prokaryotic organelle comprising magnetic mineral crystals surrounded by a phospholipid bilayer. These inclusions are biomineralized by the magnetotactic bacteria which are ubiquitous, aquatic, motile microorganisms. Magnetosomes cause cells of magnetotactic bacteria to passively align and swim along the Earth's magnetic field lines, as miniature motile compass needles. These specialized compartments consist of a phospholipid bilayer membrane surrounding magnetic crystals of magnetite (Fe3O4) or greigite (Fe3S4). The morphology of these membrane-bound crystals varies by species with a nominal magnetic domain size between 35 and 120 nm. Almost all magnetotactic bacteria arrange their magnetosomes in a chain within the cell there by maximizing the magnetic dipole moment of the cell. It is presumed that magnetotactic bacteria use magnetotaxis in conjunction with chemotaxis to locate and maintain an optimum position for growth and survival based on chemistry, redox and physiology in aquatic habitats with vertical chemical concentration and redox gradients. The biosynthesis of magnetosomes is a complex process that involves several distinct steps including cytoplasmic membrane modifications, iron uptake and transport, initiation of crystallization, crystal maturation and magnetosome chain formation. While many mechanistic details remain unresolved, magnetotactic bacteria appear to contain the genetic determinants for magnetosome biomineralization within their genomes in clusters of genes that make up what is referred to as the magnetosome gene island in some species. In addition, magnetosomes contain a unique set of proteins, not present in other cellular fractions, which control the biomineralization process. Through the development of genetic systems, proteomic and genomic work, and the use of molecular and biochemical tools, the functions of a number of magnetosome membrane proteins have been demonstrated and the molecular

  2. Lysosome-related organelles: Unusual compartments become mainstream

    PubMed Central

    Marks, Michael S.; Heijnen, Harry F. G.; Raposo, Graça

    2013-01-01

    Lysosome-related organelles (LROs) comprise a group of cell type-specific subcellular compartments with unique composition, morphology and structure that share some features with endosomes and lysosomes and that function in varied processes such as pigmentation, hemostasis, lung plasticity and immunity. In recent years, studies of genetic diseases in which LRO functions are compromised have provided new insights into the mechanisms of LRO biogenesis and the regulated secretion of LRO contents. These insights have revealed previously unappreciated specialized endosomal sorting processes in all cell types, and are expanding our views of the plasticity of the endosomal and secretory systems in adapting to cell type-specific needs. PMID:23726022

  3. The different facets of organelle interplay—an overview of organelle interactions

    PubMed Central

    Schrader, Michael; Godinho, Luis F.; Costello, Joseph L.; Islinger, Markus

    2015-01-01

    Membrane-bound organelles such as mitochondria, peroxisomes, or the endoplasmic reticulum (ER) create distinct environments to promote specific cellular tasks such as ATP production, lipid breakdown, or protein export. During recent years, it has become evident that organelles are integrated into cellular networks regulating metabolism, intracellular signaling, cellular maintenance, cell fate decision, and pathogen defence. In order to facilitate such signaling events, specialized membrane regions between apposing organelles bear distinct sets of proteins to enable tethering and exchange of metabolites and signaling molecules. Such membrane associations between the mitochondria and a specialized site of the ER, the mitochondria associated-membrane (MAM), as well as between the ER and the plasma membrane (PAM) have been partially characterized at the molecular level. However, historical and recent observations imply that other organelles like peroxisomes, lysosomes, and lipid droplets might also be involved in the formation of such apposing membrane contact sites. Alternatively, reports on so-called mitochondria derived-vesicles (MDV) suggest alternative mechanisms of organelle interaction. Moreover, maintenance of cellular homeostasis requires the precise removal of aged organelles by autophagy—a process which involves the detection of ubiquitinated organelle proteins by the autophagosome membrane, representing another site of membrane associated-signaling. This review will summarize the available data on the existence and composition of organelle contact sites and the molecular specializations each site uses in order to provide a timely overview on the potential functions of organelle interaction. PMID:26442263

  4. Morphological Examination and Phylogenetic Analyses of Phycopeltis spp. (Trentepohliales, Ulvophyceae) from Tropical China

    PubMed Central

    Zhu, Huan; Zhao, Zhijuan; Xia, Shuang; Hu, Zhengyu; Liu, Guoxiang

    2015-01-01

    During an investigation of Trentepohliales (Ulvophyceae) from tropical areas in China, four species of the genus Phycopeltis were identified: Phycopeltis aurea, P. epiphyton, P. flabellata and P. prostrata. The morphological characteristics of both young and adult thalli were observed and compared. Three species (P. flabellata, P. aurea and P. epiphyton) shared a symmetrical development with dichotomously branching vegetative cells during early stages; conversely, P. prostrata had dishevelled filaments with no dichotomously branching filaments and no symmetrical development. The adult thalli of the former three species shared common morphological characteristics, such as equally dichotomous filaments, absence of erect hair and gametangia formed in prostate vegetative filaments. Phylogenetic analyses based on SSU and ITS rDNA sequences showed that the three morphologically similar species were in a clade that was sister to a clade containing T. umbrina and T. abietina, thus confirming morphological monophyly. Conversely, Phycopeltis prostrata, a species with erect filaments, sessile gametangia on the basal erect hair, larger length/width ratio of vegetative cells and very loosely coalescent prostrate filaments, branched separately from the core Phycopeltis group and the T. umbrina and T. abietina clade. Based on morphological and molecular evidence, the genus Phycopeltis was paraphyletic. Furthermore, the traditional taxonomic criteria for Phycopeltis must be reassessed based on phylogeny using more species. A new circumscription of the Phycopeltis and the erection of new genera are recommended. PMID:25643363

  5. Preliminary examination of the effects of relative humidity on the fracture morphology of cotton flat bundles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of the relative humidity (RH) of testing conditions on stelometer cotton flat bundle strength and elongation measurements, and on the morphology of fiber fractures are presented herein. A trend is observed for stelometer strength and elongations measurements; testing in conditions with h...

  6. Organelle redox autonomy during environmental stress.

    PubMed

    Bratt, Avishay; Rosenwasser, Shilo; Meyer, Andreas; Fluhr, Robert

    2016-09-01

    Oxidative stress is generated in plants because of inequalities in the rate of reactive oxygen species (ROS) generation and scavenging. The subcellular redox state under various stress conditions was assessed using the redox reporter roGFP2 targeted to chloroplastic, mitochondrial, peroxisomal and cytosolic compartments. In parallel, the vitality of the plant was measured by ion leakage. Our results revealed that during certain physiological stress conditions the changes in roGFP2 oxidation are comparable to application of high concentrations of exogenous H2 O2 . Under each stress, particular organelles were affected. Conditions of extended dark stress, or application of elicitor, impacted chiefly on the status of peroxisomal redox state. In contrast, conditions of drought or high light altered the status of mitochondrial or chloroplast redox state, respectively. Amalgamation of the results from diverse environmental stresses shows cases of organelle autonomy as well as multi-organelle oxidative change. Importantly, organelle-specific oxidation under several stresses proceeded cell death as measured by ion leakage, suggesting early roGFP oxidation as predictive of cell death. The measurement of redox state in multiple compartments enables one to look at redox state connectivity between organelles in relation to oxidative stress as well as assign a redox fingerprint to various types of stress conditions. PMID:27037976

  7. Computational Examination of Orientation-Dependent Morphological Evolution during the Electrodeposition and Electrodissolution of Magnesium

    DOE PAGESBeta

    DeWitt, S.; Hahn, N.; Zavadil, K.; Thornton, K.

    2015-12-30

    Here a new model of electrodeposition and electrodissolution is developed and applied to the evolution of Mg deposits during anode cycling. The model captures Butler-Volmer kinetics, facet evolution, the spatially varying potential in the electrolyte, and the time-dependent electrolyte concentration. The model utilizes a diffuse interface approach, employing the phase field and smoothed boundary methods. Scanning electron microscope (SEM) images of magnesium deposited on a gold substrate show the formation of faceted deposits, often in the form of hexagonal prisms. Orientation-dependent reaction rate coefficients were parameterized using the experimental SEM images. Three-dimensional simulations of the growth of magnesium deposits yieldmore » deposit morphologies consistent with the experimental results. The simulations predict that the deposits become narrower and taller as the current density increases due to the depletion of the electrolyte concentration near the sides of the deposits. Increasing the distance between the deposits leads to increased depletion of the electrolyte surrounding the deposit. Two models relating the orientation-dependence of the deposition and dissolution reactions are presented. Finally, the morphology of the Mg deposit after one deposition-dissolution cycle is significantly different between the two orientation-dependence models, providing testable predictions that suggest the underlying physical mechanisms governing morphology evolution during deposition and dissolution.« less

  8. Computational Examination of Orientation-Dependent Morphological Evolution during the Electrodeposition and Electrodissolution of Magnesium

    SciTech Connect

    DeWitt, S.; Hahn, N.; Zavadil, K.; Thornton, K.

    2015-12-30

    Here a new model of electrodeposition and electrodissolution is developed and applied to the evolution of Mg deposits during anode cycling. The model captures Butler-Volmer kinetics, facet evolution, the spatially varying potential in the electrolyte, and the time-dependent electrolyte concentration. The model utilizes a diffuse interface approach, employing the phase field and smoothed boundary methods. Scanning electron microscope (SEM) images of magnesium deposited on a gold substrate show the formation of faceted deposits, often in the form of hexagonal prisms. Orientation-dependent reaction rate coefficients were parameterized using the experimental SEM images. Three-dimensional simulations of the growth of magnesium deposits yield deposit morphologies consistent with the experimental results. The simulations predict that the deposits become narrower and taller as the current density increases due to the depletion of the electrolyte concentration near the sides of the deposits. Increasing the distance between the deposits leads to increased depletion of the electrolyte surrounding the deposit. Two models relating the orientation-dependence of the deposition and dissolution reactions are presented. Finally, the morphology of the Mg deposit after one deposition-dissolution cycle is significantly different between the two orientation-dependence models, providing testable predictions that suggest the underlying physical mechanisms governing morphology evolution during deposition and dissolution.

  9. Examination of High Resolution Channel Topography to Determine Suitable Metrics to Characterize Morphological Complexity

    NASA Astrophysics Data System (ADS)

    Stewart, R. L.; Gaeuman, D.

    2015-12-01

    Complex bed morphology is deemed necessary to restore salmonid habitats, yet quantifiable metrics that capture channel complexity have remained elusive. This work utilizes high resolution topographic data from the 40 miles of the Trinity River of northern California to determine a suitable metric for characterizing morphological complexity at the reach scale. The study area is segregated into reaches defined by individual riffle pool units or aggregates of several consecutive units. Potential measures of complexity include rugosity and depth statistics such as standard deviation and interquartile range, yet previous research has shown these metrics are scale dependent and subject to sampling density-based bias. The effect of sampling density on the present analysis has been reduced by underrepresenting the high resolution topographic data as a 3'x 3' raster so that all areas are equally sampled. Standard rugosity, defined as the three-dimensional surface area divided by projected area, has been shown to be dependent on average depth. We therefore define R*, a empirically depth-corrected rugosity metric in which rugosity is corrected using an empirical relationship based on linear regression between the standard rugosity metric and average depth. By removing the dependence on depth using a regression based on the study reach, R* provides a measure reach scale complexity relative to the entire study area. The interquartile range of depths is also depth-dependent, so we defined a non-dimensional metric (IQR*) as the interquartile range dividing by median depth. These are calculated to develop rankings of channel complexity which, are found to closely agree with perceived channel complexity observed in the field. Current efforts combine these measures of morphological complexity with salmonid habitat suitability to evaluate the effects of channel complexity on the various life stages of salmonids. Future work will investigate the downstream sequencing of channel

  10. Inter-organelle ER-endolysosomal contact sites in metabolism and disease across evolution.

    PubMed

    Hariri, Hanaa; Ugrankar, Rupali; Liu, Yang; Henne, W Mike

    2016-01-01

    Since their initial observation, contact sites formed between different organelles have transitioned from ignored curiosities to recognized centers for the exchange of metabolites and lipids. Contact formed between the ER and endomembrane system (eg. the plasma membrane, endosomes, and lysosomes) is of particular biomedical interest, as it governs aspects of lipid metabolism, organelle identity, and cell signaling. Here, we review the field of ER-endolysosomal communication from the perspective of three model systems: budding yeast, the fruit fly D. melanogaster, and mammals. From this broad perspective, inter-organelle communication displays a consistent role in metabolic regulation that was differentially tuned during the development of complex metazoan life. We also examine the current state of understanding of lipid exchange between organelles, and discuss molecular mechanisms by which this occurs. PMID:27489577

  11. Inter-organelle ER-endolysosomal contact sites in metabolism and disease across evolution

    PubMed Central

    Hariri, Hanaa; Ugrankar, Rupali; Liu, Yang; Henne, W. Mike

    2016-01-01

    ABSTRACT Since their initial observation, contact sites formed between different organelles have transitioned from ignored curiosities to recognized centers for the exchange of metabolites and lipids. Contact formed between the ER and endomembrane system (eg. the plasma membrane, endosomes, and lysosomes) is of particular biomedical interest, as it governs aspects of lipid metabolism, organelle identity, and cell signaling. Here, we review the field of ER-endolysosomal communication from the perspective of three model systems: budding yeast, the fruit fly D. melanogaster, and mammals. From this broad perspective, inter-organelle communication displays a consistent role in metabolic regulation that was differentially tuned during the development of complex metazoan life. We also examine the current state of understanding of lipid exchange between organelles, and discuss molecular mechanisms by which this occurs. PMID:27489577

  12. A combined electrophysiological and morphological examination of episodic memory decline in amnestic mild cognitive impairment

    PubMed Central

    Hoppstädter, Michael; King, Andrea Victoria; Frölich, Lutz; Wessa, Michèle; Flor, Herta; Meyer, Patric

    2013-01-01

    Early stages of Alzheimer’s disease (AD) are characterized by neuropathological changes within the medial temporal lobe cortex (MTLC), which lead to characteristic impairments in episodic memory, i.e., amnestic mild cognitive impairment (aMCI). Here, we tested the neural correlates of this memory impairment using event-related potentials (ERPs) and voxel-based morphometry. Twenty-four participants were instructed to encode lists of words and were tested in a yes/no recognition memory task. The dual-process model of recognition memory dissociates between acontextual familiarity and recollection of contextual details. The early frontal ERP old/new effect, which is thought to represent a neural correlate of familiarity-based memory, was absent in aMCI, whereas the control group showed a significant early old/new effect at frontal electrodes. This effect was positively correlated with behavioral episodic memory performance. Analyses of brain morphology revealed a focused gray matter loss in the inferior and medial temporal lobes in aMCI versus healthy controls. Moreover, the positive correlation between gray matter volume in the MTLC and the familiarity-related early frontal old/new effect supports the notion that this effect relies upon the integrity of the MTLC. Thus, the present findings might provide a further functional marker for prodromal AD. PMID:24065918

  13. Morphological and biochemical examination of Cosmos 1887 rat heart tissue. Part 1: Ultrastructure

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Popova, I. A.; Kato, K.; Stevenson, J.; Miquel, J.; Sapp, W.

    1990-01-01

    Morphological changes were observed in the left ventricle of rat heart tissue from animals flown on the Cosmos 1887 biosatellite for 12.5 days. These tissues were compared to the synchronous and vivarium control hearts. While many normal myofibrils were observed, others exhibited ultrastructural alterations, i.e., damaged and irregular-shaped mitochondria and generalized myofibrillar edema. Analysis of variance (ANOVA) of the volume density data revealed a statistically significant increase in glycogen and a significant decrease in mitochondria compared to the synchronous and vivarium controls. Point counting indicated an increase in lipid and myeloid bodies and a decrease in microtubules, but these changes were not statistically significant. In addition, the flight animals exhibited some patchy loss of protofibrils (actin and myosin filaments) and some abnormal supercontracted myofibrils that were not seen in the controls. This study was undertaken to gain insight into the mechanistic aspects of cardiac changes in both animals and human beings as a consequence of space travel. Cardiac hypotrophy and fluid shifts have been observed after actual or simulated weightlessness and raise concerns about the functioning of the heart and circulatory system during and after travel in space.

  14. Isolation of tissue layers in hermatypic corals by N-acetylcysteine: morphological and proteomic examinations

    NASA Astrophysics Data System (ADS)

    Peng, S.-E.; Luo, Y.-J.; Huang, H.-J.; Lee, I.-T.; Hou, L.-S.; Chen, W.-N. U.; Fang, L.-S.; Chen, C.-S.

    2008-03-01

    Corals are diploblastic in body pattern and include two tissue layers, the epidermis and gastrodermis, interconnected by an acellular matrix mesoglea. During development, cells in these tissue layers differentiate morphologically and functionally. In most hermatypic corals, the gastrodermis further develops an ability to associate with microalgae dinoflagellates. This endosymbiosis occurs inside specific host gastrodermal cells, and its mechanism still remains unclear notwithstanding decades of research. The delay in progress is partly due to the difficulty in separating the gastrodermis and its symbionts from the epidermis for detailed cellular and biochemical investigations. The present study reports a simple method to separate these two tissue layers in hermatypic corals using the reducing agent, N-acetylcysteine (NAC). Efficient tissue and proteomic isolations are demonstrated by microscopy and two-dimensional SDS polyacrylamide gel electrophoresis (2D SDS-PAGE). The NAC treatment was able to separate tissue layers without inducing protein degradation. Furthermore, the sensitivity of protein detection greatly increases in the isolated tissue layers. The application of the present technique provides future research on endosymbiosis and coral development with a tool for higher accuracy and sensitivity.

  15. [Examination of ontogenetic-morphologic growth of cholinergic receptor system in isolated preparation of human trachea in vitro].

    PubMed

    Islami, Hilmi; Sukalo, Aziz; Shabani, R; Disha, M; Kutllovci, S

    2006-01-01

    Morphologic growth of cholinergic bronchial respiratory system was examined at live and dead newborns. Tracheal smooth musculature was examined at 18 experimental preparations taken by the autopsy after exiting from different factors. Samples were divided into three groups based on gestational weeks. First group: from 23-29 gestational weeks (immature, N=5); second group: from 30-37 gestational weeks (premature, N=7); third group: from 38-41 gestational weeks (mature, N=6). Based on morphological examination of isolated preparations human trachea fingings are the following: in 23-29 week are found nerve endings with axo-axonal synapses mainly at ramification phase of lungs blood vessels net, without trachea bronchial innervations with axo-axonal synapses, and with perichondrial localization. In 30-37 gestational weeks axo-xonal synapses are found in between glands acinus's and vessels net, and also emphatic choline reactivity at lung ganglions: this suggests existing of cholinergic system at alive newborns. At 38-41 gestational weeks exists a wealthy nerve neuromuscular net in smooth tracheal musculature with different vesicles. Choline reactivity is emphasized peri and intrachondrial at lamina propria, at most around sensory glands and in smooth musculature. This suggests that there is no choline reactivity at epithelium and of existence of cholinergic system in tracheal bronchial smooth musculature. PMID:16425526

  16. Cryptic organelle homology in Apicomplexan parasites: Insights from evolutionary cell biology

    PubMed Central

    Klinger, Christen M.; Nisbet, R. Ellen; Ouologuem, Dinkorma T.; Roos, David S.; Dacks, Joel B.

    2013-01-01

    The economic and clinical significance of apicomplexan parasites drives interest in their many evolutionary novelties. Distinctive intracellular organelles play key roles in parasite motility, invasion, metabolism, and replication, and understanding their relationship with the organelles of better-studied eukaryotic systems suggests potential targets for therapeutic intervention. Recent work has demonstrated divergent aspects of canonical eukaryotic components in the apicomplexa, including Golgi bodies and mitochondria. The apicoplast is a relict plastid of secondary endosymbiotic origin, harboring metabolic pathways distinct from those of host species. The inner membrane complex is derived from the cortical alveoli defining the superphylum Alveolata, but in apicomplexans functions in parasite motility and replication. Micronemes and rhoptries are associated with establishment of the intracellular niche, and define the apical complex for which the phylum is named. Morphological, cell biological and molecular evidence strongly suggest that these organelles are derived from the endocytic pathway. PMID:23932202

  17. Review on Recent Advances in the Analysis of Isolated Organelles

    PubMed Central

    Satori, Chad P.; Kostal, Vratislav; Arriaga, Edgar A.

    2012-01-01

    The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices. PMID:23107131

  18. A Eukaryote without a Mitochondrial Organelle.

    PubMed

    Karnkowska, Anna; Vacek, Vojtěch; Zubáčová, Zuzana; Treitli, Sebastian C; Petrželková, Romana; Eme, Laura; Novák, Lukáš; Žárský, Vojtěch; Barlow, Lael D; Herman, Emily K; Soukal, Petr; Hroudová, Miluše; Doležal, Pavel; Stairs, Courtney W; Roger, Andrew J; Eliáš, Marek; Dacks, Joel B; Vlček, Čestmír; Hampl, Vladimír

    2016-05-23

    The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell. PMID:27185558

  19. Mitochondrial fission: rings around the organelle

    PubMed Central

    Pon, Liza A.

    2014-01-01

    Mitochondria form a dynamic network, in which organelles fuse or divide in response to metabolic changes or cellular stress. Inhibition of these processes leads to cell dysfunction and numerous human diseases. New work from several laboratories shows that mitochondria do not divide in isolation from other cellular structures. Rather, they carry out this process in partnership with the endoplasmic reticulum (ER) and actin filaments. PMID:23578876

  20. Ciliary Extracellular Vesicles: Txt Msg Organelles.

    PubMed

    Wang, Juan; Barr, Maureen M

    2016-04-01

    Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies. PMID:26983828

  1. Organelle communication: signaling crossroads between homeostasis and disease.

    PubMed

    Bravo-Sagua, Roberto; Torrealba, Natalia; Paredes, Felipe; Morales, Pablo E; Pennanen, Christian; López-Crisosto, Camila; Troncoso, Rodrigo; Criollo, Alfredo; Chiong, Mario; Hill, Joseph A; Simmen, Thomas; Quest, Andrew F; Lavandero, Sergio

    2014-05-01

    Cellular organelles do not function as isolated or static units, but rather form dynamic contacts between one another that can be modulated according to cellular needs. The physical interfaces between organelles are important for Ca2+ and lipid homeostasis, and serve as platforms for the control of many essential functions including metabolism, signaling, organelle integrity and execution of the apoptotic program. Emerging evidence also highlights the importance of organelle communication in disorders such as Alzheimer's disease, pulmonary arterial hypertension, cancer, skeletal and cardiac muscle dysfunction. Here, we provide an overview of the current literature on organelle communication and the link to human pathologies. PMID:24534274

  2. Using a morphologic sediment budget to examine the influence of geomorphic context and disturbance history on channel morphodynamics

    NASA Astrophysics Data System (ADS)

    Lea, D. M.

    2013-12-01

    Morphology-based sediment budgets serve to highlight spatial patterns of bed material transport and storage. In this study, I constructed a sediment budget for a gravel-bed river in northern Yellowstone by using a time series of remotely sensed data to estimate volumes of erosion and deposition. To link these changes in channel morphology to the processes that drive stream dynamics, I calculated the cumulative effective energy expenditure between each pair of images as the time integral of stream power above a critical threshold for bed material entrainment; this approach accounts for different time periods and variable streamflows between successive images. This metric of flow energy is used in combination with the sediment budget to examine the influence of geomorphic context and disturbance history on channel morphodynamics. My working hypothesis is that the locations experiencing greatest volumes of erosion are dictated by the spatial distribution of larger-scale geomorphic features within the watershed. For example, significant erosion is likely to occur where the stream channel encounters Holocene landslides, debris flows, or alluvial fans and steepens to incise through these deposits. Conversely, sediment accumulation is expected to occur upstream of these depositional landforms where local channel gradient is reduced. Similarly, the cumulative energy expenditure above the critical threshold for bed material entrainment between successive images acts as a ';disturbance index' to relate the energy available to perform geomorphic work to the nature, degree, and spatial organization of channel change.

  3. Intravascular disorders of microcirculation in patients with chronic obstructive pulmonary disease: the results of clinical and morphological examination

    NASA Astrophysics Data System (ADS)

    Fiodorova, Tatiana A.

    1999-05-01

    We have evaluated the results of clinical and morphological study of microcirculation and its intravascular factors in 120 patients with chronic obstructive pulmonary diseases (COPD). Conjunctival biomicroscopy with quantitative evaluation of microcirculatory changes we performed. This data were compared with the results of laboratory study of erythrocytes and thrombocytes aggregation, some plasma hemostasis indices and morphological examination of microcirculation. The results of conjunctival biomicroscopy showed the close correlation between the clinical severity of the disease, the degree of respiratory failure and the degree of microcirculatory disorders. Progress of the disease with the development of respiratory failure and cor pulmonale was characterized by the expansion of the process of erythrocytes aggregation to the whole parts of the microcirculatory bad and was associated with perivascular hemorrhages. In some patients with severe COPD laboratory data showed chronic disseminated intravascular microcoagulation (DVS-syndrome). Intravascular platelets, erythrocytes and mixed aggregates which completely cork the vessels and compressed endothelium were uncovered by electron microscopy. Platelets membrane injuring with its degranulation was seen. This discovered correlation between microcirculatory abnormalities in lungs and in conjunctiva in patients with COPD demonstrate that this abnormalities of microcirculation are prevalent. This allows to use in clinical accessible and informative method of conjunctival biomicroscopy to estimate the condition of microcirculation in this pathology.

  4. Is Spontaneous Translocation of Polar Lipids Between Cellular Organelles Negligible?

    PubMed Central

    Somerharju, Pentti

    2015-01-01

    In most reviews addressing intracellular lipid trafficking, spontaneous diffusion of lipid monomers between the cellular organelles is considered biologically irrelevant because it is thought to be far too slow to significantly contribute to organelle biogenesis. This view is based on intervesicle transfer experiments carried out in vitro with few lipids as well as on the view that lipids are highly hydrophobic and thus cannot undergo spontaneous intermembrane diffusion at a significant rate. However, besides that single-chain lipids can translocate between vesicles in seconds, it has been demonstrated that the rate of spontaneous transfer of two-chain polar lipids can vary even 1000-fold, depending on the number of carbons and double bonds in the acyl chains. In addition, the rate of spontaneous lipid transfer can strongly depend on the experimental conditions such as vesicle composition and concentration. This review examines the studies suggesting that spontaneous lipid transfer is probably more relevant to intracellular trafficking of amphipathic lipids than commonly thought. PMID:27147824

  5. Complete evaluation of anatomy and morphology of the infertile patient in a single visit; the modern infertility pelvic ultrasound examination.

    PubMed

    Groszmann, Yvette S; Benacerraf, Beryl R

    2016-06-01

    The comprehensive "one-stop shop" ultrasound evaluation of an infertile woman, performed around cycle days 5 to 9, will reveal abundant information about the anatomy and morphology of the pelvic organs and thereby avoid costly radiation and iodinated contrast exposure. We propose a two-dimensional and three-dimensional ultrasound to examine the appearance and shape of the endometrium, endometrial cavity, myometrium, and junctional zone, to assess for müllerian duct anomalies fibroids, adenomyosis, and polyps. We then evaluate the adnexa with grayscale ultrasound and Doppler, looking for ovarian masses or cysts, and signs of tubal disease. The cul-de-sac is imaged to look for masses, endometriosis, and free fluid. We then push gently on the uterus and ovaries to assess mobility. Lack of free movement of the organs would suggest adhesions or endometriosis. The sonohysterogram then allows for more detailed evaluation of the endometrial cavity, endometrial lining, and any intracavitary lesions. Tubal patency is then assessed during the sonohysterogram in real time by introducing air and saline or contrast and imaging the tubes (HyCoSy). With this single comprehensive ultrasound examination, patients can obtain a reliable, time-efficient, minimally invasive infertility evaluation in their own clinician's office at significantly less cost and without radiation. PMID:27054310

  6. Optogenetic Control of Molecular Motors and Organelle Distributions in Cells

    PubMed Central

    Duan, Liting; Che, Daphne; Zhang, Kai; Ong, Qunxiang; Guo, Shunling; Cui, Bianxiao

    2015-01-01

    SUMMARY Intracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival and apoptosis. Here we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon blue light exposure, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven towards the cell periphery upon light-induced recruitment of kinesin, or towards the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells. PMID:25963241

  7. The Evolution of Per-cell Organelle Number.

    PubMed

    Cole, Logan W

    2016-01-01

    Organelles with their own distinct genomes, such as plastids and mitochondria, are found in most eukaryotic cells. As these organelles and their host cells have evolved, the partitioning of metabolic processes and the encoding of interacting gene products have created an obligate codependence. This relationship has played a role in shaping the number of organelles in cells through evolution. Factors such as stochastic evolutionary forces acting on genes involved in organelle biogenesis, organelle-nuclear gene interactions, and physical limitations may, to varying degrees, dictate the selective constraint that per-cell organelle number is under. In particular, coordination between nuclear and organellar gene expression may be important in maintaining gene product stoichiometry, which may have a significant role in constraining the evolution of this trait. PMID:27588285

  8. Requirements and standards for organelle genome databases

    SciTech Connect

    Boore, Jeffrey L.

    2006-01-09

    Mitochondria and plastids (collectively called organelles)descended from prokaryotes that adopted an intracellular, endosymbioticlifestyle within early eukaryotes. Comparisons of their remnant genomesaddress a wide variety of biological questions, especially when includingthe genomes of their prokaryotic relatives and the many genes transferredto the eukaryotic nucleus during the transitions from endosymbiont toorganelle. The pace of producing complete organellar genome sequences nowmakes it unfeasible to do broad comparisons using the primary literatureand, even if it were feasible, it is now becoming uncommon for journalsto accept detailed descriptions of genome-level features. Unfortunatelyno database is currently useful for this task, since they have littlestandardization and are riddled with error. Here I outline what iscurrently wrong and what must be done to make this data useful to thescientific community.

  9. Synthetic cells and organelles: compartmentalization strategies.

    PubMed

    Roodbeen, Renée; van Hest, Jan C M

    2009-12-01

    The recent development of RNA replicating protocells and capsules that enclose complex biosynthetic cascade reactions are encouraging signs that we are gradually getting better at mastering the complexity of biological systems. The road to truly cellular compartments is still very long, but concrete progress is being made. Compartmentalization is a crucial natural methodology to enable control over biological processes occurring within the living cell. In fact, compartmentalization has been considered by some theories to be instrumental in the creation of life. With the advancement of chemical biology, artificial compartments that can mimic the cell as a whole, or that can be regarded as cell organelles, have recently received much attention. The membrane between the inner and outer environment of the compartment has to meet specific requirements, such as semi-permeability, to allow communication and molecular transport over the border. The membrane can either be built from natural constituents or from synthetic polymers, introducing robustness to the capsule. PMID:19877005

  10. Biogenesis and architecture of arterivirus replication organelles.

    PubMed

    van der Hoeven, Barbara; Oudshoorn, Diede; Koster, Abraham J; Snijder, Eric J; Kikkert, Marjolein; Bárcena, Montserrat

    2016-07-15

    All eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral RNA synthesis. Arteriviruses (order Nidovirales) belong to the subset of +RNA viruses that induce double-membrane vesicles (DMVs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis C virus. In the last years, electron tomography has revealed substantial differences between the structures induced by these different virus groups. Arterivirus-induced DMVs appear to be closed compartments that are continuous with endoplasmic reticulum membranes, thus forming an extensive reticulovesicular network (RVN) of intriguing complexity. This RVN is remarkably similar to that described for the distantly related coronaviruses (also order Nidovirales) and sets them apart from other DMV-inducing viruses analysed to date. We review here the current knowledge and open questions on arterivirus replication organelles and discuss them in the light of the latest studies on other DMV-inducing viruses, particularly coronaviruses. Using the equine arteritis virus (EAV) model system and electron tomography, we present new data regarding the biogenesis of arterivirus-induced DMVs and uncover numerous putative intermediates in DMV formation. We generated cell lines that can be induced to express specific EAV replicase proteins and showed that DMVs induced by the transmembrane proteins nsp2 and nsp3 form an RVN and are comparable in topology and architecture to those formed during viral infection. Co-expression of the third EAV transmembrane protein (nsp5), expressed as part of a self-cleaving polypeptide that mimics viral polyprotein processing in infected cells, led to the formation of DMVs whose size was more homogenous and closer to what is observed upon EAV infection, suggesting a regulatory role for nsp5 in modulating membrane curvature and DMV formation. PMID

  11. Organelles on the move: insights from yeast vacuole inheritance.

    PubMed

    Weisman, Lois S

    2006-04-01

    Organelle inheritance is one of several processes that occur during cell division. Recent studies on yeast vacuole inheritance have indicated rules that probably apply to most organelle-inheritance pathways. They have uncovered a molecular mechanism for membrane-cargo transport that is partially conserved from yeast to humans. They have also shown that the transport complex, which is composed of a molecular motor and its receptor, regulates the destination and timing of vacuole movement and might coordinate organelle movement with several other organelle functions. PMID:16607287

  12. Some aspects of lunar and martian volcanism as examined with spectral, topographic, and morphologic data derived from spacecraft images

    NASA Astrophysics Data System (ADS)

    Robinson, Mark Southwick

    Utilizing newly calibrated Mariner 10 color images, the titanium abundances of lunar mare soils on the eastern limb and farside are examined. These maria are found to have TiO2 contents in the range of less than 2 to 5%. The existence of cryptomare deposits northeast of Mare Marginis is confirmed. This leads to the prediction that no high TiO2 (greater than 8 wt%) mare basalt soils will be found in regions with thickened crust (most of the lunar farside) that are yet to be examine with spectrometers, due to the greater density of high titanium magma. Utilizing Viking Orbiter and Mariner 9 data, the martian volcanoes Biblis Patera, Ceraunius Tholus, Jovis Tholus, Ulysses Patera Uranius Patera, and Uranius Tholus are analyzed. Specifically, morphologic and topographic features indicative of the eruption style (effusive vs. explosive) that formed each edifice are examined. From new digital mosaics of these volcanoes, both effusive and explosive deposits are found. It is proposed that the initial period of activity for some martian volcanoes was dominantly explosive (driven by juvenile gases), whereas later activity was mostly effusive. In support a this hypothesis, an analysis of the martian volcano Apollinaris Patera is presented. Using digital images, thermal inertia data and a new topographic model, the chronology of Apollinaris Patera is determined to have been dominated early on by explosive activity, followed by later effusive eruption. From the topographic data, its volume is estimated to be approximately 105/cu km. The volcano may have been active for approximately 107 yrs, based on its volume and an inferred rate of eruption of 1.5 x 10-2/cu km/yr. It is proposed that at least 2 x 1016 kg of juvenile water was added to the martian atmosphere as a consequence of these eruptions. Detailed examination of a multi-temporal series of Viking Orbiter color images of the Apollinaris Patera region shows, that, for a given area on the surface, the red over violet ratio

  13. Short-range inversions: rethinking organelle genome stability: template switching events during DNA replication destabilize organelle genomes.

    PubMed

    Tremblay-Belzile, Samuel; Lepage, Étienne; Zampini, Éric; Brisson, Normand

    2015-10-01

    In the organelles of plants and mammals, recent evidence suggests that genomic instability stems in large part from template switching events taking place during DNA replication. Although more than one mechanism may be responsible for this, some similarities exist between the different proposed models. These can be separated into two main categories, depending on whether they involve a single-strand-switching or a reciprocal-strand-switching event. Single-strand-switching events lead to intermediates containing Y junctions, whereas reciprocal-strand-switching creates Holliday junctions. Common features in all the described models include replication stress, fork stalling and the presence of inverted repeats, but no single element appears to be required in all cases. We review the field, and examine the ideas that several mechanisms may take place in any given genome, and that the presence of palindromes or inverted repeats in certain regions may favor specific rearrangements. PMID:26222836

  14. Exocyst-Positive Organelles and Autophagosomes Are Distinct Organelles in Plants1[OPEN

    PubMed Central

    Lin, Youshun; Ding, Yu; Wang, Juan; Shen, Jinbo; Kung, Chun Hong; Zhuang, Xiaohong; Cui, Yong; Yin, Zhao; Xia, Yiji; Lin, Hongxuan; Robinson, David G.; Jiang, Liwen

    2015-01-01

    Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner. PMID:26358417

  15. Exocyst-Positive Organelles and Autophagosomes Are Distinct Organelles in Plants.

    PubMed

    Lin, Youshun; Ding, Yu; Wang, Juan; Shen, Jinbo; Kung, Chun Hong; Zhuang, Xiaohong; Cui, Yong; Yin, Zhao; Xia, Yiji; Lin, Hongxuan; Robinson, David G; Jiang, Liwen

    2015-11-01

    Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner. PMID:26358417

  16. Assessing Morphological Awareness as a Predictor of Academic Performance and Performance on the National Physical Therapy Examination

    ERIC Educational Resources Information Center

    Moran, Kelley A.

    2012-01-01

    The primary purpose of this study was to validate a method for assessing Morphological Awareness (MA) using multimorphemic words commonly used in the academic and clinical practice settings of physical therapy. The Medical Morphology Test (MMT) was developed for this study and was compared to scores on the Nelson-Denny Reading Test (NDRT©). The…

  17. The Evolution of Per-cell Organelle Number

    PubMed Central

    Cole, Logan W.

    2016-01-01

    Organelles with their own distinct genomes, such as plastids and mitochondria, are found in most eukaryotic cells. As these organelles and their host cells have evolved, the partitioning of metabolic processes and the encoding of interacting gene products have created an obligate codependence. This relationship has played a role in shaping the number of organelles in cells through evolution. Factors such as stochastic evolutionary forces acting on genes involved in organelle biogenesis, organelle–nuclear gene interactions, and physical limitations may, to varying degrees, dictate the selective constraint that per-cell organelle number is under. In particular, coordination between nuclear and organellar gene expression may be important in maintaining gene product stoichiometry, which may have a significant role in constraining the evolution of this trait. PMID:27588285

  18. The number of symbiotic origins of organelles.

    PubMed

    Cavalier-Smith, T

    1992-01-01

    Mitochondria and chloroplasts both originated from bacterial endosymbionts. The available evidence strongly supports a single origin for mitochondria and only somewhat less strongly a single, slightly later, origin for chloroplasts. The arguments and evidence that have sometimes been presented in favor of the alternative theories of the multiple or polyphyletic origins of these two organelles are evaluated and the kinds of data that are needed to test more rigorously the monophyletic theory are discussed. Although chloroplasts probably originated only once, eukaryotic algae are polyphyletic because chloroplasts have been secondarily transferred to new lineages by the permanent incorporation of a photosynthetic eukaryotic algal cell into a phagotrophic protozoan host. How often this has happened is much less clear. It is particularly unclear whether or not the chloroplasts of typical dinoflagellates and euglenoids originated in this way from a eukaryotic symbiont: their direct divergence from the ancestral chloroplast cannot be ruled out and indeed has several arguments in its favor. The evidence for and against the view that the chloroplast of the kingdom Chromista was acquired in a single endosymbiotic event is discussed. The possibility that even the chloroplast of Chlorarachnion might have been acquired during the same symbiosis that created the cryptomonad cell, if the symbiont was a primitive alga that had chlorophyll a, b and c as well as phycobilins, is also considered. An alga with such a combination of pigments might have been ancestral to all eukaryote algae. PMID:1292670

  19. Future of nanotherapeutics: Targeting the cellular sub-organelles.

    PubMed

    Ma, Xiaowei; Gong, Ningqiang; Zhong, Lin; Sun, Jiadong; Liang, Xing-Jie

    2016-08-01

    Many diseases originate from alterations at nanoscale levels. Precise drug delivery should be achieved not only at cell level, but also at organelle level to achieve maximum therapeutic responses as well as avoiding possible toxic side effects of the drugs. However, organelles and subcellular structures are natural barriers that hampering many therapeutics from taking effects. Nanodelivery vehicle is a favorable platform to navigate across physiological barriers and to achieve selective delivery of therapeutic and diagnostic agents to intracellular targets. In this review, we have highlighted recent innovations in organelle-targeted nanomaterials designed to treat a variety of currently challenging diseases. Targeting strategies of four main kinds of organelles: mitochondria, nucleus, lysosomes and endoplasmic reticulum are discussed in detail. This review will help to clarify the intracellular nanomaterial-organelle interactions, and understand the fundamentals of organelle-targeted drug delivery strategies, which is of vital importance for the design and successful biomedical applications of nanomaterials in therapeutic treatments. At the end of this review, challenge and perspectives of organelle-targeted nanotherapy are discussed. PMID:27155363

  20. Programmed death phenomena: from organelle to organism.

    PubMed

    Skulachev, Vladimir P

    2002-04-01

    Programmed death phenomena appear to be inherent not only in living cells (apoptosis), but also in subcellular organelles (e.g., self-elimination of mitochondria, called mitoptosis), organs (organoptosis), and even whole organisms (phenoptosis). In all these cases, the "Samurai law of biology"--it is better to die than to be wrong--seems to be operative. The operation of this law helps complicated living systems avoid the risk of ruin when a system of lower hierarchic position makes a significant mistake. Thus, mitoptosis purifies a cell from damaged and hence unwanted mitochondria; apoptosis purifies a tissue from unwanted cells; and phenoptosis purifies a community from unwanted individuals. Defense against reactive oxygen species (ROS) is probably one of the primary evolutionary functions of programmed death mechanisms. So far, it seems that ROS play a key role in the mito-, apo-, organo-, and phenoptoses, which is consistent with Harman's theory of aging. Here a concept is described that tries to unite Weismann's hypothesis of aging as an adaptive programmed death mechanism and the generally accepted alternative point of view that considers aging as an inevitable result of accumulation in an organism of occasional injuries. It is suggested that injury accumulation is monitored by a system(s) actuating a phenoptotic death program when the number of injuries reaches some critical level. The system(s) in question are organized in such a way that the lethal case appears to be a result of phenoptosis long before the occasional injuries make impossible the functioning of the organism. It is stressed that for humans these cruel regulations look like an atavism that, if overcome, might dramatically prolong the human life span. PMID:11976198

  1. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    PubMed Central

    Luzio, J. Paul; Hackmann, Yvonne; Dieckmann, Nele M.G.; Griffiths, Gillian M.

    2014-01-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  2. Individual organelle pH determinations of magnetically-enriched endocytic organelles via laser-induced fluorescence detection

    PubMed Central

    Satori, Chad P.; Kostal, Vratislav; Arriaga, Edgar A.

    2011-01-01

    The analysis of biotransformations that occur in lysosomes and other endocytic organelles is critical to studies on intracellular degradation, nutrient recycling and lysosomal storage disorders. Such analyses require bioactive organelle preparations that are devoid of other contaminating organelles. Commonly used differential centrifugation techniques produce impure fractions and may not compatible with micro-scale separation platforms. Density gradient centrifugation procedures reduce the level of impurities but may compromise bioactivity. Here we report on simple magnetic setup and a procedure that produce highly enriched bioactive organelles based on their magnetic capture as they traveled through open tubes. Following capture, in-line laser-induced fluorecence detection (LIF) determined for the first time that each magnetically retained individual endocytic organelles have an acidic pH. Unlike bulk measurements, this method was suitable to describe the distributions of pH values in endocytic organelles from L6 rat myoblasts treated with dextran-coated iron oxide nanoparticles (for magnetic retention) and fluorescein/TMRM-conjugated dextran (for pH measurements by LIF). Their individual pH values ranged from 4 to 6, which is typical of bioactive endocytic organelles. These analytical procedures are of high relevance to evaluate lysosomal-related degradation pathways in aging, storage disorders and drug development. PMID:21863795

  3. Organelle acidification is important for localisation of vacuolar proteins in Saccharomyces cerevisiae.

    PubMed

    Matsumoto, Risa; Suzuki, Kuninori; Ohya, Yoshikazu

    2013-12-01

    The acidic environments in the vacuole and other acidic organelles are important for many cellular processes in eukaryotic cells. In this study, we comprehensively investigated the roles of organelle acidification in vacuolar protein localisation in Saccharomyces cerevisiae. After repressing the acidification of acidic compartments by treatment with concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), we examined the localisation of GFP-fused proteins that were predicted to localise in the vacuolar lumen or on the vacuolar membrane. Of the 73 proteins examined, 19 changed their localisation to the cytoplasmic region. Localisation changes were evaluated quantitatively using the image processing programme CalMorph. The delocalised proteins included vacuolar hydrolases, V-ATPase subunits, transporters and enzymes for membrane biogenesis, as well as proteins required for protein transport. These results suggest that many alterations in the localisation of vacuolar proteins occur after loss of the acidification of acidic compartments. PMID:23708375

  4. Proteomics of secretory and endocytic organelles in Giardia lamblia.

    PubMed

    Wampfler, Petra B; Tosevski, Vinko; Nanni, Paolo; Spycher, Cornelia; Hehl, Adrian B

    2014-01-01

    Giardia lamblia is a flagellated protozoan enteroparasite transmitted as an environmentally resistant cyst. Trophozoites attach to the small intestine of vertebrate hosts and proliferate by binary fission. They access nutrients directly via uptake of bulk fluid phase material into specialized endocytic organelles termed peripheral vesicles (PVs), mainly on the exposed dorsal side. When trophozoites reach the G2/M restriction point in the cell cycle they can begin another round of cell division or encyst if they encounter specific environmental cues. They induce neogenesis of Golgi-like organelles, encystation-specific vesicles (ESVs), for regulated secretion of cyst wall material. PVs and ESVs are highly simplified and thus evolutionary diverged endocytic and exocytic organelle systems with key roles in proliferation and transmission to a new host, respectively. Both organelle systems physically and functionally intersect at the endoplasmic reticulum (ER) which has catabolic as well as anabolic functions. However, the unusually high degree of sequence divergence in Giardia rapidly exhausts phylogenomic strategies to identify and characterize the molecular underpinnings of these streamlined organelles. To define the first proteome of ESVs and PVs we used a novel strategy combining flow cytometry-based organelle sorting with in silico filtration of mass spectrometry data. From the limited size datasets we retrieved many hypothetical but also known organelle-specific factors. In contrast to PVs, ESVs appear to maintain a strong physical and functional link to the ER including recruitment of ribosomes to organelle membranes. Overall the data provide further evidence for the formation of a cyst extracellular matrix with minimal complexity. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000694. PMID:24732305

  5. Proteomics of Secretory and Endocytic Organelles in Giardia lamblia

    PubMed Central

    Wampfler, Petra B.; Tosevski, Vinko; Nanni, Paolo; Spycher, Cornelia; Hehl, Adrian B.

    2014-01-01

    Giardia lamblia is a flagellated protozoan enteroparasite transmitted as an environmentally resistant cyst. Trophozoites attach to the small intestine of vertebrate hosts and proliferate by binary fission. They access nutrients directly via uptake of bulk fluid phase material into specialized endocytic organelles termed peripheral vesicles (PVs), mainly on the exposed dorsal side. When trophozoites reach the G2/M restriction point in the cell cycle they can begin another round of cell division or encyst if they encounter specific environmental cues. They induce neogenesis of Golgi-like organelles, encystation-specific vesicles (ESVs), for regulated secretion of cyst wall material. PVs and ESVs are highly simplified and thus evolutionary diverged endocytic and exocytic organelle systems with key roles in proliferation and transmission to a new host, respectively. Both organelle systems physically and functionally intersect at the endoplasmic reticulum (ER) which has catabolic as well as anabolic functions. However, the unusually high degree of sequence divergence in Giardia rapidly exhausts phylogenomic strategies to identify and characterize the molecular underpinnings of these streamlined organelles. To define the first proteome of ESVs and PVs we used a novel strategy combining flow cytometry-based organelle sorting with in silico filtration of mass spectrometry data. From the limited size datasets we retrieved many hypothetical but also known organelle-specific factors. In contrast to PVs, ESVs appear to maintain a strong physical and functional link to the ER including recruitment of ribosomes to organelle membranes. Overall the data provide further evidence for the formation of a cyst extracellular matrix with minimal complexity. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000694. PMID:24732305

  6. Recombination and the maintenance of plant organelle genome stability.

    PubMed

    Maréchal, Alexandre; Brisson, Normand

    2010-04-01

    Like their nuclear counterpart, the plastid and mitochondrial genomes of plants have to be faithfully replicated and repaired to ensure the normal functioning of the plant. Inability to maintain organelle genome stability results in plastid and/or mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly, deregulation of recombination in organelles results in genomic instability, often accompanied by adverse consequences for plant fitness. The recent identification of four families of proteins that prevent aberrant recombination of organelle DNA sheds much needed mechanistic light on this important process. What comes out of these investigations is a partial portrait of the recombination surveillance machinery in which plants have co-opted some proteins of prokaryotic origin but have also evolved whole new factors to keep their organelle genomes intact. These new features presumably optimized the protection of plastid and mitochondrial genomes against the particular genotoxic stresses they face. PMID:20180912

  7. Disorders of Lysosome-related Organelle Biogenesis: Clinical and Molecular Genetics

    PubMed Central

    Huizing, Marjan; Helip-Wooley, Amanda; Westbroek, Wendy; Gunay-Aygun, Meral; Gahl, William A.

    2009-01-01

    Lysosome-related organelles (LROs) are a heterogeneous group of vesicles that share various features with lysosomes, but are distinct in function, morphology, and composition. The biogenesis of LROs employs a common machinery, and genetic defects in this machinery can affect all LROs or only an individual LRO, resulting in a variety of clinical features. In this review, we discuss the main components in LRO biogenesis. We also address the function, composition and resident cell type of the major LROs. Finally, we describe the clinical characteristics of the major human LRO disorders. PMID:18544035

  8. Anti-CD antibody microarray for human leukocyte morphology examination allows analyzing rare cell populations and suggesting preliminary diagnosis in leukemia

    PubMed Central

    Khvastunova, Alina N.; Kuznetsova, Sofya A.; Al-Radi, Liubov S.; Vylegzhanina, Alexandra V.; Zakirova, Anna O.; Fedyanina, Olga S.; Filatov, Alexander V.; Vorobjev, Ivan A.; Ataullakhanov, Fazly

    2015-01-01

    We describe a method for leukocyte sorting by a microarray of anti-cluster-of-differentiation (anti-CD) antibodies and for preparation of the bound cells for morphological or cytochemical examination. The procedure results in a “sorted” smear with cells positive for certain surface antigens localised in predefined areas. The morphology and cytochemistry of the microarray-captured normal and neoplastic peripheral blood mononuclear cells are identical to the same characteristics in a smear. The microarray permits to determine the proportions of cells positive for the CD antigens on the microarray panel with high correlation with flow cytometry. Using the anti-CD microarray we show that normal granular lymphocytes and lymphocytes with radial segmentation of the nuclei are positive for CD3, CD8, CD16 or CD56 but not for CD4 or CD19. We also show that the described technique permits to obtain a pure leukemic cell population or to separate two leukemic cell populations on different antibody spots and to study their morphology or cytochemistry directly on the microarray. In cases of leukemias/lymphomas when circulating neoplastic cells are morphologically distinct, preliminary diagnosis can be suggested from full analysis of cell morphology, cytochemistry and their binding pattern on the microarray. PMID:26212756

  9. Anti-CD antibody microarray for human leukocyte morphology examination allows analyzing rare cell populations and suggesting preliminary diagnosis in leukemia.

    PubMed

    Khvastunova, Alina N; Kuznetsova, Sofya A; Al-Radi, Liubov S; Vylegzhanina, Alexandra V; Zakirova, Anna O; Fedyanina, Olga S; Filatov, Alexander V; Vorobjev, Ivan A; Ataullakhanov, Fazly

    2015-01-01

    We describe a method for leukocyte sorting by a microarray of anti-cluster-of-differentiation (anti-CD) antibodies and for preparation of the bound cells for morphological or cytochemical examination. The procedure results in a "sorted" smear with cells positive for certain surface antigens localised in predefined areas. The morphology and cytochemistry of the microarray-captured normal and neoplastic peripheral blood mononuclear cells are identical to the same characteristics in a smear. The microarray permits to determine the proportions of cells positive for the CD antigens on the microarray panel with high correlation with flow cytometry. Using the anti-CD microarray we show that normal granular lymphocytes and lymphocytes with radial segmentation of the nuclei are positive for CD3, CD8, CD16 or CD56 but not for CD4 or CD19. We also show that the described technique permits to obtain a pure leukemic cell population or to separate two leukemic cell populations on different antibody spots and to study their morphology or cytochemistry directly on the microarray. In cases of leukemias/lymphomas when circulating neoplastic cells are morphologically distinct, preliminary diagnosis can be suggested from full analysis of cell morphology, cytochemistry and their binding pattern on the microarray. PMID:26212756

  10. Association of a Nonmuscle Myosin II with Axoplasmic Organelles

    PubMed Central

    DeGiorgis, Joseph A.; Reese, Thomas S.; Bearer, Elaine L.

    2002-01-01

    Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ∼220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ∼220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single ∼220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor. PMID:11907281

  11. Systematic Structural Analyses of Attachment Organelle in Mycoplasma pneumoniae

    PubMed Central

    Matsuo, Lisa; Miyata, Makoto

    2015-01-01

    Mycoplasma pneumoniae, a human pathogenic bacterium, glides on host cell surfaces by a unique and unknown mechanism. It forms an attachment organelle at a cell pole as a membrane protrusion composed of surface and internal structures, with a highly organized architecture. In the present study, we succeeded in isolating the internal structure of the organelle by sucrose-gradient centrifugation. The negative-staining electron microscopy clarified the details and dimensions of the internal structure, which is composed of terminal button, paired plates, and bowl complex from the end of cell front. Peptide mass fingerprinting of the structure suggested 25 novel components for the organelle, and 3 of them were suggested for their involvement in the structure through their subcellular localization determined by enhanced yellow fluorescent protein (EYFP) tagging. Thirteen component proteins including the previously reported ones were mapped on the organelle systematically for the first time, in nanometer order by EYFP tagging and immunoelectron microscopy. Two, three, and six specific proteins localized specifically to the terminal button, the paired plates, and the bowl, respectively and interestingly, HMW2 molecules were aligned parallel to form the plate. The integration of these results gave the whole image of the organelle and allowed us to discuss possible gliding mechanisms. PMID:26633540

  12. Imaging trace element distributions in single organelles and subcellular features

    PubMed Central

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-01-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies. PMID:26911251

  13. Transient domain formation in membrane-bound organelles undergoing maturation

    NASA Astrophysics Data System (ADS)

    Dmitrieff, Serge; Sens, Pierre

    2013-12-01

    The membrane components of cellular organelles have been shown to segregate into domains as the result of biochemical maturation. We propose that the dynamical competition between maturation and lateral segregation of membrane components regulates domain formation. We study a two-component fluid membrane in which enzymatic reaction irreversibly converts one component into another and phase separation triggers the formation of transient membrane domains. The maximum domain size is shown to depend on the maturation rate as a power law similar to the one observed for domain growth with time in the absence of maturation, despite this time dependence not being verified in the case of irreversible maturation. This control of domain size by enzymatic activity could play a critical role in regulating exchange between organelles or within compartmentalized organelles such as the Golgi apparatus.

  14. Insights into the mechanisms of sterol transport between organelles.

    PubMed

    Mesmin, Bruno; Antonny, Bruno; Drin, Guillaume

    2013-09-01

    In cells, the levels of sterol vary greatly among organelles. This uneven distribution depends largely on non-vesicular routes of transfer, which are mediated by soluble carriers called lipid-transfer proteins (LTPs). These proteins have a domain with a hydrophobic cavity that accommodates one sterol molecule. However, a demonstration of their role in sterol transport in cells remains difficult. Numerous LTPs also contain membrane-binding elements, but it is not clear how these LTPs couple their ability to target organelles with lipid transport activity. This issue appears critical, since many sterol transporters are thought to act at contact sites between two membrane-bound compartments. Here, we emphasize that biochemical and structural studies provide precious insights into the mode of action of sterol-binding proteins. Recent studies on START, Osh/ORP and NPC proteins suggest models on how these proteins could transport sterol between organelles and, thereby, influence cellular functions. PMID:23283302

  15. Imaging trace element distributions in single organelles and subcellular features

    NASA Astrophysics Data System (ADS)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  16. Verbal Inflectional Morphology in L1 and L2 Spanish: A Frequency Effects Study Examining Storage versus Composition

    ERIC Educational Resources Information Center

    Bowden, Harriet Wood; Gelfand, Matthew P.; Sanz, Cristina; Ullman, Michael T.

    2010-01-01

    This study examines the storage versus composition of Spanish inflected verbal forms in first language (L1) and second language (L2) speakers of Spanish. L2 participants were selected to have mid-to-advanced proficiency, high classroom experience, and low immersion experience, typical of medium-to-advanced foreign language learners. Participants…

  17. Phospholipids of subcellular organelles isolated from cultured BHK cells.

    PubMed

    Brotherus, J; Renkonen, O

    1977-02-23

    Mitochondrial and nuclei were purified from cultured hamster fibroblasts (BHK21 cells) by centrifugation in sucrose gradients. The phospholipid compositions of the preparations were compared to those of the previously purified plasma membranes, endoplasmic reticulum and lysosomes. The mitochondria had a characteristically high content (approx. 16% of lipid phosphorus) of cardiolipin, which was practically absent from the other purified organelles. The nuclei were enriched in phosphatidylcholine and phosphatidylinositol (approx. 68% and 5% of lipid phosphorus, respectively). Lysobisphosphatidic acid was almost absent from the mitochondria and nuclei, as well as from the plasma membrane and endoplasmic reticulum, which suggests that this phospholipid is confined to the lysosomes of the BHK cell. The nuclei and the mitochondria contained relatively little sphingomyelin, a characteristic lipid of the plasma membrane. The distributions of the total cellular phospholipid and protein between the various organelles were calculated and compared to the corresponding data estimated for the rat liver. The BHK cell contained relatively more phospholipids in the nucleus and the lysosomes than the liver. All the organelles of the BHK cell contained less protein per phospholipid than the equivalent organelles of the liver. PMID:836856

  18. Osmotic regulation of Rab-mediated organelle docking

    PubMed Central

    Brett, Christopher L.; Merz, Alexey J.

    2009-01-01

    SUMMARY Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1–6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, while hypertonic gradients cause vacuoles to fragment on a slower time scale [7–11]. Here, we analyze the influence of osmotic strength on homotypic fusion of isolated yeast vacuoles. Consistent with previously reported in vivo results, we find that decreases in osmolyte concentration increase the rate and extent of vacuole fusion in vitro, while increases in osmolyte concentration prevent fusion. Unexpectedly, our results reveal that osmolytes regulate fusion by inhibiting early, Rab-dependent docking or predocking events, not late events. Our experiments reveal an organelle-autonomous pathway that may control organelle surface to volume ratio, size and copy number: decreasing the osmolyte concentration in the cytoplasmic compartment accelerates Rab-mediated docking and fusion. Fusion, by altering the organelle surface-to-enclosed volume relationship, in turn reduces the risk of membrane rupture. PMID:18619842

  19. Osmotic regulation of Rab-mediated organelle docking.

    PubMed

    Brett, Christopher L; Merz, Alexey J

    2008-07-22

    Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1-6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, whereas hypertonic gradients cause vacuoles to fragment on a slower time scale [7-11]. Here, we analyze the influence of osmotic strength on homotypic fusion of isolated yeast vacuoles. Consistent with previously reported in vivo results, we find that decreases in osmolyte concentration increase the rate and extent of vacuole fusion in vitro, whereas increases in osmolyte concentration prevent fusion. Unexpectedly, our results reveal that osmolytes regulate fusion by inhibiting early Rab-dependent docking or predocking events, not late events. Our experiments reveal an organelle-autonomous pathway that may control organelle surface-to-volume ratio, size, and copy number: Decreasing the osmolyte concentration in the cytoplasmic compartment accelerates Rab-mediated docking and fusion. By altering the relationship between the organelle surface and its enclosed volume, fusion in turn reduces the risk of membrane rupture. PMID:18619842

  20. Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

    PubMed Central

    Kharkwal, Himanshu; Furgiuele, Sara Shanda; Smith, Caitlin G.

    2015-01-01

    ABSTRACT UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid-membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCE Membrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to

  1. Off to the Organelles - Killing Cancer Cells with Targeted Gold Nanoparticles

    PubMed Central

    Kodiha, Mohamed; Wang, Yi Meng; Hutter, Eliza; Maysinger, Dusica; Stochaj, Ursula

    2015-01-01

    Gold nanoparticles (AuNPs) are excellent tools for cancer cell imaging and basic research. However, they have yet to reach their full potential in the clinic. At present, we are only beginning to understand the molecular mechanisms that underlie the biological effects of AuNPs, including the structural and functional changes of cancer cells. This knowledge is critical for two aspects of nanomedicine. First, it will define the AuNP-induced events at the subcellular and molecular level, thereby possibly identifying new targets for cancer treatment. Second, it could provide new strategies to improve AuNP-dependent cancer diagnosis and treatment. Our review summarizes the impact of AuNPs on selected subcellular organelles that are relevant to cancer therapy. We focus on the nucleus, its subcompartments, and mitochondria, because they are intimately linked to cancer cell survival, growth, proliferation and death. While non-targeted AuNPs can damage tumor cells, concentrating AuNPs in particular subcellular locations will likely improve tumor cell killing. Thus, it will increase cancer cell damage by photothermal ablation, mechanical injury or localized drug delivery. This concept is promising, but AuNPs have to overcome multiple hurdles to perform these tasks. AuNP size, morphology and surface modification are critical parameters for their delivery to organelles. Recent strategies explored all of these variables, and surface functionalization has become crucial to concentrate AuNPs in subcellular compartments. Here, we highlight the use of AuNPs to damage cancer cells and their organelles. We discuss current limitations of AuNP-based cancer research and conclude with future directions for AuNP-dependent cancer treatment. PMID:25699096

  2. Threshold-free method for three-dimensional segmentation of organelles

    NASA Astrophysics Data System (ADS)

    Chan, Yee-Hung M.; Marshall, Wallace F.

    2012-03-01

    An ongoing challenge in the field of cell biology is to how to quantify the size and shape of organelles within cells. Automated image analysis methods often utilize thresholding for segmentation, but the calculated surface of objects depends sensitively on the exact threshold value chosen, and this problem is generally worse at the upper and lower zboundaries because of the anisotropy of the point spread function. We present here a threshold-independent method for extracting the three-dimensional surface of vacuoles in budding yeast whose limiting membranes are labeled with a fluorescent fusion protein. These organelles typically exist as a clustered set of 1-10 sphere-like compartments. Vacuole compartments and center points are identified manually within z-stacks taken using a spinning disk confocal microscope. A set of rays is defined originating from each center point and radiating outwards in random directions. Intensity profiles are calculated at coordinates along these rays, and intensity maxima are taken as the points the rays cross the limiting membrane of the vacuole. These points are then fit with a weighted sum of basis functions to define the surface of the vacuole, and then parameters such as volume and surface area are calculated. This method is able to determine the volume and surface area of spherical beads (0.96 to 2 micron diameter) with less than 10% error, and validation using model convolution methods produce similar results. Thus, this method provides an accurate, automated method for measuring the size and morphology of organelles and can be generalized to measure cells and other objects on biologically relevant length-scales.

  3. Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum.

    PubMed

    Wu, S S; Platt, K A; Ratnayake, C; Wang, T W; Ting, J T; Huang, A H

    1997-11-11

    The monolayer tapetum cells of the maturing flowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by flotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g/cm(3) and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g/cm(3) and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of floral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface. PMID:11038591

  4. Morphological and electrophysiological examination of olfactory sensory neurons during the early developmental prolarval stage of the sea lamprey Petromyzon marinus L

    USGS Publications Warehouse

    Zielinski, B.S.; Fredricks, Keith; McDonald, R.; Zaidi, A.U.

    2005-01-01

    This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage. ?? 2006 Springer Science + Business Media, LLC.

  5. Verbal Inflectional Morphology in L1 and L2 Spanish: A Frequency Effects Study Examining Storage versus Composition

    PubMed Central

    Bowden, Harriet Wood; Gelfand, Matthew P.; Sanz, Cristina; Ullman, Michael T.

    2009-01-01

    This study examines the storage vs. composition of Spanish inflected verbal forms in L1 and L2 speakers of Spanish. L2 participants were selected to have mid-to-advanced proficiency, high classroom experience, and low immersion experience, typical of medium-to-advanced foreign language learners. Participants were shown the infinitival forms of verbs from either Class I (the default class, which takes new verbs) or Classes II and III (non-default classes), and were asked to produce either first-person singular present-tense or imperfect forms, in separate tasks. In the present tense, the L1 speakers showed inflected-form frequency effects (i.e., higher frequency forms were produced faster, which is taken as a reflection of storage) for stem-changing (irregular) verb-forms from both Class I (e.g., pensar-pienso) and Classes II and III (e.g., perder-pierdo), as well as for non-stem-changing (regular) forms in Classes II/III (e.g., vender-vendo), in which the regular transformation does not appear to constitute a default. In contrast, Class I regulars (e.g., pescar-pesco), whose non-stem-changing transformation constitutes a default (e.g., it is applied to new verbs), showed no frequency effects. L2 speakers showed frequency effects for all four conditions (Classes I and II/III, regulars and irregulars). In the imperfect tense, the L1 speakers showed frequency effects for Class II/III (-ía-suffixed) but not Class I (-aba-suffixed) forms, even though both involve non-stem-change (regular) default transformations. The L2 speakers showed frequency effects for both types of forms. The pattern of results was not explained by a wide range of potentially confounding experimental and statistical factors, and does not appear to be compatible with single-mechanism models, which argue that all linguistic forms are learned and processed in associative memory. The findings are consistent with a dual-system view in which both verb class and regularity influence the storage vs

  6. Cadmium Stress Disrupts the Endomembrane Organelles and Endocytosis during Picea wilsonii Pollen Germination and Tube Growth

    PubMed Central

    Feng, Yu; Li, Xue; Wei, Qian; Sheng, Xianyong

    2014-01-01

    As one of the most severe pollutants, cadmium has been reported to be harmful to plant cells, but the effects of cadmium on gymnosperm pollen germination and tube growth and the mechanism of this involvement are still unclear. Here, we report that cadmium not only strongly inhibited P. wilsonii pollen germination and tube growth, but also significantly altered tube morphology in a dose-dependent manner. Time-lapse images obtained with a laser scanning confocal microscope revealed that endocytosis was dramatically inhibited by cadmium stress. Further investigation with ER-Tracker dye indicated that cadmium stress reduced the number of the Golgi apparatus, and induced dilation of ER. Additionally, Lyso-Tracker staining showed that cadmium distinctly promoted the formation of acidic organelles in pollen tubes, likely derived from the dilated ER. Taken together, our studies indicated that P. wilsonii pollens were highly susceptible to cadmium stress, and that cadmium stress strongly inhibited pollen germination and tube growth by disrupting the endomembrane organelles, inhibiting endo/exocytosis, and forming acidic vacuoles, resulting in swollen tube tips and irregularly broadened tube diameters. These findings provide a new insight into the effects of cadmium toxicity on the tip growth of pollen tubes. PMID:24722362

  7. Amyloplast sedimentation and organelle saltation in living corn columella cells

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Suyemoto, M. M.; Leopold, A. C.

    1986-01-01

    Amyloplast sedimentation during gravistimulation and organelle movements was studied in living central rootcap cells of Zea mays L. cv. Merit. Cells from sectioned roots were viewed with a horizontally-mounted videomicroscope. The kinetics of gravity-induced amyloplast sedimentation were comparable to those calculated from experiments using fixed material. Individual amyloplasts fell at an average velocity of 5.5 micrometers min-1; the maximal velocity of fall measured was 18.0 micrometers min-1. Amyloplasts often rotated, sometimes rose in the cytoplasm, and occasionally underwent sudden rapid movements as fast as 58 micrometers min-1. Saltations of other organelles were frequently observed. This appears to be the first report of cytoplasmic streaming in the presumptive statocytes of roots.

  8. Organelle transport along microtubules - the role of KIFs.

    PubMed

    Hirokawa, N

    1996-04-01

    Organelle transporters are very important for cellular morphogenesis and other cellular functions, conveying and targeting important materials to the correct destination, often at considerable velocities. One of the first proteins to be identified as a motor was kinesin, and recently at least 10 new kinesin superfamily proteins (KIFs) have been described. Characterization of some of them reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Here, Nobutaka Hirokawa reviews what is known about the kinesin superfamily and discusses how a study of the different types of motors is helping to elucidate the mechanism of mechanical force generation. PMID:15157476

  9. Updating Our View of Organelle Genome Nucleotide Landscape

    PubMed Central

    Smith, David Roy

    2012-01-01

    Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT) content. Recent studies, however, have uncovered guanine and cytosine (GC)-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits. PMID:22973299

  10. Imaging trace element distributions in single organelles and subcellular features

    DOE PAGESBeta

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-25

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cdmore » (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators.We find it could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.« less

  11. GOBASE—a database of organelle and bacterial genome information

    PubMed Central

    O'Brien, Emmet A.; Zhang, Yue; Yang, LiuSong; Wang, Eric; Marie, Veronique; Lang, B. Franz; Burger, Gertraud

    2006-01-01

    The organelle genome database GOBASE is now in its twelfth release, and includes 350 000 mitochondrial sequences and 118 000 chloroplast sequences, roughly a 3-fold expansion since previously documented. GOBASE also includes a fully reannotated genome sequence of Rickettsia prowazekii, one of the closest bacterial relatives of mitochondria, and will shortly expand to contain more data from bacteria from which organelles originated. All these sequences are now accessible through a single unified interface. Enhancements to the functionality of GOBASE include addition of pages for RNA structures and a page compiling data about the taxonomic distribution of organelle-encoded genes; incorporation of Gene Ontology terms; addition of features deduced from incomplete annotations to sequences in GenBank; marking of type examples in cases where single genes in single species are oversampled within GenBank; and addition of graphics illustrating gene structure and the position of neighbouring genes on a sequence. The database has been reimplemented in PostgreSQL to facilitate development and maintenance, and structural modifications have been made to speed up queries, particularly those related to taxonomy. The GOBASE database can be queried at and inquiries should be directed to gobase@bch.umontreal.ca. PMID:16381962

  12. Whole-Genome Hitchhiking on an Organelle Mutation.

    PubMed

    Flood, Pádraic J; van Heerwaarden, Joost; Becker, Frank; de Snoo, C Bastiaan; Harbinson, Jeremy; Aarts, Mark G M

    2016-05-23

    Strong selection on a beneficial mutation can cause a selective sweep, which fixes the mutation in the population and reduces the genetic variation in the region flanking the mutation [1-3]. These flanking regions have increased in frequency due to their physical association with the selected loci, a phenomenon called "genetic hitchhiking" [4]. Theoretically, selection could extend the hitchhiking to unlinked parts of the genome, to the point that selection on organelles affects nuclear genome diversity. Such indirect selective sweeps have never been observed in nature. Here we show that strong selection on a chloroplast gene in the wild plant species Arabidopsis thaliana has caused widespread and lasting hitchhiking of the whole nuclear genome. The selected allele spread more than 400 km along the British railway network, reshaping the genetic composition of local populations. This demonstrates that selection on organelle genomes can significantly reduce nuclear genetic diversity in natural populations. We expect that organelle-mediated genetic draft is a more common occurrence than previously realized and needs to be considered when studying genome evolution. PMID:27133865

  13. New molecular mechanisms of inter-organelle lipid transport.

    PubMed

    Drin, Guillaume; von Filseck, Joachim Moser; Čopič, Alenka

    2016-04-15

    Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated. PMID:27068959

  14. Muscle intermediate filaments and their links to membranes and membranous organelles

    SciTech Connect

    Capetanaki, Yassemi . E-mail: ycapetanaki@bioacademy.gr; Bloch, Robert J.; Kouloumenta, Asimina; Mavroidis, Manolis; Psarras, Stelios

    2007-06-10

    Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.

  15. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

    PubMed Central

    Shim, Sang-Hee; Xia, Chenglong; Zhong, Guisheng; Babcock, Hazen P.; Vaughan, Joshua C.; Huang, Bo; Wang, Xun; Xu, Cheng; Bi, Guo-Qiang; Zhuang, Xiaowei

    2012-01-01

    Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes. PMID:22891300

  16. Mechanisms of organelle division and inheritance and their implications regarding the origin of eukaryotic cells

    PubMed Central

    KUROIWA, Tsuneyoshi

    2010-01-01

    Mitochondria and plastids have their own DNAs and are regarded as descendants of endosymbiotic prokaryotes. Organellar DNAs are not naked in vivo but are associated with basic proteins to form DNA-protein complexes (called organelle nuclei). The concept of organelle nuclei provides a new approach to explain the origin, division, and inheritance of organelles. Organelles divide using organelle division rings (machineries) after organelle-nuclear division. Organelle division machineries are a chimera of the FtsZ (filamentous temperature sensitive Z) ring of bacterial origin and the eukaryotic mechanochemical dynamin ring. Thus, organelle division machineries contain a key to solve the origin of organelles (eukaryotes). The maternal inheritance of organelles developed during sexual reproduction and it is also probably intimately related to the origin of organelles. The aims of this review are to describe the strategies used to reveal the dynamics of organelle division machineries, and the significance of the division machineries and maternal inheritance in the origin and evolution of eukaryotes. PMID:20467212

  17. Myosin-Va and dynamic actin oppose microtubules to drive long-range organelle transport.

    PubMed

    Evans, Richard D; Robinson, Christopher; Briggs, Deborah A; Tooth, David J; Ramalho, Jose S; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V; Hume, Alistair N

    2014-08-01

    In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the "highways and local roads" model for transport along microtubule and actin tracks. The "cooperative capture" model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning. PMID:25065759

  18. The Journey of the Organelle: Teamwork and Regulation in Intracellular Transport

    PubMed Central

    Barlan, Kari; Rossow, Molly J.; Gelfand, Vladimir I.

    2013-01-01

    Specific subsets of biochemical reactions in eukaryotic cells are restricted to individual membrane compartments, or organelles. Cells, therefore, face the monumental task of moving the products of those reactions between individual organelles. Because of the high density of the cytoplasm and the large size of membrane organelles, simple diffusion is grossly insufficient for this task. Proper trafficking between membrane organelles thus relies on cytoskeletal elements and the activity of motor proteins, that act both in transport of membrane compartments and as tethering agents to ensure their proper distribution and to facilitate organelle interactions. PMID:23510681

  19. Scanning ion images; analysis of pharmaceutical drugs at organelle levels

    NASA Astrophysics Data System (ADS)

    Larras-Regard, E.; Mony, M.-C.

    1995-05-01

    With the ion analyser IMS 4F used in microprobe mode, it is possible to obtain images of fields of 10 × 10 [mu]m2, corresponding to an effective magnification of 7000 with lateral resolution of 250 nm, technical characteristics that are appropriate for the size of cell organelles. It is possible to characterize organelles by their relative CN-, P- and S- intensities when the tissues are prepared by freeze fixation and freeze substitution. The recognition of organelles enables correlation of the tissue distribution of ebselen, a pharmaceutical drug containing selenium. The various metabolites characterized in plasma, bile and urine during biotransformation of ebselen all contain selenium, so the presence of the drug and its metabolites can be followed by images of Se. We were also able to detect the endogenous content of Se in tissue, due to the increased sensitivity of ion analysis in microprobe mode. Our results show a natural occurrence of Se in the border corresponding to the basal lamina of cells of proximal but not distal tubules of the kidney. After treatment of rats with ebselen, an additional site of Se is found in the lysosomes. We suggest that in addition to direct elimination of ebselen and its metabolites by glomerular filtration and urinary elimination, a second process of elimination may occur: Se compounds reaching the epithelial cells via the basal lamina accumulate in lysosomes prior to excretion into the tubular fluid. The technical developments of using the IMS 4F instrument in the microprobe mode and the improvement in preparation of samples by freeze fixation and substitution further extend the limit of ion analysis in biology. Direct imaging of trace elements and molecules marked with a tracer make it possible to determine their targets by comparison with images of subcellular structures. This is a promising advance in the study of pathways of compounds within tissues, cells and the whole organism.

  20. Evolution of the bacterial organelle responsible for magnetotaxis.

    PubMed

    Lefèvre, Christopher T; Wu, Long-Fei

    2013-10-01

    There are few examples of protein- and lipid-bounded organelles in bacteria that are encoded by conserved gene clusters and lead to a specific function. The magnetosome chain represents one of these rare examples and is responsible for magnetotaxis in magnetotactic bacteria (MTB), a behavior thought to aid in finding their optimal growth conditions. The origin and evolution of the magnetotaxis is still a matter of debate. Recent breakthroughs in isolation, cultivation, single-cell separation, and whole-genome sequencing have generated abundant data that give new insights into the biodiversity and evolution of MTB. PMID:23948365

  1. Organelle DNA polymorphism in apple cultivars and rootstocks.

    PubMed

    Ishikawa, S; Kato, S; Imakawa, S; Mikami, T; Shimamoto, Y

    1992-05-01

    Restriction fragment length polymorphisms (RFLPs) have been used to detect chloroplast (cp) and mitochondrial (mt) DNA variation among 18 apple cultivars and three rootstocks. The distribution of RFLP patterns allowed the assignment of these genotypes into three groups of cytoplasmic relatedness. Our results also demonstrate maternal inheritance of cp- and mtDNAs in apple. Thus, the organelle DNA assay provides a convenient and reliable method to assess cytoplasmic diversity within the apple germ-plasm collection and to trace the maternal lineages involved in the evolution of apple. PMID:24202920

  2. Ligand-directed profiling of organelles with internalizing phage libraries

    PubMed Central

    Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

  3. The lipid droplet—a well-connected organelle

    PubMed Central

    Gao, Qiang; Goodman, Joel M.

    2015-01-01

    Our knowledge of inter-organellar communication has grown exponentially in recent years. This review focuses on the interactions that cytoplasmic lipid droplets have with other organelles. Twenty-five years ago droplets were considered simply particles of coalesced fat. Ten years ago there were hints from proteomics studies that droplets might interact with other structures to share lipids and proteins. Now it is clear that the droplets interact with many if not most cellular structures to maintain cellular homeostasis and to buffer against insults such as starvation. The evidence for this statement, as well as probes to understand the nature and results of droplet interactions, are presented. PMID:26322308

  4. Detection, imaging, and kinetics of sub-micron organelles of chondrocytes by multiple beam interference microscopy

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Medina, Honorio; Barboza, J. M.; Colantuoni, Gladys; Quintero, Maritza

    2004-07-01

    Chondrocytes, obtained from testosterone treated human articular cartilage, were examined by a recently developed Multiple Beam Interference Microscopy (MBIM) attached to a confocal set up, Video-enhanced differential interference microphotography and also by cinematography. In the MBIM, the intensity of the transmitted pattern is given by the Airy function which increases the contrast dramatically as the coefficient of the reflectance of the parallel plates increases. Moreover, in this configuration, the beam passes several times through a specific organelle and increases its optical path difference both because of the increase in the trajectory and refractive index (high density) of the organelle. The improved contrast enhances the resolving power of the system and makes visible several structural details of sub micron dimensions like nucleolus, retraction fibers, podia, etc. which are not possible to reveal with such a clarity by conventional techniques such as bright field, phase contrast or DIC. This technique permits to detect the oscillatory and rotational motions of unstained cilia for the first time. The frequency of oscillations was found to be 0.8 Hz.

  5. Function of metabolic and organelle networks in crowded and organized media

    PubMed Central

    Aon, Miguel A.; Cortassa, Sonia

    2015-01-01

    (Macro)molecular crowding and the ability of the ubiquitous cytoskeleton to dynamically polymerize–depolymerize are prevalent cytoplasmic conditions in prokaryotic and eukaryotic cells. Protein interactions, enzymatic or signaling reactions - single, sequential or in complexes - whole metabolic pathways and organelles can be affected by crowding, the type and polymeric status of cytoskeletal proteins (e.g., tubulin, actin), and their imparted organization. The self-organizing capability of the cytoskeleton can orchestrate metabolic fluxes through entire pathways while its fractal organization can frame the scaling of activities in several levels of organization. The intracellular environment dynamics (e.g., biochemical reactions) is dominated by the orderly cytoskeleton and the intrinsic randomness of molecular crowding. Existing evidence underscores the inherent capacity of intracellular organization to generate emergent global behavior. Yet unknown is the relative impact on cell function provided by organelle or functional compartmentation based on transient proteins association driven by weak interactions (quinary structures) under specific environmental challenges or functional conditions (e.g., hypoxia, division, differentiation). We propose a qualitative, integrated structural–functional model of cytoplasmic organization based on a modified version of the Sierspinsky–Menger–Mandelbrot sponge, a 3D representation of a percolation cluster, and examine its capacity to accommodate established experimental facts. PMID:25653618

  6. A morphological and histological examination of the pan-tropical spotted dolphin (Stenella attenuata) and the spinner dolphin (Stenella longirostris) adrenal gland.

    PubMed

    Clark, L S; Cowan, D F; Pfeiffer, D C

    2008-04-01

    The morphology and histology of the cetacean adrenal gland are poorly understood. Therefore, this study examined 32 pairs of adrenal glands from 18 pan-tropical spotted dolphins (Stenella attenuata) and 14 spinner dolphins (Stenella longirostris). In both species, the cortex was pseudolobulated and contained a typical mammalian zonation. Medullary protrusions (0-3 per section) and a medullary band were identified in both species. For S. attenuata, no statistical differences were found in the cortex to medulla (CM) ratio or the percent cross-sectional area (PCA) of the adrenal glands compared with sex or sexual maturity. The mean CM ratio for S. attenuata was 2.34 and the PCA was 64.4% cortex, 29.4% medulla and 6.2%'other'. 'Other' indicates blood vessels, connective tissue and the gland capsule itself. For S. longirostris, there was no statistical difference in the CM ratio compared with sexual maturity. However, a statistical difference was found between the CM ratio and sex, suggesting sexual dimorphism (female CM ratio = 2.46 and males = 3.21). No statistical differences were found in the PCA of S. longirostris adrenal glands by sexual maturity. However, a statistical difference was found between the PCA by sex. Female S. longirostris adrenal glands consisted of 65.0% cortex, 27.3% medulla and 7.7% 'other', whereas male adrenal glands consisted of 71.7% cortex, 22.7% medulla and 5.6% 'other'. PMID:18070242

  7. Observation of in vivo morphologic changes after carbon dioxide ablative fractional laser in a mouse model using noninvasive imaging modalities and comparison with histologic examination.

    PubMed

    Yoo, Kwang Ho; Kwon, Tae Rin; Kim, So Young; Song, Yi Seop; Cheon, Young Sook; Kim, Yu Mi; Yeo, In Kwon; Ko, Eun Jung; Li, Kapsok; Kim, Myeung Nam; Kim, Beom Joon

    2014-01-01

    Ablative fractional carbon dioxide (CO2) lasers have been widely used for several types of cosmetic dermatosis. A number of previous studies have evaluated this technique in animals or human beings by observing morphologic changes using an invasive modality such as skin biopsy. In this study, we assessed in vivo skin changes after CO2 ablative fractional laser treatment in a mouse model using noninvasive imaging modalities (Folliscope(®) and Visioscan 98(®)), and each results was compared with data from histologic examination. An ablative fractional CO2 laser was applied with different pulse energy between 7 to 35 mJ/microspot. As results of above methods, we also confirmed that the CO2 ablative fractional laser generated injuries with increasing width and depth with increasing pulse energy. Although numerous papers have described application of this laser in vivo skin specimens, our study evaluated the feasibility of using relative noninvasive imaging modalities for assessing the outcome of laser ablation. Based on our data, we suggest that these technologies may be useful alternative modalities for assessing laser ablation that are easier to perform and less invasive than skin biopsy. PMID:25041574

  8. The Amyloid Precursor Protein of Alzheimer's Disease Clusters at the Organelle/Microtubule Interface on Organelles that Bind Microtubules in an ATP Dependent Manner.

    PubMed

    Stevenson, James W; Conaty, Eliza A; Walsh, Rylie B; Poidomani, Paul J; Samoriski, Colin M; Scollins, Brianne J; DeGiorgis, Joseph A

    2016-01-01

    The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer's disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer's protein tau has a microtubule-based function. PMID:26814888

  9. The Amyloid Precursor Protein of Alzheimer’s Disease Clusters at the Organelle/Microtubule Interface on Organelles that Bind Microtubules in an ATP Dependent Manner

    PubMed Central

    Stevenson, James W.; Conaty, Eliza A.; Walsh, Rylie B.; Poidomani, Paul J.; Samoriski, Colin M.; Scollins, Brianne J.; DeGiorgis, Joseph A.

    2016-01-01

    The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer’s disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer’s protein tau has a microtubule-based function. PMID:26814888

  10. The organelle of differentiation in embryos: the cell state splitter.

    PubMed

    Gordon, Natalie K; Gordon, Richard

    2016-01-01

    The cell state splitter is a membraneless organelle at the apical end of each epithelial cell in a developing embryo. It consists of a microfilament ring and an intermediate filament ring subtending a microtubule mat. The microtubules and microfilament ring are in mechanical opposition as in a tensegrity structure. The cell state splitter is bistable, perturbations causing it to contract or expand radially. The intermediate filament ring provides metastability against small perturbations. Once this snap-through organelle is triggered, it initiates signal transduction to the nucleus, which changes gene expression in one of two readied manners, causing its cell to undergo a step of determination and subsequent differentiation. The cell state splitter also triggers the cell state splitters of adjacent cells to respond, resulting in a differentiation wave. Embryogenesis may be represented then as a bifurcating differentiation tree, each edge representing one cell type. In combination with the differentiation waves they propagate, cell state splitters explain the spatiotemporal course of differentiation in the developing embryo. This review is excerpted from and elaborates on "Embryogenesis Explained" (World Scientific Publishing, Singapore, 2016). PMID:26965444

  11. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    PubMed Central

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  12. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria. PMID:27088225

  13. Geometric modeling of subcellular structures, organelles, and multiprotein complexes

    PubMed Central

    Feng, Xin; Xia, Kelin; Tong, Yiying; Wei, Guo-Wei

    2013-01-01

    SUMMARY Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multi-protein complexes have emerged as a leading interest in structural biology. Geometric modeling not only provides visualizations of shapes for large biomolecular complexes but also fills the gap between structural information and theoretical modeling, and enables the understanding of function, stability, and dynamics. This paper introduces a suite of computational tools for volumetric data processing, information extraction, surface mesh rendering, geometric measurement, and curvature estimation of biomolecular complexes. Particular emphasis is given to the modeling of cryo-electron microscopy data. Lagrangian-triangle meshes are employed for the surface presentation. On the basis of this representation, algorithms are developed for surface area and surface-enclosed volume calculation, and curvature estimation. Methods for volumetric meshing have also been presented. Because the technological development in computer science and mathematics has led to multiple choices at each stage of the geometric modeling, we discuss the rationales in the design and selection of various algorithms. Analytical models are designed to test the computational accuracy and convergence of proposed algorithms. Finally, we select a set of six cryo-electron microscopy data representing typical subcellular complexes to demonstrate the efficacy of the proposed algorithms in handling biomolecular surfaces and explore their capability of geometric characterization of binding targets. This paper offers a comprehensive protocol for the geometric modeling of subcellular structures, organelles, and multiprotein complexes. PMID:23212797

  14. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

    PubMed Central

    Mackinder, Luke C. M.; Meyer, Moritz T.; Mettler-Altmann, Tabea; Chen, Vivian K.; Mitchell, Madeline C.; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S.; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C.

    2016-01-01

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency. PMID:27166422

  15. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

    PubMed

    Mackinder, Luke C M; Meyer, Moritz T; Mettler-Altmann, Tabea; Chen, Vivian K; Mitchell, Madeline C; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C

    2016-05-24

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency. PMID:27166422

  16. Analysis of the Sam50 translocase of Excavate organisms supports evolution of divergent organelles from a common endosymbiotic event

    PubMed Central

    Kay, Christopher J.; Lawler, Karen; Kerr, Ian D.

    2013-01-01

    As free-living organisms the ancestors of mitochondria and plastids encoded complete genomes, proteomes and metabolomes. As these symbionts became organelles all these aspects were reduced – genomes have degenerated with the host nucleus now encoding the most of the remaining endosymbiont proteome, while the metabolic processes of the symbiont have been streamlined to the functions of the emerging organelle. By contrast, the topology of the endosymbiont membrane has been preserved, necessitating the development of complex pathways for membrane insertion and translocation. In this study, we examine the characteristics of the endosymbiont-derived β-barrel insertase Sam501 in the excavate super-group. A candidate is further characterized in Trichomonas vaginalis, an unusual eukaryote possessing degenerate hydrogen-producing mitochondria called hydrogenosomes. This information supports a mitochondriate eukaryotic common ancestor with a similarly evolved β-barrel insertase, which has continued to be conserved in degenerate mitochondria. PMID:24147756

  17. Differential recall of derived and inflected word forms in working memory: examining the role of morphological information in simple and complex working memory tasks.

    PubMed

    Service, Elisabet; Maury, Sini

    2014-01-01

    Working memory (WM) has been described as an interface between cognition and action, or a system for access to a limited amount of information needed in complex cognition. Access to morphological information is needed for comprehending and producing sentences. The present study probed WM for morphologically complex word forms in Finnish, a morphologically rich language. We studied monomorphemic (boy), inflected (boy+'s), and derived (boy+hood) words in three tasks. Simple span, immediate serial recall of words, in Experiment 1, is assumed to mainly rely on information in the focus of attention. Sentence span, a dual task combining sentence reading with recall of the last word (Experiment 2) or of a word not included in the sentence (Experiment 3) is assumed to involve establishment of a search set in long-term memory for fast activation into the focus of attention. Recall was best for monomorphemic and worst for inflected word forms with performance on derived words in between. However, there was an interaction between word type and experiment, suggesting that complex span is more sensitive to morphological complexity in derivations than simple span. This was explored in a within-subjects Experiment 4 combining all three tasks. An interaction between morphological complexity and task was replicated. Both inflected and derived forms increased load in WM. In simple span, recall of inflectional forms resulted in form errors. Complex span tasks were more sensitive to morphological load in derived words, possibly resulting from interference from morphological neighbors in the mental lexicon. The results are best understood as involving competition among inflectional forms when binding words from input into an output structure, and competition from morphological neighbors in secondary memory during cumulative retrieval-encoding cycles. Models of verbal recall need to be able to represent morphological as well as phonological and semantic information. PMID:25642181

  18. Differential recall of derived and inflected word forms in working memory: examining the role of morphological information in simple and complex working memory tasks

    PubMed Central

    Service, Elisabet; Maury, Sini

    2015-01-01

    Working memory (WM) has been described as an interface between cognition and action, or a system for access to a limited amount of information needed in complex cognition. Access to morphological information is needed for comprehending and producing sentences. The present study probed WM for morphologically complex word forms in Finnish, a morphologically rich language. We studied monomorphemic (boy), inflected (boy+’s), and derived (boy+hood) words in three tasks. Simple span, immediate serial recall of words, in Experiment 1, is assumed to mainly rely on information in the focus of attention. Sentence span, a dual task combining sentence reading with recall of the last word (Experiment 2) or of a word not included in the sentence (Experiment 3) is assumed to involve establishment of a search set in long-term memory for fast activation into the focus of attention. Recall was best for monomorphemic and worst for inflected word forms with performance on derived words in between. However, there was an interaction between word type and experiment, suggesting that complex span is more sensitive to morphological complexity in derivations than simple span. This was explored in a within-subjects Experiment 4 combining all three tasks. An interaction between morphological complexity and task was replicated. Both inflected and derived forms increased load in WM. In simple span, recall of inflectional forms resulted in form errors. Complex span tasks were more sensitive to morphological load in derived words, possibly resulting from interference from morphological neighbors in the mental lexicon. The results are best understood as involving competition among inflectional forms when binding words from input into an output structure, and competition from morphological neighbors in secondary memory during cumulative retrieval-encoding cycles. Models of verbal recall need to be able to represent morphological as well as phonological and semantic information. PMID:25642181

  19. Quantifying morphological features of actin cytoskeletal filaments in plant cells based on mathematical morphology.

    PubMed

    Kimori, Yoshitaka; Hikino, Kazumi; Nishimura, Mikio; Mano, Shoji

    2016-01-21

    By quantifying the morphological properties of biological structures, we can better evaluate complex shapes and detect subtle morphological changes in organisms. In this paper, we propose a shape analysis method based on morphological image processing, and apply it to image analysis of actin cytoskeletal filaments in root hair cells of Arabidopsis thaliana. In plant cells, the actin cytoskeletal filaments have critical roles in various cellular processes such as vesicle trafficking and organelle motility. The dynamics of vesicles and organelles in plant cells depend on actin cytoskeletal filaments, regulating cell division and cell enlargement. To better understand the actin-dependent organelle motility, we attempted to quantify the organization of actin filaments in the root hair cells of the root hair defective 3 (rhd3) mutant. RHD3 is involved in actin organization, and its defect has been reported to affect the dynamics of various vesicles and organelles. We measured three shape features of the actin filaments in wild-type and mutant plants. One feature (thickness) was depicted on a grayscale; the others (describing the complexity of the filament network patterns in two-dimensional space) were depicted as binary features. The morphological phenotypes of the cytoskeletal filaments clearly differed between wild-type and mutant. Subtle variations of filament morphology among the mutants were detected and statistically quantified. PMID:26551157

  20. Artificial Organelles: Reactions inside Protein-Polymer Supramolecular Assemblies.

    PubMed

    Garni, Martina; Einfalt, TomaŽ; Lomora, Mihai; Car, Anja; Meier, Wolfgang; Palivan, Cornelia G

    2016-01-01

    Reactions inside confined compartments at the nanoscale represent an essential step in the development of complex multifunctional systems to serve as molecular factories. In this respect, the biomimetic approach of combining biomolecules (proteins, enzymes, mimics) with synthetic membranes is an elegant way to create functional nanoreactors, or even simple artificial organelles, that function inside cells after uptake. Functionality is provided by the specificity of the biomolecule(s), whilst the synthetic compartment provides mechanical stability and robustness. The availability of a large variety of biomolecules and synthetic membranes allows the properties and functionality of these reaction spaces to be tailored and adjusted for building complex self-organized systems as the basis for molecular factories. PMID:27363371

  1. Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs

    NASA Astrophysics Data System (ADS)

    Wang, Lihong V.; Hu, Song

    2012-03-01

    Photoacoustic tomography (PAT) can create multiscale multicontrast images of living biological structures ranging from organelles to organs. This emerging technology overcomes the high degree of scattering of optical photons in biological tissue by making use of the photoacoustic effect. Light absorption by molecules creates a thermally induced pressure jump that launches ultrasonic waves, which are received by acoustic detectors to form images. Different implementations of PAT allow the spatial resolution to be scaled with the desired imaging depth in tissue while a high depth-to-resolution ratio is maintained. As a rule of thumb, the achievable spatial resolution is on the order of 1/200 of the desired imaging depth, which can reach up to 7 centimeters. PAT provides anatomical, functional, metabolic, molecular, and genetic contrasts of vasculature, hemodynamics, oxygen metabolism, biomarkers, and gene expression. We review the state of the art of PAT for both biological and clinical studies and discuss future prospects.

  2. Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis.

    PubMed

    Symens, Nathalie; Soenen, Stefaan J; Rejman, Joanna; Braeckmans, Kevin; De Smedt, Stefaan C; Remaut, Katrien

    2012-01-01

    The nucleocytoplasmic partitioning of nanoparticles as a result of cell division is highly relevant to the field of nonviral gene delivery. We reviewed the literature on the intracellular distribution of cell organelles (the endosomal vesicles, Golgi apparatus, endoplasmic reticulum and nucleus), foreign macromolecules (dextrans and plasmid DNA) and inorganic nanoparticles (gold, quantum dot and iron oxide) during mitosis. For nonviral gene delivery particles (lipid- or polymer-based), indirect proof of nuclear entry during mitosis is provided. We also describe how retroviruses and latent DNA viruses take advantage of mitosis to transfer their viral genome and segregate their episomes into the host daughter nuclei. Based on this knowledge, we propose strategies to improve nonviral gene delivery in dividing cells with the ultimate goal of designing nonviral gene delivery systems that are as efficient as their viral counterparts but non-immunogenic, non-oncogenic and easy and inexpensive to prepare. PMID:22210278

  3. Phase transitions and size scaling of membrane-less organelles

    PubMed Central

    2013-01-01

    The coordinated growth of cells and their organelles is a fundamental and poorly understood problem, with implications for processes ranging from embryonic development to oncogenesis. Recent experiments have shed light on the cell size–dependent assembly of membrane-less cytoplasmic and nucleoplasmic structures, including ribonucleoprotein (RNP) granules and other intracellular bodies. Many of these structures behave as condensed liquid-like phases of the cytoplasm/nucleoplasm. The phase transitions that appear to govern their assembly exhibit an intrinsic dependence on cell size, and may explain the size scaling reported for a number of structures. This size scaling could, in turn, play a role in cell growth and size control. PMID:24368804

  4. Mitochondria as signaling organelles in the vascular endothelium

    PubMed Central

    Quintero, Marisol; Colombo, Sergio L.; Godfrey, Andrew; Moncada, Salvador

    2006-01-01

    Vascular endothelial cells are highly glycolytic and consume relatively low amounts of oxygen (O2) compared with other cells. We have confirmed that oxidative phosphorylation is not the main source of ATP generation in these cells. We also show that at a low O2 concentration (<1%) endogenous NO plays a key role in preventing the accumulation of the α-subunit of hypoxia-inducible factor 1. At higher O2 concentrations (1–3%) NO facilitates the production of mitochondrial reactive oxygen species. This production activates the AMP-activated protein kinase by a mechanism independent of nucleotide concentrations. Thus, the primary role of mitochondria in vascular endothelial cells may not be to generate ATP but, under the control of NO, to act as signaling organelles using either O2 or O2-derived species as signaling molecules. Diversion of O2 away from endothelial cell mitochondria by NO might also facilitate oxygenation of vascular smooth muscle cells. PMID:16565215

  5. Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum.

    PubMed

    Lavoie, Christine; Roy, Line; Lanoix, Joël; Taheri, Mariam; Young, Robin; Thibault, Geneviève; Farah, Carol Abi; Leclerc, Nicole; Paiement, Jacques

    2011-06-01

    The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions. PMID:21536318

  6. The microsporidian polar tube: A highly specialised invasion organelle

    PubMed Central

    Xu, Yanji; Weiss, Louis M.

    2011-01-01

    All of the members of the Microsporidia possess a unique, highly specialised structure, the polar tube. This article reviews the available data on the organisation, structure and function of this invasion organelle. It was over 100 years ago that Thelohan accurately described the microsporidian polar tube and the triggering of its discharge. In the spore, the polar tube is connected at the anterior end, and then coils around the sporoplasm. Upon appropriate environmental stimulation the polar tube rapidly discharges out of the spore pierces a cell membrane and serves as a conduit for sporoplasm passage into the new host cell. The mechanism of germination of spores, however, remains to be definitively determined. In addition, further studies on the characterisation of the early events in the rupture of the anterior attachment complex, eversion of the polar tube as well as the mechanism of host cell attachment and penetration are needed in order to clarify the function and assembly of this structure. The application of immunological and molecular techniques has resulted in the identification of three polar tube proteins referred to as PTP1, PTP2 and PTP3. The interactions of these identified proteins in the formation and function of the polar tube remain to be determined. Data suggest that PTP1 is an O-mannosylated glycoprotein, a post-translational modification that may be important for its function. With the availability of the Encephalitozoon cuniculi genome it is now possible to apply proteomic techniques to the characterisation of the components of the microsporidian spore and invasion organelle. PMID:16005007

  7. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    PubMed

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying; Naleway, John Joseph

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  8. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

    PubMed Central

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  9. An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria

    PubMed Central

    Borg, Sarah; Popp, Felix; Hofmann, Julia; Leonhardt, Heinrich; Rothbauer, Ulrich

    2015-01-01

    ABSTRACT  Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular “nanotraps,” which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW1-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria. Importance   Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular “nanotraps” in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for

  10. Chemical Shift Images of Organelles in Leydig cells of Mice Testes

    NASA Astrophysics Data System (ADS)

    Ejima, T.; Neichi, Y.; Yanagihara, M.; Kado, M.; Ishino, M.; Yasuda, K.; Tamotsu, S.

    2013-10-01

    Soft X-ray transmission images of Leydig cells of mice testes changing incident wavelength were observed with the use of a contact microscope. After normalization of transmission images, absorbance images were obtained and compared with a visible differential interference image. Some organelles were identified by the image comparison, and absorption spectra of the organelles were obtained from the absorbance images. The absorption spectra show that peak structures are different depending on the observed organelles. The structures and the positions of organelles were clearly identified at C-K absorption.

  11. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions.

    PubMed

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  12. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions

    PubMed Central

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L.; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  13. Developmental changes and organelle biogenesis in the reproductive organs of thermogenic skunk cabbage (Symplocarpus renifolius)

    PubMed Central

    Ito-Inaba, Yasuko; Sato, Mayuko; Masuko, Hiromi; Hida, Yamato; Toyooka, Kiminori; Watanabe, Masao; Inaba, Takehito

    2009-01-01

    Sex-dependent thermogenesis during reproductive organ development in the inflorescence is a characteristic feature of some of the protogynous arum species. One such plant, skunk cabbage (Symplocarpus renifolius), can produce massive heat during the female stage but not during the subsequent male stage in which the stamen completes development, the anthers dehisce, and pollen is released. Unlike other thermogenic species, skunk cabbage belongs to the bisexual flower group. Although recent studies have identified the spadix as the thermogenic organ, it remains unclear how individual tissues or intracellular structures are involved in thermogenesis. In this study, reproductive organ development and organelle biogenesis were examined during the transition from the female to the male stage. During the female stage, the stamens exhibit extensive structural changes including changes in organelle structure and density. They accumulate high levels of mitochondrial proteins, including possible thermogenic factors, alternative oxidase, and uncoupling protein. By contrast, the petals and pistils do not undergo extensive changes during the female stage. However, they contain a larger number of mitochondria than during the male stage in which they develop large cytoplasmic vacuoles. Comparison between female and male spadices suggests that mitochondrial number rather than their level of activity correlates with thermogenesis. Their spadices, even in the male, contain a larger amount of mitochondria that had greater oxygen consumption, compared with non-thermogenic plants. Taken together, our data suggest that the extensive maturation process in stamens produces massive heat through increased metabolic activities. The possible mechanisms by which petal and pistil metabolism may affect thermogenesis are also discussed. PMID:19640927

  14. Modeling organelle transport in branching dendrites with a variable cross-sectional area

    PubMed Central

    2010-01-01

    The purpose of this paper is to develop a method for calculating organelle transport in dendrites with a non-uniform cross-sectional area that depends on the distance from the neuron soma. The model is based on modified Smith–Simmons equations governing molecular motor-assisted organelle transport. The developed method is then applied to simulating organelle transport in branching dendrites with two particular microtubule (MT) orientations reported from experiments. It is found that the rate of organelle transport toward a dendrite’s growth cone heavily depends on the MT orientation, and since there is experimental evidence that the MT orientation in a particular region of a dendrite may depend on the dendrite’s developmental stage, the obtained results suggest that a rearrangement of the MT structure may depend on the amount of organelles needed at the growth cone. PMID:21886345

  15. Mouse oocytes differentiate through organelle enrichment from sister cyst germ cells.

    PubMed

    Lei, Lei; Spradling, Allan C

    2016-04-01

    Oocytes differentiate in diverse species by receiving organelles and cytoplasm from sister germ cells while joined in germline cysts or syncytia. Mouse primordial germ cells form germline cysts, but the role of cysts in oogenesis is unknown. We find that mouse germ cells receive organelles from neighboring cyst cells and build a Balbiani body to become oocytes, whereas nurselike germ cells die. Organelle movement, Balbiani body formation, and oocyte fate determination are selectively blocked by low levels of microtubule-dependent transport inhibitors. Membrane breakdown within the cyst and an apoptosis-like process are associated with organelle transfer into the oocyte, events reminiscent of nurse cell dumping in Drosophila We propose that cytoplasmic and organelle transport plays an evolutionarily conserved and functionally important role in mammalian oocyte differentiation. PMID:26917595

  16. Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

    PubMed Central

    Lundqvist, J. Anders; Sahlin, Frida; Åberg, Maria A. I.; Strömberg, Anette; Eriksson, Peter S.; Orwar, Owe

    1998-01-01

    We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed. PMID:9724707

  17. Mitochondrial redox and pH signaling occurs in axonal and synaptic organelle clusters

    PubMed Central

    Breckwoldt, Michael O.; Armoundas, Antonis A.; Aon, Miguel A.; Bendszus, Martin; O’Rourke, Brian; Schwarzländer, Markus; Dick, Tobias P.; Kurz, Felix T.

    2016-01-01

    Redox switches are important mediators in neoplastic, cardiovascular and neurological disorders. We recently identified spontaneous redox signals in neurons at the single mitochondrion level where transients of glutathione oxidation go along with shortening and re-elongation of the organelle. We now have developed advanced image and signal-processing methods to re-assess and extend previously obtained data. Here we analyze redox and pH signals of entire mitochondrial populations. In total, we quantified the effects of 628 redox and pH events in 1797 mitochondria from intercostal axons and neuromuscular synapses using optical sensors (mito-Grx1-roGFP2; mito-SypHer). We show that neuronal mitochondria can undergo multiple redox cycles exhibiting markedly different signal characteristics compared to single redox events. Redox and pH events occur more often in mitochondrial clusters (medium cluster size: 34.1 ± 4.8 μm2). Local clusters possess higher mitochondrial densities than the rest of the axon, suggesting morphological and functional inter-mitochondrial coupling. We find that cluster formation is redox sensitive and can be blocked by the antioxidant MitoQ. In a nerve crush paradigm, mitochondrial clusters form sequentially adjacent to the lesion site and oxidation spreads between mitochondria. Our methodology combines optical bioenergetics and advanced signal processing and allows quantitative assessment of entire mitochondrial populations. PMID:27000952

  18. L1 and L2 Word Recognotion in Finnish. Examining L1 Effects on L2 Processing of Morphological Complexity and Morphophonological Transparency

    ERIC Educational Resources Information Center

    Vainio, Seppo; Anneli, Pajunen; Hyona, Jukka

    2014-01-01

    This study investigated the effect of the first language (L1) on the visual word recognition of inflected nouns in second language (L2) Finnish by native Russian and Chinese speakers. Case inflection is common in Russian and in Finnish but nonexistent in Chinese. Several models have been posited to describe L2 morphological processing. The unified…

  19. The Gas Vacuole - an Early Organelle of Prokaryote Motility

    NASA Astrophysics Data System (ADS)

    Staley, James T.

    1980-06-01

    Several lines of evidence suggest that the gas vesicle may have been an early organelle of prokaryote motility. First, it is found in bacteria that are thought to be representatives of primitive groups. Second, it is a simple structure, and the structure alone imparts the function of motility. Thirdly, it is widely distributed amongst prokaryotes, having been found in the purple and green sulfur photosynthetic bacteria, cyanobacteria, methanogenic bacteria, obligate and facultative anaerobic heterotrophic bacteria, as well as aerobic heterotrophic bacteria that divide by budding and binary transverse fission. Recent evidence suggests that in some bacteria the genes for gas vesicle synthesis occur on plasmids. Thus, the wide distribution of this characteristic could be due to recent evolution and rapid dispersal, though early evolution is not precluded. Though the gas vesicle structure itself appears to be highly conserved among the various groups of bacteria, it seems doubtful that the regulatory mechanism to control its synthesis could be the same for the diverse gas vacuolate bacterial groups.

  20. Nanopreparations for Organelle-Specific Delivery in Cancer

    PubMed Central

    Biswas, Swati; Torchilin, Vladimir P.

    2014-01-01

    To efficiently deliver therapeutics into cancer cells, a number of strategies have been recently investigated. The toxicity associated with the administration of chemotherapeutic drugs due to their random interactions throughout the body necessitates the development of drug-encapsulating nanopreparations that significantly mask, or reduce, the toxic side effects of the drugs. In addition to reduced side effects associated with drug encapsulation, nanocarriers preferentially accumulate in tumors as a result of its abnormally leaky vasculature via the Enhanced Permeability and Retention (EPR) effect. However, simple passive nanocarrier delivery to the tumor site is unlikely to be enough to elicit a maximum therapeutic response as the drug-loaded carriers must reach the intracellular target sites. Therefore, efficient translocation of the nanocarrier through the cell membrane is necessary for cytosolic delivery of the cargo. However, Crossing the cell membrane barrier and reaching cytosol might still not be enough for achieving maximum therapeutic benefit, which necessitates the delivery of drugs directly to intracellular targets, such as bringing pro-apoptotic drugs to mitochondria, nucleic acid therapeutics to nuclei, and lysosomal enzymes to defective lysosomes. In this review, we discuss the strategies developed for tumor targeting, cytosolic delivery via cell membrane translocation, and finally organelle-specific targeting, which may be applied for developing highly efficacious, truly multifunctional, cancer-targeted nanopreparations. PMID:24270008

  1. Modularity of a carbon-fixing protein organelle

    PubMed Central

    Bonacci, Walter; Teng, Poh K.; Afonso, Bruno; Niederholtmeyer, Henrike; Grob, Patricia; Silver, Pamela A.; Savage, David F.

    2012-01-01

    Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO2-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO2. Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO2 in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures. PMID:22184212

  2. Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria

    PubMed Central

    Chahales, Peter; Thanassi, David G.

    2015-01-01

    Bacteria assemble a wide range of adhesive proteins, termed adhesins, to mediate binding to receptors and colonization of surfaces. For pathogenic bacteria, adhesins are critical for early stages of infection, allowing the bacteria to initiate contact with host cells, colonize different tissues, and establish a foothold within the host. The adhesins expressed by a pathogen are also critical for bacterial-bacterial interactions and the formation of bacterial communities such as biofilms. The ability to adhere to host tissues is particularly important for bacteria that colonize sites such as the urinary tract, where the flow of urine functions to maintain sterility by washing away non-adherent pathogens. Adhesins vary from monomeric proteins that are directly anchored to the bacterial surface to polymeric, hairlike fibers that extend out from the cell surface. These latter fibers are termed pili or fimbriae, and were among the first identified virulence factors of uropathogenic Escherichia coli. Studies since then have identified a range of both pilus and non-pilus adhesins that contribute to bacterial colonization of the urinary tract, and have revealed molecular details of the structures, assembly pathways, and functions of these adhesive organelles. In this review, we describe the different types of adhesins expressed by both Gram-negative and Gram-positive uropathogens, what is known about their structures, how they are assembled on the bacterial surface, and the functions of specific adhesins in the pathogenesis of urinary tract infections. PMID:26542038

  3. The murine cardiac 26S proteasome: an organelle awaiting exploration.

    PubMed

    Gomes, Aldrin V; Zong, Chenggong; Edmondson, Ricky D; Berhane, Beniam T; Wang, Guang-Wu; Le, Steven; Young, Glen; Zhang, Jun; Vondriska, Thomas M; Whitelegge, Julian P; Jones, Richard C; Joshua, Irving G; Thyparambil, Sheeno; Pantaleon, Dawn; Qiao, Joe; Loo, Joseph; Ping, Peipei

    2005-06-01

    Multiprotein complexes have been increasingly recognized as essential functional units for a variety of cellular processes, including the protein degradation system. Selective degradation of proteins in eukaryotes is primarily conducted by the ubiquitin proteasome system. The current knowledge base, pertaining to the proteasome complexes in mammalian cells, relies largely upon information gained in the yeast system, where the 26S proteasome is hypothesized to contain a 20S multiprotein core complex and one or two 19S regulatory complexes. To date, the molecular structure of the proteasome system, the proteomic composition of the entire 26S multiprotein complexes, and the specific designated function of individual components within this essential protein degradation system in the heart remain virtually unknown. A functional proteomic approach, employing multidimensional chromatography purification combined with liquid chromatography tandem mass spectrometry and protein chemistry, was utilized to explore the murine cardiac 26S proteasome system. This article presents an overview on the subject of protein degradation in mammalian cells. In addition, this review shares the limited information that has been garnered thus far pertaining to the molecular composition, function, and regulation of this important organelle in the cardiac cells. PMID:16093497

  4. Modularity of a carbon-fixing protein organelle.

    PubMed

    Bonacci, Walter; Teng, Poh K; Afonso, Bruno; Niederholtmeyer, Henrike; Grob, Patricia; Silver, Pamela A; Savage, David F

    2012-01-10

    Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures. PMID:22184212

  5. MLT1 links cytoskeletal asymmetry to organelle placement in Chlamydomonas

    PubMed Central

    Mittelmeier, Telsa M.; Thompson, Mark D.; Lamb, Mary Rose; Lin, Huawen; Dieckmann, Carol L.

    2015-01-01

    Asymmetric placement of the photosensory eyespot organelle in Chlamydomonas is patterned by mother-daughter differences between the two basal bodies, which template the anterior flagella. Each basal body is associated with two bundled microtubule rootlets, one with two microtubules and one with four, forming a cruciate pattern. In wild type cells, the single eyespot is positioned at the equator in close proximity to the plus end of the daughter rootlet comprising four microtubules, the D4. Here we identify mutations in two linked loci, MLT1 and MLT2, which cause multiple eyespots. Antiserum raised against MLT1 localized the protein along the D4 rootlet microtubules, from the basal bodies to the eyespot. MLT1 associates immediately with the new D4 as it extends during cell division, before microtubule acetylation. MLT1 is a low-complexity protein of over 300,000 daltons. The expression or stability of MLT1 is dependent on MLT2, predicted to encode a second large, low-complexity protein. MLT1 was not restricted to the D4 rootlet in cells with the vfl2-220 mutation in the gene encoding the basal body-associated protein centrin. The cumulative data highlight the role of mother-daughter basal body differences in establishing asymmetry in associated rootlets, and suggest that eyespot components are directed to the correct location by MLT1 on the D4 microtubules. PMID:25809438

  6. Lung Surfactant and Organelles after an Exposure to Dibenzoxazepine (CR)

    PubMed Central

    Pattle, R. E.; Schock, C.; Dirnhuber, P.; Creasey, J. M.

    1974-01-01

    Rats were exposed to a heavy dosage of the sensory irritant dibenz (b.f.)-1,4 oxazepine (CR). No change in the lung surfactant could be detected by the methods used. Electron micrography showed that the ordinary lamellated osmiophilic bodies (LOPBs) and their precursors were unaffected. Bodies containing both mitochondrial cristae and dense osmiophilic whorls (“mitochondrial lamellated bodies”, or MLBs) were found in the type II cells of some animals up to 15 days after the exposure. These whorls originate from the bounding membranes and cristae; serial sections show that they usually abut on the boundary of the organelle. A large proportion of the mitochondria in any cell may be affected by this process. Unequivocal evidence that the MLBs finally evolve into LOPBs without cristae was not obtained in this series; the ultimate fate of the MLBs and the cells containing them is uncertain. The MLBs may perhaps act as an emergency source of surfactant. ImagesFigs. 9-10Figs. 6-8Figs. 1-2Figs. 3-5 PMID:4479334

  7. Monitoring cell survival after extraction of a single subcellular organelle using optical trapping and pulsed-nitrogen laser ablation.

    PubMed

    Shelby, J Patrick; Edgar, J Scott; Chiu, Daniel T

    2005-01-01

    This paper characterizes cell viability in three different cell lines--Chinese hamster ovary cells (CHO), neuroblastoma cells fused with glialoma cells (NG108-15) and murine embryonic stem cells (ES-D3)--after N2 laser disruption of the cell membrane and removal, via optical trapping, of a single subcellular organelle. Morphological changes and viability (as determined by live/dead fluorescent stains) of the cell were monitored every half hour over a 4-h period postsurgery. The ability of the cell to survive organelle extraction was found to depend both on the conditions under which surgery was performed and on the cell type. The average viability after surgery for CHO cells was approximately 80%, for NG 108 cells it was approximately 30% and for ES-D3 cells postsurgery viability was approximately 10%. From over 600 surgeries we found the survival of the cell is determined almost exclusively within the first hour postsurgery regardless of cell line. The optimal pulse energy for N2 laser ablation was approximately 0.7 microJ. The N2 pulse produced an approximately 1-3 microm hole in the cell membrane and proved to be the primary source of cell death in those cells that did not survive the procedure. PMID:15850426

  8. Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae.

    PubMed Central

    Smith, M G; Simon, V R; O'Sullivan, H; Pon, L A

    1995-01-01

    During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread. Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells. Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast. These organelles display low levels of movement. During meiosis I, we observed a threefold increase in mitochondrial motility. Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures. Mitochondria do not co-localize with microtubules. Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation. In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility. In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures. The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures. These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast. Images PMID:8573793

  9. Sphingosine Kinase Regulates Microtubule Dynamics and Organelle Positioning Necessary for Proper G1/S Cell Cycle Transition in Trypanosoma brucei

    PubMed Central

    Pasternack, Deborah A.; Sharma, Aabha I.; Olson, Cheryl L.

    2015-01-01

    ABSTRACT Sphingolipids are important constituents of cell membranes and also serve as mediators of cell signaling and cell recognition. Sphingolipid metabolites such as sphingosine-1-phosphate and ceramide regulate signaling cascades involved in cell proliferation and differentiation, autophagy, inflammation, and apoptosis. Little is known about how sphingolipids and their metabolites function in single-celled eukaryotes. In the present study, we investigated the role of sphingosine kinase (SPHK) in the biology of the protozoan parasite Trypanosoma brucei, the agent of African sleeping sickness. T. brucei SPHK (TbSPHK) is constitutively but differentially expressed during the life cycle of T. brucei. Depletion of TbSPHK in procyclic-form T. brucei causes impaired growth and attenuation in the G1/S phase of the cell cycle. TbSPHK-depleted cells also develop organelle positioning defects and an accumulation of tyrosinated α-tubulin at the elongated posterior end of the cell, known as the “nozzle” phenotype, caused by other molecular perturbations in this organism. Our studies indicate that TbSPHK is involved in G1-to-S cell cycle progression, organelle positioning, and maintenance of cell morphology. Cytotoxicity assays using TbSPHK inhibitors revealed a favorable therapeutic index between T. brucei and human cells, suggesting TbSPHK to be a novel drug target. PMID:26443455

  10. Does lake size matter? Combining morphology and process modeling to examine the contribution of lake classes to population-scale processes

    USGS Publications Warehouse

    Winslow, Luke A.; Read, Jordan S.; Hanson, Paul C.; Stanley, Emily H.

    2014-01-01

    With lake abundances in the thousands to millions, creating an intuitive understanding of the distribution of morphology and processes in lakes is challenging. To improve researchers’ understanding of large-scale lake processes, we developed a parsimonious mathematical model based on the Pareto distribution to describe the distribution of lake morphology (area, perimeter and volume). While debate continues over which mathematical representation best fits any one distribution of lake morphometric characteristics, we recognize the need for a simple, flexible model to advance understanding of how the interaction between morphometry and function dictates scaling across large populations of lakes. These models make clear the relative contribution of lakes to the total amount of lake surface area, volume, and perimeter. They also highlight the critical thresholds at which total perimeter, area and volume would be evenly distributed across lake size-classes have Pareto slopes of 0.63, 1 and 1.12, respectively. These models of morphology can be used in combination with models of process to create overarching “lake population” level models of process. To illustrate this potential, we combine the model of surface area distribution with a model of carbon mass accumulation rate. We found that even if smaller lakes contribute relatively less to total surface area than larger lakes, the increasing carbon accumulation rate with decreasing lake size is strong enough to bias the distribution of carbon mass accumulation towards smaller lakes. This analytical framework provides a relatively simple approach to upscaling morphology and process that is easily generalizable to other ecosystem processes.

  11. Systematic status of the caridean families Gnathophyllidae Dana and Hymenoceridae Ortmann (Crustacea: Decapoda): a further examination based on molecular and morphological data

    NASA Astrophysics Data System (ADS)

    Gan, Zhibin; Li, Xinzheng; Kou, Qi; Chan, Tinyam; Chu, Kahou; Huang, Hui

    2015-01-01

    The four palaemonoid (sub)families Anchistioididae, Gnathophyllidae, Hymenoceridae, and Pontoniinae are similar in morphology, and all live in marine habitats. Their systematic relationships are controversial. In this study, we used sequences from a mitochondrial ribosomal gene (16S rRNA) and three nuclear genes (H3, NaK, and enolase) to explore the phylogenetic relationships of these four taxa. Our tree based on 43 species belonging to 28 genera shows that Gnathophyllidae and Hymenoceridae are nested within Pontoniinae. This result is consistent with evidence from larval morphology. The defining characteristics of Gnathophyllidae and Hymenoceridae, a vestigial or missing mandibular incisor process and a broadened third maxilliped, can also be found in Pontoniinae; conversely, on the basis of published species descriptions, gnathophyllids and hymenocerids meet most of the defining characteristics of Pontoniinae. The peculiar form of the third maxilliped in gnathophyllids and hymenocerids might be the result of adaptive evolution, as these particular features are also present in pontoniines. According to our phylogenetic tree, Anchistioididae are more remote from Pontoniinae, which is consistent with the distinct morphological differences in the pleopods. The pontoniine genera analyzed (together with Gnathophyllidae and Hymenoceridae) are divided into two clades. The members of Clade I exhibit primordial characteristics similar to those of the Palaemoninae, and might be direct descendants of the ancestor of the Pontoniinae; members of Clade II are more specialized.

  12. Ion Channels in Plant Bioenergetic Organelles, Chloroplasts and Mitochondria: From Molecular Identification to Function.

    PubMed

    Carraretto, Luca; Teardo, Enrico; Checchetto, Vanessa; Finazzi, Giovanni; Uozumi, Nobuyuki; Szabo, Ildiko

    2016-03-01

    Recent technical advances in electrophysiological measurements, organelle-targeted fluorescence imaging, and organelle proteomics have pushed the research of ion transport a step forward in the case of the plant bioenergetic organelles, chloroplasts and mitochondria, leading to the molecular identification and functional characterization of several ion transport systems in recent years. Here we focus on channels that mediate relatively high-rate ion and water flux and summarize the current knowledge in this field, focusing on targeting mechanisms, proteomics, electrophysiology, and physiological function. In addition, since chloroplasts evolved from a cyanobacterial ancestor, we give an overview of the information available about cyanobacterial ion channels and discuss the evolutionary origin of chloroplast channels. The recent molecular identification of some of these ion channels allowed their physiological functions to be studied using genetically modified Arabidopsis plants and cyanobacteria. The view is emerging that alteration of chloroplast and mitochondrial ion homeostasis leads to organelle dysfunction, which in turn significantly affects the energy metabolism of the whole organism. Clear-cut identification of genes encoding for channels in these organelles, however, remains a major challenge in this rapidly developing field. Multiple strategies including bioinformatics, cell biology, electrophysiology, use of organelle-targeted ion-sensitive probes, genetics, and identification of signals eliciting specific ion fluxes across organelle membranes should provide a better understanding of the physiological role of organellar channels and their contribution to signaling pathways in plants in the future. PMID:26751960

  13. Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

    PubMed Central

    Beznoussenko, Galina V.; Pilyugin, Sergei S.; Geerts, Willie J. C.; Kozlov, Michael M.; Burger, Koert N. J.; Luini, Alberto; Derganc, Jure; Mironov, Alexander A.

    2015-01-01

    Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant. PMID:25761238

  14. Hypoxia signaling pathways: modulators of oxygen-related organelles

    PubMed Central

    Schönenberger, Miriam J.; Kovacs, Werner J.

    2015-01-01

    Oxygen (O2) is an essential substrate in cellular metabolism, bioenergetics, and signaling and as such linked to the survival and normal function of all metazoans. Low O2 tension (hypoxia) is a fundamental feature of physiological processes as well as pathophysiological conditions such as cancer and ischemic diseases. Central to the molecular mechanisms underlying O2 homeostasis are the hypoxia-inducible factors-1 and -2 alpha (HIF-1α and EPAS1/HIF-2α) that function as master regulators of the adaptive response to hypoxia. HIF-induced genes promote characteristic tumor behaviors, including angiogenesis and metabolic reprogramming. The aim of this review is to critically explore current knowledge of how HIF-α signaling regulates the abundance and function of major O2-consuming organelles. Abundant evidence suggests key roles for HIF-1α in the regulation of mitochondrial homeostasis. An essential adaptation to sustained hypoxia is repression of mitochondrial respiration and induction of glycolysis. HIF-1α activates several genes that trigger mitophagy and represses regulators of mitochondrial biogenesis. Several lines of evidence point to a strong relationship between hypoxia, the accumulation of misfolded proteins in the endoplasmic reticulum, and activation of the unfolded protein response. Surprisingly, although peroxisomes depend highly on molecular O2 for their function, there has been no evidence linking HIF signaling to peroxisomes. We discuss our recent findings that establish HIF-2α as a negative regulator of peroxisome abundance and suggest a mechanism by which cells attune peroxisomal function with O2 availability. HIF-2α activation augments peroxisome turnover by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We discuss potential mechanisms by which HIF-2α might trigger pexophagy and place special emphasis on the potential pathological implications of HIF-2α-mediated pexophagy for human health. PMID:26258123

  15. MORPHOLOGICAL CONTROL OF MITOCHONDRIAL BIOENERGETICS

    PubMed Central

    Yu, Tianzheng; Wang, Li; Yoon, Yisang

    2015-01-01

    The major function of mitochondria is production and supply of cellular energy. Mitochondria are highly dynamic organelles undergoing frequent shape changes via fission and fusion. Many studies have elucidated the molecular components mediating fission and fusion and their regulatory mechanisms, and mitochondrial shape change is now recognized as an essential cellular process that is closely associated with functional states of mitochondria. This review updates the recent advancements in fission and fusion mechanisms, and discusses the bi-directional relationship between mitochondrial morphology and energetic states in physio-pathological settings. PMID:25553448

  16. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments

    NASA Astrophysics Data System (ADS)

    Huber, Matthias C.; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R.; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M.

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

  17. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments.

    PubMed

    Huber, Matthias C; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials. PMID:25362355

  18. Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

    NASA Astrophysics Data System (ADS)

    Phelps, Mandy S.; Sturtevant, Drew; Chapman, Kent D.; Verbeck, Guido F.

    2016-02-01

    We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern.

  19. Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study.

    PubMed

    Zhao, C M; Chen, D; Lintunen, M; Panula, P; Håkanson, R

    1999-12-01

    ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm

  20. Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria

    PubMed Central

    Jorda, Julien; Lopez, David; Wheatley, Nicole M; Yeates, Todd O

    2013-01-01

    Bacterial microcompartment (MCP) organelles are cytosolic, polyhedral structures consisting of a thin protein shell and a series of encapsulated, sequentially acting enzymes. To date, different microcompartments carrying out three distinct types of metabolic processes have been characterized experimentally in various bacteria. In the present work, we use comparative genomics to explore the existence of yet uncharacterized microcompartments encapsulating a broader set of metabolic pathways. A clustering approach was used to group together enzymes that show a strong tendency to be encoded in chromosomal proximity to each other while also being near genes for microcompartment shell proteins. The results uncover new types of putative microcompartments, including one that appears to encapsulate B12-independent, glycyl radical-based degradation of 1,2-propanediol, and another potentially involved in amino alcohol metabolism in mycobacteria. Preliminary experiments show that an unusual shell protein encoded within the glycyl radical-based microcompartment binds an iron-sulfur cluster, hinting at complex mechanisms in this uncharacterized system. In addition, an examination of the computed microcompartment clusters suggests the existence of specific functional variations within certain types of MCPs, including the alpha carboxysome and the glycyl radical-based microcompartment. The findings lead to a deeper understanding of bacterial microcompartments and the pathways they sequester. PMID:23188745

  1. A host cell membrane microdomain is a critical factor for organelle discharge by Toxoplasma gondii.

    PubMed

    Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Matsubara, Ryuma; Aonuma, Hiroka; Nagamune, Kisaburo

    2016-10-01

    Host cell microdomains are involved in the attachment, entry, and replication of intracellular microbial pathogens. Entry into the host cell of Toxoplasma gondii and the subsequent survival of this protozoan parasite are tightly coupled with the proteins secreted from organelle called rhoptry. The rhoptry proteins are rapidly discharged into clusters of vesicles, called evacuoles, which are then delivered to parasitophorous vacuoles (PVs) or nucleus. In this study, we examined the roles of two host cell microdomain components, cholesterol and glycosylphosphatidylinositol (GPI), in evacuole formation. The acute depletion of cholesterol from the host cell plasma membrane blocked evacuole formation but not invasion. Whereas the lack of host cell GPI also altered evacuole formation but not invasion, instead inducing excess evacuole formation. The latter effect was not influenced by the evacuole-inhibiting effects of host cell cholesterol depletion, indicating the independent roles of host GPI and cholesterol in evacuole formation. In addition, the excess formation of evacuoles resulted in the enhanced recruitment of host mitochondria and endoplasmic reticulum to PVs, which in turn stimulated the growth of the parasite. PMID:27217289

  2. Multiple organelle-targeting signals in the N-terminal portion of peroxisomal membrane protein PMP70.

    PubMed

    Iwashita, Shohei; Tsuchida, Masashi; Tsukuda, Miwa; Yamashita, Yukari; Emi, Yoshikazu; Kida, Yuichiro; Komori, Masayuki; Kashiwayama, Yoshinori; Imanaka, Tsuneo; Sakaguchi, Masao

    2010-04-01

    Most membrane proteins are recognized by a signal recognition particle and are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, whereas almost all peroxisomal membrane proteins are posttranslationally targeted to the destination. Here we examined organelle-targeting properties of the N-terminal portions of the peroxisomal isoform of the ABC transporter PMP70 (ABCD3) using enhanced green fluorescent protein (EGFP) fusion. When the N-terminal 80 amino acid residue (N80)-segment preceding transmembrane segment (TM) 1 was deleted and the TM1-TM2 region was fused to EGFP, the TM1 segment induced ER-targeting and integration in COS cells. When the N80-segment was fused to EGFP, the fusion protein was targeted to the outer mitochondrial membrane. When both the N80-segment and the following TM1-TM2 region were present, the fusion located exclusively to the peroxisome. The full-length PMP70 molecule was clearly located in the ER in the absence of the N80-segment, even when multiple peroxisome-targeting signals were retained. We concluded that the TM1 segment possesses a sufficient ER-targeting function and that the N80-segment is critical for suppressing the ER-targeting function to allow the TM1-TM2 region to localize to the peroxisome. Cooperation of the organelle-targeting signals enables PMP70 to correctly target to peroxisomal membranes. PMID:20007743

  3. Glucocorticoid effect on repair processes in vascular connective tissue. Morphological examination and biochemical studies on collagen RNA and DNA in rabbit aorta.

    PubMed

    Manthorpe, R; Garbarsch, C; Lorenzen, I

    1975-10-01

    Male rabbits were injured by a single mechanical dilatation of the aorta and then injected with prednisone 2 mg/kg saline for 14 days or starved. Morphological studies and biochemical measurements of the collagen metabolism, the content of alpha-amino nitrogen, RNA, DNA, water and fat, and the aorta to serum ratio of 125I-albumin were performed on the intima-media layer of the descending thoracic aorta. Prednisone inhibited the intimal thickening. In the media the infiltration by mononuclear cells, the proliferation and regeneration of the smooth muscle cells and the calcification were reduced. Prednisone caused a decrease in 0.45 M NaCl soluble collagen as well as in the dialysable and non-dialysable 14C-hydroxyproline fractions. The total amount of collagen, elastin and alpha-amino nitrogen was unchanged, whereas the 14C-proline incorporation in the non-dialysable protein fraction was inhibited to a greater extent than the 14C-hydroxyproline synthesis. The findings indicate that prednisone inhibits the biosynthesis of collagen, which is inhibited to a greater extent than the general protein synthesis. Prednisone increased the dialysable to non-dialysable 14C-hydroxyproline ratio consistent with a relative increase in the catabolism of newly synthesized collagen. The aortic content of RNA and DNA was reduced consistent with the inhibition of protein synthesis and cell proliferation. Finally prednisone decreased the aortic content of water when related to the wet weight and increased the aortic content of fat. The aorta to serum ratio of 125I-albumin was not influenced by prednisone. It is concluded that administration of glucocorticoid for 14 days exerts an inhibitory action on the histological reaction to injury as well as on the biosynthesis of collagen of the repair processes in vascular connective tissue. A comparison with the effects of prednisone on undamaged rabbit aorta (Manthorpe et al. 1974) demonstrates that the metabolism of collagen of vascular

  4. Diagnostic correlation between RET proto-oncogene mutation, imaging techniques, biochemical markers and morphological examination in MEN2A syndrome: case report and literature review.

    PubMed

    Sovrea, Alina Simona; Dronca, Eleonora; Galatâr, Mihaela; Radian, Serban; Vornicescu, Corina; Georgescu, Carmen

    2014-01-01

    Multiple endocrine neoplasia type 2 (MEN2) is a rare autosomal dominant monogenic disorder caused mostly by missense mutations in the RET (REarranged during Transfection) proto-oncogene on chromosome 10q11.2. MEN2A represents more than 50% of all MEN2 cases, having a regular pattern with medullary thyroid carcinoma (MTC) incidence of 90-100%, bilateral pheochromocytoma (PCC) incidence of 40-50% and primary hyperparathyroidism (HPT) incidence of 10-25%. Until recently, the diagnosis of MTC was most frequently based on fine-needle aspiration of thyroid nodules, after an ultrasound examination and endocrine evaluation of serum calcitonin levels. Nowadays, RET gene screening (starting with exons 10 and 11) is a mandatory test used for identification of both symptomatic and non-symptomatic MTC carriers or for exclusion of healthy individuals from subsequent periodical clinical/biochemical screening. In this context, and in the idea of PCC preceding MTC, the early detection of germline RET mutations are highly suggestive for hereditary disease. PCC diagnosis is established in classical manner by abdominal ultrasound imaging or computed tomography confirming the presence of adrenal gland masses, elevated plasma metanephrines and normetanephrines values and histopathological examination. Additional HPT diagnosis is acknowledged by serum ionized calcium and parathormone levels. Here we report a hereditary case of MEN2A in a two-generation Romanian family, along with data presenting the importance of correlative plurifactorial diagnostic scheme in this syndrome and a short literature review. PMID:24969991

  5. Mitochondria and hydrogenosomes are two forms of the same fundamental organelle.

    PubMed Central

    Embley, T Martin; van der Giezen, Mark; Horner, David S; Dyal, Patricia L; Foster, Peter

    2003-01-01

    Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention. PMID:12594927

  6. Synthesis of cellular organelles containing nano-magnets stunts growth of magnetotactic bacteria.

    PubMed

    Naresh, Mohit; Hasija, Vivek; Sharma, Megha; Mittal, Aditya

    2010-07-01

    Magnetotactic bacteria are unique prokaryotes possessing the feature of cellular organelles called magnetosomes (membrane bound 40-50 nm vesicles entrapping a magnetic nano-crystal of magnetite or greigite). The obvious energetic impact of sophisticated eukaryotic-like membrane-bound organelle assembly on a presumably simpler prokaryotic system is not addressed in literature. In this work, while presenting evidence of direct coupling of carbon source consumption to synthesis of magnetosomes, we provide the first experimentally derived estimate of energy for organelle synthesis by Magnetospirillum gryphiswaldense as approximately 5 nJoules per magnetosome. Considering our estimate of approximately 0.2 microJoules per bacterial cell as the energy required for growth, we show that the energetic load of organelle synthesis results in stunting of cell growth. We also show that removal of soluble iron or sequestration by exogenous compounds in the bacterial cell cultures reverses the impact of the excess metabolic load exerted during magnetosomal synthesis. Thus, by taking advantage of the magnetotactic bacterial system we present the first experimental evidence for the presumed energy consumption during assembly of naturally occurring sub-100 nm intra-cellular organelles. PMID:21128392

  7. Lipid droplet organelle distribution in populations of dividing cells studied by simulation

    NASA Astrophysics Data System (ADS)

    Dalhaimer, Paul

    2013-06-01

    One of the key questions in cell biology is how organelles are passed from parent to daughter cells. To help address this question, I used Brownian dynamics to simulate lipid droplets as model organelles in populations of dividing cells. Lipid droplets are dynamic bodies that can form both de novo and by fission, they can also be depleted. The quantitative interplay among these three events is unknown but would seem crucial for controlling droplet distribution in populations of dividing cells. Surprisingly, of the three main events studied: biogenesis, fission, and depletion, the third played the key role in maintaining droplet organelle number—and to a lesser extent volume—in populations of dividing cells where formation events would have seemed paramount. In the case of lipid droplets, this provides computational evidence that they must be sustained, most likely through contacts with the endoplasmic reticulum. The findings also agree with video microscopy experiments over much shorter timescales where droplet depletion in fission yeast cells was not observed. In general, this work shows that organelle maintenance is invaluable and lack thereof cannot necessarily be compensated for by organelle formation. This study provides a time-accurate, physical-based template for long-term cell division studies.

  8. Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

    2016-04-01

    The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

  9. New Organelles by Gene Duplication in a Biophysical Model of Eukaryote Endomembrane Evolution

    PubMed Central

    Ramadas, Rohini; Thattai, Mukund

    2013-01-01

    Extant eukaryotic cells have a dynamic traffic network that consists of diverse membrane-bound organelles exchanging matter via vesicles. This endomembrane system arose and diversified during a period characterized by massive expansions of gene families involved in trafficking after the acquisition of a mitochondrial endosymbiont by a prokaryotic host cell >1.8 billion years ago. Here we investigate the mechanistic link between gene duplication and the emergence of new nonendosymbiotic organelles, using a minimal biophysical model of traffic. Our model incorporates membrane-bound compartments, coat proteins and adaptors that drive vesicles to bud and segregate cargo from source compartments, and SNARE proteins and associated factors that cause vesicles to fuse into specific destination compartments. In simulations, arbitrary numbers of compartments with heterogeneous initial compositions segregate into a few compositionally distinct subsets that we term organelles. The global structure of the traffic system (i.e., the number, composition, and connectivity of organelles) is determined completely by local molecular interactions. On evolutionary timescales, duplication of the budding and fusion machinery followed by loss of cross-interactions leads to the emergence of new organelles, with increased molecular specificity being necessary to maintain larger organellar repertoires. These results clarify potential modes of early eukaryotic evolution as well as more recent eukaryotic diversification. PMID:23746528

  10. Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles

    PubMed Central

    Colcombet, Jean; Lopez-Obando, Mauricio; Heurtevin, Laure; Bernard, Clément; Martin, Karine; Berthomé, Richard; Lurin, Claire

    2013-01-01

    Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles. PMID:24037373

  11. Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo.

    PubMed

    Melchionda, Manuela; Pittman, Jon K; Mayor, Roberto; Patel, Sandip

    2016-03-28

    Increasing evidence implicates Ca(2+) in the control of cell migration. However, the underlying mechanisms are incompletely understood. Acidic Ca(2+) stores are fast emerging as signaling centers. But how Ca(2+) is taken up by these organelles in metazoans and the physiological relevance for migration is unclear. Here, we identify a vertebrate Ca(2+)/H(+)exchanger (CAX) as part of a widespread family of homologues in animals. CAX is expressed in neural crest cells and required for their migration in vivo. It localizes to acidic organelles, tempers evoked Ca(2+) signals, and regulates cell-matrix adhesion during migration. Our data provide new molecular insight into how Ca(2+) is handled by acidic organelles and link this to migration, thereby underscoring the role of noncanonical Ca(2+) stores in the control of Ca(2+)-dependent function. PMID:27002171

  12. Membrane contact sites between pathogen-containing compartments and host organelles.

    PubMed

    Dumoux, Maud; Hayward, Richard D

    2016-08-01

    Intracellular pathogens survive and replicate within specialised membrane-bound compartments that can be considered as pseudo-organelles. Using the obligate intracellular bacterium Chlamydia as an illustrative example, we consider the modes of lipid transport between pathogen-containing compartments and host organelles, including the formation of static membrane contact sites. We discuss how lipid scavenging can be mediated via the reprogramming of cellular transporters at these interfaces and describe recent data suggesting that pathogen effectors modulate the formation of specific membrane contacts. Further study of these emerging mechanisms is likely to yield new insights into the cell biology of lipid transport and organelle communication, which highlights potential new targets and strategies for future therapeutics. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26825687

  13. Artificially-induced organelles are optimal targets for optical trapping experiments in living cells.

    PubMed

    López-Quesada, C; Fontaine, A-S; Farré, A; Joseph, M; Selva, J; Egea, G; Ludevid, M D; Martín-Badosa, E; Montes-Usategui, M

    2014-07-01

    Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

  14. Artificially-induced organelles are optimal targets for optical trapping experiments in living cells

    PubMed Central

    López-Quesada, C.; Fontaine, A.-S.; Farré, A.; Joseph, M.; Selva, J.; Egea, G.; Ludevid, M. D.; Martín-Badosa, E.; Montes-Usategui, M.

    2014-01-01

    Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

  15. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

    NASA Astrophysics Data System (ADS)

    Aumiller, William M.; Keating, Christine D.

    2016-02-01

    Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

  16. Lipidomics Analyses Reveal Temporal and Spatial Lipid Organization and Uncover Daily Oscillations in Intracellular Organelles.

    PubMed

    Aviram, Rona; Manella, Gal; Kopelman, Naama; Neufeld-Cohen, Adi; Zwighaft, Ziv; Elimelech, Meytar; Adamovich, Yaarit; Golik, Marina; Wang, Chunyan; Han, Xianlin; Asher, Gad

    2016-05-19

    Cells have evolved mechanisms to handle incompatible processes through temporal organization by circadian clocks and by spatial compartmentalization within organelles defined by lipid bilayers. Recent advances in lipidomics have led to identification of plentiful lipid species, yet our knowledge regarding their spatiotemporal organization is lagging behind. In this study, we quantitatively characterized the nuclear and mitochondrial lipidome in mouse liver throughout the day, upon different feeding regimens, and in clock-disrupted mice. Our analyses revealed potential connections between lipid species within and between lipid classes. Remarkably, we uncovered diurnal oscillations in lipid accumulation in the nucleus and mitochondria. These oscillations exhibited opposite phases and readily responded to feeding time. Furthermore, we found that the circadian clock coordinates the phase relation between the organelles. In summary, our study provides temporal and spatial depiction of lipid organization and reveals the presence and coordination of diurnal rhythmicity in intracellular organelles. PMID:27161994

  17. Fat(al) attraction: Picornaviruses Usurp Lipid Transfer at Membrane Contact Sites to Create Replication Organelles.

    PubMed

    van der Schaar, Hilde M; Dorobantu, Cristina M; Albulescu, Lucian; Strating, Jeroen R P M; van Kuppeveld, Frank J M

    2016-07-01

    All viruses that carry a positive-sense RNA genome (+RNA), such as picornaviruses, hepatitis C virus, dengue virus, and SARS- and MERS-coronavirus, confiscate intracellular membranes of the host cell to generate new compartments (i.e., replication organelles) for amplification of their genome. Replication organelles (ROs) are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors that create a unique lipid microenvironment optimal for genome replication. While some lipids may be locally synthesized de novo, other lipids are shuttled towards ROs. In picornavirus-infected cells, lipids are exchanged at membrane contact sites between ROs and other organelles. In this paper, we review recent advances in our understanding of how picornaviruses exploit host membrane contact site machinery to generate ROs, a mechanism that is used by some other +RNA viruses as well. PMID:27020598

  18. Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules.

    PubMed

    Serpinskaya, Anna S; Tuphile, Karine; Rabinow, Leonard; Gelfand, Vladimir I

    2014-01-01

    Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the biological role of some of the proteins binding to the microtubule track is to regulate cargo transport along these tracks. PMID:24163433

  19. A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles | Office of Cancer Genomics

    Cancer.gov

    Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells.

  20. Transcriptional changes of mouse splenocyte organelle components following acute infection with Toxoplasma gondii.

    PubMed

    He, Jun-Jun; Ma, Jun; Li, Fa-Cai; Song, Hui-Qun; Xu, Min-Jun; Zhu, Xing-Quan

    2016-08-01

    Toxoplasmosis is a globally spread zoonosis. The pathogen Toxoplasma gondii can hijack cellular organelles of host for replication. Although a number of important cellular life events are controlled by cell organelles, very little is known of the transcriptional changes of host cellular organelles after infection with T. gondii. Herein, we performed RNA-sequencing (RNA-seq) and bioinformatics analyses to study the global organelle component changes. It was found that many transcripts of the mouse spleen cellular organelle components were altered by acute T. gondii infection with the RH strain (Type I). Most differentially expressed transcripts of mitochondrial components were downregulated, especially those involved in biosynthetic and metabolic processes. Moreover, mitochondria based apoptosis process was downregulated. In terms of cytoskeleton, most differentially expressed transcript of cytoskeleton components were also downregulated, including septin cytoskeleton, cytoskeleton organization, centrosome and myosin. For endolysosomal system, ion transporters were downregulated at mRNA level, whereas the cytolytic components were increased, such as granzymes, Rab27a and perforin1 (Prf1). The main transcripts of Golgi apparatus components involved in sialylation or vesicle-mediated transportation were downregulated, while immune related components were upregulated. For endoplasmic reticulum (ER), posttranslational modification, drug metabolism and material transportation related transcripts were downregulated. In addition, T. gondii antigen cross-presentation by MHC-I complex could be downregulated by the downregulation of CD76 and ubiquitination related transcripts. The present study, for the first time, described the transcriptional changes of the mouse spleen cellular organelles following acute T. gondii infection, which provides a foundation to study the interaction between T. gondii and host cells at the sub-cellular level. PMID:27132051

  1. General morphology and ultrastructure of the female reproductive apparatus of Trichomalopsis shirakii crawford (Hymenoptera, Pteromalidae).

    PubMed

    Mao, Ning; Tang, Pu; Tian, Hong-Wei; Shi, Min; Chen, Xue-Xin

    2016-07-01

    The morphology and ultrastructure of the female reproductive system were examined for a larval-pupal parasitoid Trichomalopsis shirakii Crawford of Oulema oryzae Kuwayama using light and electron microscopes. The reproductive system includes two ovaries, two pairs of accessory glands, an unbranched venom gland, a large venom reservoir and a Dufour gland. Each ovariole contains follicles and oocytes at different stages of maturation. A fibrous layer covers the surface of mature egg. The accessory glands are made up of a layer of secretory cells surrounded by muscle fibers. In these secretory cells, numerous mitochondria, electron-dense secretory granules and vesicles filled with dense granular particles are present. These granular particles appear as virus-like particles (VLPs). The venom gland consists of a single layer of secretory cells which are organelle rich with abundant rough endoplasmic reticulum, mitochondria and vesicular organelles, a layer of duct cells and an inner intima. The reservoir consists of a muscular sheath, epidermal cells with few organelles and an intima layer. The Dufour gland has a relatively large lumen surrounded by a single layer of columnar epithelial cells which are characterized by clusters of smooth endoplasmic reticulum and lipid droplets. Aside from the venom, the fibrous layer coating the egg and the granular particles which may be VLPs have been discovered in our study. They may serve as one of the parasitoid-associated factors in their host-parasitoid relationship and play a role in host immune suppression. Microsc. Res. Tech. 79:625-636, 2016. © 2016 Wiley Periodicals, Inc. PMID:27151249

  2. A single origin of the photosynthetic organelle in different Paulinella lineages

    PubMed Central

    Yoon, Hwan Su; Nakayama, Takuro; Reyes-Prieto, Adrian; Andersen, Robert A; Boo, Sung Min; Ishida, Ken-ichiro; Bhattacharya, Debashish

    2009-01-01

    Background Gaining the ability to photosynthesize was a key event in eukaryotic evolution because algae and plants form the base of the food chain on our planet. The eukaryotic machines of photosynthesis are plastids (e.g., chloroplast in plants) that evolved from cyanobacteria through primary endosymbiosis. Our knowledge of plastid evolution, however, remains limited because the primary endosymbiosis occurred more than a billion years ago. In this context, the thecate "green amoeba" Paulinella chromatophora is remarkable because it very recently (i.e., minimum age of ≈ 60 million years ago) acquired a photosynthetic organelle (termed a "chromatophore"; i.e., plastid) via an independent primary endosymbiosis involving a Prochlorococcus or Synechococcus-like cyanobacterium. All data regarding P. chromatophora stem from a single isolate from Germany (strain M0880/a). Here we brought into culture a novel photosynthetic Paulinella strain (FK01) and generated molecular sequence data from these cells and from four different cell samples, all isolated from freshwater habitats in Japan. Our study had two aims. The first was to compare and contrast cell ultrastructure of the M0880/a and FK01 strains using scanning electron microscopy. The second was to assess the phylogenetic diversity of photosynthetic Paulinella to test the hypothesis they share a vertically inherited plastid that originated in their common ancestor. Results Comparative morphological analyses show that Paulinella FK01 cells are smaller than M0880/a and differ with respect to the number of scales per column. There are more distinctive, multiple fine pores on the external surface of FK01 than in M0880/a. Molecular phylogenetic analyses using multiple gene markers demonstrate these strains are genetically distinct and likely comprise separate species. The well-supported monophyly of the Paulinella chromatophora strains analyzed here using plastid-encoded 16S rRNA suggests strongly that they all share a

  3. High-throughput imaging of heterogeneous cell organelles with an X-ray laser

    SciTech Connect

    Hantke, Max, F.

    2014-11-17

    Preprocessed detector images that were used for the paper "High-throughput imaging of heterogeneous cell organelles with an X-ray laser". The CXI file contains the entire recorded data - including both hits and blanks. It also includes down-sampled images and LCLS machine parameters. Additionally, the Cheetah configuration file is attached that was used to create the pre-processed data.

  4. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

    PubMed Central

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T.; Texier, Yves; van Beersum, Sylvia E. C.; Horn, Nicola; Willer, Jason R.; Mans, Dorus A.; Dougherty, Gerard; Lamers, Ideke J. C.; Coene, Karlien L. M.; Arts, Heleen H.; Betts, Matthew J.; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J. F.; Marcelis, Carlo L.; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M.; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A.; Toedt, Grischa; van Dam, Teunis J. P.; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L.; Blacque, Oliver E.; Gibson, Toby J.; Huynen, Martijn A.; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E.; Franco, Brunella; Giles, Rachel H.; Ueffing, Marius; Russell, Robert B.; Roepman, Ronald; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Danecek, Petr; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Reghan Foley, A.; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; Joyce, Chris; McCarthy, Shane; Mitchison, Hannah M.; Muddyman, Dawn; Muntoni, Francesco; O'Rahilly, Stephen; Onoufriadis, Alexandros; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter; Schmidts, Miriam; Schoenmakers, Nadia; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Whittall, Ros; Williamson, Kathy

    2016-01-01

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

  5. A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts.

    PubMed

    Chat, Joëlle; Decroocq, Stéphane; Petit, Rémy J

    2003-04-22

    Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences. PMID:12737655

  6. A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts.

    PubMed Central

    Chat, Joëlle; Decroocq, Stéphane; Petit, Rémy J

    2003-01-01

    Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences. PMID:12737655

  7. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.

    PubMed

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T; Texier, Yves; van Beersum, Sylvia E C; Horn, Nicola; Willer, Jason R; Mans, Dorus A; Dougherty, Gerard; Lamers, Ideke J C; Coene, Karlien L M; Arts, Heleen H; Betts, Matthew J; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J F; Marcelis, Carlo L; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A; Toedt, Grischa; van Dam, Teunis J P; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L; Blacque, Oliver E; Gibson, Toby J; Huynen, Martijn A; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E; Franco, Brunella; Giles, Rachel H; Ueffing, Marius; Russell, Robert B; Roepman, Ronald

    2016-01-01

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

  8. In situ observation of cellular organelles with a contact x-ray microscope

    NASA Astrophysics Data System (ADS)

    Kado, M.; Kishimoto, M.; Tamotsu, S.; Yasuda, K.; Shinohara, K.

    2013-10-01

    A contact x-ray microscope coupled with a highly intense laser plasma soft x-ray source has been developed and in situ observations of cellular organelles have been conducted. The soft x-rays were generated by irradiating a high power laser pulse onto a thin-foiled gold target and about 1.3×1015 photons/sr were obtained, which allowed the inner structures of live wet biological cells to be imaged. Single shot flash imaging is a key technique to image cellular organelles of live biological cells avoiding degradation of the spatial resolution of the images resulting from motion blur and radiation damage. The use of a fluorescence microscope to identify cellular organelles in conjunction with the soft x-ray microscope has allowed several cellular organelles to be identified precisely in the soft x-ray images. Combining the fluorescence microscope with the soft x-ray microscope will be very useful and will establish the technique as a powerful tool to analyze living function of biological cells.

  9. Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

    PubMed Central

    Nott, Timothy J.; Petsalaki, Evangelia; Farber, Patrick; Jervis, Dylan; Fussner, Eden; Plochowietz, Anne; Craggs, Timothy D.; Bazett-Jones, David P.; Pawson, Tony; Forman-Kay, Julie D.; Baldwin, Andrew J.

    2015-01-01

    Summary Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles. PMID:25747659

  10. Biogenesis of the crystalloid organelle in Plasmodium involves microtubule-dependent vesicle transport and assembly

    PubMed Central

    Saeed, Sadia; Tremp, Annie Z.; Dessens, Johannes T.

    2015-01-01

    Malaria parasites possess unique subcellular structures and organelles. One of these is the crystalloid, a multivesicular organelle that forms during the parasite’s development in vector mosquitoes. The formation and function of these organelles remain poorly understood. A family of six conserved and modular proteins named LCCL-lectin adhesive-like proteins (LAPs), which have essential roles in sporozoite transmission, localise to the crystalloids. In this study we analyse crystalloid formation using transgenic Plasmodium berghei parasites expressing GFP-tagged LAP3. We show that deletion of the LCCL domain from LAP3 causes retarded crystalloid development, while knockout of LAP3 prevents formation of the organelle. Our data reveal that the process of crystalloid formation involves active relocation of endoplasmic reticulum-derived vesicles to common assembly points via microtubule-dependent transport. Inhibition of microtubule-dependent cargo transport disrupts this process and replicates the LCCL domain deletion mutant phenotype in wildtype parasites. These findings provide the first clear insight into crystalloid biogenesis, demonstrating a fundamental role for the LAP family in this process, and identifying the crystalloid and its formation as potential targets for malaria transmission control. PMID:25900212

  11. Lens fibre cell differentiation and organelle loss: many paths lead to clarity

    PubMed Central

    Wride, Michael A.

    2011-01-01

    The programmed removal of organelles from differentiating lens fibre cells contributes towards lens transparency through formation of an organelle-free zone (OFZ). Disruptions in OFZ formation are accompanied by the persistence of organelles in lens fibre cells and can contribute towards cataract. A great deal of work has gone into elucidating the nature of the mechanisms and signalling pathways involved. It is apparent that multiple, parallel and redundant pathways are involved in this process and that these pathways form interacting networks. Furthermore, it is possible that the pathways can functionally compensate for each other, for example in mouse knockout studies. This makes sense given the importance of lens clarity in an evolutionary context. Apoptosis signalling and proteolytic pathways have been implicated in both lens fibre cell differentiation and organelle loss, including the Bcl-2 and inhibitor of apoptosis families, tumour necrosis factors, p53 and its regulators (such as Mdm2) and proteolytic enzymes, including caspases, cathepsins, calpains and the ubiquitin–proteasome pathway. Ongoing approaches being used to dissect the molecular pathways involved, such as transgenics, lens-specific gene deletion and zebrafish mutants, are discussed here. Finally, some of the remaining unresolved issues and potential areas for future studies are highlighted. PMID:21402582

  12. Membrane trafficking and organelle biogenesis in Giardia lamblia: use it or lose it.

    PubMed

    Faso, Carmen; Hehl, Adrian B

    2011-04-01

    The secretory transport capacity of Giardia trophozoites is perfectly adapted to the changing environment in the small intestine of the host and is able to deploy essential protective surface coats as well as molecules which act on epithelia. These lumen-dwelling parasites take up nutrients by bulk endocytosis through peripheral vesicles or by receptor-mediated transport. The environmentally-resistant cyst form is quiescent but poised for activation following stomach passage. Its versatility and fidelity notwithstanding, the giardial trafficking systems appear to be the product of a general secondary reduction process geared towards minimization of all components and machineries identified to date. Since membrane transport is directly linked to organelle biogenesis and maintenance, less complexity also means loss of organelle structures and functions. A case in point is the Golgi apparatus which is missing as a steady-state organelle system. Only a few basic Golgi functions have been experimentally demonstrated in trophozoites undergoing encystation. Similarly, mitochondrial remnants have reached a terminally minimized state and appear to be functionally restricted to essential iron-sulfur protein maturation processes. Giardia's minimized organization combined with its genetic tractability provides unique opportunities to study basic principles of secretory transport in an uncluttered cellular environment. Not surprisingly, Giardia is gaining increasing attention as a model for the investigation of gene regulation, organelle biogenesis, and export of simple but highly protective cell wall biopolymers, a hallmark of all perorally transmitted protozoan and metazoan parasites. PMID:21296082

  13. Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

    PubMed Central

    Reisen, Daniel; Hanson, Maureen R

    2007-01-01

    Background Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2), which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins. Results We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1), myosin XI-6 (At MYA2), myosin XI-8 (At XI-B), myosin XI-15 (At XI-I), myosin XI-16 (At XI-J) and myosin XI-17 (At XI-K) were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2), previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria. Conclusion 6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and/or organelles when

  14. Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells

    PubMed Central

    2012-01-01

    Background In the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells. Results We used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles. Conclusions To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution

  15. Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens.

    PubMed

    Millet, Coralie O M; Williams, Catrin F; Hayes, Anthony J; Hann, Anthony C; Cable, Joanne; Lloyd, David

    2013-10-01

    In some eukaryotes, mitochondria have become modified during evolution to yield derived organelles (MDOs) of a similar size (hydrogenosomes), or extremely reduced to produce tiny cellular vesicles (mitosomes). The current study provides evidence for the presence of MDOs in the highly infectious fish pathogen Spironucleus vortens, an organism that produces H₂ and is shown here to have no detectable cytochromes. Transmission electron microscopy (TEM) reveals that S. vortens trophozoites contain electron-dense, membranous structures sometimes with an electron-dense core (200 nm-1 μm), resembling the hydrogenosomes previously described in other protists from habitats deficient in O₂. Confocal microscopy establishes that these organelles exhibit autofluorescence emission spectra similar to flavoprotein constituents previously described for mitochondria and also present in hydrogenosomes. These organelles possess a membrane potential and are labelled by a fluorescently labeled antibody against Fe-hydrogenase from Blastocystis hominis. Heterologous antibodies raised to mitochondrial proteins frataxin and Isu1, also exhibit a discrete punctate pattern of localization in S. vortens; however these labelled structures are distinctly smaller (90-150 nm) than hydrogenosomes as observed previously in other organisms. TEM confirms the presence of double-membrane bounded organelles of this smaller size. In addition, strong background immunostaining occurs in the cytosol for frataxin and Isu1, and labelling by anti-ferredoxin antibody is generally distributed and not specifically localized except for at the anterior polar region. This suggests that some of the functions traditionally attributed to such MDOs may also occur elsewhere. The specialized parasitic life-style of S. vortens may necessitate more complex intracellular compartmentation of redox reactions than previously recognized. Control of infection requires biochemical characterization of redox-related organelles

  16. Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance.

    PubMed

    Chan, Yee-Hung M; Reyes, Lorena; Sohail, Saba M; Tran, Nancy K; Marshall, Wallace F

    2016-05-01

    It has long been noted that larger animals have larger organs compared to smaller animals of the same species, a phenomenon termed scaling [1]. Julian Huxley proposed an appealingly simple model of "relative growth"-in which an organ and the whole body grow with their own intrinsic rates [2]-that was invoked to explain scaling in organs from fiddler crab claws to human brains. Because organ size is regulated by complex, unpredictable pathways [3], it remains unclear whether scaling requires feedback mechanisms to regulate organ growth in response to organ or body size. The molecular pathways governing organelle biogenesis are simpler than organogenesis, and therefore organelle size scaling in the cell provides a more tractable case for testing Huxley's model. We ask the question: is it possible for organelle size scaling to arise if organelle growth is independent of organelle or cell size? Using the yeast vacuole as a model, we tested whether mutants defective in vacuole inheritance, vac8Δ and vac17Δ, tune vacuole biogenesis in response to perturbations in vacuole size. In vac8Δ/vac17Δ, vacuole scaling increases with the replicative age of the cell. Furthermore, vac8Δ/vac17Δ cells continued generating vacuole at roughly constant rates even when they had significantly larger vacuoles compared to wild-type. With support from computational modeling, these results suggest there is no feedback between vacuole biogenesis rates and vacuole or cell size. Rather, size scaling is determined by the relative growth rates of the vacuole and the cell, thus representing a cellular version of Huxley's model. PMID:27151661

  17. Desulfovibrio magneticus RS-1 contains an iron- and phosphorus-rich organelle distinct from its bullet-shaped magnetosomes

    PubMed Central

    Byrne, Meghan E.; Ball, David A.; Guerquin-Kern, Jean-Luc; Rouiller, Isabelle; Wu, Ting-Di; Downing, Kenneth H.; Vali, Hojatollah; Komeili, Arash

    2010-01-01

    Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the α-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle. PMID:20566879

  18. Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

    PubMed Central

    Rubio, Noemi; Coupienne, Isabelle; Di Valentin, Emmanuel; Heirman, Ingeborg; Grooten, Johan; Piette, Jacques; Agostinis, Patrizia

    2012-01-01

    Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H2O2 in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis. PMID:22889744

  19. The Mitochondrial Antioxidants MitoE2 and MitoQ10 Increase Mitochondrial Ca2+ Load upon Cell Stimulation by Inhibiting Ca2+ Efflux from the Organelle

    PubMed Central

    Leo, Sara; Szabadkai, György; Rizzuto, Rosario

    2009-01-01

    Mitochondrial reactive oxygen species (ROS) production is recognized as a major pathogenic event in a number of human diseases, and mitochondrial scavenging of ROS appears a promising therapeutic approach. Recently, two mitochondrial antioxidants have been developed; conjugating α-tocopherol and the ubiquinol moiety of coenzyme Q to the lipophilic triphenylphosphonium cation (TPP+), denominated MitoE2 and MitoQ10, respectively. We have investigated the effect of these compounds on mitochondrial Ca2+ homeostasis, which controls processes as diverse as activation of mitochondrial dehydrogenases and pro-apoptotic morphological changes of the organelle. We demonstrate that treatment of HeLa cells with both MitoE2 and MitoQ10 induces (albeit with different efficacy) a major enhancement of the increase in matrix Ca2+ concentration triggered by cell stimulation with the inositol 1,4,5-trisphosphate-generating agonist histamine. The effect is a result of the inhibition of Ca2+ efflux from the organelle and depends on the TPP+ moiety of these compounds. Overall, the data identify an effect independent of their antioxidant activity, that on the one hand may be useful in addressing disorders in which mitochondrial Ca2+ handling is impaired (e.g., mitochondrial diseases) and on the other may favor mitochondrial Ca2+ overload and thus increase cell sensitivity to apoptosis (thus possibly counteracting the benefits of the antioxidant activity). PMID:19076448

  20. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    SciTech Connect

    Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

    2010-05-07

    Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

  1. Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles.

    PubMed

    Zheng, Jiameng; Won Han, Sang; Munnik, Teun; Rojas-Pierce, Marcela

    2014-08-13

    Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol-3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol-3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells. PMID:25119109

  2. Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles

    PubMed Central

    Zheng, Jiameng; Han, Sang Won; Munnik, Teun; Rojas-Pierce, Marcela

    2014-01-01

    Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells. PMID:25482812

  3. Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles.

    PubMed

    Zheng, Jiameng; Han, Sang Won; Munnik, Teun; Rojas-Pierce, Marcela

    2014-01-01

    Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells. PMID:25482812

  4. Biogenesis and subcellular organization of the magnetosome organelles of magnetotactic bacteria

    PubMed Central

    Greene, Shannon E.; Komeili, Arash

    2013-01-01

    Bacterial cells, like their eukaryotic counterparts, are capable of constructing lipid-based organelles that carry out essential biochemical functions. The magnetosomes of magnetotactic bacteria are one such compartment that is quickly becoming a model for exploring the process of organelle biogenesis in bacteria. Magnetosomes consist of a lipid-bilayer compartment that houses a magnetic crystal. By arranging magnetosomes into chains within the cell, magnetotactic bacteria create an internal compass that is used for navigation along magnetic fields. Over the past decade, a number of studies have elucidated the possible factors involved in the formation of the magnetosome membrane and biomineralization of magnetic minerals. Here, we highlight some of these recent advances with a particular focus on the cell biology of magnetosome formation. PMID:22726584

  5. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells.

    PubMed

    Kuzmin, Andrey N; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N

    2016-01-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging. PMID:27339882

  6. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    PubMed Central

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  7. Toxoplasma Rhoptries: Unique Secretory Organelles and Source of Promising Vaccine Proteins for Immunoprevention of Toxoplasmosis

    PubMed Central

    Dlugonska, Henryka

    2008-01-01

    Toxoplasma gondii is an obligate intracellular protozoan parasite classified in the phylum Apicomplexa, which includes numerous notable human and animal pathogens (Plasmodium species, Cryptosporidium species, Neospora caninum, etc.). The invasive stages of apicomplexans are characterized by the presence of an apical complex composed of specialized cytoskeletal and secretory organelles, including rhoptries. Rhoptries, unique apical secretory organelles shared exclusively by all apicomplexan parasites, are known to be involved in an active parasite's penetration into the host cell associated with the biogenesis of specific intracellular compartment, parasitophorous vacuole in which the parasite multiplies intensively, avoiding intracellular killing. Due to the key biological role of rhoptries, rhoptry proteins have recently become vaccine candidates for the prevention of several parasitoses, toxoplasmosis among them. The article presents current data on T. gondii rhoptries biology and new approaches to the development of effective vaccines against toxoplasmosis using rhoptry antigens. PMID:18670609

  8. Organelle biogenesis and interorganellar connections: Better in contact than in isolation.

    PubMed

    Daniele, Tiziana; Schiaffino, Maria Vittoria

    2014-01-01

    Membrane contact sites (MCSs) allow the exchange of molecules and information between organelles, even when their membranes cannot fuse directly. In recent years, a number of functions have been attributed to these contacts, highlighting their critical role in cell homeostasis. Although inter-organellar connections typically involve the endoplasmic reticulum (ER), we recently reported the presence of a novel MCSs between melanosomes and mitochondria. Melanosome-mitochondrion contacts appear mediated by fibrillar bridges resembling the protein tethers linking mitochondria and the ER, both for their ultrastructural features and the involvement of Mitofusin 2. The frequency of these connections correlates spatially and timely with melanosome biogenesis, suggesting a functional link between the 2 processes and in general that organelle biogenesis in the secretory pathway requires interorganellar crosstalks at multiple steps. Here, we summarize the different functions attributed to MCSs, and discuss their possible relevance for the newly identified melanosome-mitochondrion liaison. PMID:25346798

  9. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    NASA Astrophysics Data System (ADS)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-06-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ~95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

  10. Evaluation of predictions of the stochastic model of organelle production based on exact distributions

    PubMed Central

    Craven, C Jeremy

    2016-01-01

    We present a reanalysis of the stochastic model of organelle production and show that the equilibrium distributions for the organelle numbers predicted by this model can be readily calculated in three different scenarios. These three distributions can be identified as standard distributions, and the corresponding exact formulae for their mean and variance can therefore be used in further analysis. This removes the need to rely on stochastic simulations or approximate formulae (derived using the fluctuation dissipation theorem). These calculations allow for further analysis of the predictions of the model. On the basis of this we question the extent to which the model can be used to conclude that peroxisome biogenesis is dominated by de novo production when Saccharomyces cerevisiae cells are grown on glucose medium. DOI: http://dx.doi.org/10.7554/eLife.10167.001 PMID:26783763

  11. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.

    PubMed

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  12. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-06-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions.

  13. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    NASA Astrophysics Data System (ADS)

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-06-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

  14. Organelle-Specific Activity-Based Protein Profiling in Living Cells

    SciTech Connect

    Wiedner, Susan D.; Anderson, Lindsey N.; Sadler, Natalie C.; Chrisler, William B.; Kodali, Vamsi K.; Smith, Richard D.; Wright, Aaron T.

    2014-02-06

    A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.

  15. Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa.

    PubMed

    Gilmore, Simon; Peakall, Rod; Robertson, James

    2007-10-25

    Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis. PMID:17293071

  16. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis

    PubMed Central

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R.

    2016-01-01

    ABSTRACT Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  17. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    PubMed Central

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-01-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions. PMID:24957323

  18. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    PubMed Central

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-01-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging. PMID:27339882

  19. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis.

    PubMed

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R

    2016-05-01

    Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  20. A stereological study on organelle distribution in human oocytes at prophase I.

    PubMed

    Pires-Luís, Ana Sílvia; Rocha, Eduardo; Bartosch, Carla; Oliveira, Elsa; Silva, Joaquina; Barros, Alberto; Sá, Rosália; Sousa, Mário

    2016-06-01

    The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation. PMID:26170179

  1. Nanohole Array-Directed Trapping of Mammalian Mitochondria Enabling Single Organelle Analysis.

    PubMed

    Kumar, Shailabh; Wolken, Gregory G; Wittenberg, Nathan J; Arriaga, Edgar A; Oh, Sang-Hyun

    2015-12-15

    We present periodic nanohole arrays fabricated in free-standing metal-coated nitride films as a platform for trapping and analyzing single organelles. When a microliter-scale droplet containing mitochondria is dispensed above the nanohole array, the combination of evaporation and capillary flow directs individual mitochondria to the nanoholes. Mammalian mitochondria arrays were rapidly formed on chip using this technique without any surface modification steps, microfluidic interconnects, or external power sources. The trapped mitochondria were depolarized on chip using an ionophore with results showing that the organelle viability and behavior were preserved during the on-chip assembly process. Fluorescence signal related to mitochondrial membrane potential was obtained from single mitochondria trapped in individual nanoholes revealing statistical differences between the behavior of polarized vs depolarized mammalian mitochondria. This technique provides a fast and stable route for droplet-based directed localization of organelles-on-a-chip with minimal limitations and complexity, as well as promotes integration with other optical or electrochemical detection techniques. PMID:26593329

  2. Axonal transport of organelles visualized by light microscopy: cinemicrographic and computer analysis.

    PubMed

    Forman, D S; Padjen, A L; Siggins, G R

    1977-11-11

    Rapid movements of intra-axonal organelles in acutely isolated single myelinated fibers from bullfrog sciatic nerve were visualized by dark-field microscopy. The movements were recorded by cinemicrography, and analyzed by computer-based methods. The movements are saltatory and bidirectional, but each particle moves mainly in a single direction. For more than 90% of the particles, the predominant movement direction is retrograde, i.e. toward the cell body. Quantitative measurements on a variety of parameters of the organelle movements are presented. Different particles in the same axon show a broad range of mean speeds. The average mean speed of movement in the retrograde direction at 28 degrees C was 1.08 micrometer/sec (S.D. - 0.41), equivalent to an axonal transport rate of 93 mm/day. Disperse distributions were also found for other parameters such as the instantaneous velocities of individual particles. Quantal velocities, periodic movement patterns, and specific 'channels' were not detected. When the data from a population of particles is treated statistically, the average mean speed, the distribution of velocities, and other statistical parameters are found to be similar in different axons studied at the same temperature. Direct microscopical observation of axonal organelle movement is a technique which provides information about axonal transport which is different from and complementary to that obtained from enzyme accumulation of radioactive tracer methods. PMID:72584

  3. Viral Reorganization of the Secretory Pathway Generates Distinct Organelles for RNA Replication

    PubMed Central

    Hsu, Nai-Yun; Ilnytska, Olha; Belov, Georgiy; Santiana, Marianita; Chen, Ying-Han; Takvorian, Peter M.; Pau, Cyrilla; van der Schaar, Hilde; Kaushik-Basu, Neerja; Balla, Tamas; Cameron, Craig E.; Ehrenfeld, Ellie; van Kuppeveld, Frank J.M.; Altan-Bonnet, Nihal

    2010-01-01

    SUMMARY Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid micro-environment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIβ inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication. PMID:20510927

  4. New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

    PubMed

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

    Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. PMID:27241217

  5. Towards understanding the evolution and functional diversification of DNA-containing plant organelles.

    PubMed

    Leister, Dario

    2016-01-01

    Plastids and mitochondria derive from prokaryotic symbionts that lost most of their genes after the establishment of endosymbiosis. In consequence, relatively few of the thousands of different proteins in these organelles are actually encoded there. Most are now specified by nuclear genes. The most direct way to reconstruct the evolutionary history of plastids and mitochondria is to sequence and analyze their relatively small genomes. However, understanding the functional diversification of these organelles requires the identification of their complete protein repertoires - which is the ultimate goal of organellar proteomics. In the meantime, judicious combination of proteomics-based data with analyses of nuclear genes that include interspecies comparisons and/or predictions of subcellular location is the method of choice. Such genome-wide approaches can now make use of the entire sequences of plant nuclear genomes that have emerged since 2000. Here I review the results of these attempts to reconstruct the evolution and functions of plant DNA-containing organelles, focusing in particular on data from nuclear genomes. In addition, I discuss proteomic approaches to the direct identification of organellar proteins and briefly refer to ongoing research on non-coding nuclear DNAs of organellar origin (specifically, nuclear mitochondrial DNA and nuclear plastid DNA). PMID:26998248

  6. Helical repeats modular proteins are major players for organelle gene expression.

    PubMed

    Hammani, Kamel; Bonnard, Géraldine; Bouchoucha, Ayoub; Gobert, Anthony; Pinker, Franziska; Salinas, Thalia; Giegé, Philippe

    2014-05-01

    Mitochondria and chloroplasts are often described as semi-autonomous organelles because they have retained a genome. They thus require fully functional gene expression machineries. Many of the required processes going all the way from transcription to translation have specificities in organelles and arose during eukaryote history. Most factors involved in these RNA maturation steps have remained elusive for a long time. The recent identification of a number of novel protein families including pentatricopeptide repeat proteins, half-a-tetratricopeptide proteins, octotricopeptide repeat proteins and mitochondrial transcription termination factors has helped to settle long-standing questions regarding organelle gene expression. In particular, their functions have been related to replication, transcription, RNA processing, RNA editing, splicing, the control of RNA turnover and translation throughout eukaryotes. These families of proteins, although evolutionary independent, seem to share a common overall architecture. For all of them, proteins contain tandem arrays of repeated motifs. Each module is composed of two to three α-helices and their succession forms a super-helix. Here, we review the features characterising these protein families, in particular, their distribution, the identified functions and mode of action and propose that they might share similar substrate recognition mechanisms. PMID:24021622

  7. A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles

    PubMed Central

    Wang, Chensu; Wang, Yiguang; Li, Yang; Bodemann, Brian; Zhao, Tian; Ma, Xinpeng; Huang, Gang; Hu, Zeping; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming

    2015-01-01

    Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells. Deployment in cells allows quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. Together, these biological applications indicate the robustness and adaptability of this nanotechnology-enabled ‘detection and perturbation' strategy. PMID:26437053

  8. Involvement of Rab6a in organelle rearrangement and cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Ma, Rujun; Zhang, Jiaqi; Liu, Xiaohui; Li, Ling; Liu, Honglin; Rui, Rong; Gu, Ling; Wang, Qiang

    2016-01-01

    Rab GTPases have been reported to define the identity and transport routes of vesicles. Rab6 is one of the most extensively studied Rab proteins involved in regulating organelle trafficking and integrity maintenance. However, to date, the function of Rab6 in mammalian oocytes has not been addressed. Here we report severe disorganization of endoplasmic reticulum upon specific knockdown of Rab6a in mouse oocytes. In line with this finding, intracellular Ca2+ stores are accordingly reduced in Rab6a-depleted oocytes. Furthermore, in these oocytes, we observe the absence of cortical granule free domain, which is a kind of special organelle in matured oocytes and its exocytosis is calcium dependent. On the other hand, following Rab6a knockdown, the prominent defects of cytoskeletal structures are detected during oocyte meiosis. In particular, the majority of Rab6a-depleted oocytes fail to form the actin cap, and the frequency of spindle defects and chromosome misalignment is significantly elevated. In summary, our data reveal that Rab6a not only participates in modulating the organization of oocyte organelles, but also is a novel regulator of meiotic apparatus in mammalian oocytes. PMID:27030207

  9. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

    SciTech Connect

    Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand

    2010-09-07

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

  10. Surface Organelles Assembled by Secretion Systems of Gram-Negative Bacteria: Diversity in Structure and Function

    PubMed Central

    Thanassi, David G.; Bliska, James B.; Christie, Peter J.

    2012-01-01

    Gram-negative bacteria express a wide variety of organelles on their cell surface. These surface structures may be the end products of secretion systems, such as the hair-like fibers assembled by the chaperone/usher and type IV pilus pathways, which generally function in adhesion to surfaces and bacterial-bacterial and bacterial-host interactions. Alternatively, the surface organelles may be integral components of the secretion machinery itself, such as the needle complex and pilus extensions formed by the type III and type IV secretion systems, which function in the delivery of bacterial effectors inside host cells. Bacterial surface structures perform functions critical for pathogenesis and have evolved to withstand forces exerted by the external environment and cope with defenses mounted by the host immune system. Given their essential roles in pathogenesis and exposed nature, bacterial surface structures also make attractive targets for therapeutic intervention. This review will describe the structure and function of surface organelles assembled by four different Gram-negative bacterial secretion systems: the chaperone/usher pathway, the type IV pilus pathway, and the type III and type IV secretion systems. PMID:22545799

  11. Organelle sedimentation in gravitropic roots of Limnobium is restricted to the elongation zone

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Kim, D.; Stein, B.

    1994-01-01

    Roots of the aquatic angiosperm Limnobium spongia (Bosc) Steud. were evaluated by light and electron microscopy to determine the distribution of organelle sedimentation towards gravity. Roots of Limnobium are strongly gravitropic. The rootcap consists of only two layers of cells. Although small amyloplasts are present in the central cap cells, no sedimentation of any organelle, including amyloplasts, was found. In contrast, both amyloplasts and nuclei sediment consistently and completely in cells of the elongation zone. Sedimentation occurs in one cell layer of the cortex just outside the endodermis. Sedimentation of both amyloplasts and nuclei begins in cells that are in their initial stages of elongation and persists at least to the level of the root where root hairs emerge. This is the first modern report of the presence of sedimentation away from, but not in, the rootcap. It shows that sedimentation in the rootcap is not necessary for gravitropic sensing in at least one angiosperm. If amyloplast sedimentation is responsible for gravitropic sensing, then the site of sensing in Limnobium roots is the elongation zone and not the rootcap. These data do not necessarily conflict with the hypothesis that sensing occurs in the cap in other roots, since Limnobium roots are exceptional in rootcap origin and structure, as well as in the distribution of organelle sedimentation. Similarly, if nuclear sedimentation is involved in gravitropic sensing, then nuclear mass would function in addition to, not instead of, that of amyloplasts.

  12. Towards understanding the evolution and functional diversification of DNA-containing plant organelles

    PubMed Central

    Leister, Dario

    2016-01-01

    Plastids and mitochondria derive from prokaryotic symbionts that lost most of their genes after the establishment of endosymbiosis. In consequence, relatively few of the thousands of different proteins in these organelles are actually encoded there. Most are now specified by nuclear genes. The most direct way to reconstruct the evolutionary history of plastids and mitochondria is to sequence and analyze their relatively small genomes. However, understanding the functional diversification of these organelles requires the identification of their complete protein repertoires – which is the ultimate goal of organellar proteomics. In the meantime, judicious combination of proteomics-based data with analyses of nuclear genes that include interspecies comparisons and/or predictions of subcellular location is the method of choice. Such genome-wide approaches can now make use of the entire sequences of plant nuclear genomes that have emerged since 2000. Here I review the results of these attempts to reconstruct the evolution and functions of plant DNA-containing organelles, focusing in particular on data from nuclear genomes. In addition, I discuss proteomic approaches to the direct identification of organellar proteins and briefly refer to ongoing research on non-coding nuclear DNAs of organellar origin (specifically, nuclear mitochondrial DNA and nuclear plastid DNA). PMID:26998248

  13. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

    NASA Astrophysics Data System (ADS)

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  14. Plant vacuole morphology and vacuolar trafficking

    PubMed Central

    Zhang, Chunhua; Hicks, Glenn R.; Raikhel, Natasha V.

    2014-01-01

    Plant vacuoles are essential organelles for plant growth and development, and have multiple functions. Vacuoles are highly dynamic and pleiomorphic, and their size varies depending on the cell type and growth conditions. Vacuoles compartmentalize different cellular components such as proteins, sugars, ions and other secondary metabolites and play critical roles in plants response to different biotic/abiotic signaling pathways. In this review, we will summarize the patterns of changes in vacuole morphology in certain cell types, our understanding of the mechanisms of plant vacuole biogenesis, and the role of SNAREs and Rab GTPases in vacuolar trafficking. PMID:25309565

  15. Evaluation of Organelle Changes in Promastigotes of Unresponsive Leishmania Tropica to Meglumine Antimoniate in Comparison with Sensitive and Standard Isolates by Electron Microscopy

    PubMed Central

    Bahreini, Mitra; Bolorizadeh, Mehdi; Dabiri, Shahriar; Sharifi, Iraj

    2015-01-01

    Background: The control of leishmaniasis faces serious challenges because of resistance to the first-line antimonial drugs. We aimed to evaluate the differences in organelle changes of cultivated promastigotes obtained from skin lesions of sensitive and unresponsive isolates to meglumine antimoniate (Glucantime) by electron microscopy. Material and Methods: This study was done in Bam city, southeastern Iran, in which the incidence of disease has sharply increased since the earthquake in 2003. The samples were taken from 66 patients who were referred to the cutaneous leishmaniasis (CL) treatment center in Bam. A questionnaire was completed for each individual, recording their demographic characteristics and CL status. The scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by Giemsa, and examined under a compound light microscope for amastigote form simultaneously. To prepare the specimens for transmission electron imaging, promastigotes were centrifuged and resuspened. Results: Transmission electron microscopic study of the cultivated promastigotes revealed that there were alterations in the organelles and structures of sensitive isolates compared with unresponsive and standard ones. Organelles and structures such as mitochondria, kinetoplast, microtubules, cytoplasmic vacuoles, plasma membrane and vesicles were studied. The alterations such as disintegration of kinetoplast into thin filaments and condensation of kinetoplast DNA core, changes in size, number and location of vesicles and microtubules were observed. We noted intense cytoplasmic vacuolization, and considerable swelling of mitochondria. Conclusion: The significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism. PMID:26120175

  16. The molecular mechanism of organelle transport along microtubules: the identification and characterization of KIFs (kinesin superfamily proteins).

    PubMed

    Hirokawa, N

    1996-10-01

    In the cells various kinds of organelles are transported and distributed to their proper destinations in the cell. Organelle transports are very important for cellular morphogenesis and functions, with the conveying and targeting of essential materials to their correct destination being conducted, often at considerable velocities. Recently we have identified at least 10 new microtubule-associated motor proteins named as KIFs (kinesin superfamily proteins). Their characterization reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Molecular cell biological approaches combining multidisciplinary methods such as new electron microscopy, biochemistry, immunocytochemistry, biophysics, molecular biology and molecular genetics have revealed precise mechanisms of organelle transports in the cells by KIFs. PMID:9118241

  17. Musical morphology.

    PubMed

    Chakravarty, M Mallar; Vuust, Peter

    2009-07-01

    Morphologic measures have long been used to determine the patho-anatomical signature of different neurologic disorders. However, these measures can also be used to determine effects of specific learning tasks and quantifiable human abilities on cerebral structure. Musicians provide interesting opportunities for this type of analysis as their various skills, such as rhythmic ability and pitch and harmony discrimination (acquired through years of practicing and playing) can be quantified and compared using distinct morphologic analyses. Here, we review magnetic resonance imaging-based morphologic analyses in the music and neuroscience literature and provide some results from our own analysis of rhythmic ability in a cohort of musicians. PMID:19673757

  18. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole

    PubMed Central

    Miranda, Kildare; Pace, Douglas A.; Cintron, Roxana; Rodrigues, Juliany C.F.; Fang, Jianmin; Smith, Alyssa; Rohloff, Peter; Coelho, Elvis; de Haas, Felix; de Souza, Wanderley; Coppens, Isabelle; Sibley, L. David; Moreno, Silvia N. J.

    2010-01-01

    Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. Like most apicomplexans, Toxoplasma gondii possesses several plant-like features, such as the chloroplast-like organelle, the apicoplast. We describe and characterize a novel organelle in T. gondii tachyzoites, which is visible by light microscopy, and possesses a broad similarity to the plant vacuole. Electron tomography shows the interaction of this vacuole with other organelles. The presence of a plant-like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, a cathepsin L-like protease (TgCPL), an aquaporin (TgAQP1), as well as Ca2+/H+ and Na+/H+ exchange activities, supports similarity to the plant vacuole. Biochemical characterization of TgVP1 in enriched fractions shows a functional similarity to the respective plant enzyme. The organelle is a Ca2+ store and appears to have protective effects against salt stress potentially linked to its sodium transport activity. In intracellular parasites, the organelle fragments, with some markers co-localizing with the late endosomal marker, Rab7, suggesting its involvement with the endocytic pathway. Studies on the characterization of this novel organelle will be relevant to the identification of novel targets for chemotherapy against T. gondii and other apicomplexan parasites as well. PMID:20398214

  19. Vacuoles in sperm head are not associated with head morphology, DNA damage and reproductive success.

    PubMed

    Fortunato, Adriana; Boni, Raffaele; Leo, Rita; Nacchia, Giuseppina; Liguori, Francesca; Casale, Sofia; Bonassisa, Paolo; Tosti, Elisabetta

    2016-02-01

    In this retrospective study of 873 men enrolled for assisted reproduction techniques, relationships between sperm quality parameters, motile sperm organelle morphology examination (MSOME), DNA damage and live birth rate were evaluated. The presence of vacuoles in the sperm heads was detected by MSOME. Either chromatin decondensation or DNA fragmentation was used to study DNA damage. Results show that age significantly affected some of the examined parameters. In particular, sperm concentration was positively correlated (R = 0.088; P = 0.01) and chromatin decondensation was negatively correlated (R = -0.102; P = 0.003) with age. Furthermore, live birth rate was significantly lower in men aged 40 years or older (P < 0.02) compared with the younger age groups. The presence of sperm head vacuoles was not associated with head morphology, main sperm quality parameters, DNA fragmentation and live birth rate. Considering sperm heads in relation to the shape (normal/abnormal) and vacuoles (presence/absence), no significant variations in the occurrence of vacuoles in either normal or abnormal heads were found. These data suggest that vacuoles are physiological features that do not alter sperm functionality, and it seems that MSOME is not necessary for increasing the success of assisted reproduction techniques. PMID:26655650

  20. Optical tweezers for single molecule force spectroscopy on bacterial adhesion organelles

    NASA Astrophysics Data System (ADS)

    Andersson, Magnus; Axner, Ove; Uhlin, Bernt Eric; Fällman, Erik

    2006-08-01

    Instrumentation and methodologies for single molecule force spectroscopy on bacterial adhesion organelles by the use of force measuring optical tweezers have been developed. A thorough study of the biomechanical properties of fimbrial adhesion organelles expressed by uropathogenic E. coli, so-called pili, is presented. Steady-state as well as dynamic force measurements on P pili, expressed by E. coli causing pyelonephritis, have revealed, among other things, various unfolding and refolding properties of the helical structure of P pili, the PapA rod. Based on these properties an energy landscape model has been constructed by which specific biophysical properties of the PapA rod have been extracted, e.g. the number of subunits, the length of a single pilus, bond lengths and activation energies for bond opening and closure. Moreover, long time repetitive measurements have shown that the rod can be unfolded and refolded repetitive times without losing its intrinsic properties. These properties are believed to be of importance for the bacteria's ability to maintain close contact with host cells during initial infections. The results presented are considered to be of importance for the field of biopolymers in general and the development of new pharmaceuticals towards urinary tract infections in particular. The results show furthermore that the methodology can be used to gain knowledge of the intrinsic biomechanical function of adhesion organelles. The instrumentation is currently used for characterization of type 1 pili, expressed by E. coli causing cystitis, i.e. infections in the bladder. The first force spectrometry investigations of these pili will be presented.

  1. Redistribution of Golgi Stacks and Other Organelles during Mitosis and Cytokinesis in Plant Cells1[w

    PubMed Central

    Nebenführ, Andreas; Frohlick, Jennifer A.; Staehelin, L. Andrew

    2000-01-01

    We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean α-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the “Golgi belt,” accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly. PMID:10982429

  2. Monte Carlo analysis of obstructed diffusion in three dimensions: application to molecular diffusion in organelles.

    PubMed

    Olveczky, B P; Verkman, A S

    1998-05-01

    Molecular transport in the aqueous lumen of organelles involves diffusion in a confined compartment with complex geometry. Monte Carlo simulations of particle diffusion in three dimensions were carried out to evaluate the influence of organelle structure on diffusive transport and to relate experimental photobleaching data to intrinsic diffusion coefficients. Two organelle structures were modeled: a mitochondria-like long closed cylinder containing fixed luminal obstructions of variable number and size, and an endoplasmic reticulum-like network of interconnected cylinders of variable diameter and density. Trajectories were computed in each simulation for >10(5) particles, generally for >10(5) time steps. Computed time-dependent concentration profiles agreed quantitatively with analytical solutions of the diffusion equation for simple geometries. For mitochondria-like cylinders, significant slowing of diffusion required large or wide single obstacles, or multiple obstacles. In simulated spot photobleaching experiments, a approximately 25% decrease in apparent diffusive transport rate (defined by the time to 75% fluorescence recovery) was found for a single thin transverse obstacle occluding 93% of lumen area, a single 53%-occluding obstacle of width 16 lattice points (8% of cylinder length), 10 equally spaced 53% obstacles alternately occluding opposite halves of the cylinder lumen, or particle binding to walls (with mean residence time = 10 time steps). Recovery curve shape with obstacles showed long tails indicating anomalous diffusion. Simulations also demonstrated the utility of measurement of fluorescence depletion at a spot distant from the bleach zone. For a reticulum-like network, particle diffusive transport was mildly reduced from that in unobstructed three-dimensional space. In simulated photobleaching experiments, apparent diffusive transport was decreased by 39-60% in reticular structures in which 90-97% of space was occluded. These computations provide

  3. Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles

    PubMed Central

    Dobrowolski, Radek; De Robertis, Edward M.

    2012-01-01

    Signal transduction and endocytosis are intertwined processes. The internalization of ligand-activated receptors by endocytosis has classically been thought to attenuate signals by targeting receptors for degradation in lysosomes, but it can also maintain signals in early signalling endosomes. In both cases, localization to multivesicular endosomes en route to lysosomes is thought to terminate signalling. However, during WNT signal transduction, sequestration of the enzyme glycogen synthase kinase 3 (GSK3) inside multivesicular endosomes results in the stabilization of many cytosolic proteins. Thus, the role of endocytosis during signal transduction may be more diverse than anticipated, and multivesicular endosomes may constitute a crucial signalling organelle. PMID:22108513

  4. Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling

    PubMed Central

    Zhu, Qian; Du, Zengmin; Hu, Aiguo; Yang, Yi

    2015-01-01

    Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function. PMID:25923693

  5. Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling.

    PubMed

    Wang, Jianhua; Zhao, Yuzheng; Wang, Chao; Zhu, Qian; Du, Zengmin; Hu, Aiguo; Yang, Yi

    2015-01-01

    Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function. PMID:25923693

  6. Observation of organelle by a laser plasma x-ray microscope

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Kishimoto, Maki; Ishino, Masahiko; Tamotsu, Satoshi; Yasuda, Keiko; Shinohara, Kunio

    2012-07-01

    Contact x-ray microscopy has a potential to image wet biological specimens in natural condition. It is very important to identify obtained features in the x-ray images, since x-ray microscopes have potential to image features that have not been visualized yet. We have proposed to compare the x-ray images of the biological specimens with the fluorescence images and to identify the features found in the x-ray images. We have succeeded to observe fine structures of the cellular organelles such as mitochondria by the soft x-ray microscope.

  7. Kinesin and dynein superfamily proteins in organelle transport and cell division.

    PubMed

    Hirokawa, N; Noda, Y; Okada, Y

    1998-02-01

    Microtubule-associated motor proteins of the kinesin and dynein superfamilies play important roles in cellular mechanisms such as organelle transport and mitosis. Identification and characterization of new family members (in particular KIFC2, 16 new KIFs, XKlp2 and XKCM1 of the kinesin superfamily, and DHC2 and DHC3 of the dynein superfamily) and further characterization of known family members have improved our understanding of these cellular mechanisms. Sophisticated biophysical and structural analyses of monomeric and dimeric motor proteins have contributed to elucidating the mechanisms behind motor protein motility and polarity. PMID:9484596

  8. Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells.

    PubMed

    Kang, Kongsoo; Takahara, Michiyo; Sakaue, Haruka; Sakaguchi, Masao

    2016-05-01

    Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we estimated the extent of CP-domain folding in the cytosolic space. When fused to mitochondrial hydrophobic multispanning membrane protein ABCB10, more than half of the EGFP remained in the cytoplasm, whereas most of the CP-portion was in the mitochondrial fraction. When fused to the endoplasmic reticulum (ER) signal, the cleaved EGFP was observed only in the ER fraction, confirming that the CP-domain cannot fold on the cytoplasmic side during cotranslational ER translocation. Thus, import of the ABCB10 molecule was not as tightly coupled with chain elongation as ER translocation. Use of this probe to quantitatively examine stop-translocation at the ER translocon in living cells revealed that positively charged residues on the translocating nascent chain stall at the ER translocon. PMID:26711239

  9. Multiclassifier combinatorial proteomics of organelle shadows at the example of mitochondria in chromatin data

    PubMed Central

    Kustatscher, Georg; Grabowski, Piotr

    2016-01-01

    Subcellular localization is an important aspect of protein function, but the protein composition of many intracellular compartments is poorly characterized. For example, many nuclear bodies are challenging to isolate biochemically and thus remain inaccessible to proteomics. Here, we explore covariation in proteomics data as an alternative route to subcellular proteomes. Rather than targeting a structure of interest biochemically, we target it by machine learning. This becomes possible by taking data obtained for one organelle and searching it for traces of another organelle. As an extreme example and proof‐of‐concept we predict mitochondrial proteins based on their covariation in published interphase chromatin data. We detect about ⅓ of the known mitochondrial proteins in our chromatin data, presumably most as contaminants. However, these proteins are not present at random. We show covariation of mitochondrial proteins in chromatin proteomics data. We then exploit this covariation by multiclassifier combinatorial proteomics to define a list of mitochondrial proteins. This list agrees well with different databases on mitochondrial composition. This benchmark test raises the possibility that, in principle, covariation proteomics may also be applicable to structures for which no biochemical isolation procedures are available. PMID:26510496

  10. Three-Dimensional Spot Detection in Ratiometric Fluorescence Imaging For Measurement of Subcellular Organelles

    PubMed Central

    Lau, William W.; Johnson, Calvin A.; Lioi, Sara; Mindell, Joseph A.

    2014-01-01

    Lysosomes are subcellular organelles playing a vital role in the endocytosis process of the cell. Lysosomal acidity is an important factor in assuring proper functioning of the enzymes within the organelle, and can be assessed by labeling the lysosomes with pH-sensitive fluorescence probes. To enhance our understanding of the acidification mechanisms, the goal of this work is to develop a method that can accurately detect and characterize the acidity of each lysosome captured in ratiometric fluorescence images. We present an algorithm that utilizes the h-dome transformation and reconciles spots detected independently from two wavelength channels. We evaluated our algorithm using simulated images for which the exact locations were known. The h-dome algorithm achieved an f-score as high as 0.890. We also computed the fluorescence ratios from lysosomes in live HeLa cell images with known lysosomal pHs. Using leave-one-out cross-validation, we demonstrated that the new algorithm was able to achieve much better pH prediction accuracy than the conventional method. PMID:25621319

  11. A Non-sulfated Chondroitin Stabilizes Membrane Tubulation in Cnidarian Organelles*

    PubMed Central

    Adamczyk, Patrizia; Zenkert, Claudia; Balasubramanian, Prakash G.; Yamada, Shuhei; Murakoshi, Saori; Sugahara, Kazuyuki; Hwang, Jung Shan; Gojobori, Takashi; Holstein, Thomas W.; Özbek, Suat

    2010-01-01

    Membrane tubulation is generally associated with rearrangements of the cytoskeleton and other cytoplasmic factors. Little is known about the contribution of extracellular matrix components to this process. Here, we demonstrate an essential role of proteoglycans in the tubulation of the cnidarian nematocyst vesicle. The morphogenesis of this extrusive organelle takes place inside a giant post-Golgi vesicle, which topologically represents extracellular space. This process includes the formation of a complex collagenous capsule structure that elongates into a long tubule, which invaginates after its completion. We show that a non-sulfated chondroitin appears as a scaffold in early morphogenesis of all nematocyst types in Hydra and Nematostella. It accompanies the tubulation of the vesicle membrane forming a provisional tubule structure, which after invagination matures by collagen incorporation. Inhibition of chondroitin synthesis by β-xylosides arrests nematocyst morphogenesis at different stages of tubule outgrowth resulting in retention of tubule material and a depletion of mature capsules in the tentacles of hydra. Our data suggest a conserved role of proteoglycans in the stabilization of a membrane protrusion as an essential step in organelle morphogenesis. PMID:20538610

  12. Extension of the concept of an anomalous water component to images of T-cell organelles.

    PubMed

    Tychinsky, Vladimir P

    2014-12-01

    Microscopic images of a living cell are the main source of information on its functional state. Modern interference microscopy techniques allow the numerical parameters of cell images to be obtained with an accuracy not available with other methods. Quantitative analysis of phase images of T lymphocytes (TCs) in different functional states demonstrated that variations of the properties of intracellular water should be taken into account. This conclusion agrees with the current view that the physical parameters of water, including the refractive index (RI) of a water layer, depend on the hydrophilicity and other characteristics of the adjacent surface. Application of this concept to phase images of TCs showed that the contribution of the fourth phase of water (4-water) or the structured water component, which has an increased RI, should be considered. The proportion of 4-water depends on the functional state of the cell determined by the culture medium composition. Normally, the proportion of 4-water in organelles is as high as 30%; it is considerably lower in organelles of cells with inhibited metabolism. PMID:25500678

  13. Cytosolic organelles shape calcium signals and exo-endocytotic responses of chromaffin cells.

    PubMed

    García, Antonio G; Padín, Fernando; Fernández-Morales, José C; Maroto, Marcos; García-Sancho, Javier

    2012-01-01

    The concept of stimulus-secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca(2+)) entry and this generates a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers the exocytotic release of catecholamines. The control of the [Ca(2+)](c) signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca(2+) from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca(2+) extrusion mechanisms, such as the plasma membrane Ca(2+)-stimulated ATPase, and the Na(+)/Ca(2+) exchanger. Computation of the rates of Ca(2+) fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca(2+) transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca(2+)](c) transients occurring at different cell compartments, and generated by redistribution and release of Ca(2+) by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo-endocytotic activities with energy production and protein synthesis. PMID:22209033

  14. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters

    NASA Astrophysics Data System (ADS)

    Nott, Timothy J.; Craggs, Timothy D.; Baldwin, Andrew J.

    2016-06-01

    Membraneless organelles are cellular compartments made from drops of liquid protein inside a cell. These compartments assemble via the phase separation of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an organic solvent than like water. One of the most-stable biological structures known, the DNA double helix, can be melted once inside the liquid droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in organic chemistry is to utilize different solvents to influence the behaviour of molecules and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles.

  15. A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus.

    PubMed Central

    Wang, S P; Sharma, P L; Schoenlein, P V; Ely, B

    1993-01-01

    Mutations having pleiotropic effects on polar organelle development (pod) in Caulobacter crescentus have been identified and shown to occur in at least 13 genes scattered throughout the genome. Mutations at each locus affect a unique combination of polar traits, suggesting that complex interactions occur among these genes. The DNA sequence of one of these genes, pleC, indicates that it is homologous to members of the family of histidine protein kinase genes. Membes of this family include the senor components of the bacterial two-component regulatory systems. Furthermore, in vitro experiments demonstrated that the PleC protein was capable of autophosphorylation. These results suggest that the PleC protein (and perhaps the proteins encoded by the other pod genes as well) regulates the expression of genes involved in polar organelle development through the phosphorylation of key regulatory proteins. The use of a phosphorelay system cued to internal changes in the cell would provide a mechanism for coordinating major changes in gene expression with the completion of specific cell cycle events. Images PMID:8421698

  16. Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells

    PubMed Central

    Rangel, Roberto; Guzman-Rojas, Liliana; le Roux, Lucia G.; Staquicini, Fernanda I.; Hosoya, Hitomi; Barbu, E. Magda; Ozawa, Michael G.; Nie, Jing; Jr, Kenneth Dunner; Langley, Robert R.; Sage, E. Helene; Koivunen, Erkki; Gelovani, Juri G.; Lobb, Roy R.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2012-01-01

    Phage display screening allows the study of functional protein–protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development. PMID:22510693

  17. Oxidative Stress in the Healthy and Wounded Hepatocyte: A Cellular Organelles Perspective

    PubMed Central

    Mello, Tommaso; Zanieri, Francesca; Ceni, Elisabetta; Galli, Andrea

    2016-01-01

    Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte's organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte. PMID:26788252

  18. Evolution and significance of the Lon gene family in Arabidopsis organelle biogenesis and energy metabolism

    PubMed Central

    Rigas, Stamatis; Daras, Gerasimos; Tsitsekian, Dikran; Alatzas, Anastasios; Hatzopoulos, Polydefkis

    2014-01-01

    Lon is the first identified ATP-dependent protease highly conserved across all kingdoms. Model plant species Arabidopsis thaliana has a small Lon gene family of four members. Although these genes share common structural features, they have distinct properties in terms of gene expression profile, subcellular targeting and substrate recognition motifs. This supports the notion that their functions under different environmental conditions are not necessarily redundant. This article intends to unravel the biological role of Lon proteases in energy metabolism and plant growth through an evolutionary perspective. Given that plants are sessile organisms exposed to diverse environmental conditions and plant organelles are semi-autonomous, it is tempting to suggest that Lon genes in Arabidopsis are paralogs. Adaptive evolution through repetitive gene duplication events of a single archaic gene led to Lon genes with complementing sets of subfunctions providing to the organism rapid adaptability for canonical development under different environmental conditions. Lon1 function is adequately characterized being involved in mitochondrial biogenesis, modulating carbon metabolism, oxidative phosphorylation and energy supply, all prerequisites for seed germination and seedling establishment. Lon is not a stand-alone proteolytic machine in plant organelles. Lon in association with other nuclear-encoded ATP-dependent proteases builds up an elegant nevertheless, tight interconnected circuit. This circuitry channels properly and accurately, proteostasis and protein quality control among the distinct subcellular compartments namely mitochondria, chloroplasts, and peroxisomes. PMID:24782883

  19. Release from myosin V via regulated recruitment of an E3 Ub ligase controls organelle localization

    PubMed Central

    Yau, Richard G.; Peng, Yutian; Valiathan, Rajeshwari R.; Birkeland, Shanda R.; Wilson, Thomas E.; Weisman, Lois S.

    2014-01-01

    Summary Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin-V, Myo2, binds the vacuole specific adaptor, Vac17, to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 which recruits the E3 ligase, Dma1, to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism which detaches myosin-V from select cargoes. PMID:24636257

  20. Oxidative Stress in the Healthy and Wounded Hepatocyte: A Cellular Organelles Perspective.

    PubMed

    Mello, Tommaso; Zanieri, Francesca; Ceni, Elisabetta; Galli, Andrea

    2016-01-01

    Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte's organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte. PMID:26788252

  1. Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

    NASA Astrophysics Data System (ADS)

    Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

    2005-04-01

    Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

  2. Cell organelles at uncoated cryofractured surfaces as viewed with the scanning electron microscope.

    PubMed

    Woods, P S; Ledbetter, M C

    1976-06-01

    A method of direct visualization of cell organelles by scanning electron microscopy (SEM) is described. Plant and animal tissues fixed in glutaraldehyde and osmium tetroxide are treated with the ligand thiocarbohydrazide and a second osmium tetroxide solution, to increase their osmium content. Tissues are then dehydrated, infiltrated with an epoxy monomer, and together solidified with dry ice and fractured. The pieces are transferred to pure acetone, critical-point dried, attached to stubs with silver paint and viewed by SEM. The ligating procedure increases the osmium concentration at its original bonding site sufficiently to render the tissue electrically conductive, thus obviating the need for metallic coating. he organelles at the fractured surface are revaled in relation to their osmium incorporation rather than by surface irregularities as with coating methods. The image derived from the uncoated surface approaches in resolution that of transmission electron micrographs of thin sections. A protion of the image arising from a small distance below the surface, while at progressively lower resolution, provides some 3-dimensional information about cell fine structure. PMID:777015

  3. Extension of the concept of an anomalous water component to images of T-cell organelles

    NASA Astrophysics Data System (ADS)

    Tychinsky, Vladimir P.

    2014-12-01

    Microscopic images of a living cell are the main source of information on its functional state. Modern interference microscopy techniques allow the numerical parameters of cell images to be obtained with an accuracy not available with other methods. Quantitative analysis of phase images of T lymphocytes (TCs) in different functional states demonstrated that variations of the properties of intracellular water should be taken into account. This conclusion agrees with the current view that the physical parameters of water, including the refractive index (RI) of a water layer, depend on the hydrophilicity and other characteristics of the adjacent surface. Application of this concept to phase images of TCs showed that the contribution of the fourth phase of water (4-water) or the structured water component, which has an increased RI, should be considered. The proportion of 4-water depends on the functional state of the cell determined by the culture medium composition. Normally, the proportion of 4-water in organelles is as high as 30% it is considerably lower in organelles of cells with inhibited metabolism.

  4. From Endosymbiont to Host-Controlled Organelle: The Hijacking of Mitochondrial Protein Synthesis and Metabolism

    PubMed Central

    Gabaldón, Toni; Huynen, Martijn A

    2007-01-01

    Mitochondria are eukaryotic organelles that originated from the endosymbiosis of an alpha-proteobacterium. To gain insight into the evolution of the mitochondrial proteome as it proceeded through the transition from a free-living cell to a specialized organelle, we compared a reconstructed ancestral proteome of the mitochondrion with the proteomes of alpha-proteobacteria as well as with the mitochondrial proteomes in yeast and man. Overall, there has been a large turnover of the mitochondrial proteome during the evolution of mitochondria. Early in the evolution of the mitochondrion, proteins involved in cell envelope synthesis have virtually disappeared, whereas proteins involved in replication, transcription, cell division, transport, regulation, and signal transduction have been replaced by eukaryotic proteins. More than half of what remains from the mitochondrial ancestor in modern mitochondria corresponds to translation, including post-translational modifications, and to metabolic pathways that are directly, or indirectly, involved in energy conversion. Altogether, the results indicate that the eukaryotic host has hijacked the proto-mitochondrion, taking control of its protein synthesis and metabolism. PMID:17983265

  5. Apical Organelle Secretion by Toxoplasma Controls Innate and Adaptive Immunity and Mediates Long-Term Protection.

    PubMed

    Sloves, Pierre-Julien; Mouveaux, Thomas; Ait-Yahia, Saliha; Vorng, Han; Everaere, Laetitia; Sangare, Lamba Omar; Tsicopoulos, Anne; Tomavo, Stanislas

    2015-11-01

    Apicomplexan parasites have unique apical rhoptry and microneme secretory organelles that are crucial for host infection, although their role in protection against Toxoplasma gondii infection is not thoroughly understood. Here, we report a novel function of the endolysosomal T. gondii sortilin-like receptor (TgSORTLR), which mediates trafficking to functional apical organelles and their subsequent secretion of virulence factors that are critical to the induction of sterile immunity against parasite reinfection. We further demonstrate that the T. gondii armadillo repeats-only protein (TgARO) mutant, which is deficient only in apical secretion of rhoptries, is also critical in mounting protective immunity. The lack of TgSORTLR and TgARO proteins completely inhibited T-helper 1-dependent adaptive immunity and compromised the function of natural killer T-cell-mediated innate immunity. Our findings reveal an essential role for apical secretion in promoting sterile protection against T. gondii and provide strong evidence for rhoptry-regulated discharge of antigens as a key effector for inducing protective immunity. PMID:25910629

  6. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters.

    PubMed

    Nott, Timothy J; Craggs, Timothy D; Baldwin, Andrew J

    2016-06-01

    Membraneless organelles are cellular compartments made from drops of liquid protein inside a cell. These compartments assemble via the phase separation of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an organic solvent than like water. One of the most-stable biological structures known, the DNA double helix, can be melted once inside the liquid droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in organic chemistry is to utilize different solvents to influence the behaviour of molecules and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles. PMID:27219701

  7. Visualization of volatile substances in different organelles with an atmospheric-pressure mass microscope.

    PubMed

    Harada, Takahiro; Yuba-Kubo, Akiko; Sugiura, Yuki; Zaima, Nobuhiro; Hayasaka, Takahiro; Goto-Inoue, Naoko; Wakui, Masatoshi; Suematsu, Makoto; Takeshita, Kengo; Ogawa, Kiyoshi; Yoshida, Yoshikazu; Setou, Mitsutoshi

    2009-11-01

    We have developed a mass microscope (mass spectrometry imager with spatial resolution higher than the naked eye) equipped with an atmospheric pressure ion-source chamber for laser desorption/ionization (AP-LDI) and a quadrupole ion trap time-of-flight (QIT-TOF) analyzer. The optical microscope combined with the mass spectrometer permitted us to precisely determine the relevant tissue region prior to performing imaging mass spectrometry (IMS). An ultraviolet laser tightly focused with a triplet lens was used to achieve high spatial resolution. An atmospheric pressure ion-source chamber enables us to analyze fresh samples with minimal loss of intrinsic water or volatile compounds. Mass-microscopic AP-LDI imaging of freshly cut ginger rhizome sections revealed that 6-gingerol ([M + K](+)at m/z 333.15, positive mode; [M - H](-) at m/z 293.17, negative mode) and the monoterpene ([M + K](+) at m/z 191.09), which are the compounds related to pungency and flavor, respectively, were localized in oil drop-containing organelles. AP-LDI-tandem MS/MS analyses were applied to compare authentic signals from freshly cut ginger directly with the standard reagent. Thus, our atmosphere-imaging mass spectrometer enabled us to monitor a quality of plants at the organelle level. PMID:19788281

  8. The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.

    PubMed Central

    Steinberg, G; Schliwa, M

    1995-01-01

    The "conventional" kinesins comprise a conserved family of molecular motors for organelle transport that have been identified in various animal species. Organelle motors from other phyla have not yet been analyzed at the molecular level. Here we report the identification, biochemical and immunological characterization, and molecular cloning of a cytoplasmic motor in a "lower" eukaryote, the Ascomycete fungus Neurospora crassa. This motor, termed Nkin (for Neurospora kinesin), exhibits several unique structural and functional features, including a high rate of microtubule transport, a lack of copurifying light chains, a second P-loop motif, and an overall sequence organization reminiscent of a kinesin-like protein. However, a greater than average sequence homology in the motor domain and the presence of a highly conserved region in the C-terminus identify Nkin as a distant relative of the family of conventional kinesins. A molecular phylogenetic analysis suggests Nkin to have diverged early in the evolution of this family of motors. The discovery of Nkin may help identify domains important for specific biological functions in conventional kinesins. Images PMID:8589459

  9. Mitochondrial Morphology in Metabolic Diseases

    PubMed Central

    Galloway, Chad A.

    2013-01-01

    Abstract Significance: Mitochondria are the cellular energy-producing organelles and are at the crossroad of determining cell life and death. As such, the function of mitochondria has been intensely studied in metabolic disorders, including diabetes and associated maladies commonly grouped under all-inclusive pathological condition of metabolic syndrome. More recently, the altered metabolic profiles and function of mitochondria in these ailments have been correlated with their aberrant morphologies. This review describes an overview of mitochondrial fission and fusion machineries, and discusses implications of mitochondrial morphology and function in these metabolic maladies. Recent Advances: Mitochondria undergo frequent morphological changes, altering the mitochondrial network organization in response to environmental cues, termed mitochondrial dynamics. Mitochondrial fission and fusion mediate morphological plasticity of mitochondria and are controlled by membrane-remodeling mechanochemical enzymes and accessory proteins. Growing evidence suggests that mitochondrial dynamics play an important role in diabetes establishment and progression as well as associated ailments, including, but not limited to, metabolism–secretion coupling in the pancreas, nonalcoholic fatty liver disease progression, and diabetic cardiomyopathy. Critical Issues: While mitochondrial dynamics are intimately associated with mitochondrial bioenergetics, their cause-and-effect correlation remains undefined in metabolic diseases. Future Directions: The involvement of mitochondrial dynamics in metabolic diseases is in its relatively early stages. Elucidating the role of mitochondrial dynamics in pathological metabolic conditions will aid in defining the intricate form–function correlation of mitochondria in metabolic pathologies and should provide not only important clues to metabolic disease progression, but also new therapeutic targets. Antioxid. Redox Signal. 19, 415–430. PMID:22793999

  10. QUANTITATIVE MORPHOLOGY

    EPA Science Inventory

    Abstract: In toxicology, the role of quantitative assessment of brain morphology can be understood in the context of two types of treatment-related alterations. One type of alteration is specifically associated with treatment and is not observed in control animals. Measurement ...

  11. Morphological Analysis of Rubus Seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genus Rubus exhibits morphological diversity and a wide range of reproductive systems and habitats. We examined seed coat ultrastructural morphology of seed accessions of 10 subgenera preserved at the US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Reposito...

  12. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants

    PubMed Central

    Morley, Stewart A.; Nielsen, Brent L.

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles. PMID:26870072

  13. Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle diversification mechanism and intragenus phylotyping strategy in microalgae

    PubMed Central

    2013-01-01

    Background Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes. Results Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5 Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species. Conclusion This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy

  14. The Role of Organelle Stresses in Diabetes Mellitus and Obesity: Implication for Treatment

    PubMed Central

    Chang, Yi-Cheng; Hee, Siow-Wey; Hsieh, Meng-Lun; Jeng, Yung-Ming; Chuang, Lee-Ming

    2015-01-01

    The type 2 diabetes pandemic in recent decades is a huge global health threat. This pandemic is primarily attributed to the surplus of nutrients and the increased prevalence of obesity worldwide. In contrast, calorie restriction and weight reduction can drastically prevent type 2 diabetes, indicating a central role of nutrient excess in the development of diabetes. Recently, the molecular links between excessive nutrients, organelle stress, and development of metabolic disease have been extensively studied. Specifically, excessive nutrients trigger endoplasmic reticulum stress and increase the production of mitochondrial reactive oxygen species, leading to activation of stress signaling pathway, inflammatory response, lipogenesis, and pancreatic beta-cell death. Autophagy is required for clearance of hepatic lipid clearance, alleviation of pancreatic beta-cell stress, and white adipocyte differentiation. ROS scavengers, chemical chaperones, and autophagy activators have demonstrated promising effects for the treatment of insulin resistance and diabetes in preclinical models. Further results from clinical trials are eagerly awaited. PMID:26613076

  15. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation. PMID:24489879

  16. [Methods of substances and organelles introduction in living cell for cell engineering technologies].

    PubMed

    Nikitin, V A

    2007-01-01

    We have presented the classification of more than 40 methods of genetic material, substances and organelles introduction into a living cell. Each of them has its characteristic advantages, disadvantages and limitations with respect to cell viability, transfer efficiency, general applicability, and technical requirements. It this article we have enlarged on the description of our developments of several new and improved approaches, methods and devices of the direct microinjection into a single cell and cell microsurgery with the help of glass micropipettes. The problem of low efficiency of mammalian cloning is discussed with emphasis on the necessity of expertizing of each step of single cell reconstruction to begin with microsurgical manipulations and necessity of the development of such methods of single cell resonstruction that could minimize the possible damage of the cell. PMID:17926558

  17. Biochemistry of the Phagosome: The Challenge to Study a Transient Organelle

    PubMed Central

    Nüsse, Oliver

    2011-01-01

    Phagocytes are specialized cells of the immune system, designed to engulf and destroy harmful microorganisms inside the newly formed phagosome. The latter is an intracellular organelle that is transformed into a toxic environment within minutes and disappears once the pathogen is destroyed. Reactive oxygen species and reactive nitrogen species are produced inside the phagosome. Intracellular granules or lysosomes of the phagocyte fuse with the phagosome and liberate their destructive enzymes. This process of phagocytosis efficiently protects against most infections; however, some microorganisms avoid their destruction and cause severe damage. To understand such failure of phagosomal killing, we need to learn more about the actual destruction process in the phagosome. This paper summarizes methods to investigate the biochemistry of the phagosome and discusses some of their limitations. In accordance with the nature of the phagosome, the issue of localization and temporal dynamics is emphasized, and recent developments are highlighted. PMID:22194668

  18. B Chromosomes of Aegilops speltoides Are Enriched in Organelle Genome-Derived Sequences

    PubMed Central

    Ruban, Alevtina; Fuchs, Jörg; Marques, André; Schubert, Veit; Soloviev, Alexander; Raskina, Olga; Badaeva, Ekaterina; Houben, Andreas

    2014-01-01

    B chromosomes (Bs) are dispensable components of the genome exhibiting non-Mendelian inheritance. Chromosome counts and flow cytometric analysis of the grass species Aegilops speltoides revealed a tissue-type specific distribution of the roughly 570 Mbp large B chromosomes. To address the question whether organelle-to-nucleus DNA transfer is a mechanism that drives the evolution of Bs, in situ hybridization was performed with labelled organellar DNA. The observed B-specific accumulation of chloroplast- and mitochondria-derived sequences suggests a reduced selection against the insertion of organellar DNA in supernumerary chromosomes. The distribution of B-localised organellar-derived sequences and other sequences differs between genotypes of different geographical origins. PMID:24587288

  19. Organelle-specific injury to melanin-containing cells in human skin by pulsed laser irradiation

    SciTech Connect

    Murphy, G.F.; Shepard, R.S.; Paul, B.S.; Menkes, A.; Anderson, R.R.; Parrish, J.A.

    1983-12-01

    Physical models predict that ultraviolet laser radiation of appropriately brief pulses can selectively alter melanin-containing cellular targets in human skin. Skin of normal human volunteers was exposed to brief (20 nanosecond) 351-nm wave length pulses from a XeF excimer laser, predicting that those cells containing the greatest quantities of melanized melanosomes (lower half of the epidermis) would be selectively damaged. Transmission electron microscopy revealed the earliest cellular alteration to be immediate disruption of melanosomes, both within melanocytes and basal keratinocytes. This disruption was dose dependent and culminated in striking degenerative changes in these cells. Superficial keratinocytes and Langerhans cells were not affected. It was concluded that the XeF excimer laser is capable of organelle-specific injury to melanosomes. These findings may have important clinical implications in the treatment of both benign and malignant pigmented lesions by laser radiations of defined wave lengths and pulse durations.

  20. Influence of organelle geometry on the apparent binding kinetics of peripheral membrane proteins

    NASA Astrophysics Data System (ADS)

    Hoffmann, Julia; Fickentscher, Rolf; Weiss, Matthias

    2015-02-01

    Information processing in living cells frequently involves an exchange of peripheral membrane proteins between the cytosol and organelle membranes. The typical time scale τ of these association-dissociation cycles is commonly quantified in vivo via fluorescence recovery after photobleaching (FRAP). Contrary to common assumptions, we show here that τ values determined by FRAP depend on the size and number of target structures. Hence, FRAP times alone are insufficient to draw conclusions about the proteins' binding kinetics. In contrast, extracting primary molecular association and dissociation rates from FRAP approaches provides a size-independent and therefore robust measure for the proteins' binding kinetics. We support our theoretical considerations with experiments on the small GTPase Arf-1 that transiently associates with Golgi membranes: While Arf-1 recovery times in untreated cells and in cells with disrupted microtubules are significantly different, the molecular kinetic rates are shown to be the same in both cases.

  1. The dynamic subcellular localization of ERK: mechanisms of translocation and role in various organelles.

    PubMed

    Wainstein, Ehud; Seger, Rony

    2016-04-01

    The dynamic subcellular localization of ERK in resting and stimulated cells plays an important role in its regulation. In resting cells, ERK localizes in the cytoplasm, and upon stimulation, it translocates to its target substrates and organelles. ERK signaling initiated from different places in resting cells has distinct outcomes. In this review, we summarize the mechanisms of ERK1/2 translocation to the nucleus and mitochondria, and of ERK1c to the Golgi. We also show that ERK1/2 translocation to the nucleus is a useful anti cancer target. Unraveling the complex subcellular localization of ERK and its dynamic changes upon stimulation provides a better understanding of the regulation of ERK signaling and may result in the development of new strategies to combat ERK-related diseases. PMID:26827288

  2. Symbiotic theory of the origin of eukaryotic organelles; criteria for proof.

    PubMed

    Margulis, L

    1975-01-01

    The purpose of a scientific theory is to unite apparently disparate observations into a coherent set of generalizations with predictive power. Historical theories, which necessarily treat complex irreversible events, can never be directly tested. However they certainly can lead to predictions. The 'extreme' version of the serial endosymbiotic theory argues that three classes of eukaryotic organelles had free-living ancestors: mitochondria, basal bodies/flagella/cilia [(9 + 2) homologues] and photosynthetic plastids. Many lines of evidence support this theory and can be interpreted in relation to one another on the basis of this theory. Even if this theory should eventually be proved wrong it has the real advantage of generating a large number of unique experimentally verifiable hypotheses. PMID:822529

  3. MicroRNAs in the intracellular space, regulation of organelle specific pathways in health and disease.

    PubMed

    Nguyen, Thao T; Brenu, Ekua W; Staines, Don R; Marshall-Gradisnik, Sonya M

    2014-01-01

    MicroRNAs (miRNA) are small (~22 nucleotide] non-coding RNA molecules originally characterised as nonsense or junk DNA. Emerging research suggests that these molecules have diverse regulatory roles in an array of molecular, cellular and physiological processes. MiRNAs are versatile and highly stable molecules, therefore, they are able to exist as intracellular or extracellular miRNAs. The purpose of this paper is to review the function and role of miRNAs in the intracellular space with specific focus on the interactions between miRNAs and organelles such as the mitochondria and the rough endoplasmic reticulum. Understanding the role of miRNAs in the intracellular space may be vital in understanding the mechanism of certain diseases. PMID:25541912

  4. Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite.

    PubMed

    Santos, Jorge M; Duarte, Neuza; Kehrer, Jessica; Ramesar, Jai; Avramut, M Cristina; Koster, Abraham J; Dessens, Johannes T; Frischknecht, Friedrich; Chevalley-Maurel, Séverine; Janse, Chris J; Franke-Fayard, Blandine; Mair, Gunnar R

    2016-06-28

    Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid-a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony-is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission. PMID:27303037

  5. Metabolic Interplay between Peroxisomes and Other Subcellular Organelles Including Mitochondria and the Endoplasmic Reticulum

    PubMed Central

    Wanders, Ronald J. A.; Waterham, Hans R.; Ferdinandusse, Sacha

    2016-01-01

    Peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways which include: (1.) etherphospholipid biosynthesis; (2.) fatty acid beta-oxidation; (3.) bile acid synthesis; (4.) docosahexaenoic acid (DHA) synthesis; (5.) fatty acid alpha-oxidation; (6.) glyoxylate metabolism; (7.) amino acid degradation, and (8.) ROS/RNS metabolism. The importance of peroxisomes for human health and development is exemplified by the existence of a large number of inborn errors of peroxisome metabolism in which there is an impairment in one or more of the metabolic functions of peroxisomes. Although the clinical signs and symptoms of affected patients differ depending upon the enzyme which is deficient and the extent of the deficiency, the disorders involved are usually (very) severe diseases with neurological dysfunction and early death in many of them. With respect to the role of peroxisomes in metabolism it is clear that peroxisomes are dependent on the functional interplay with other subcellular organelles to sustain their role in metabolism. Indeed, whereas mitochondria can oxidize fatty acids all the way to CO2 and H2O, peroxisomes are only able to chain-shorten fatty acids and the end products of peroxisomal beta-oxidation need to be shuttled to mitochondria for full oxidation to CO2 and H2O. Furthermore, NADH is generated during beta-oxidation in peroxisomes and beta-oxidation can only continue if peroxisomes are equipped with a mechanism to reoxidize NADH back to NAD+, which is now known to be mediated by specific NAD(H)-redox shuttles. In this paper we describe the current state of knowledge about the functional interplay between peroxisomes and other subcellular compartments notably the mitochondria and endoplasmic reticulum for each of the metabolic pathways in which peroxisomes are involved. PMID:26858947

  6. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism

    PubMed Central

    Toledo, Daniel A. M.; Roque, Natália R.; Teixeira, Lívia; Milán-Garcés, Erix A.; Carneiro, Alan B.; Almeida, Mariana R.; Andrade, Gustavo F. S.; Martins, Jefferson S.; Pinho, Roberto R.; Freire-de-Lima, Célio G.; Bozza, Patrícia T.; D’Avila, Heloisa

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas’ disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  7. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism.

    PubMed

    Toledo, Daniel A M; Roque, Natália R; Teixeira, Lívia; Milán-Garcés, Erix A; Carneiro, Alan B; Almeida, Mariana R; Andrade, Gustavo F S; Martins, Jefferson S; Pinho, Roberto R; Freire-de-Lima, Célio G; Bozza, Patrícia T; D'Avila, Heloisa; Melo, Rossana C N

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas' disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  8. Pseudomonas aeruginosa Type IV Pilus Expression in Neisseria gonorrhoeae: Effects of Pilin Subunit Composition on Function and Organelle Dynamics▿ †

    PubMed Central

    Winther-Larsen, Hanne C.; Wolfgang, Matthew C.; van Putten, Jos P. M.; Roos, Norbert; Aas, Finn Erik; Egge-Jacobsen, Wolfgang M.; Maier, Berenike; Koomey, Michael

    2007-01-01

    Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilAPAK pilin subunit in N. gonorrhoeae. We show here that, although PilAPAK pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilAPAK pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilAPAK pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilAPAK pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilAPAK pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa. PMID:17573479

  9. Mitochondria Change Dynamics and Morphology during Grapevine Leaf Senescence

    PubMed Central

    Bodner, Martina; La Rocca, Nicoletta; De Michele, Roberto; Carimi, Francesco; Schiavo, Fiorella Lo; Zottini, Michela

    2014-01-01

    Leaf senescence is the last stage of development of an organ and is aimed to its ordered disassembly and nutrient reallocation. Whereas chlorophyll gradually degrades during senescence in leaves, mitochondria need to maintain active to sustain the energy demands of senescing cells. Here we analysed the motility and morphology of mitochondria in different stages of senescence in leaves of grapevine (Vitis vinifera), by stably expressing a GFP (green fluorescent protein) reporter targeted to these organelles. Results show that mitochondria were less dynamic and markedly changed morphology during senescence, passing from the elongated, branched structures found in mature leaves to enlarged and sparse organelles in senescent leaves. Progression of senescence in leaves was not synchronous, since changes in mitochondria from stomata were delayed. Mitochondrial morphology was also analysed in grapevine cell cultures. Mitochondria from cells at the end of their growth curve resembled those from senescing leaves, suggesting that cell cultures might represent a useful model system for senescence. Additionally, senescence-associated mitochondrial changes were observed in plants treated with high concentrations of cytokinins. Overall, morphology and dynamics of mitochondria might represent a reliable senescence marker for plant cells. PMID:25009991

  10. Ear examination

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003340.htm Ear examination To use the sharing features on this page, ... ear References King EF, Couch ME. History, physical examination, and the preoperative evaluation. In: Flint PW, Haughey ...

  11. Microscopy with spatial filtering for monitoring subcellular morphology

    NASA Astrophysics Data System (ADS)

    Zheng, Jing-Yi

    Dynamic alteration in organelle morphology is an important indicator of cellular function and many efforts have been made to monitor the subcellular morphology. Optical scatter imaging (OSI), which combines light scattering spectroscopy with microscopic imaging, was developed to non-invasively track real-time changes in particle morphology in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter (OSIR, optical scatter imaging ratio) at each pixel in the full field of view. For spherical particles, the OSIR was shown to decrease monotonically with diameter. In living cells, we reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl- xL transmembrane domain, but could not be observed by fluorescence or DIC images1. However, the initial design was based on Mie theory of scattering by spheres, and hence only adequate for measuring spherical particles. This limits the applicability of OSI to cellular functional studies involving organelles, which are naturally non-spherical. In this project, we aim to enhance the current capability of the existing optical scatter microscope to assess size and shape information for both spherical and non-spherical particles, and eventually apply this technique for monitoring and quantifying subcellular morphology within living cells. To reach this goal, we developed an improved system, in which the variable diameter iris is replaced with a digital micromirror device and adopted the concept of Gabor filtering to extend our assessment of morphology to the characterization of particle shape and orientation. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This

  12. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    SciTech Connect

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A.; Solecki, David J.

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.

  13. Insights into the biogenesis of lysosome-related organelles from the study of the Hermansky-Pudlak syndrome.

    PubMed

    Bonifacino, Juan S

    2004-12-01

    Lysosome-related organelles (LROs) are a family of cell-type-specific organelles that include melanosomes, platelet dense bodies, and cytotoxic T cell granules. The name, LRO, recognizes the fact that all of these organelles contain subsets of lysosomal proteins in addition to cell-type-specific proteins. The recent identification of genetic disorders that cause combined defects in several of these organelles indicates that they share common biogenetic pathways. Studies of one of these disorders, the Hermansky-Pudlak syndrome (HPS), have provided helpful insights into the molecular machinery involved in LRO biogenesis. HPS is a genetically heterogeneous disorder caused by mutations in any of 7 genes in humans and 15 genes in mice. These genes encode subunits of 4 multi-protein complexes named AP-3, BLOC-1, BLOC-2 and BLOC-3, in addition to miscellaneous components of the general protein trafficking machinery. The AP-3 complex is a coat protein involved in vesicle formation and cargo selection in the endosomal-lysosomal system. One of these cargo molecules is the melanosomal enzyme, tyrosinase, the missorting of which may explain the defective melanosomes in AP-3-deficient humans and mice. The function of the BLOC complexes is unknown, although they are thought to mediate either vesicle tethering/fusion or cytoplasmic dispersal of LROs. Further studies of these complexes should contribute to the elucidation of the mechanisms of LRO biogenesis and the pathogenesis of HPS. PMID:15838104

  14. Exocytosis of Endothelial Lysosome-Related Organelles Hair-Triggers a Patchy Loss of Glycocalyx at the Onset of Sepsis.

    PubMed

    Zullo, Joseph A; Fan, Jie; Azar, Tala T; Yen, Wanyi; Zeng, Min; Chen, Jun; Ratliff, Brian B; Song, Jun; Tarbell, John M; Goligorsky, Michael S; Fu, Bingmei M

    2016-02-01

    Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. Here, we tested a hypothesis that patchy degradation of ESG occurs early in sepsis and is a result of exocytosis of lysosome-related organelles. Time-lapse video microscopy revealed that exocytosis of Weibel-Palade bodies and secretory lysosomes occurred a few minutes after application of lipopolysaccharides to endothelial cells. Two therapeutic maneuvers, a nitric oxide intermediate, NG-hydroxy-l-arginine, and culture media conditioned by endothelial progenitor cells reduced the motility of lysosome-related organelles. Confocal and stochastic optical reconstruction microscopy confirmed the patchy loss of ESG simultaneously with the exocytosis of lysosome-related organelles and Weibel-Palade bodies in cultured endothelial cells and mouse aorta. The loss of ESG was blunted by pretreatment with NG-hydroxy-l-arginine or culture media conditioned by endothelial progenitor cells. Moreover, these treatments resulted in a significant reduction in deaths of septic mice. Our data support the hypothesis assigning to stress-induced exocytosis of these organelles the role of a hair-trigger for local degradation of ESG that initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later rates of morbidity and mortality. PMID:26683662

  15. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    DOE PAGESBeta

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A.; Solecki, David J.

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

  16. Quantitative description of the spatial arrangement of organelles in a polarised secretory epithelial cell: the salivary gland acinar cell

    PubMed Central

    MAYHEW, TERRY M.

    1999-01-01

    Previous quantitative descriptions of cellular ultrastructure have focused on spatial content (volume, surface area and number of organelles and membrane domains). It is possible to complement such descriptions by also quantifying spatial arrangements. Hitherto, applications of stereological methods for achieving this (notably, estimation of covariance and pair correlation functions) have been confined to organ and tissue levels. This study explores 3-dimensional subcellular arrangements of key organelles within acinar cells of rabbit parotid salivary glands, highly polarised epithelial cells specialised for exocrine secretion of α-amylase. It focuses on spatial arrangements of secretion product stores (zymogen granules), rough endoplasmic reticulum (RER) and mitochondria. Systematic random samples of electron microscopical fields of view from 3 rabbits were analysed using test grids bearing linear dipole probes of different sizes. Unbiased estimates of organelle volume densities were obtained by point counting and estimates of covariance and pair correlation functions by dipole counting. Plots of pair correlation functions against dipole length identified spatial arrangement differences between organelle types. Volumes within RER and mitochondrial compartments were positively correlated with themselves at distances below 4 μm and 2 μm respectively but were essentially randomly arranged at longer distances. In sharp contrast, zymogen granules were not randomly arranged. They were clustered at distances below 6–7 μm and more widely scattered at greater distances. These findings provide quantitative confirmation of the polarised arrangement of zymogen granules within acinar cells and further support for the relative invariance of biological organisation between subjects. PMID:10337960

  17. Not All the Organelles of Living Cells Are Equal! Or Are They? Engaging Students in Deep Learning and Conceptual Change

    ERIC Educational Resources Information Center

    Cherif, Abour H.; Siuda, JoElla Eaglin; Jedlicka, Dianne M.; Bondoc, Jasper Marc; Movahedzadeh, Farahnaz

    2016-01-01

    The cell is the fundamental basis for understanding biology much like the atom is the fundamental basis for understanding physics. Understanding biology requires the understanding of the fundamental functions performed by components within each cell. These components, or organelles, responsible for both maintenance and functioning of the cell…

  18. Imaging Implicit Morphological Processing: Evidence from Hebrew

    ERIC Educational Resources Information Center

    Bick, Atira S.; Frost, Ram; Goelman, Gadi

    2010-01-01

    Is morphology a discrete and independent element of lexical structure or does it simply reflect a fine-tuning of the system to the statistical correlation that exists among orthographic and semantic properties of words? Hebrew provides a unique opportunity to examine morphological processing in the brain because of its rich morphological system.…

  19. Platelets: production, morphology and ultrastructure.

    PubMed

    Thon, Jonathan N; Italiano, Joseph E

    2012-01-01

    Platelets are anucleate, discoid cells, roughly 2-3 μm in diameter that function primarily as regulators of hemostasis, but also play secondary roles in angiogensis and innate immunity. Although human adults contain nearly one trillion platelets in circulation that are turned over every 8-10 days, our understanding of the mechanisms involved in platelet production is still incomplete. Platelets stem from large (30-100 μm) nucleated cells called megakaryocytes that reside primarily in the bone marrow. During maturation megakaryocytes extend long proplatelet elongations into sinusoidal blood vessels from which platelets ultimately release. During this process, platelets develop a number of distinguishable structural elements including: a delimited plasma membrane; invaginations of the surface membrane that form the open canalicular system (OCS); a closed-channel network of residual endoplasmic reticulum that form the dense tubular system (DTS); a spectrin-based membrane skeleton; an actin-based cytoskeletal network; a peripheral band of microtubules; and numerous organelles including α-granules, dense-granules, peroxisomes, lysosomes, and mitochondria. Proplatelet elongation and platelet production is an elaborate and complex process that defines the morphology and ultrastructure of circulating platelets, and is critical in understanding their increasingly numerous and varied biological functions. PMID:22918725

  20. Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia.

    PubMed

    Stefanic, Sasa; Palm, Daniel; Svärd, Staffan G; Hehl, Adrian B

    2006-03-17

    During encystation Giardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport. PMID:16407213

  1. Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach.

    PubMed

    Sohn, Catherine S; Cheng, Tim T; Drummond, Michael L; Peng, Eric D; Vermont, Sarah J; Xia, Dong; Cheng, Stephen J; Wastling, Jonathan M; Bradley, Peter J

    2011-01-01

    Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen. PMID:21483743

  2. Diminished organelle motion in murine Kupffer cells during the erythrocytic stage of malaria

    PubMed Central

    Bellows, Charles F.; Molina, Ramon M.; Brain, Joseph D.

    2011-01-01

    Parasitized erythrocytes are ingested by murine hepatic macrophages during malaria infection. We non-invasively monitored how this altered the motion of intracellular phagosomes in Kupffer cells using magnetometry. Submicrometric γFe2O3 particles were injected prior to malaria infection. They were cleared from the blood, primarily by Kupffer cells, and retained within their phagosomes. The mice were periodically magnetized. After removing this external magnet, the aligned iron particles created a remnant magnetic field (RMF) which then decayed (relaxation), reflecting the motion of particle-containing phagosomes. After baseline measurements of relaxation, the mice were injected intravenously with Plasmodium chabaudi-parasitized or normal murine red blood cells (RBCs). During the next 15 days, relaxation measurements, parasitaemia and haematocrit values were monitored. At 6 days post injection with 3 × 107 parasitized RBCs, relaxation rates had decreased. At this time, all mice had parasitaemias greater than 58 per cent and haematocrits less than 20 per cent. At day 7, while the parasitaemias were declining, the rate of relaxation continued to decrease. Throughout the experiment, relaxation remained constant in animals injected with normal RBCs. Electron microscopy revealed Kupffer cells filled with damaged and parasitized erythrocytes, and haemoglobin degradation pigment. We conclude that ingestion and metabolism of parasitized erythrocytes by liver macrophages during malaria infection decreases their organelle motion with likely consequences of compromised host defences. PMID:21068031

  3. Compartmentalization Approaches in Soft Matter Science: From Nanoreactor Development to Organelle Mimics.

    PubMed

    Schoonen, Lise; van Hest, Jan C M

    2016-02-10

    Compartmentalization is an essential feature found in living cells to ensure that biological processes occur without being affected by undesired external influences. Over the years many scientists have designed self-assembled soft matter structures that mimic these natural catalytic compartments. The rationale behind this research is threefold. First of all, compartmentalization leads to the creation of a secluded environment for the catalytic species, which solves compatibility issues and which can improve catalyst efficiency and selectivity. Secondly, nano- and micro-compartments are constructed with the aim to obtain microenvironments that more closely mimic the cellular architecture. These biomimetic platforms are used to attain a better understanding of how cellular processes are executed. Thirdly, natural design rules are applied to create biomolecular assemblies with unusual functionality, which for example are used as artificial organelles. Here, recent developments will be discussed regarding these compartmentalized catalytic systems, with a selected number of illustrative examples to demonstrate which strategies have been followed, and to show to what extent the ambitious goals of this field of science have been reached. The focus here is on the field of soft matter science, covering the wide spectrum from polymeric assemblies to protein nanocages. PMID:26509964

  4. Single-Axonal Organelle Analysis Method Reveals New Protein–Motor Associations

    PubMed Central

    2012-01-01

    Axonal transport of synaptic vesicle proteins is required to maintain neurons’ ability to communicate via synaptic transmission. Neurotransmitter-containing synaptic vesicles are assembled at synaptic terminals via highly regulated endocytosis of membrane proteins. These synaptic vesicle membrane proteins are synthesized in the cell body and transported to synapses in carrier vesicles that make their way down axons via microtubule-based transport utilizing kinesin molecular motors. Identifying the cargos that each kinesin motor protein carries from the cell bodies to the synapse is key to understanding both diseases caused by motor protein dysfunction and how synaptic vesicles are assembled. However, obtaining a bulk sample of axonal transport complexes from central nervous system (CNS) neurons to use for identification of their contents has posed a challenge to researchers. To obtain axonal carrier vesicles from primary cultured neurons, we fabricated a microfluidic chip designed to physically isolate axons from dendrites and cell bodies and developed a method to remove bulk axonal samples and label their contents. Synaptic vesicle protein carrier vesicles in these samples were labeled with antibodies to the synaptic vesicle proteins p38, SV2A, and VAMP2, and the anterograde axonal transport motor KIF1A, after which antibody overlap was evaluated using single-organelle TIRF microscopy. This work confirms a previously discovered association between KIF1A and p38 and shows that KIF1A also transports SV2A- and VAMP2-containing carrier vesicles. PMID:23421679

  5. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle

    PubMed Central

    Kerfeld, Cheryl A.

    2016-01-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  6. Chloroplast envelope protein targeting fidelity is independent of cytosolic components in dual organelle assays

    PubMed Central

    Kriechbaumer, Verena; Abell, Ben M.

    2012-01-01

    The general mechanisms of intracellular protein targeting are well established, and depend on a targeting sequence in the protein, which is recognized by a targeting factor. Once a membrane protein is delivered to the correct organelle its targeting sequence can be recognized by receptors and a translocase, leading to membrane insertion. However, the relative contribution of each step for generating fidelity and efficiency of the overall process has not been systematically addressed. Here, we use tail-anchored (TA) membrane proteins in cell-free competitive targeting assays to chloroplasts to show that targeting can occur efficiently and with high fidelity in the absence of all cytosolic components, suggesting that chloroplast envelope protein targeting is primarily dependent on events at the outer envelope. Efficiency of targeting was increased by the addition of complete cytosol, and by Hsp70 or Hsp90, depending on the protein, but none of these cytosolic components influenced the fidelity of targeting. Our results suggest that the main role of targeting factors in chloroplast localization is to increase targeting efficiency by maintaining recognition competency at the outer envelope. PMID:22783268

  7. Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation.

    PubMed

    Ramadass, Mahalakshmi; Catz, Sergio D

    2016-09-01

    Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion. PMID:27558339

  8. Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

    PubMed Central

    Suresh, Harsha Garadi; da Silveira dos Santos, Aline Xavier; Kukulski, Wanda; Tyedmers, Jens; Riezman, Howard; Bukau, Bernd; Mogk, Axel

    2015-01-01

    Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes. PMID:25761633

  9. Organization of cytoskeletal elements and organelles preceding growth cone emergence from an identified neuron in situ.

    PubMed

    Lefcort, F; Bentley, D

    1989-05-01

    The purpose of this study was to investigate the arrangement of cytoskeletal elements and organelles in an identified neuron in situ at the site of emergence of its growth cone just before and concurrent with the onset of axonogenesis. The Ti1 pioneer neurons are the first pair of afferent neurons to differentiate in embryonic grasshopper limbs. They arise at the distal tip of the limb bud epithelium, the daughter cells of a single precursor cell, the Pioneer Mother Cell (PMC). Using immunohistochemical markers, we characterized the organization of microtubules, centrosomes, Golgi apparatus, midbody, actin filaments, and chromatin from mitosis in the PMC through axonogenesis in the Tils. Just before and concurrent with the onset of axonogenesis, a characteristic arrangement of tubulin, actin filaments, and Golgi apparatus is localized at the proximal pole of the proximal pioneer neuron. The growth cone of the proximal cell stereotypically arises from this site. Although the distal cell's axon generally grows proximally, occasionally it arises from its distal pole; in such limbs, the axons from the sister cells extend from mirror symmetric locations on their somata. In the presence of cytochalasin D, the PMC undergoes nuclear division but not cytokinesis and although other neuronal phenotypes are expressed, axongenesis is inhibited. Our data suggest that intrinsic information determines the site of growth cone emergence of an identified neuron in situ. PMID:2654140

  10. Extending the Limited Transfer Window Hypothesis to Inter-organelle DNA Migration

    PubMed Central

    Smith, David Roy

    2011-01-01

    Mitochondrial genomes often contain large amounts of plastid DNA (ptDNA)-derived sequences (MTPTs). It has been suggested that the intercompartmental transfer of ptDNA is greatly reduced in species with only a single plastid per cell (monoplastidic) as compared with those with many plastids per cell (polyplastidic). This hypothesis has not been applied to the movement of DNA from plastids to mitochondria. By analyzing the organelle genomes from diverse mono- and polyplastidic taxa, I show that MTPTs are restricted to the mitochondrial genomes of species with many plastids per cell and are absent from those with one plastid per cell or with monoplastidic meristematic systems. Moreover, the most bloated mitochondrial genomes that were explored had the largest MTPT contents. These data, like previous results on ptDNA-derived sequences in nuclear genomes, support the hypothesis that plastid number and the forces governing the expansion and contraction of noncoding mitochondrial DNA (mtDNA) influence MTPT abundance. I also show that plastid genomes are depauperate in mtDNA-derived sequences (PTMTs), irrespective of the number of mitochondria per cell and plastid genome size, which may reflect the lack of a DNA uptake system in plastids. PMID:21803764

  11. Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

    PubMed Central

    Pramanik, Smritimoy; Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2014-01-01

    Important biological reactions involving nucleic acids occur near the surface of membranes such as the nuclear membrane (NM) and rough endoplasmic reticulum (ER); however, the interactions between biomembranes and nucleic acids are poorly understood. We report here that transcription was facilitated in solution with liposomes, which mimic a biomembrane surface, relative to the reaction in a homogeneous aqueous solution when the template was able to form a G-quadruplex. The G-quadruplex is known to be an inhibitor of transcription, but the stability of the G-quadruplex was decreased at the liposome surface because of unfavourable enthalpy. The destabilization of the G-quadruplex was greater at the surface of NM- and ER-mimicking liposomes than at the surfaces of liposomes designed to mimic other organelles. Thermodynamic analyses revealed that the G-rich oligonucleotides adopted an extended structure at the liposome surface, whereas in solution the compact G-quadruplex was formed. Our data suggest that changes in structure and stability of nucleic acids regulate biological reactions at membrane surfaces. PMID:25336617

  12. Unraveling the Secrets of Bacterial Adhesion Organelles Using Single-Molecule Force Spectroscopy

    NASA Astrophysics Data System (ADS)

    Axner, Ove; Björnham, Oscar; Castelain, Mickaël; Koutris, Efstratios; Schedin, Staffan; Fällman, Erik; Andersson, Magnus

    Many types of bacterium express micrometer-long attachment organelles (so-called pili) whose role is to mediate adhesion to host tissue. Until recently, little was known about their function in the adhesion process. Force-measuring optical tweezers (FMOT) have since then been used to unravel the biomechanical properties of various types of pili, primarily those from uropathogenic E. coli, in particular their force-vs.-elongation response, but lately also some properties of the adhesin are situated at the distal end of the pilus. This knowledge provides an understanding of how piliated bacteria can sustain external shear forces caused by rinsing processes, e.g., urine flow. It has been found that many types of pilus exhibit unique and complex force-vs.-elongation responses. It has been conjectured that their dissimilar properties impose significant differences in their ability to sustain external forces and that different types of pilus therefore have dissimilar predisposition to withstand different types of rinsing conditions. An understanding of these properties is of high importance since it can serve as a basis for finding new means to combat bacterial adhesion, including that caused by antibiotic-resistance bacteria. This work presents a review of the current status of the assessment of biophysical properties of individual pili on single bacteria exposed to strain/stress, primarily by the FMOT technique. It also addresses, for the first time, how the elongation and retraction properties of the rod couple to the adhesive properties of the tip adhesin.

  13. Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles.

    PubMed

    Prentki, M; Wollheim, C B

    1984-10-15

    The role of Ca2+ in secretagogue-induced insulin release is documented not only by the measurements of 45Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca2+, [Ca2+]i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca2+]i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca2+ homeostasis by organelles from a rat insulinoma, was investigated with a Ca2+ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca2+ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca2+]i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca2+ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release. PMID:6436050

  14. Shifts in oxidation states of cerium oxide nanoparticles detected inside intact hydrated cells and organelles

    SciTech Connect

    Szymanski, Craig J.; Munusamy, Prabhakaran; Mihai, Cosmin; Xie, Yumei; Hu, Dehong; Gilles, Marry K.; Tyliszczak, T.; Thevuthasan, Suntharampillai; Baer, Donald R.; Orr, Galya

    2015-09-01

    Cerium oxide nanoparticles (CNPs) have been shown to induce diverse biological effects, ranging from toxic to beneficial. The beneficial effects have been attributed to the potential antioxidant activity of CNPs via certain redox reactions, depending on their oxidation state or Ce3+/Ce4+ ratio. However, this ratio is strongly dependent on the environment and age of the nanoparticles and it is unclear whether and how the complex intracellular environment impacts this ratio and the possible redox reactions of CNPs. To identify any changes in the oxidation state of CNPs in the intracellular environment and better understand their intracellular reactions, we directly quantified the oxidation states of CNPs outside and inside intact hydrated cells and organelles using correlated scanning transmission x-ray and super resolution fluorescence microscopies. By analyzing hundreds of small CNP aggregates, we detected a shift to a higher Ce3+/Ce4+ ratio in CNPs inside versus outside the cells, indicating a net reduction of CNPs in the intracellular environment. We further found a similar ratio in the cytoplasm and in the lysosomes, indicating that the net reduction occurs earlier in the internalization pathway. Together with oxidative stress and toxicity measurements, our observations identify a net reduction of CNPs in the intracellular environment, which is consistent with their involvement in potentially beneficial oxidation reactions, but also point to interactions that can negatively impact the health of cells.

  15. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole. PMID:27311484

  16. Investigation of the organelle pathology of skeletal muscle in chronic alcoholism.

    PubMed Central

    Martin, F C; Slavin, G; Levi, A J; Peters, T J

    1984-01-01

    The muscle abnormalities associated with chronic alcohol consumption were studied by applying histological and biochemical techniques to tissue obtained by percutaneous needle biopsy from the quadriceps muscles of 41 patients. Measurement of the fibre size showed atrophy of both type I (p less than 0.05) and type II (p less than 0.001) fibres. The degree of atrophy was more severe for type II fibres (33% reduction in median diameter) than type I (17%). Marker enzyme activities for the principal organelles were assayed. Compared with biopsy specimens from non-alcoholic controls, no differences were found in the activities of lysosomal, mitochondrial, peroxisomal, cytosolic, sarcolemmal, or sarcoplasmic reticulum enzymes, expressed per microgram DNA. A reduction in the protein to DNA ratio was evident in severely atrophic biopsies, and this was associated with a significant reduction of myofibrillary Ca2+-ATPase activity. These results suggest a selective loss of type II fibre myofibrillary protein and do not confirm earlier suggestions of specific mitochondrial damage. Images PMID:6707228

  17. Expression of 1L-Myoinositol-1-Phosphate Synthase in Organelles1

    PubMed Central

    Lackey, Kimberly Helms; Pope, Patricia Marie; Johnson, Margaret Dean

    2003-01-01

    We have studied the expression of 1l-myoinositol-1-phosphate synthase (MIPS; EC 5.5.1.4) in developing organs of Phaseolus vulgaris to define genetic controls that spatially regulate inositol phosphate biosynthesis. MIPS, the pivotal biosynthetic enzyme in inositol metabolism, is the only enzyme known to catalyze the conversion of glucose 6-phosphate to inositol phosphate. It is found in unicellular and multicellular eukaryotes and has been isolated as a soluble enzyme from both. Thus, it is widely accepted that inositol phosphate biosynthesis is largely restricted to the cytosol. Here, we report findings that suggest the enzyme is also expressed in membrane-bound organelles. Microscopic and biochemical analyses detected MIPS expression in plasma membranes, plastids, mitochondria, endoplasmic reticula, nuclei, and cell walls of bean. To address mechanisms by which the enzyme could be targeted to or through membranes, MIPS genes were analyzed for sorting signals within primary structures and upstream open reading frames that we discovered through our sequence analyses. Comprehensive computer analyses revealed putative transit peptides that are predicted to target the enzyme to different cellular compartments. Reverse transcriptase PCR experiments suggest that these putative targeting peptides are expressed in bean roots and leaves. PMID:12913178

  18. Structure and Mechanisms of a Protein-Based Organelle in Escherichia coli

    SciTech Connect

    Tanaka, Shiho; Sawaya, Michael R.; Yeates, Todd O.

    2010-08-18

    Many bacterial cells contain proteinaceous microcompartments that act as simple organelles by sequestering specific metabolic processes involving volatile or toxic metabolites. Here we report the three-dimensional (3D) crystal structures, with resolutions between 1.65 and 2.5 angstroms, of the four homologous proteins (EutS, EutL, EutK, and EutM) that are thought to be the major shell constituents of a functionally complex ethanolamine utilization (Eut) microcompartment. The Eut microcompartment is used to sequester the metabolism of ethanolamine in bacteria such as Escherichia coli and Salmonella enterica. The four Eut shell proteins share an overall similar 3D fold, but they have distinguishing structural features that help explain the specific roles they play in the microcompartment. For example, EutL undergoes a conformational change that is probably involved in gating molecular transport through shell protein pores, whereas structural evidence suggests that EutK might bind a nucleic acid component. Together these structures give mechanistic insight into bacterial microcompartments.

  19. Assembly, Properties and Function of Synthetic Phase-Separated RNA/Protein Organelles

    NASA Astrophysics Data System (ADS)

    Taylor, Nicole; Elbaum, Shana; Stone, Howard; Brangwynne, Clifford

    2015-03-01

    Non-membrane bound RNA/protein (RNP) bodies play a key role in cellular RNA processing steps. Many RNA helicases, required for RNA processing, are key components of RNPs. Consistent with this, a purified RNA helicase, Laf-1, exhibits a salt and protein concentration dependent phase separation in vitro, resulting in liquid-like droplets. We use such synthetic RNPs to study the biophysics of RNP assembly, and to elucidate the link between their physical properties and function. To accomplish this, we are developing custom microfluidic devices to measure biophysical properties, nucleation and growth kinetics, and RNA processing function of droplets. We measure droplet viscosity by applying a shear stress to protein droplets that adhere to the channel wall; measurements are consistent with those taken using a particle microrheology approach. We also monitor and control protein droplet nucleation using oil/water emulsions. Our results provide a new platform for addressing how the cell regulates organelle assembly and properties through protein, RNA, and ATP concentration. We anticipate that these findings will offer insight into the contribution of RNPs in key RNA processing functions in the cell.

  20. Lipid Droplets: A Key Cellular Organelle Associated with Cancer Cell Survival under Normoxia and Hypoxia.

    PubMed

    Koizume, Shiro; Miyagi, Yohei

    2016-01-01

    The Warburg effect describes the phenomenon by which cancer cells obtain energy from glycolysis even under normoxic (O₂-sufficient) conditions. Tumor tissues are generally exposed to hypoxia owing to inefficient and aberrant vasculature. Cancer cells have multiple molecular mechanisms to adapt to such stress conditions by reprogramming the cellular metabolism. Hypoxia-inducible factors are major transcription factors induced in cancer cells in response to hypoxia that contribute to the metabolic changes. In addition, cancer cells within hypoxic tumor areas have reduced access to serum components such as nutrients and lipids. However, the effect of such serum factor deprivation on cancer cell biology in the context of tumor hypoxia is not fully understood. Cancer cells are lipid-rich under normoxia and hypoxia, leading to the increased generation of a cellular organelle, the lipid droplet (LD). In recent years, the LD-mediated stress response mechanisms of cancer cells have been revealed. This review focuses on the production and functions of LDs in various types of cancer cells in relation to the associated cellular environment factors including tissue oxygenation status and metabolic mechanisms. This information will contribute to the current understanding of how cancer cells adapt to diverse tumor environments to promote their survival. PMID:27589734

  1. High-throughput imaging of heterogeneous cell organelles with an X-ray laser

    NASA Astrophysics Data System (ADS)

    Hantke, Max F.; Hasse, Dirk; Maia, Filipe R. N. C.; Ekeberg, Tomas; John, Katja; Svenda, Martin; Loh, N. Duane; Martin, Andrew V.; Timneanu, Nicusor; Larsson, Daniel S. D.; van der Schot, Gijs; Carlsson, Gunilla H.; Ingelman, Margareta; Andreasson, Jakob; Westphal, Daniel; Liang, Mengning; Stellato, Francesco; Deponte, Daniel P.; Hartmann, Robert; Kimmel, Nils; Kirian, Richard A.; Seibert, M. Marvin; Mühlig, Kerstin; Schorb, Sebastian; Ferguson, Ken; Bostedt, Christoph; Carron, Sebastian; Bozek, John D.; Rolles, Daniel; Rudenko, Artem; Epp, Sascha; Chapman, Henry N.; Barty, Anton; Hajdu, Janos; Andersson, Inger

    2014-12-01

    We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.

  2. Selective molecular transport through the protein shell of a bacterial microcompartment organelle.

    PubMed

    Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; Sawaya, Michael R; Sinha, Sharmistha; Yeates, Todd O; Bobik, Thomas A

    2015-03-10

    Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins. PMID:25713376

  3. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

    PubMed

    Erbilgin, Onur; Sutter, Markus; Kerfeld, Cheryl A

    2016-03-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  4. Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

    PubMed Central

    Huang, Guozhong; Ulrich, Paul N.; Storey, Melissa; Johnson, Darryl; Tischer, Julie; Tovar, Javier A.; Moreno, Silvia N. J.; Orlando, Ron; Docampo, Roberto

    2014-01-01

    Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate. PMID:25503798

  5. Advances and New Concepts in Alcohol-Induced Organelle Stress, Unfolded Protein Responses and Organ Damage

    PubMed Central

    Ji, Cheng

    2015-01-01

    Alcohol is a simple and consumable biomolecule yet its excessive consumption disturbs numerous biological pathways damaging nearly all organs of the human body. One of the essential biological processes affected by the harmful effects of alcohol is proteostasis, which regulates the balance between biogenesis and turnover of proteins within and outside the cell. A significant amount of published evidence indicates that alcohol and its metabolites directly or indirectly interfere with protein homeostasis in the endoplasmic reticulum (ER) causing an accumulation of unfolded or misfolded proteins, which triggers the unfolded protein response (UPR) leading to either restoration of homeostasis or cell death, inflammation and other pathologies under severe and chronic alcohol conditions. The UPR senses the abnormal protein accumulation and activates transcription factors that regulate nuclear transcription of genes related to ER function. Similarly, this kind of protein stress response can occur in other cellular organelles, which is an evolving field of interest. Here, I review recent advances in the alcohol-induced ER stress response as well as discuss new concepts on alcohol-induced mitochondrial, Golgi and lysosomal stress responses and injuries. PMID:26047032

  6. Exosomes as Intercellular Signaling Organelles Involved in Health and Disease: Basic Science and Clinical Applications

    PubMed Central

    Corrado, Chiara; Raimondo, Stefania; Chiesi, Antonio; Ciccia, Francesco; De Leo, Giacomo; Alessandro, Riccardo

    2013-01-01

    Cell to cell communication is essential for the coordination and proper organization of different cell types in multicellular systems. Cells exchange information through a multitude of mechanisms such as secreted growth factors and chemokines, small molecules (peptides, ions, bioactive lipids and nucleotides), cell-cell contact and the secretion of extracellular matrix components. Over the last few years, however, a considerable amount of experimental evidence has demonstrated the occurrence of a sophisticated method of cell communication based on the release of specialized membranous nano-sized vesicles termed exosomes. Exosome biogenesis involves the endosomal compartment, the multivesicular bodies (MVB), which contain internal vesicles packed with an extraordinary set of molecules including enzymes, cytokines, nucleic acids and different bioactive compounds. In response to stimuli, MVB fuse with the plasma membrane and vesicles are released in the extracellular space where they can interact with neighboring cells and directly induce a signaling pathway or affect the cellular phenotype through the transfer of new receptors or even genetic material. This review will focus on exosomes as intercellular signaling organelles involved in a number of physiological as well as pathological processes and their potential use in clinical diagnostics and therapeutics. PMID:23466882

  7. Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics.

    PubMed

    Piro, Amalia; Serra, Ilia Anna; Spadafora, Antonia; Cardilio, Monica; Bianco, Linda; Perrotta, Gaetano; Santos, Rui; Mazzuca, Silvia

    2015-12-01

    Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses. PMID:26444578

  8. Mathematical morphology for shape description

    NASA Astrophysics Data System (ADS)

    Schmitt, M.

    2002-03-01

    We first examine the measurements one can perform on the space of compact and convex sets. A famous theorem, due to Hadwiger (1957), shows that any measurement with nice properties, namely additivity, is a linear combination of Minkowski functionals. Then, some useful formulae, linking measurements in different dimensions of space are derived.In the second step, we make use of the morphological operations transforming sets into sets. These sets are then measured using the previous measurements. The most famous attempt yields the concept of granulomerties and their extensions.In the last part, we examine a way to build morphological random sets which are compatible with morphological operators like erosions or openings and apply it to the most famous example in morphology, the Boolean model, describing objects located at random.

  9. Effects of Selenium on Morphological Changes in Candida utilis ATCC 9950 Yeast Cells.

    PubMed

    Kieliszek, Marek; Błażejak, Stanisław; Bzducha-Wróbel, Anna; Kurcz, Agnieszka

    2016-02-01

    This paper presents the results of microscopic examinations of the yeast cells cultured in yeast extract-peptone-dextrose (YPD) media supplemented with sodium selenite(IV). The analysis of the morphological changes in yeast cells aimed to determine whether the selected selenium doses and culturing time may affect this element accumulation in yeast cell structures in a form of inorganic or organic compounds, as a result of detoxification processes. The range of characteristic morphological changes in yeasts cultivated in experimental media with sodium selenite(IV) was observed, including cell shrinkage and cytoplasm thickening of the changes within vacuole structure. The processes of vacuole disintegration were observed in aging yeast cells in culturing medium, which may indicate the presence of so-called ghost cells lacking intracellular organelles The changes occurring in the morphology of yeasts cultured in media supplemented with sodium selenite were typical for stationary phase of yeast growth. From detailed microscopic observations, larger surface area of the cell (6.03 μm(2)) and yeast vacuole (2.17 μm(2)) were noticed after 24-h culturing in the medium with selenium of 20 mg Se(4+)/L. The coefficient of shape of the yeast cells cultured in media enriched with sodium selenite as well as in the control YPD medium ranged from 1.02 to 1.22. Elongation of cultivation time (up to 48 and 72 h) in the media supplemented with sodium selenite caused a reduction in the surface area of the yeast cell and vacuole due to detoxification processes. PMID:26166197

  10. The Flagellum Attachment Zone: 'The Cellular Ruler' of Trypanosome Morphology.

    PubMed

    Sunter, Jack D; Gull, Keith

    2016-04-01

    A defining feature of Trypanosoma brucei cell shape is the lateral attachment of the flagellum to the cell body, mediated by the flagellum attachment zone (FAZ). The FAZ is a complex cytoskeletal structure that connects the flagellum skeleton through two membranes to the cytoskeleton. The FAZ acts as a 'cellular ruler' of morphology by regulating cell length and organelle position and is therefore critical for both cell division and life cycle differentiations. Here we provide an overview of the advances in our understanding of the composition, assembly, and function of the FAZ. PMID:26776656

  11. Ear examination

    MedlinePlus

    King EF, Couch ME. History, physical examination, and the preoperative evaluation. In: Flint PW, Haughey BH, Lund LJ, et al, eds. Cummings Otolaryngology: Head & Neck Surgery . 6th ed. Philadelphia, ...

  12. [Examination procedures].

    PubMed

    Vassault, A; Arnaud, J; Szymanovicz, A

    2010-12-01

    Examination procedures have to be written for each examination according to the standard requirements. Using CE marked devices, technical inserts can be used, but because of their lack of homogeneity, it could be easier to document their use as a standard procedure. Document control policy applies for those procedures, the content of which could be as provided in this document. Electronic manuals can be used as well. PMID:21613016

  13. Serial block-face scanning electron microscopy for three-dimensional analysis of morphological changes in mitochondria regulated by Cdc48p/p97 ATPase.

    PubMed

    Miyazaki, Naoyuki; Esaki, Masatoshi; Ogura, Teru; Murata, Kazuyoshi

    2014-08-01

    Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae. We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells. PMID:24893221

  14. Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

    PubMed Central

    Swanson, SJ; Bethke, PC; Jones, RL

    1998-01-01

    Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione. PMID:9596630

  15. Mitochondrial and plastid genomes of the colonial green alga Gonium pectorale give insights into the origins of organelle DNA architecture within the volvocales.

    PubMed

    Hamaji, Takashi; Smith, David R; Noguchi, Hideki; Toyoda, Atsushi; Suzuki, Masahiro; Kawai-Toyooka, Hiroko; Fujiyama, Asao; Nishii, Ichiro; Marriage, Tara; Olson, Bradley J S C; Nozaki, Hisayoshi

    2013-01-01

    Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion. PMID:23468928

  16. Ricinosomes: an organelle for developmentally regulated programmed cell death in senescing plant tissues

    NASA Astrophysics Data System (ADS)

    Gietl, C.; Schmid, M.

    2001-02-01

    This review describes aspects of programmed cell death (PCD). Present research maps the enzymes involved and explores the signal transduction pathways involved in their synthesis. A special organelle (the ricinosome) has been discovered in the senescing endosperm of germinating castor beans (Ricinus communis) that develops at the beginning of PCD and delivers large amounts of a papain-type cysteine endopeptidase (CysEP) in the final stages of cellular disintegration. Castor beans store oil and proteins in a living endosperm surrounding the cotyledons. These stores are mobilized during germination and transferred into the cotyledons. PCD is initiated after this transfer is complete. The CysEP is synthesized in the lumen of the endoplasmic reticulum (ER) where it is retained by its C-terminal KDEL peptide as a rather inactive pro-enzyme. Large number of ricinosomes bud from the ER at the same time as the nuclear DNA is characteristically fragmented during PCD. The mitochondria, glyoxysomes and ribosomes are degraded in autophagic vacuoles, while the endopeptidase is activated by removal of the propeptide and the KDEL tail and enters the cytosol. The endosperm dries and detaches from the cotyledons. A homologous KDEL-tailed cysteine endopeptidase has been found in several senescing tissues; it has been localized in ricinosomes of withering day-lily petals and dying seed coats. Three genes for a KDEL-tailed cysteine endopeptidase have been identified in Arabidopsis. One is expressed in senescing ovules, the second in the vascular vessels and the third in maturing siliques. These genes open the way to exploring PCD in plants.

  17. Polymeric Nucleic Acid Vehicles Exploit Active Inter-Organelle Trafficking Mechanisms

    PubMed Central

    Fichter, Katye M.; Ingle, Nilesh. P.; McLendon, Patrick M.; Reineke, Theresa M.

    2013-01-01

    Materials that self-assemble with nucleic acids into nanocomplexes (polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect, (which slowly degrades via hydrolysis) and linear PEI, (which does not rapidly hydrolyze) to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h, and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical “proton sponge mechanism”. PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides (ODNs) to the Golgi and endoplasmic reticulum, which may be a route taken for nuclear delivery. However, Glycofect polyplexes demonstrated higher colocalization with the ER than PEI polyplexes and colocalization experiments indicate retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery. PMID:23234474

  18. Interaction of lipid bodies with other cell organelles in the maturing pollen of Magnolia x soulangeana (Magnoliaceae).

    PubMed

    Dinis, Augusto M; Coutinho, A Pereira

    2009-12-01

    The pollen grain maturation in Magnolia x soulangeana was studied ultrastructurally and cytochemically using both the light and transmission electron microscope. Emphasis was given on the storage lipid bodies of the vegetative cell (VC) and their interaction with other cell organelles. Stereological analysis of electron micrographs was performed to evaluate the variation in volume density (V(V)), surface density, and surface-to-volume ratio (S/V) of various cell organelles during pollen maturation. The size and numerical density of the lipid bodies, and their frequency of association with other cell organelles, were also determined. It was noted that during pollen ontogeny and maturation, the lipid bodies changed their pattern of distribution in the VC cytoplasm, which may be a good marker for the succeeding stages of pollen development. Also, the size, osmiophily, and V(V) of the lipid bodies were progressively reduced during pollen maturation whereas the S/V was significantly increased. This seems to indicate that the lipid bodies are mobilized in part during this period of pollen maturation. In particular, the intermediate and mature pollen showed a high percentage of lipid bodies establishing a physical contact with either glyoxysomes, either protein storage vacuoles, or small vesicles presumably originated from dictyosomes. This physical contact was found in both the chemically fixed and rapid freeze-fixed pollen indicating that it is neither artifactual nor casual. On the basis of this intimate association with other cell organelles and the morphometric analysis performed, we suggest that the mobilization of lipid bodies is likely mediated not only by glyoxysomes but also by other catabolic pathways involving the interaction of lipid bodies with either protein storage vacuoles or small Golgi vesicles. PMID:19763782

  19. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

  20. A morphometric analysis of the redistribution of organelles in columella cells of horizontally-oriented roots of Zea mays

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1986-01-01

    In order to determine what structural changes in graviperceptive cells are associated with onset of root gravicurvature, the redistribution of organelles in columella cells of horizontally-oriented, graviresponding roots of Zea mays has been quantified. Root gravicurvature began by 15 min after reorientation, and did not involve significant changes in the (i) volume of individual columella cells or amyloplasts, (ii) relative volume of any cellular organelle, (iii) number of amyloplasts per columella cell, or (iv) surface area of cellular location of endoplasmic reticulum. Sedimentation of amyloplasts began within 1 to 2 min after reorientation, and was characterized by an intensely staining area of cytoplasm adjacent to the sedimenting amyloplasts. By 5 min after reorientation, amyloplasts were located in the lower distal corner of columella cells, and, by 15 min after reorientation, overlaid the entire length of the lower cell wall. No consistent contact between amyloplasts and any cellular structure was detected at any stage of gravicurvature. Centrally-located nuclei initially migrated upward in columella cells of horizontally-oriented roots, after which they moved to the proximal ends of the cells by 15 min after reorientation. No significant pattern of redistribution of vacuoles, mitochondria, dictyosomes, or hyaloplasm was detected that correlated with the onset of gravicurvature. These results indicate that amyloplasts and nuclei are the only organelles whose movements correlate positively with the onset of gravicurvature by primary roots of this cultivar of Zea mays.

  1. PRIMARY PEPTIDE SEQUENCES FROM SQUID MUSCLE AND OPTIC LOBE MYOSIN IIs: A STRATEGY TO IDENTIFY AN ORGANELLE MYOSIN

    PubMed Central

    MEDEIROS, NELSON A.; REESE, THOMAS S.; JAFFE, HOWARD; DEGIORGIS, JOSEPH A.; BEARER, ELAINE L.

    2013-01-01

    The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60–95%) and chick pectoralis muscle (31–83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50–60%, identities), and some also matched unconventional myosins (33–50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles. PMID:9878103

  2. Development and disintegration of tapetum-specific lipid-accumulating organelles, elaioplasts and tapetosomes, in Arabidopsis thaliana and Brassica napus.

    PubMed

    Suzuki, Toshiya; Tsunekawa, Sonomi; Koizuka, Chie; Yamamoto, Kanta; Imamura, Jun; Nakamura, Kenzo; Ishiguro, Sumie

    2013-06-01

    The pollen coat covering the surface of pollen grains has many important roles for pollination. In Brassicaceae plants, the pollen coat components are synthesized and temporarily accumulated in two tapetum-specific organelles, the elaioplast and the tapetosome. Although many biochemical and electron microscopic analyses have been attempted, the structure and biogenesis of these organelles have not been fully elucidated. To resolve this problem, we performed live imaging of these organelles using two markers, FIB1a-GFP and GRP17-GFP. FIB1a is an Arabidopsis fibrillin, a structural protein of elaioplast plastoglobules. In transgenic Arabidopsis, fluorescence of FIB1a-GFP appeared in young elaioplasts, in which small plastoglobules were developing. However, the fluorescence disappeared in later stages, while enlargement of plastoglobules continued. GRP17 is an Arabidopsis oleopollenin, an oleosin-like protein in tapetosomes. Fluorescence microscopy of GRP17-GFP expressed in Arabidopsis and Brassica napus revealed that tapetosomes do not contain oleopollenin-coated vesicles but have an outer envelope, indicating that the tapetosome structure is distinct from seed oil bodies. Visualization of GRP17-GFP also demonstrated that the tapetal cells become protoplasts and migrate into locules before pollen coat formation, and provided live imaging of the foot formation between pollen grains and stigmatic papilla cells. PMID:23602096

  3. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair[C][W

    PubMed Central

    Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand

    2010-01-01

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway. PMID:20551348

  4. Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection.

    PubMed

    Sloves, Pierre-Julien; Delhaye, Stephane; Mouveaux, Thomas; Werkmeister, Elisabeth; Slomianny, Christian; Hovasse, Agnes; Dilezitoko Alayi, Tchilabalo; Callebaut, Isabelle; Gaji, Rajshekhar Y; Schaeffer-Reiss, Christine; Van Dorsselear, Alain; Carruthers, Vern B; Tomavo, Stanislas

    2012-05-17

    Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles. TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma. PMID:22607804

  5. Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha

    PubMed Central

    Fokina, Anastasia V.; Chechenova, Maria B.; Karginov, Azamat V.; Ter-Avanesyan, Michael D.; Agaphonov, Michael O.

    2015-01-01

    Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole. PMID:26717478

  6. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  7. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    SciTech Connect

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-04-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of approx. = 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4/sup 0/C or in medium containing 5 ..mu..M colchicine or nocodazole at 37/sup 0/C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37/sup 0/C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures.

  8. Characterization of the major proteins in gamma particles, cytoplasmic organelles in Blastocladiella emersonii zoospores.

    PubMed

    Hohn, T M; Lovett, J S; Bracker, C E

    1984-04-01

    The gamma particles of Blastocladiella emersonii are 0.5-micron (diameter), electron dense, membrane-enclosed organelles in the cytoplasm of zoospores that have been reported (E.C. Cantino and G.L. Mills, in P. Lemke, ed., Viruses and Plasmids in Fungi, 1979, and R.B. Myers and E.C. Cantino, in A. Wolsky (ed.), Monographs in Developmental Biology, 1974) to store the enzyme chitin synthetase. These particles were isolated from zoospores, and the two major proteins were purified for an analysis of their composition and function. The lower-molecular-weight protein (apparent molecular weight, 41,000) was insoluble in aqueous buffers, had an unusual, very basic amino acid composition, and comprised the characteristic electron-dense inclusions seen in micrographs of sections of fixed and stained gamma particles. After dispersal of the gamma particle membranes with detergent, the higher-molecular-weight protein (apparent molecular weight, 43,000) and a third minor protein (apparent molecular weight, 45,000) sedimented through sucrose cushions with the 41 kilodalton inclusion body protein but were dissociated from it by sonication in buffer containing 7 M urea. Together, the two major proteins represent 60 to 70% of the total protein in the gamma particle and 2.9% of the total protein in zoospores. Tests with specific antisera showed that the two major proteins were not antigenically related, a result consistent with the differences in amino acid composition. When zoospore lysates were centrifuged in sucrose density gradients, the major gamma particle proteins and chitin synthetase activity migrated to regions of different density. Proteins from sporulating thalli and germinating zoospores were separated by gel electrophoresis, and the two major gamma particle proteins were detected by reaction with specific antisera after electrophoretic transfer to nitrocellulose filters. Neither protein could be found in growth phase cells; the appearance and disappearances of both

  9. Nonlinear electromagnetic responses of active membrane protein complexes in live cells and organelles

    NASA Astrophysics Data System (ADS)

    Nawarathna, Dharmakirthi

    observed, possibly due to the F0 domain of ATP synthase. Finally, harmonics generated by chloroplasts, the plant organelles responsible for photosynthesis, were measured, which are similar in structure and function to mitochondria, depend dramatically on incident light, and vanish in the absence of light. Using spinach chloroplasts, light sensitive peaks were detected in the range of 0--12 kHz, again suggesting that these harmonics are indicative of electron processes in the light harvesting complexes, reaction center, and/or photosynthetic electron transport chain.

  10. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    SciTech Connect

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; Grimwood, Jane; Grigoriev, Igor V.; Van Etten, James L.; Keeling, Patrick J.

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.

  11. Morphologic criteria and detection of apoptosis.

    PubMed

    Saraste, A

    1999-05-01

    Apoptosis is an organized, energy dependent process, which leads to cell death. Its definition is based on distinct morphological features [10] and demonstration of internucleosomal DNA degradation [27], executed by selectively activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include chromatic margination, nuclear condensation and fragmentation, and condensation of the cell with preservation of organelles. The process is followed by fragmentation of the cell into membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis characteristically occurs in insolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell death, a prototype of which is cell death due to ischemia (oncosis), is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles and rupture of the plasma membrane [15]. Groups of necrotic cells and inflammation are found in tissues [10, 15]. The significance of apoptosis has mostly been studied using the TUNEL assay that detects DNA strand breaks in tissue sections and allows quantification of apoptotic cells by light microscopy [6]. Common experience seems to be that the TUNEL assay is prone to false positive or negative findings. This has been explained by the dependence of the staining kinetics on the reagent concentration [17], fixation of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To obtain reliable and reproducible results, TUNEL assay should be carefully standardized by using tissue sections treated with DNAse (positive control of apoptosis). Quantification of apoptosis should include enough microscopic fields and identification of the cell type undergoing apoptosis

  12. Comparison of Long-Term Storage in Chemical Fixatives on Morphology and Anatomy of Super-Dwarf Wheat

    NASA Technical Reports Server (NTRS)

    Bubenheim, David L.; Campbell, W. F.; Salisbury, F. B.; Hole, P. S.; Gillespie, L.; Levbinskikh, M.; Kliss, Mark H. (Technical Monitor)

    1996-01-01

    Wheat plants (Triticum aestivum L. cv Super-Dwarf) are grown in the microgravity of space and harvested for morphological and anatomical comparison with those exposed to gravity on earth. Such plants are subjected to relatively long periods of storage in chemical fixatives. Examination, evaluation and verification that the integrity of the vascular system is maintained for extended periods of storage in fixatives are required. McDowell and Trump's [4% Formaldehyde -- 1 % Glutaraldehyde (4F: 1 G)] or Variant I [(Russian Fixative): Formalin: Acetic Acid: Alcohol] fixatives, adjusted to pH 7.2, were placed in Aclam(TM), FilmORap(TM), or FilmOSun(TM) plastic bags on April 4, 1994. Wheat seedlings were harvested on days 9, 28, and 68 and preserved in these fixatives. Subsamples of leaves and/or seeds were taken from these stocks after various times in storage, dehydrated, and embedded in Spurr's, LR White's or Unicryl resin. Semithin (1 mm) and thin (50-70 nm) sections were examined by light and transmission electron microscopy. In a few sections, we have observed a slight plasmolysis of the cytostol in leaf tissue fixed with the Variant I, but overall there seem to be no major artifacts in the anatomical structure. The plasmalemma and other organelles appeared normal in the McDowell and Trump fixative. Use of differential chromophores suggests that LR White or Unicryl resins may give greater flexibility for enzyme localizations at both the light and electron microscopical levels.

  13. Reconceptualizing the chlamydial inclusion as a pathogen-specified parasitic organelle: an expanded role for Inc proteins

    PubMed Central

    Moore, Elizabeth R.; Ouellette, Scot P.

    2014-01-01

    Chlamydia is an obligate intracellular pathogen that develops in the host cell in a vacuole termed the chlamydial inclusion. The prevailing concept of the chlamydial inclusion is of a parasitophorous vacuole. Here, the inclusion is the recipient of one-way host-pathogen interactions thus draining nutrients from the cell and negatively impacting it. While Chlamydia orchestrates some aspects of cell function, recent data indicate host cells remain healthy up until, and even after, chlamydial egress. Thus, while Chlamydia relies on the host cell for necessary metabolites, the overall function of the host cell, during chlamydial growth and development, is not grossly disturbed. This is consistent with the obligate intracellular organism's interest to maintain viability of its host. To this end, Chlamydia expresses inclusion membrane proteins, Incs, which serve as molecular markers for the inclusion membrane. Incs also contribute to the physical structure of the inclusion membrane and facilitate host-pathogen interactions across it. Given the function of Incs and the dynamic interactions that occur at the inclusion membrane, we propose that the inclusion behaves similarly to an organelle-albeit one that benefits the pathogen. We present the hypothesis that the chlamydial inclusion acts as a pathogen-specified parasitic organelle. This representation integrates the inclusion within existing subcellular trafficking pathways to divert a subset of host-derived metabolites thus maintaining host cell homeostasis. We review the known interactions of the chlamydial inclusion with the host cell and discuss the role of Inc proteins in the context of this model and how this perspective can impact the study of these proteins. Lessons learnt from the chlamydial pathogen-specified parasitic organelle can be applied to other intracellular pathogens. This will increase our understanding of how intracellular pathogens engage the host cell to establish their unique developmental niches

  14. Identification of a New Vertebrate Nucleoporin, Nup188, with the Use of a Novel Organelle Trap Assay

    PubMed Central

    Miller, Brian R.; Powers, Maureen; Park, Minkyu; Fischer, Wolfgang; Forbes, Douglass J.

    2000-01-01

    The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleoporins. We have developed a novel two-step “organelle trap” assay involving affinity selection and in vitro pore assembly. In the first step, soluble proteins derived from Xenopus egg extracts are applied to a column containing a ligand of interest. The bound proteins are then tagged by biotinylation and eluted. In the second step, potential nucleoporins are selected for by virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The incorporated proteins are then recognized by their biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and has been shown to directly bind three known nucleoporins from Xenopus extract, Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues. Under reduced-stringency conditions, three additional proteins bind to WGA–Sepharose and are revealed by the organelle trap assay. We identified all three as partner nucleoporins. Two were discovered to be Xenopus Nup93 and Nup205. The third is a novel vertebrate nucleoporin, Nup188. This new vertebrate protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly via the N-acetylglucosamine–modified nucleoporins. A gene encoding human Nup188 was also identified. The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein–protein interactions illustrates the power of the two-step organelle trap assay and identifies new building blocks for constructing the nuclear pore. PMID:11029043

  15. Export of cyst wall material and Golgi organelle neogenesis in Giardia lamblia depend on endoplasmic reticulum exit sites.

    PubMed

    Faso, Carmen; Konrad, Christian; Schraner, Elisabeth M; Hehl, Adrian B

    2013-04-01

    Giardia lamblia parasitism accounts for the majority of cases of parasitic diarrheal disease, making this flagellated eukaryote the most successful intestinal parasite worldwide. This organism has undergone secondary reduction/elimination of entire organelle systems such as mitochondria and Golgi. However, trophozoite to cyst differentiation (encystation) requires neogenesis of Golgi-like secretory organelles named encystation-specific vesicles (ESVs), which traffic, modify and partition cyst wall proteins produced exclusively during encystation. In this work we ask whether neogenesis of Golgi-related ESVs during G. lamblia differentiation, similarly to Golgi biogenesis in more complex eukaryotes, requires the maintenance of distinct COPII-associated endoplasmic reticulum (ER) subdomains in the form of ER exit sites (ERES) and whether ERES are also present in non-differentiating trophozoites. To address this question, we identified conserved COPII components in G. lamblia cells and determined their localization, quantity and dynamics at distinct ERES domains in vegetative and differentiating trophozoites. Analogous to ERES and Golgi biogenesis, these domains were closely associated to early stages of newly generated ESV. Ectopic expression of non-functional Sar1 GTPase variants caused ERES collapse and, consequently, ESV ablation, leading to impaired parasite differentiation. Thus, our data show how ERES domains remain conserved in G. lamblia despite elimination of steady-state Golgi. Furthermore, the fundamental eukaryotic principle of ERES to Golgi/Golgi-like compartment correspondence holds true in differentiating Giardia presenting streamlined machinery for secretory organelle biogenesis and protein trafficking. However, in the Golgi-less trophozoites ERES exist as stable ER subdomains, likely as the sole sorting centres for secretory traffic. PMID:23094658

  16. Magnetic Fractionation and Proteomic Dissection of Cellular Organelles Occupied by the Late Replication Complexes of Semliki Forest Virus

    PubMed Central

    Varjak, Margus; Saul, Sirle; Arike, Liisa; Lulla, Aleksei; Peil, Lauri

    2013-01-01

    Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection. PMID:23864636

  17. The circadian bioluminescence rhythm of Gonyaulax is related to daily variations in the number of light-emitting organelles.

    PubMed

    Fritz, L; Morse, D; Hastings, J W

    1990-02-01

    The number of scintillons, which are cellular organelles responsible for light emission in the marine alga Gonyaulax, were counted by both immunofluorescence and electron microscopic methods and found to vary tenfold between subjective day and subjective night. The number of scintillons peaks during the subjective night, as does stimulated bioluminescence (flashing). Furthermore, the number drops sharply at the time of the maximal spontaneous bioluminescence (glow), which occurs at the end of the night phase, suggesting that the breakdown of scintillons may be responsible for this mode of emission. PMID:2196272

  18. Universals in Morphological Representation: Evidence from Italian.

    ERIC Educational Resources Information Center

    Orsolini, Margherita; Marslen-Wilson, William

    1997-01-01

    Examines the dual mechanism hypothesis, which argues that the apparent contrast, in English inflectional morphology, between a rule-based default procedure, generating regular past-tense forms, and listed irregular forms stored in an associative net, reflects universal constraints on the properties of possible morphological systems. The study…

  19. Phagocytic properties and organelle motility of pulmonary macrophages from smokers and nonsmokers estimated in vitro by magnetometric means.

    PubMed

    Im Hof, V; Klauser, M; Gehr, P

    1990-02-01

    Monolayer cultures of pulmonary macrophages (PM) from 11 smokers (S) and 9 nonsmokers (NS) were incubated with submicrometric ferromagnetic Fe3O4 particles for one hour. After magnetization by an externally applied pulse magnetic field, the aligned Fe3O4 particles, located in the phagosomes and secondary lysosomes of the PM, emanated a remanent magnetic field which decayed with time. The initial field strength, B0, which is proportional to the amount of phagocytosed Fe3O4 particles, was 13.24 nT (SE 0.79 nT) in S and 11.74 nT (SE 1.39 nT) in NS. Ten nT correspond to roughly 4 micrograms Fe3O4. The initial rate of decay of the remanent field (during the first 60 s after magnetization), lambda 0, is proportional to the rate of particle misalignment. Therefore, lambda 0, is hypothesized to be an estimate of organelle motility. It was found to be the same in S and in NS, being 3.14 x 10(-3).s-1 (SE 0.18 x 10(-3).s-1) in S and 3.17 x 10(-3).s-1) (SE 0.22 x 10(-3).s-1) in NS. These results suggest that, in vitro, there is no difference in phagocytic activity and organelle motility in PM from S and NS. PMID:2311741

  20. Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

    PubMed Central

    Calvo-Garrido, Javier; Carilla-Latorre, Sergio; Lázaro-Diéguez, Francisco; Egea, Gustavo

    2008-01-01

    Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1− Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1− cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised. PMID:18550798

  1. Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells

    PubMed Central

    Kobayashi, Yuki; Kanesaki, Yu; Tanaka, Ayumi; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Tanaka, Kan

    2009-01-01

    Eukaryotic cells arose from an ancient endosymbiotic association of prokaryotes, with plant cells harboring 3 genomes as the remnants of such evolution. In plant cells, plastid and mitochondrial DNA replication [organelle DNA replication (ODR)] occurs in advance of the subsequent cell cycles composed of nuclear DNA replication (NDR) and cell division. However, the mechanism by which replication of these genomes with different origins is coordinated is largely unknown. Here, we show that NDR is regulated by a tetrapyrrole signal in plant cells, which has been suggested as an organelle-to-nucleus retrograde signal. In synchronized cultures of the primitive red alga Cyanidioschyzon merolae, specific inhibition of A-type cyclin-dependent kinase (CDKA) prevented NDR but not ODR after onset of the cell cycle. In contrast, inhibition of ODR by nalidixic acid also resulted in inhibition of NDR, indicating a strict dependence of NDR on ODR. The requirement of ODR for NDR was bypassed by addition of the tetrapyrrole intermediates protoporphyrin IX (ProtoIX) or Mg-ProtoIX, both of which activated CDKA without inducing ODR. This scheme was also observed in cultured tobacco cells (BY-2), where inhibition of ODR by nalidixic acid prevented CDKA activation and NDR, and these inhibitions were circumvented by Mg-ProtoIX without inducing ODR. We thus show that tetrapyrrole-mediated organelle–nucleus replicational coupling is an evolutionary conserved process among plant cells. PMID:19141634

  2. Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism of Mycoplasma pneumoniae

    PubMed Central

    Kawamoto, Akihiro; Matsuo, Lisa; Kato, Takayuki; Yamamoto, Hiroki

    2016-01-01

    ABSTRACT Mycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding. PMID:27073090

  3. Disruption of cellular homeostasis induces organelle stress and triggers apoptosis like cell-death pathways in malaria parasite

    PubMed Central

    Rathore, S; Datta, G; Kaur, I; Malhotra, P; Mohmmed, A

    2015-01-01

    A regulated protein turnover machinery in the cell is essential for effective cellular homeostasis; any interference with this system induces cellular stress and alters the normal functioning of proteins important for cell survival. In this study, we show that persistent cellular stress and organelle dysfunction because of disruption of cellular homeostasis in human malaria parasite Plasmodium falciparum, leads to apoptosis-like cell death. Quantitative global proteomic analysis of the stressed parasites before onset of cell death, showed upregulation of a number of proteins involved in cellular homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. However, persistent stress on parasites cause structural abnormalities in endoplasmic reticulum and mitochondria, subsequently a cascade of reactions are initiated in parasites including rise in cytosolic calcium levels, loss of mitochondrial membrane potential and activation of VAD-FMK-binding proteases. We further show that activation of VAD-FMK-binding proteases in the parasites leads to degradation of phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), a known target of metacaspases, as well as degradation of other components of spliceosomal complex. Loss of spliceosomal machinery impairs the mRNA splicing, leading to accumulation of unprocessed RNAs in the parasite and thus dysregulate vital cellular functions, which in turn leads to execution of apoptosis-like cell death. Our results establish one of the possible mechanisms of instigation of cell death by organelle stress in Plasmodium. PMID:26136076

  4. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    PubMed

    Tjaden, Joachim; Haferkamp, Ilka; Boxma, Brigitte; Tielens, Aloysius G M; Huynen, Martijn; Hackstein, Johannes H P

    2004-03-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently. PMID:14982636

  5. A Set of Organelle-Localizable Reactive Molecules for Mitochondrial Chemical Proteomics in Living Cells and Brain Tissues.

    PubMed

    Yasueda, Yuki; Tamura, Tomonori; Fujisawa, Alma; Kuwata, Keiko; Tsukiji, Shinya; Kiyonaka, Shigeki; Hamachi, Itaru

    2016-06-22

    Protein functions are tightly regulated by their subcellular localization in live cells, and quantitative evaluation of dynamically altered proteomes in each organelle should provide valuable information. Here, we describe a novel method for organelle-focused chemical proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive molecules (MRMs) were designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where protein labeling is facilitated by the condensation effect. The combination of this selective labeling and liquid chromatography-mass spectrometry (LC-MS) based proteomics technology facilitated identification of mitochondrial proteomes and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of mitochondrial proteins whose expression levels change significantly during an oxidant-induced apoptotic process was performed by combination of this MRMs-based method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical proteomics to elucidate mitochondria-associated biological events and diseases. PMID:27228550

  6. Experimental impact crater morphology

    NASA Astrophysics Data System (ADS)

    Dufresne, A.; Poelchau, M. H.; Hoerth, T.; Schaefer, F.; Thoma, K.; Deutsch, A.; Kenkmann, T.

    2012-04-01

    The research group MEMIN (Multidisciplinary Experimental and Impact Modelling Research Network) is conducting impact experiments into porous sandstones, examining, among other parameters, the influence of target pore-space saturation with water, and projectile velocity, density and mass, on the cratering process. The high-velocity (2.5-7.8 km/s) impact experiments were carried out at the two-stage light-gas gun facilities of the Fraunhofer Institute EMI (Germany) using steel, iron meteorite (Campo del Cielo IAB), and aluminium projectiles with Seeberg Sandstone as targets. The primary objectives of this study within MEMIN are to provide detailed morphometric data of the experimental craters, and to identify trends and characteristics specific to a given impact parameter. Generally, all craters, regardless of impact conditions, have an inner depression within a highly fragile, white-coloured centre, an outer spallation (i.e. tensile failure) zone, and areas of arrested spallation (i.e. spall fragments that were not completely dislodged from the target) at the crater rim. Within this general morphological framework, distinct trends and differences in crater dimensions and morphological characteristics are identified. With increasing impact velocity, the volume of craters in dry targets increases by a factor of ~4 when doubling velocity. At identical impact conditions (steel projectiles, ~5km/s), craters in dry and wet sandstone targets differ significantly in that "wet" craters are up to 76% larger in volume, have depth-diameter ratios generally below 0.19 (whereas dry craters are almost consistently above this value) at significantly larger diameters, and their spallation zone morphologies show very different characteristics. In dry craters, the spall zone surfaces dip evenly at 10-20° towards the crater centre. In wet craters, on the other hand, they consist of slightly convex slopes of 10-35° adjacent to the inner depression, and of sub-horizontal tensile

  7. Destruction of Tissue, Cells and Organelles in Type 1 Diabetic Rats Presented at Macromolecular Resolution

    PubMed Central

    Ravelli, Raimond B. G.; Kalicharan, Ruby D.; Avramut, M. Cristina; Sjollema, Klaas A.; Pronk, Joachim W.; Dijk, Freark; Koster, Abraham J.; Visser, Jeroen T. J.; Faas, Frank G. A.; Giepmans, Ben N. G.

    2013-01-01

    Finding alternatives for insulin therapy and making advances in etiology of type 1 diabetes benefits from a full structural and functional insight into Islets of Langerhans. Electron microscopy (EM) can visualize Islet morphology at the highest possible resolution, however, conventional EM only provides biased snapshots and lacks context. We developed and employed large scale EM and compiled a resource of complete cross sections of rat Islets during immuno-destruction to provide unbiased structural insight of thousands of cells at macromolecular resolution. The resource includes six datasets, totalling 25.000 micrographs, annotated for cellular and ultrastructural changes during autoimmune diabetes. Granulocytes are attracted to the endocrine tissue, followed by extravasation of a pleiotrophy of leukocytes. Subcellullar changes in beta cells include endoplasmic reticulum stress, insulin degranulation and glycogen accumulation. Rare findings include erythrocyte extravasation and nuclear actin-like fibers. While we focus on a rat model of autoimmune diabetes, our approach is general applicable. PMID:23652855

  8. Stone Morphology: Implication for Pathogenesis

    NASA Astrophysics Data System (ADS)

    Daudon, Michel; Jungers, Paul; Bazin, Dominique

    2008-09-01

    Urinary stones of similar crystalline composition as identified by X-ray diffraction or Fourier transform infrared spectroscopy (FT-IR) may exhibit distinct structures, which are reflected in distinctive stone morphology. Among factors involved in stone morphology—some reflecting lithogenic activity, others depending on the crystal shape, or on the propensity of crystalline phases to form large aggregates, finally the environment where the stone is growing—all of these factors influence the inner structure of the stone and its superficial characteristics. We present here examples of the clinical interest of refined morphologic examination of stones, in addition to X-ray diffraction or FT-IR identification of its components. Such combination of methods allows the identification of specific etiologies among calcium oxalate stones, especially a morphological type pathognomonic of primary hyperoxaluria and other types related to distinct conditions of stone formation. Among phosphatic stones—in addition to stone composition, which must be considered not only on the basis of the main component, but also taking into account the minor crystalline phases, which often are clinically relevant—morphological types also contribute to diagnosis of the underlying etiology, especially for stones related to distal tubular acidosis. Finally, common purine stones also exhibit different morphologies related to stone composition and etiology: two main structures for uric acid and two for ammonium urate help to distinguish risk factors and lithogenic conditions involved in the formation of these calculi. Morphologic examination is a simple, rapid and cheap method that points to specific diseases or lithogenic factors. Better awareness of its clinical relevance should lead to wider utilization.

  9. Imaging of fine structures of cellular organelles in hydrated biological cells by a soft x-ray microscope combined with a fluorescence microscope

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Kishimoto, Maki; Tamotsu, Satoshi; Yasuda, Keiko; Aoyama, Masato; Shinohara, Kunio

    2013-09-01

    We have proposed and developed a new hybrid microscopy system using a soft x-ray microscope and a fluorescence microscope imaging the same biological cells at the nearly same time. Combining the powerful advantages such as high spatial resolution of the soft x-ray microscope and the accurate organelle identification of the fluorescence microscope, we can observe fine structures of the cellular organelles in live hydrated biological cells in situ. Staining the cells with several fluorescent dyes such as Mito-tracker, Phalloidin, and DAPI, the soft x-ray images of the cells have been directly compared with the fluorescent images and the cellular organelles such as mitochondria, actin filaments, and chromosomes in the soft x-ray images have been clearly identified. Since the soft x-ray microscope has higher spatial resolution than that of the fluorescence microscope, not only shape of the cellular organelles but also the fine structures of the cellular organelles of the live biological cells have been clearly observed for the first time.

  10. Of Organelles and Octagons: What Do Preservice Secondary Teachers Learn from Interdisciplinary Teaching?

    ERIC Educational Resources Information Center

    Spalding, Elizabeth

    2002-01-01

    Examined the impact of an interdisciplinary teacher project on the learning of preservice secondary teachers enrolled in a graduate level teacher education program. Data on four successive cohorts of students indicated that participants successfully created and implemented interdisciplinary curriculum and were quite positive about the experience.…

  11. KIFC2 is a novel neuron-specific C-terminal type kinesin superfamily motor for dendritic transport of multivesicular body-like organelles.

    PubMed

    Saito, N; Okada, Y; Noda, Y; Kinoshita, Y; Kondo, S; Hirokawa, N

    1997-03-01

    We have cloned two novel C-terminal motor domain-type kinesin superfamily motor proteins (KIFCs) from mouse brain by utilizing a KIFC-specific consensus sequence. The first protein was the murine homologue of CHO2 antigen, a member of the kar3-type mitotic motor subfamily, and we designated this protein KIFC1. The other protein, KIFC2 (792 amino acids), is novel, with no significant similarity to known kinesin superfamily proteins (KIFs). KIFC2 was specifically expressed in adult neurons, and was immunofluorescently localized to punctate structures in cell bodies and dendrites, but was not detected in axons. Electron microscopic analysis of the immunoisolated KIFC2-bound organelles revealed that KIFC2 associates with multivesicular body (mvb)-like organelles, suggesting that KIFC2 functions as the motor for the transport of mvb-like organelles in dendrites. PMID:9115736

  12. Microscopy with spatial filtering for sorting particles and monitoring subcellular morphology

    NASA Astrophysics Data System (ADS)

    Zheng, Jing-Yi; Qian, Zhen; Pasternack, Robert M.; Boustany, Nada N.

    2009-02-01

    Optical scatter imaging (OSI) was developed to non-invasively track real-time changes in particle morphology with submicron sensitivity in situ without exogenous labeling, cell fixing, or organelle isolation. For spherical particles, the intensity ratio of wide-to-narrow angle scatter (OSIR, Optical Scatter Image Ratio) was shown to decrease monotonically with diameter and agree with Mie theory. In living cells, we recently reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl-xL transmembrane domain, and could not be observed by fluorescence or differential interference contrast images. Here we further extend the ability of morphology assessment by adopting a digital micromirror device (DMD) for Fourier filtering. When placed in the Fourier plane the DMD can be used to select scattering intensities at desired combination of scattering angles. We designed an optical filter bank consisting of Gabor-like filters with various scales and rotations based on Gabor filters, which have been widely used for localization of spatial and frequency information in digital images and texture analysis. Using a model system consisting of mixtures of polystyrene spheres and bacteria, we show how this system can be used to sort particles on a microscopic slide based on their size, orientation and aspect ratio. We are currently applying this technique to characterize the morphology of subcellular organelles to help understand fundamental biological processes.

  13. High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

    NASA Astrophysics Data System (ADS)

    Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

    2014-09-01

    Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

  14. A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4.

    PubMed

    Galletta, Brian J; Fagerstrom, Carey J; Schoborg, Todd A; McLamarrah, Tiffany A; Ryniawec, John M; Buster, Daniel W; Slep, Kevin C; Rogers, Gregory C; Rusan, Nasser M

    2016-01-01

    The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein-protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a 'domain-level' centrosome interactome using direct protein-protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes. PMID:27558293

  15. Comparative study of the cytoplasmic organelles of epithelial cell lines derived from human carcinomas and nonmalignant tissues

    SciTech Connect

    Springer, E.L.

    1980-03-01

    The cytoplasmic organelles of 16 human epithelial cell lines have been characterized by electron microscopy. The cell lines were derived from normal, nonmalignant tissues of cancerous organs and from primary and metastatic carcinomas. Mitochondrial pleomorphism was expressed slightly by normal, to variable degrees by lines derived from nonmalignant tissues of cancerous organs, and to a much greater extent by all lines derived from malignant tissues. Hypertrophied mitochondria and longitudinal cristal arrangement were found in almost all the malignant lines, but not in any lines derived from nonmalignant tissues of cancerous organs or from normal tissues. All the lines appeared differentiate and showed slightly to moderately developed Golgi and smooth and rough endoplasmic reticula. There were no significant ultrastructural differences in cells at different passage levels or subconfluent and confluent tumor cells; however, more tight junctions were observed in confluent than in subconfluent normal cells.

  16. A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4

    PubMed Central

    Galletta, Brian J.; Fagerstrom, Carey J.; Schoborg, Todd A.; McLamarrah, Tiffany A.; Ryniawec, John M.; Buster, Daniel W.; Slep, Kevin C.; Rogers, Gregory C.; Rusan, Nasser M.

    2016-01-01

    The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membrane-bound nature of the centrosome dictates that protein–protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a ‘domain-level' centrosome interactome using direct protein–protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes. PMID:27558293

  17. CD4+ T Cells and Toll-Like Receptors Recognize Salmonella Antigens Expressed in Bacterial Surface Organelles

    PubMed Central

    Bergman, Molly A.; Cummings, Lisa A.; Barrett, Sara L. Rassoulian; Smith, Kelly D.; Lara, J. Cano; Aderem, Alan; Cookson, Brad T.

    2005-01-01

    A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition. PMID:15731032

  18. New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development

    PubMed Central

    Schnell, Danny J.

    2014-01-01

    The translocons at the outer (TOC) and inner (TIC) envelope membranes of chloroplasts mediate the targeting and import of several thousand nuclear encoded preproteins that are required for organelle biogenesis and homeostasis. The cytosolic events in preprotein targeting remain largely unknown, although cytoplasmic chaperones have been proposed to facilitate delivery to the TOC complex. Preprotein recognition is mediated by the TOC GTPase receptors, Toc159 and Toc34. The receptors constitute a GTP-regulated switch, which initiates membrane translocation via Toc75, a member of the OMP85 (Outer Membrane Protein 85)/TpsB (two partner secretion system B) family of bacterial, plastid and mitochondrial β-barrel outer membrane proteins. The TOC receptor systems have diversified to recognize distinct sets of preproteins, thereby maximizing the efficiency of targeting in response to changes in gene expression during developmental and physiological events that impact organelle function. The TOC complex interacts with the TIC translocon to allow simultaneous translocation of preproteins across the envelope. Two inner membrane complexes, the Tic110 and 1 MDa complexes, have both been implicated as constituents of the TIC translocon, and it remains to be determined how they interact to form the TIC channel and assemble the import-associated chaperone network in the stroma that drives import across the envelope membranes. This review will focus on recent developments in our understanding of the mechanisms and diversity of the TOC-TIC systems. Our goal is to incorporate these recent studies with previous work and present updated or revised models for the function of TOC-TIC in protein import. PMID:25174336

  19. C3-C4 intermediacy in grasses: organelle enrichment and distribution, glycine decarboxylase expression, and the rise of C2 photosynthesis.

    PubMed

    Khoshravesh, Roxana; Stinson, Corey R; Stata, Matt; Busch, Florian A; Sage, Rowan F; Ludwig, Martha; Sage, Tammy L

    2016-05-01

    Photorespiratory glycine shuttling and decarboxylation in bundle sheath (BS) cells exhibited by C2 species is proposed to be the evolutionary bridge to C4 photosynthesis in eudicots. To evaluate this in grasses, we compare anatomy, cellular localization of glycine decarboxylase (GDC), and photosynthetic physiology of a suspected C2 grass, Homolepis aturensis, with these traits in known C2 grasses, Neurachne minor and Steinchisma hians, and C3 S laxum that is sister to S hians We also use publicly available genome and RNA-sequencing data to examine the evolution of GDC subunits and enhance our understanding of the evolution of BS-specific GDC expression in C2 and C4 grasses. Our results confirm the identity of H aturensis as a C2 species; GDC is confined predominantly to the organelle-enriched BS cells in H aturensis and S hians and to mestome sheath cells of N minor Phylogenetic analyses and data obtained from immunodetection of the P-subunit of GDC are consistent with the hypothesis that the BS dominant levels of GDC in C2 and C4 species are due to changes in expression of a single GLDP gene in M and BS cells. All BS mitochondria and peroxisomes and most chloroplasts in H aturensis and S hians are situated centripetally in a pattern identical to C2 eudicots. In S laxum, which has C3-like gas exchange patterns, mitochondria and peroxisomes are positioned centripetally as they are in S hians This subcellular phenotype, also present in eudicots, is posited to initiate a facilitation cascade leading to C2 and C4 photosynthesis. PMID:27073202

  20. C3–C4 intermediacy in grasses: organelle enrichment and distribution, glycine decarboxylase expression, and the rise of C2 photosynthesis

    PubMed Central

    Khoshravesh, Roxana; Stinson, Corey R.; Stata, Matt; Busch, Florian A.; Sage, Rowan F.; Ludwig, Martha; Sage, Tammy L.

    2016-01-01

    Photorespiratory glycine shuttling and decarboxylation in bundle sheath (BS) cells exhibited by C2 species is proposed to be the evolutionary bridge to C4 photosynthesis in eudicots. To evaluate this in grasses, we compare anatomy, cellular localization of glycine decarboxylase (GDC), and photosynthetic physiology of a suspected C2 grass, Homolepis aturensis, with these traits in known C2 grasses, Neurachne minor and Steinchisma hians, and C3 S. laxum that is sister to S. hians. We also use publicly available genome and RNA-sequencing data to examine the evolution of GDC subunits and enhance our understanding of the evolution of BS-specific GDC expression in C2 and C4 grasses. Our results confirm the identity of H. aturensis as a C2 species; GDC is confined predominantly to the organelle-enriched BS cells in H. aturensis and S. hians and to mestome sheath cells of N. minor. Phylogenetic analyses and data obtained from immunodetection of the P-subunit of GDC are consistent with the hypothesis that the BS dominant levels of GDC in C2 and C4 species are due to changes in expression of a single GLDP gene in M and BS cells. All BS mitochondria and peroxisomes and most chloroplasts in H. aturensis and S. hians are situated centripetally in a pattern identical to C2 eudicots. In S. laxum, which has C3-like gas exchange patterns, mitochondria and peroxisomes are positioned centripetally as they are in S. hians. This subcellular phenotype, also present in eudicots, is posited to initiate a facilitation cascade leading to C2 and C4 photosynthesis. PMID:27073202

  1. Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis1[OPEN

    PubMed Central

    Madison, Stephanie L.; Buchanan, Matthew L.; Glass, Jeremiah D.; McClain, Tarah F.; Park, Eunsook; Nebenführ, Andreas

    2015-01-01

    Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes. PMID:26358416

  2. KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B.

    PubMed

    Noda, Y; Sato-Yoshitake, R; Kondo, S; Nangaku, M; Hirokawa, N

    1995-04-01

    Kinesin is known as a representative cytoskeletal motor protein that is engaged in cell division and axonal transport. In addition to the mutant assay, recent advances using the PCR cloning technique have elucidated the existence of many kinds of kinesin-related proteins in yeast, Drosophila, and mice. We previously cloned five different members of kinesin superfamily proteins (KIFs) in mouse brain (Aizawa, H., Y. Sekine, R. Takemura, Z. Zhang, M. Nangaku, and N. Hirokawa. 1992. J. Cell Biol. 119:1287-1296) and demonstrated that one of them, KIF3A, is an anterograde motor (Kondo, S., R. Sato-Yashitake, Y. Noda, H. Aizawa, T. Nakata, Y. Matsuura, and N. Hirokawa. J. Cell Biol. 1994. 125:1095-1107). We have now characterized another axonal transport motor, KIF2. Different from other KIFs, KIF2 is a central type motor, since its motor domain is located in the center of the molecule. Recombinant KIF2 exists as a dimer with a bigger head and plus-end directionally moves microtubules at a velocity of 0.47 +/- 0.11 microns/s, which is two thirds that of kinesin's. Immunocytological examination showed that native KIF2 is abundant in developing axons and that it accumulates in the proximal region of the ligated nerves after a 20-h ligation. Soluble KIF2 exists without a light chain, and KIF2's associated-vesicles, immunoprecipitated by anti-KIF2 antibody, are different from those carried by existing motors such as kinesin and KIF3A. They are also distinct from synaptic vesicles, although KIF2 is accumulated in so-called synaptic vesicle fractions and embryonal growth cone particles. Our results strongly suggest that KIF2 functions as a new anterograde motor, being specialized for a particular group of membranous organelles involved in fast axonal transport. PMID:7535303

  3. An Electrophysiological Investigation of Early Effects of Masked Morphological Priming

    ERIC Educational Resources Information Center

    Morris, Joanna; Grainger, Jonathan; Holcomb, Phillip J.

    2008-01-01

    This experiment examined event-related responses to targets preceded by semantically transparent morphologically related primes (e.g., farmer-farm), semantically opaque primes with an apparent morphological relation (corner-corn), and orthographically, but not morphologically, related primes (scandal-scan) using the masked priming technique…

  4. Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA.

    PubMed

    Apitz, Janina; Weihe, Andreas; Pohlheim, Frank; Börner, Thomas

    2013-02-01

    While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes. PMID:23053540

  5. Ultrastructural insights into morphology and reproductive mode of Blastocystis hominis.

    PubMed

    Zhang, Xu; Zhang, Siwei; Qiao, Jiying; Wu, Xiaomin; Zhao, Liming; Liu, Yansheng; Fan, Xiaojun

    2012-03-01

    To understand well the morphology and reproductive mode of Blastocystis hominis, with the help of transmission electron microscopy and scanning electron microscopy the ultrastructural details of B. hominis from fresh diarrheal specimens and cultured strains were observed. In both fecal samples and culture conditions, there were vacuolar and granular forms. In diarrhea, it exists in multivacuolar, avacuolar, and amoeboid forms. In the in vitro culture, vacuolar form could transform to granular form. The most commonly noticed structure on the cell surface was surface coat with diversity in appearance (the funiform, lamellar, filiform, and floccose in different thickness) and distributions. Three modes of reproduction were confirmed, they were binary fission, plasmotomy, and budding. Under the impact of host's response, the ultrastructures of surface coat, nucleus, and mitochondrion-like organelle sometimes changed. PMID:21845408

  6. Observation of Organelles in Leydig Cells by Contact Soft X-Ray Microscopy with a Laser Plasma X-Ray Source

    NASA Astrophysics Data System (ADS)

    Kado, M.; Ishino, M.; Tamotsu, S.; Yasuda, K.; Kishimoto, M.; Nishikino, M.; Kinjo, Y.; Shinohara, K.

    2011-09-01

    We observed the same biological specimens for comparison of the images by contact soft x-ray microscopy with a laser plasma x-ray source with those by confocal laser microscopy. Images of wet Leydig cells were directly comparable for organelles and showed that actin filaments and mitochondria were clearly identified in the soft x-ray images.

  7. Biochemical Characterization of the Bi-lobe Reveals a Continuous Structural Network Linking the Bi-lobe to Other Single-copied Organelles in Trypanosoma brucei*

    PubMed Central

    Gheiratmand, Ladan; Brasseur, Anais; Zhou, Qing; He, Cynthia Y.

    2013-01-01

    Trypanosoma brucei, a unicellular parasite, contains several single-copied organelles that duplicate and segregate in a highly coordinated fashion during the cell cycle. In the procyclic stage, a bi-lobed structure is found adjacent to the single ER exit site and Golgi apparatus, forming both stable and dynamic association with other cytoskeletal components including the basal bodies that seed the flagellum and the flagellar pocket collar that is critical for flagellar pocket biogenesis. To further understand the bi-lobe and its association with adjacent organelles, we performed proteomic analyses on the immunoisolated bi-lobe complex. Candidate proteins were localized to the flagellar pocket, the basal bodies, a tripartite attachment complex linking the basal bodies to the kinetoplast, and a segment of microtubule quartet linking the flagellar pocket collar and bi-lobe to the basal bodies. These results supported an extensive connection among the single-copied organelles in T. brucei, a strategy employed by the parasite for orderly organelle assembly and inheritance during the cell cycle. PMID:23235159

  8. Morphology of Treponema pallidum.

    PubMed

    Ovcinnikov, N M; Delektorskij, V V

    1966-01-01

    In recent years many investigations have been carried out on the morphology of Treponema pallidum by means of the electron microscope, and the use of ultra-thin sections has shown up a number of structural details. However, there is still need for much more evidence before the internal structure of treponemes can be elucidated fully and the functions of the structures interpreted. To provide such evidence, the authors have examined under the electron microscope negative-stained treponemes and ultra-thin sections, using both cultivated strains and treponemes obtained direct from syphilids in people suffering from fresh secondary syphilis. It has been shown that treponemes have a complex structure. T. pallidum has a two-layered outer wall, a cytoplasmic membrane proper, cytoplasm and a bunch of fibrils following a different path in different places on the treponeme. The sites of insertion of the fibrils (the basal granules) were investigated; structures similar to mesosomes and nucleoids were found. Cysts and granular forms are described. PMID:5332527

  9. Fish Manoeuvres and Morphology

    NASA Astrophysics Data System (ADS)

    Singh, Kiran; Pedley, Timothy

    2008-11-01

    The extraordinary manoeuvrability observed in many fish is attributed to their inherent flexibility, which might be enhanced by the use of appendages like fins. The aim of this work is to understand the role of morphological adaptations, such as body shape and deployment of median fins, on manoeuvrability and internal body dynamics. The 3d vortex lattice numerical method was employed to analyse the hydrodynamics for arbitrary body planforms of infinitesimal thickness. The internal structure of the body due to the combined skeletal system and soft tissue, is represented as an active Euler-Bernoulli beam, in which the time-dependent bending moment distribution is calculated from body inertia and the hydrodynamic pressure difference across the body. C-turns are the manoeuvre of choice for this work and the response for three different species of fish are examined. Angelfish(Pterophyllum eimekei), pike (Esox sp) and tuna (Thunnus albacares) were chosen for their differences in body profile, median fin use and manoeuvrability. Net direction change and bending moment response to prescribed backbone flexure are calculated and used to interpret the influence of body profile on manoeuvrability and muscle work done. Internal stresses may be computed from anatomical data on muscle fibre distribution and recruitment. To the future, it is intended to extend this work to other typical manoeuvres, such as fast starts for which muscle activation patterns have been measured quite widely.

  10. Electrodynamic eigenmodes in cellular morphology.

    PubMed

    Cifra, M

    2012-09-01

    Eigenmodes of the spherical and ellipsoidal dielectric electromagnetic resonator have been analysed. The sizes and shape of the resonators have been chosen to represent the shape of the interphase and dividing animal cell. Electromagnetic modes that have shape exactly suitable for positioning of the sufficiently large organelles in cell (centrosome, nucleus) have been identified. We analysed direction and magnitude of dielectrophoretic force exerted on large organelles by electric field of the modes. We found that the TM(1m1) mode in spherical resonator acts by centripetal force which drags the large organelles which have higher permittivity than the cytosol to the center of the cell. TM-kind of mode in the ellipsoidal resonator acts by force on large polarizable organelles in a direction that corresponds to the movement of the centrosomes (also nucleus) observed during the cell division, i.e. to the foci of the ellipsoidal cell. Minimal required force (10(-16) N), gradient of squared electric field and corresponding energy (10(-16) J) of the mode have been calculated to have biological significance within the periods on the order of time required for cell division. Minimal required energy of the mode, in order to have biological significance, can be lower in the case of resonance of organelle with the field of the cellular resonator mode. In case of sufficient energy in the biologically relevant mode, electromagnetic field of the mode will act as a positioning or steering mechanism for centrosome and nucleus in the cell, thus contribute to the spatial and dynamical self-organization in biological systems. PMID:22750075

  11. Small organelle, big responsibility: the role of centrosomes in development and disease

    PubMed Central

    Chavali, Pavithra L.; Pütz, Monika; Gergely, Fanni

    2014-01-01

    The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. PMID:25047622

  12. Control of magnetite nanocrystal morphology in magnetotactic bacteria by regulation of mms7 gene expression.

    PubMed

    Yamagishi, Ayana; Tanaka, Masayoshi; Lenders, Jos J M; Thiesbrummel, Jarla; Sommerdijk, Nico A J M; Matsunaga, Tadashi; Arakaki, Atsushi

    2016-01-01

    Living organisms can produce inorganic materials with unique structure and properties. The biomineralization process is of great interest as it forms a source of inspiration for the development of methods for production of diverse inorganic materials under mild conditions. Nonetheless, regulation of biomineralization is still a challenging task. Magnetotactic bacteria produce chains of a prokaryotic organelle comprising a membrane-enveloped single-crystal magnetite with species-specific morphology. Here, we describe regulation of magnetite biomineralization through controlled expression of the mms7 gene, which plays key roles in the control of crystal growth and morphology of magnetite crystals in magnetotactic bacteria. Regulation of the expression level of Mms7 in bacterial cells enables switching of the crystal shape from dumbbell-like to spherical. The successful regulation of magnetite biomineralization opens the door to production of magnetite nanocrystals of desired size and morphology. PMID:27417732

  13. Control of magnetite nanocrystal morphology in magnetotactic bacteria by regulation of mms7 gene expression

    PubMed Central

    Yamagishi, Ayana; Tanaka, Masayoshi; Lenders, Jos J. M.; Thiesbrummel, Jarla; Sommerdijk, Nico A. J. M.; Matsunaga, Tadashi; Arakaki, Atsushi

    2016-01-01

    Living organisms can produce inorganic materials with unique structure and properties. The biomineralization process is of great interest as it forms a source of inspiration for the development of methods for production of diverse inorganic materials under mild conditions. Nonetheless, regulation of biomineralization is still a challenging task. Magnetotactic bacteria produce chains of a prokaryotic organelle comprising a membrane-enveloped single-crystal magnetite with species-specific morphology. Here, we describe regulation of magnetite biomineralization through controlled expression of the mms7 gene, which plays key roles in the control of crystal growth and morphology of magnetite crystals in magnetotactic bacteria. Regulation of the expression level of Mms7 in bacterial cells enables switching of the crystal shape from dumbbell-like to spherical. The successful regulation of magnetite biomineralization opens the door to production of magnetite nanocrystals of desired size and morphology. PMID:27417732

  14. The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

    SciTech Connect

    Naranmandura, Hua; Xu, Shi; Koike, Shota; Pan, Li Qiang; Chen, Bin; Wang, Yan Wei; Rehman, Kanwal; Wu, Bin; Chen, Zhe; Suzuki, Noriyuki

    2012-05-01

    The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase in

  15. The Flagellum Attachment Zone: ‘The Cellular Ruler’ of Trypanosome Morphology

    PubMed Central

    Sunter, Jack D.; Gull, Keith

    2016-01-01

    A defining feature of Trypanosoma brucei cell shape is the lateral attachment of the flagellum to the cell body, mediated by the flagellum attachment zone (FAZ). The FAZ is a complex cytoskeletal structure that connects the flagellum skeleton through two membranes to the cytoskeleton. The FAZ acts as a ‘cellular ruler’ of morphology by regulating cell length and organelle position and is therefore critical for both cell division and life cycle differentiations. Here we provide an overview of the advances in our understanding of the composition, assembly, and function of the FAZ. PMID:26776656

  16. The Reciprocal Relations between Morphological Processes and Reading

    ERIC Educational Resources Information Center

    Kruk, Richard S.; Bergman, Krista

    2013-01-01

    Reciprocal relations between emerging morphological processes and reading skills were examined in a longitudinal study tracking children from Grade 1 through Grade 3. The aim was to examine predictive relationships between productive morphological processing involving composing and decomposing of inflections and derivations, reading ability for…

  17. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

    PubMed

    Park, Soonhong; Ahuja, Malini; Kim, Min Seuk; Brailoiu, G Cristina; Jha, Archana; Zeng, Mei; Baydyuk, Maryna; Wu, Ling-Gang; Wassif, Christopher A; Porter, Forbes D; Zerfas, Patricia M; Eckhaus, Michael A; Brailoiu, Eugen; Shin, Dong Min; Muallem, Shmuel

    2016-02-01

    Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV. PMID:26682800

  18. Systematic analysis of plant mitochondrial and chloroplast small RNAs suggests organelle-specific mRNA stabilization mechanisms

    PubMed Central

    Ruwe, Hannes; Wang, Gongwei; Gusewski, Sandra; Schmitz-Linneweber, Christian

    2016-01-01

    Land plant organellar genomes encode a small number of genes, many of which are essential for respiration and photosynthesis. Organellar gene expression is characterized by a multitude of RNA processing events that lead to stable, translatable transcripts. RNA binding proteins (RBPs), have been shown to generate and protect transcript termini and eventually induce the accumulation of short RNA footprints. We applied knowledge of such RBP-derived footprints to develop software (sRNA miner) that enables identification of RBP footprints, or other clusters of small RNAs, in organelles. We used this tool to determine mitochondrial and chloroplast cosRNAs (clustered organellar sRNAs) in Arabidopsis. We found that in mitochondria, cosRNAs coincide with transcript 3′-ends, but are largely absent from 5′-ends. In chloroplasts this bias is absent, suggesting a different mode of 5′ processing, possibly owing to different sets of RNases. Furthermore, we identified a large number of cosRNAs that represent silenced insertions of mitochondrial DNA in the nuclear genome of Arabidopsis. Steady-state RNA analyses demonstrate that cosRNAs display differential accumulation during development. Finally, we demonstrate that the chloroplast RBP PPR10 associates in vivo with its cognate cosRNA. A hypothetical role of cosRNAs as competitors of mRNAs for PPR proteins is discussed. PMID:27235415

  19. Dividing organelle tracks into Brownian and motor-driven intervals by variational maximization of the Bayesian evidence.

    PubMed

    Martin, Matthew J; Smelser, Amanda M; Holzwarth, George

    2016-04-01

    Many organelles and vesicles in live cells move in a start-stop manner when observed for ~10 s by optical microscopy. Changes in velocity and directional persistence of such particles are a potentially rich source of insight into the mechanisms leading to the start and stop states. Unbiased assessment of the most probable number of states, the properties of each state, and the most probable state for the particle at each moment can be accomplished by variational Bayesian methods combined with a hidden Markov model and a Gaussian mixture model. Our track analysis method, "vbTRACK", applied this combination of methods to particle velocity v or changes in the direction of travel evaluated from simulated tracks and from tracks of peroxisomes in live cells. When tested with numerical data, vbTRACK reliably determined the number of states, the mean and variance of the velocity or the direction of travel for each state, and the most probable state during each frame. When applied to the tracks of peroxisomes in live cells, some tracks separated into two states, one with high velocity and directionality, the other approximately Brownian. Other tracks of particles in live cells separated into several diffusive states with distinct diffusion constants. PMID:26538332

  20. The SM Protein Car/Vps33A Regulates SNARE-mediated Trafficking to Lysosomes and Lysosome-related Organelles

    PubMed Central

    Akbar, Mohammed A.; Ray, Sanchali

    2009-01-01

    The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes. PMID:19158398

  1. Metabolic network motifs can provide novel insights into evolution: The evolutionary origin of Eukaryotic organelles as a case study

    PubMed Central

    Shellman, Erin R.; Chen, Yu; Lin, Xiaoxia; Burant, Charles F.; Schnell, Santiago

    2014-01-01

    Phylogenetic trees are typically constructed using genetic and genomic data, and provide robust evolutionary relationships of species from the genomic point of view. We present an application of network motif mining and analysis of metabolic pathways that when used in combination with phylogenetic trees can provide a more complete picture of evolution. By using distributions of three-node motifs as a proxy for metabolic similarity, we analyze the ancestral origin of Eukaryotic organelles from the metabolic point of view to illustrate the application of our motif mining and analysis network approach. Our analysis suggests that the hypothesis of an early proto-Eukaryote could be valid. It also suggests that a δ- or ε-Proteobacteria may have been the endosymbiotic partner that gave rise to modern mitochondria. Our evolutionary analysis needs to be extended by building metabolic network reconstructions of species from the phylum Crenarchaeota, which is considered to be a possible archaeal ancestor of the eukaryotic cell. In this paper, we also propose a methodology for constructing phylogenetic trees that incorporates metabolic network signatures to identify regions of genomically-estimated phylogenies that may be spurious. We find that results generated from our approach are consistent with a parallel phylogenetic analysis using the method of feature frequency profiles. PMID:25462333

  2. Rediscovery of the nucleolinus, a dynamic RNA-rich organelle associated with the nucleolus, spindle, and centrosomes

    PubMed Central

    Alliegro, Mary Anne; Henry, Jonathan J.; Alliegro, Mark C.

    2010-01-01

    The nucleolinus is an RNA-rich compartment, closely apposed to or embedded within the nucleolus. Discovered over 150 y ago, fewer than two dozen articles have been published on the nucleolinus, probably because complex histochemical stains are required for its visualization in the great majority of cells. The nucleolinus has been reported in invertebrate oocytes, mammalian and amphibian epithelial cells, neurons, and several transformed cell lines. A prominent nucleolinus, clearly visible with transmitted light microscopes at 10× magnification, is present in each oocyte of the surf clam, Spisula solidissima. We observed a consistent relationship between the nucleolinus and the developing meiotic apparatus following Spisula oocyte activation. Through sonication and sucrose gradient fractionation of purified oocyte nuclei, we isolated nucleolini, extracted their RNA, and prepared an in situ riboprobe (NLi-1), which is associated specifically with the nucleolinus, confirming its unique composition. Other in situ observations revealed a NLi-1 and nucleolinar association with the developing spindle and centrosomes. Laser microsurgery that targeted the nucleolinus resulted in failed meiotic cell division in parthenogenetically activated oocytes and failed mitosis in fertilized oocytes. Although the nucleolinus may be a forgotten organelle, its demonstrated role in spindle formation suggests it deserves renewed attention. PMID:20643950

  3. The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.

    PubMed

    Pertl, Heidi; Schulze, Waltraud X; Obermeyer, Gerhard

    2009-11-01

    As a first step in understanding the membrane-related dynamics during pollen grain germination and subsequent tube growth, the changes in protein abundance of membrane and membrane-associated proteins of 5 different membrane/organelle fractions were studied at physiologically important stages (0, 10, 30, 60, and 240 min) of Lilium longiflorum pollen in vitro culture. Proteins of each fraction and time point were identified by 'shot-gun' proteomics (LC-MS/MS). Analysis of more than 270 identified proteins revealed an increase in the abundance of proteins involved in cytoskeleton, carbohydrate and energy metabolism, as well as ion transport before pollen grain germination (10-30 min), whereas proteins involved in membrane/protein trafficking, signal transduction, stress response and protein biosynthesis decreased in abundance during this time. Proteins of amino acids and lipids/steroids metabolism, proteolysis, transcription, cell wall biosynthesis as well as nutrient transport showed a time-independent abundance profile. These spatiotemporal patterns were confirmed by immunodetection of specific proteins of the cellular processes membrane/protein trafficking and ion transport. Our results reveal major protein rearrangements at endomembranes and the plasma membrane before and as the pollen grains start tube growth. The spatiotemporal protein abundance changes correlate with the underlying developmental and physiological processes of the germinating pollen grain. PMID:19799449

  4. Cytoplasmic membrane is the target organelle for transition metal mediated damage induced by paraquat in Escherichia coli

    SciTech Connect

    Kohen, R.; Chevion, M.

    1988-04-05

    Bacterial survival indicates that copper or iron is an essential mediator in paraquat toxicity in Escherichia coli. In this study the authors have identified the cytoplasmic membrane as a target organelle in metal-mediated paraquat toxicity and have demonstrated the complete correlation of the membrane damage with the levels of adventitious copper (or iron). The extent of membrane damage was related by use of four parameters: (a) the level of cellular ATP, (b) the level of cellular potassium, (c) the cellular capacity to accumulate and retain radiolabeled leucine, and (d) the cellular integrity as reflected by transmission electron microscopy (TEM). Exposure of bacterial cells to a combination of paraquat and copper caused a marked decline in parameters a, b, and c. This decline was found to occur in parallel with, or even to precede, the sharp loss of survival of E. coli under the same conditions. Likewise, TEM micrographs clearly indicated alternations in cellular structure that possibly reflect sites of detachment of the cytoplasmic membrane from the bacterial capsule. In contradistinction, copper alone or paraquat alone could not bring about similar changes in cellular structure. These findings are in accord with the suggested site-specific metal-mediated Haber-Weiss mechanism for paraquat toxicity and support our notion that specific chelators of transition metals could reduce or prevent the biological deleterious effects of this herbicide.

  5. Systematic analysis of plant mitochondrial and chloroplast small RNAs suggests organelle-specific mRNA stabilization mechanisms.

    PubMed

    Ruwe, Hannes; Wang, Gongwei; Gusewski, Sandra; Schmitz-Linneweber, Christian

    2016-09-01

    Land plant organellar genomes encode a small number of genes, many of which are essential for respiration and photosynthesis. Organellar gene expression is characterized by a multitude of RNA processing events that lead to stable, translatable transcripts. RNA binding proteins (RBPs), have been shown to generate and protect transcript termini and eventually induce the accumulation of short RNA footprints. We applied knowledge of such RBP-derived footprints to develop software (sRNA miner) that enables identification of RBP footprints, or other clusters of small RNAs, in organelles. We used this tool to determine mitochondrial and chloroplast cosRNAs (clustered organellar sRNAs) in Arabidopsis. We found that in mitochondria, cosRNAs coincide with transcript 3'-ends, but are largely absent from 5'-ends. In chloroplasts this bias is absent, suggesting a different mode of 5' processing, possibly owing to different sets of RNases. Furthermore, we identified a large number of cosRNAs that represent silenced insertions of mitochondrial DNA in the nuclear genome of Arabidopsis. Steady-state RNA analyses demonstrate that cosRNAs display differential accumulation during development. Finally, we demonstrate that the chloroplast RBP PPR10 associates in vivo with its cognate cosRNA. A hypothetical role of cosRNAs as competitors of mRNAs for PPR proteins is discussed. PMID:27235415

  6. Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts.

    PubMed

    Näther, Daniela J; Rachel, Reinhard; Wanner, Gerhard; Wirth, Reinhard

    2006-10-01

    Pyrococcus furiosus ("rushing fireball") was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100 degrees C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces. PMID:16980494

  7. Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum.

    PubMed

    Portera-Cailliau, C; Price, D L; Martin, L J

    1997-02-01

    Glutamate-induced excitotoxicity is a clinically relevant degenerative process that causes selective neuronal death by mechanisms that remain unclear. Cell death is usually classified as apoptotic or necrotic based on biochemical and morphological criteria. Excitotoxic lesions in the adult rat striatum result in neuronal death associated with apoptotic DNA laddering despite a necrotic appearance of neurons ultrastructurally. This suggests that apoptosis and necrosis may not be mutually exclusive modes of cell death. Here, we characterized normal developmental cell death in the newborn rat brain with respect to DNA fragmentation patterns and ultrastructural morphology to establish a standard for apoptosis in the nervous system, and we concluded that it is essentially indistinguishable from apoptosis described in other tissues. We then investigated whether brain maturity could influence the morphology of neuronal death in vivo in the excitotoxically lesioned newborn rat forebrain. Kainic acid induced DNA laddering and death of neurons exhibiting a variety of morphologies, ranging from necrosis to apoptosis. In neurons that were dying by apoptosis, morphologic changes were characterized by a highly ordered sequence of organelle abnormalities, with swelling of endoplasmic reticulum and Golgi vesiculation preceding most nuclear changes and mitochondrial disruption. We concluded that brain maturity influences the morphologic phenotype of neurodegeneration and that excitotoxic neuronal death in the immature brain is not a uniform event but, rather, a continuum of apoptotic, necrotic, and overlapping morphologies. This excitotoxic paradigm might prove useful for analyzing the mechanisms that govern cell death under physiological and pathological conditions. PMID:9120055

  8. Recruitment of PI4KIIIβ to Coxsackievirus B3 Replication Organelles Is Independent of ACBD3, GBF1, and Arf1

    PubMed Central

    Dorobantu, Cristina M.; van der Schaar, Hilde M.; Ford, Lauren A.; Strating, Jeroen R. P. M.; Ulferts, Rachel; Fang, Ying; Belov, George

    2014-01-01

    ABSTRACT Members of the Enterovirus (poliovirus [PV], coxsackieviruses, and human rhinoviruses) and Kobuvirus (Aichi virus) genera in the Picornaviridae family rely on PI4KIIIβ (phosphatidylinositol-4-kinase IIIβ) for efficient replication. The small membrane-anchored enteroviral protein 3A recruits PI4KIIIβ to replication organelles, yet the underlying mechanism has remained elusive. Recently, it was shown that kobuviruses recruit PI4KIIIβ through interaction with ACBD3 (acyl coenzyme A [acyl-CoA]-binding protein domain 3), a novel interaction partner of PI4KIIIβ. Therefore, we investigated a possible role for ACBD3 in recruiting PI4KIIIβ to enterovirus replication organelles. Although ACBD3 interacted directly with coxsackievirus B3 (CVB3) 3A, its depletion from cells by RNA interference did not affect PI4KIIIβ recruitment to replication organelles and did not impair CVB3 RNA replication. Enterovirus 3A was previously also proposed to recruit PI4KIIIβ via GBF1/Arf1, based on the known interaction of 3A with GBF1, an important regulator of secretory pathway transport and a guanine nucleotide exchange factor (GEF) of Arf1. However, our results demonstrate that inhibition of GBF1 or Arf1 either by pharmacological inhibition or depletion with small interfering RNA (siRNA) treatment did not affect the ability of 3A to recruit PI4KIIIβ. Furthermore, we show that a 3A mutant that no longer binds GBF1 was capable of recruiting PI4KIIIβ, even in ACBD3-depleted cells. Together, our findings indicate that unlike originally envisaged, coxsackievirus recruits PI4KIIIβ to replication organelles independently of ACBD3 and GBF1/Arf1. IMPORTANCE A hallmark of enteroviral infection is the generation of new membranous structures to support viral RNA replication. The functionality of these “replication organelles” depends on the concerted actions of both viral nonstructural proteins and co-opted host factors. It is thus essential to understand how these structures are

  9. Head-neck domain of Arabidopsis myosin XI, MYA2, fused with GFP produces F-actin patterns that coincide with fast organelle streaming in different plant cells

    PubMed Central

    Walter, Nadine; Holweg, Carola L

    2008-01-01

    Background The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis. Results We expressed the GFP-fused head-neck domain of MYA2 in epidermal cells of various plant species and found that it associated with F-actin. By comparison to other markers such as fimbrin and talin, we revealed that the myosin-labeled F-actin was of a lower quality and absent from the fine microfilament arrays at the cell cortex. However, it colocalized with cytoplasmic (transvacuolar) F-actin in areas coinciding with the tracks of fast organelles. This observation correlates well with the proposed function of myosin XI in organelle trafficking. The fact that organelle streaming was reduced in cells expressing the GFP-MYA2-head6IQ indicated that the functionless motor protein inhibits endogenous myosins. Furthermore, co-expression of the GFP-MYA2-head6IQ with other F-actin markers disrupted its attachment to F-actin. In nuclei, the GFP-myosin associated with short bundles of F-actin. Conclusion The localization of the head of MYA2 in living plant cells, as investigated here for the first time, suggests a close linkage between this myosin XI and cytoplasmic microfilaments that support the rapid streaming of organelles such as peroxisomes

  10. Elemental mapping of Neuromelanin organelles of human Substantia Nigra: correlative ultrastructural and chemical analysis by analytical transmission electron microscopy and nano-secondary ion mass spectrometry.

    PubMed

    Biesemeier, Antje; Eibl, Oliver; Eswara, Santhana; Audinot, Jean-Nicolas; Wirtz, Tom; Pezzoli, Gianni; Zucca, Fabio A; Zecca, Luigi; Schraermeyer, Ulrich

    2016-07-01

    Neuromelanin (NM) is a compound which highly accumulates mainly in catecholamine neurons of the substantia nigra (SN), and is contained in organelles (NM-containing organelles) with lipid bodies and proteins. These neurons selectively degenerate in Parkinson's disease and NM can play either a protective or toxic role. NM-containing organelles of SN were investigated by Analytical Electron Microscopy (AEM) and Nano-Secondary Ion Mass Spectrometry (NanoSIMS) within human tissue sections with respect to ultrastructure and elemental composition. Within the NM-containing organelle, the single NM granules and lipid bodies had sizes of about 200-600 nm. Energy-Dispersive X-ray microanalysis spectra of the NM granules and lipid bodies were acquired with 100 nm beam diameter in AEM, NanoSIMS yielded elemental maps with a lateral resolution of about 150 nm. AEM yielded the quantitative elemental composition of NM granules and bound metals, e.g., iron with a mole fraction of about 0.15 atomic percent. Chemical analyses by AEM and NanoSIMS were consistent at the subcellular level so that nanoSIMS measurements have been quantitated. In NM granules of SN from healthy subjects, a significant amount of S, Fe, and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved detection limits of nanoSIMS offer new possibilities for chemical mapping, high-sensitivity trace element detection, and reduced acquisition times. Variations between individual NM granules can now be investigated effectively and quantitatively by NanoSIMS mapping Cu and Fe. This should yield new insight into the changes in chemical composition of NM pigments during healthy aging and disease. Neuromelanin-containing organelles of dopamine neurons in normal human substantia nigra were investigated by analytical electron mircoscopy and secondary ion mass spectroscopy (NanoSIMS) yielding the ultrastructure and elemental composition. In neuromelanin

  11. Coevolution of Morphology and Virulence in Candida Species ▿

    PubMed Central

    Thompson, Delma S.; Carlisle, Patricia L.; Kadosh, David

    2011-01-01

    Many of the major human fungal pathogens are known to undergo morphological changes, which in certain cases are associated with virulence. Although there has been an intense research focus on morphology in fungi, very little is known about how morphology evolved in conjunction with a variety of other virulence properties. However, several recent important discoveries, primarily in Candida species, are beginning to shed light on this important area and answer many longstanding questions. In this minireview, we first provide a description of the major fungal morphologies, as well as the roles of morphology and morphology-associated gene expression in virulence. Next, focusing largely on Candida species, we examine the evolutionary relationships among specific morphological forms. Finally, drawing on recent findings, we begin to address the question of how specific morphological changes came to be associated with virulence of Candida species during evolution. PMID:21764907

  12. Morphological neural networks

    SciTech Connect

    Ritter, G.X.; Sussner, P.

    1996-12-31

    The theory of artificial neural networks has been successfully applied to a wide variety of pattern recognition problems. In this theory, the first step in computing the next state of a neuron or in performing the next layer neural network computation involves the linear operation of multiplying neural values by their synaptic strengths and adding the results. Thresholding usually follows the linear operation in order to provide for nonlinearity of the network. In this paper we introduce a novel class of neural networks, called morphological neural networks, in which the operations of multiplication and addition are replaced by addition and maximum (or minimum), respectively. By taking the maximum (or minimum) of sums instead of the sum of products, morphological network computation is nonlinear before thresholding. As a consequence, the properties of morphological neural networks are drastically different than those of traditional neural network models. In this paper we consider some of these differences and provide some particular examples of morphological neural network.

  13. Phenol homeostasis is ensured in vanilla fruit by storage under solid form in a new chloroplast-derived organelle, the phenyloplast.

    PubMed

    Brillouet, Jean-Marc; Verdeil, Jean-Luc; Odoux, Eric; Lartaud, Marc; Grisoni, Michel; Conéjéro, Geneviève

    2014-06-01

    A multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a 'phenyloplast'. PMID:24683183

  14. Symbiosomes: temporary moonlighting organelles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrogen fixation is the most important biological process on earth, second only to photosynthesis. The enzyme, nitrogenase, which catalyzes the reduction of atmospheric dinitrogen to ammonium, is encoded into the genomes of a few members of the a-, ß-and '-proteobacteria. The primary route of fixe...

  15. Color-Coded Organelles.

    ERIC Educational Resources Information Center

    McLaughlin, Esther; And Others

    1994-01-01

    Describes how red beets can be used to demonstrate a variety of membrane phenomena. Some of the activities include observation of vacuoles; vacuoles in intact cells; isolation of vacuoles in physiological studies; demonstration of membrane integrity; and demonstration of ion diffusion and active transport with purified vacuoles. (ZWH)

  16. Fractionation of Subcellular Organelles.

    PubMed

    Graham, John M

    2015-01-01

    This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients. Without proper consideration of these critical ancillary procedures, the resolving power of the gradient can be easily compromised. PMID:26621372

  17. Dual-Emissive Cyclometalated Iridium(III) Polypyridine Complexes as Ratiometric Biological Probes and Organelle-Selective Bioimaging Reagents.

    PubMed

    Zhang, Kenneth Yin; Liu, Hua-Wei; Tang, Man-Chung; Choi, Alex Wing-Tat; Zhu, Nianyong; Wei, Xi-Guang; Lau, Kai-Chung; Lo, Kenneth Kam-Wing

    2015-07-01

    In this Article, we present a series of cyclometalated iridium(III) polypyridine complexes of the formula [Ir(N^C)2(N^N)](PF6) that showed dual emission under ambient conditions. The structures of the cyclometalating and diimine ligands were changed systematically to investigate the effects of the substituents on the dual-emission properties of the complexes. On the basis of the photophysical data, the high-energy (HE) and low-energy (LE) emission features of the complexes were assigned to triplet intraligand ((3)IL) and triplet charge-transfer ((3)CT) excited states, respectively. Time-dependent density functional theory (TD-DFT) calculations supported these assignments and indicated that the dual emission resulted from the interruption of the communication between the higher-lying (3)IL and the lower-lying (3)CT states by a triplet amine-to-ligand charge-transfer ((3)NLCT) state. Also, the avidin-binding properties of the biotin complexes were studied by emission titrations, and the results showed that the dual-emissive complexes can be utilized as ratiometric probes for avidin. Additionally, all the complexes exhibited efficient cellular uptake by live HeLa cells. The MTT and Annexin V assays confirmed that no cell death and early apoptosis occurred during the cell imaging experiments. Interestingly, laser-scanning confocal microscopy revealed that the complexes were selectively localized on the cell membrane, mitochondria, or both, depending on the nature of the substituents of the ligands. The results of this work will contribute to the future development of dual-emissive transition metal complexes as ratiometric probes and organelle-selective bioimaging reagents. PMID:26087119

  18. Rapid neurite outgrowth in neurosecretory cells and neurons is sustained by the exocytosis of a cytoplasmic organelle, the enlargeosome.

    PubMed

    Racchetti, Gabriella; Lorusso, Anna; Schulte, Carsten; Gavello, Daniela; Carabelli, Valentina; D'Alessandro, Rosalba; Meldolesi, Jacopo

    2010-01-15

    Neurite outgrowth is known as a slow (days) process occurring in nerve cells and neurons during neurotrophin treatment and upon transfer to culture, respectively. Using Y27632, a drug that induces activation of Rac1, a downstream step of the neurotrophin signaling cascade, we have identified a new form of outgrowth, which is rapid (<1 hour) and extensive (>500 microm(2) surface enlargement/single cell/first hour). However, this outgrowth takes place only in cells (PC12-27 and SH-SY5Y cells, and embryonic and neonatal neurons) rich in an exocytic organelle, the enlargeosome. Golgi vesicles, TGN vesicles and endosomes are not involved. The need for enlargeosomes for plasma-membrane expansion was confirmed by the appearance of their marker, Ahnak, at the cell surface and by the dependence of neurite outgrowth on VAMP4, the vSNARE of enlargeosome exocytosis. In enlargeosome-rich cells, VAMP4 downregulation also attenuated the slow outgrowth induced by nerve growth factor (NGF). Similar to NGF-induced neurite outgrowth in enlargeosome-lacking cells, the new, rapid, Y27632-induced process required microtubules. Other properties of neurite outgrowth in cells lacking enlargeosomes - such as dependence on VAMP7, on microfilaments, on gene transcription and on protein synthesis, and blockade of mitoses and accumulation of neuronal markers - were not evident. The enlargeosome-sustained process might be useful for the rapid neurite outgrowth at peculiar stages and/or conditions of nerve and neuronal cells. However, its properties and its physiological and pathological role remain to be investigated. PMID:20026640

  19. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy

    PubMed Central

    Lee, Sooyeon; Sato, Yutaka; Nixon, Ralph A.

    2012-01-01

    In the hallmark neuritic dystrophy of Alzheimer’s disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers, Rab7 and LAMP1, and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated upon fusion with bi-directionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, APP, and neurofilament protein hyperphosphorylation. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn, largely cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD. PMID:21613495

  20. Inducible Knockdown of MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 Reveals Roles of Galactolipids in Organelle Differentiation in Arabidopsis Cotyledons1[W][OPEN

    PubMed Central

    Fujii, Sho; Kobayashi, Koichi; Nakamura, Yuki; Wada, Hajime

    2014-01-01

    Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of thylakoid membranes and is essential for chloroplast biogenesis in plants. In Arabidopsis (Arabidopsis thaliana), MGDG is predominantly synthesized by inner envelope-localized MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (MGD1); its knockout causes albino seedlings. Because of the lethal phenotype of the null MGD1 mutant, functional details of MGDG synthesis at seedling development have remained elusive. In this study, we used an inducible gene-suppression system to investigate the impact of MGDG synthesis on cotyledon development. We created transgenic Arabidopsis lines that express an artificial microRNA targeting MGD1 (amiR-MGD1) under the control of a dexamethasone-inducible promoter. The induction of amiR-MGD1 resulted in up to 75% suppression of MGD1 expression, although the resulting phenotypes related to chloroplast development were diverse, even within a line. The strong MGD1 suppression by continuous dexamethasone treatment caused substantial decreases in galactolipid content in cotyledons, leading to severe defects in the formation of thylakoid membranes and impaired photosynthetic electron transport. Time-course analyses of the MGD1 suppression during seedling germination revealed that MGDG synthesis at the very early germination stage is particularly important for chloroplast biogenesis. The MGD1 suppression down-regulated genes associated with the photorespiratory pathway in peroxisomes and mitochondria as well as those responsible for photosynthesis in chloroplasts and caused high expression of genes for the glyoxylate cycle. MGD1 function may link galactolipid synthesis with the coordinated transcriptional regulation of chloroplasts and other organelles during cotyledon greening. PMID:25253888

  1. Organelle DNA rearrangement mapping reveals U-turn-like inversions as a major source of genomic instability in Arabidopsis and humans

    PubMed Central

    Zampini, Éric; Lepage, Étienne; Tremblay-Belzile, Samuel; Truche, Sébastien; Brisson, Normand

    2015-01-01

    Failure to maintain organelle genome stability has been linked to numerous phenotypes, including variegation and cytosolic male sterility (CMS) in plants, as well as cancer and neurodegenerative diseases in mammals. Here we describe a next-generation sequencing approach that precisely maps and characterizes organelle DNA rearrangements in a single genome-wide experiment. In addition to displaying global portraits of genomic instability, it surprisingly unveiled an abundance of short-range rearrangements in Arabidopsis thaliana and human organelles. Among these, short-range U-turn-like inversions reach 25% of total rearrangements in wild-type Arabidopsis plastids and 60% in human mitochondria. Furthermore, we show that replication stress correlates with the accumulation of this type of rearrangement, suggesting that U-turn-like rearrangements could be the outcome of a replication-dependent mechanism. We also show that U-turn-like rearrangements are mostly generated using microhomologies and are repressed in plastids by Whirly proteins WHY1 and WHY3. A synergistic interaction is also observed between the genes for the plastid DNA recombinase RECA1 and those encoding plastid Whirly proteins, and the triple mutant why1why3reca1 accumulates almost 60 times the WT levels of U-turn-like rearrangements. We thus propose that the process leading to U-turn-like rearrangements may constitute a RecA-independent mechanism to restart stalled forks. Our results reveal that short-range rearrangements, and especially U-turn-like rearrangements, are a major factor of genomic instability in organelles, and this raises the question of whether they could have been underestimated in diseases associated with mitochondrial dysfunction. PMID:25800675

  2. ALHAMBRA survey: morphological classification

    NASA Astrophysics Data System (ADS)

    Pović, M.; Huertas-Company, M.; Márquez, I.; Masegosa, J.; Aguerri, J. A. López; Husillos, C.; Molino, A.; Cristóbal-Hornillos, D.

    2015-03-01

    The Advanced Large Homogeneous Area Medium Band Redshift Astronomical (ALHAMBRA) survey is a photometric survey designed to study systematically cosmic evolution and cosmic variance (Moles et al. 2008). It employs 20 continuous medium-band filters (3500 - 9700 Å), plus JHK near-infrared (NIR) bands, which enable measurements of photometric redshifts with good accuracy. ALHAMBRA covers > 4 deg2 in eight discontinuous regions (~ 0.5 deg2 per region), of theseseven fields overlap with other extragalactic, multiwavelength surveys (DEEP2, SDSS, COSMOS, HDF-N, Groth, ELAIS-N1). We detect > 600.000 sources, reaching the depth of R(AB) ~ 25.0, and photometric accuracy of 2-4% (Husillos et al., in prep.). Photometric redshifts are measured using the Bayesian Photometric Redshift (BPZ) code (Benítez et al. 2000), reaching one of the best accuracies up to date of δz/z <= 1.2% (Molino et al., in prep.). To deal with the morphological classification of galaxies in the ALHAMBRA survey (Pović et al., in prep.), we used the galaxy Support Vector Machine code (galSVM; Huertas-Company 2008, 2009), one of the new non-parametric methods for morphological classification, specially useful when dealing with low resolution and high-redshift data. To test the accuracy of our morphological classification we used a sample of 3000 local, visually classified galaxies (Nair & Abraham 2010), moving them to conditions typical of our ALHAMBRA data (taking into account the background, redshift and magnitude distributions, etc.), and measuring their morphology using galSVM. Finally, we measured the morphology of ALHAMBRA galaxies, obtaining for each source seven morphological parameters (two concentration indexes, asymmetry, Gini, M20 moment of light, smoothness, and elongation), probability if the source belongs to early- or late-type, and its error. Comparing ALHAMBRA morph COSMOS/ACS morphology (obtained with the same method) we expect to have qualitative separation in two main morphological

  3. Efficiency of Organelle Capture by Microtubules as a Function of Centrosome Nucleation Capacity: General Theory and the Special Case of Polyspermia

    PubMed Central

    Maly, Ivan V.

    2012-01-01

    Transport of organelles along microtubules is essential for the cell metabolism and morphogenesis. The presented analysis derives the probability that an organelle of a given size comes in contact with the microtubule aster. The question is asked how this measure of functionality of the microtubule aster is controlled by the centrosome. A quantitative model is developed to address this question. It is shown that for the given set of cellular parameters, such as size and total tubulin content, a centrosome nucleation capacity exists that maximizes the probability of the organelle capture. The developed general model is then applied to the capture of the female pronucleus by microtubules assembled on the sperm centrosome, following physiologically polyspermic fertilization. This application highlights an unintuitive reflection of nonlinearity of the nucleated polymerization of the cellular pool of tubulin. The prediction that the sperm centrosome should lower its nucleation capacity in the face of the competition from the other sperm is a stark illustration of the new optimality principle. Overall, the model calls attention to the capabilities of the centrosomal pathway of regulation of the transport-related functionality of the microtubule cytoskeleton. It establishes a quantitative and conceptual framework that can guide experiment design and interpretation. PMID:22662187

  4. Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells.

    PubMed

    Shomorony, A; Pfeifer, C R; Aronova, M A; Zhang, G; Cai, T; Xu, H; Notkins, A L; Leapman, R D

    2015-08-01

    A combination of two-dimensional (2D) and three-dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and β cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a β cell. PMID:26139222

  5. PREPACT 2.0: Predicting C-to-U and U-to-C RNA Editing in Organelle Genome Sequences with Multiple References and Curated RNA Editing Annotation

    PubMed Central

    Lenz, Henning; Knoop, Volker

    2013-01-01

    RNA editing is vast in some genetic systems, with up to thousands of targeted C-to-U and U-to-C substitutions in mitochondria and chloroplasts of certain plants. Efficient prognoses of RNA editing in organelle genomes will help to reveal overlooked cases of editing. We present PREPACT 2.0 (http://www.prepact.de) with numerous enhancements of our previously developed Plant RNA Editing Prediction & Analysis Computer Tool. Reference organelle transcriptomes for editing prediction have been extended and reorganized to include 19 curated mitochondrial and 13 chloroplast genomes, now allowing to distinguish RNA editing sites from “pre-edited” sites. Queries may be run against multiple references and a new “commons” function identifies and highlights orthologous candidate editing sites congruently predicted by multiple references. Enhancements to the BLASTX mode in PREPACT 2.0 allow querying of complete novel organelle genomes within a few minutes, identifying protein genes and candidate RNA editing sites simultaneously without prior user analyses. PMID:23362369

  6. Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles

    PubMed Central

    Barriere, Herve; Bagdany, Miklos; Bossard, Florian; Okiyoneda, Tsukasa; Wojewodka, Gabriella; Gruenert, Dieter; Radzioch, Danuta

    2009-01-01

    Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr+/+ and cftr−/− mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase–generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease. PMID:19420138

  7. GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca(2+) Dynamics in High-Ca(2+) Organelles.

    PubMed

    Navas-Navarro, Paloma; Rojo-Ruiz, Jonathan; Rodriguez-Prados, Macarena; Ganfornina, María Dolores; Looger, Loren L; Alonso, María Teresa; García-Sancho, Javier

    2016-06-23

    Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca(2+) at high concentrations. Here we describe a ratiometric low-affinity Ca(2+) sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca(2+) concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca(2+) signals inside the organelle. The use of the sensor was demonstrated under three experimental paradigms: (1) ER Ca(2+) oscillations in cultured astrocytes, (2) ex vivo functional mapping of cholinergic receptors triggering ER Ca(2+) release in acute hippocampal slices from transgenic mice, and (3) in vivo sarcoplasmic reticulum Ca(2+) dynamics in the muscle of transgenic flies. Our results provide proof of the suitability of the new biosensors to monitor Ca(2+) dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca(2+) signaling in animal models of health and disease. PMID:27291400

  8. Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells

    PubMed Central

    SHOMORONY, A.; PFEIFER, C.R.; ARONOVA, M.A.; ZHANG, G.; CAI, T.; XU, H.; NOTKINS, A.L.

    2015-01-01

    Summary A combination of two‐dimensional (2D) and three‐dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and β cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a β cell. PMID:26139222

  9. A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan

    PubMed Central

    de la Loza, M. C. Díaz; Wellinger, R. E.

    2009-01-01

    DNA is susceptible of being damaged by chemicals, UV light or gamma irradiation. Nuclear DNA damage invokes both a checkpoint and a repair response. By contrast, little is known about the cellular response to mitochondrial DNA damage. We designed an experimental system that allows organelle-specific DNA damage targeting in Saccharomyces cerevisiae. DNA damage is mediated by a toxic topoisomerase I allele which leads to the formation of persistent DNA single-strand breaks. We show that organelle-specific targeting of a toxic topoisomerase I to either the nucleus or mitochondria leads to nuclear DNA damage and cell death or to loss of mitochondrial DNA and formation of respiration-deficient ‘petite’ cells, respectively. In wild-type cells, toxic topoisomerase I–DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs. We reasoned that targeting of topoisomerase I to the mitochondria of top1Δ cells should lead to petite formation in the presence of camptothecin. Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation. Our findings demonstrate that drug modifications can lead to organelle-specific DNA damage and thus opens new perspectives on the role of mitochondrial DNA-damage in cancer treatment. PMID:19151088

  10. The force induced by organelles' weight in the microfilament is in the range of 0.1-1 pN

    NASA Astrophysics Data System (ADS)

    Yang, Chun; Wei, Dong; Zhuang, Feng Y.

    It has been well documented that a microgravity environment can bring about many changes in cell metabolism. Can mammalian cells feel the gravity directly? At present, arguments surrounding the problem are difficult to be answered through experiments. However, using finite element simulation to estimate the force exerted on the microfilament meshwork model, we demonstrated a possible way through which gravity acts on the cytoskeleton system. This system, which includes microfilaments, microtubules, and intermediate filaments, is responsible for the retention of cell shape and plays a role in many aspects related to cell proliferation and function. Many organelles, such as ribosomes and nucleus, are deposited, hinged, or attached on the cytoskeleton system. The weight of organelles can deform the cytoskeleton system and can induce force in it. Simulation results showed that the force induced by organelles' weight in the microfilament is in the range of 0.1-1 pN. The magnitude of the force is near the single Van der Waals bond force between the proteins, which is large enough to influence the hinge motion of proteins.

  11. Morphology and Dynamics of the Endocytic Pathway in Dictyostelium discoideumV⃞

    PubMed Central

    Neuhaus, Eva M.; Almers, Wolfhard; Soldati, Thierry

    2002-01-01

    Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study. D. discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging. Herein, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multivesicular bodies, and the specialized secretory lysosomes. In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane along side macropinocytosis, which is the major fluid phase uptake process. This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way reminiscent of mechanisms and structures documented in leukocytes. Finally, egestion of fluid phase from the secretory lysosomes was directly observed. PMID:11950947

  12. Oocyte morphology from primordial to early tertiary follicles of yak.

    PubMed

    Yu, S J; Yong, Y H; Cui, Y

    2010-10-01

    The objective of this study was to investigate the developmental morphology of yak oocytes from the primordial follicle to the tertiary follicle. Yak oocytes from resting primordial (n = 6), activated primordial (n = 12), primary (n = 9), secondary (n = 7) and early tertiary (n = 5) follicles were processed and analysed by light and transmission electron microscopy. The resting primordial follicular oocyte was characterized by relatively smooth surface on the oolemma, the accumulation of free and organelle-related smooth (SER) and rough endoplasmic reticulum (RER), round or oval mitochondria, and polyribosomes on the surface of the RER and throughout the ooplasm. The activated primordial follicular oocyte was dominated by numerous coated pits and coated vesicles on the oolemma, and round mitochondria. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, polyribosome, Golgi complexes and mitochondria with distinct cristae. During the secondary follicular stage, formation of the zona pellucida, development of a desmosome-like connection between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and elongated mitochondria in nearly all oocytes were seen. In the early tertiary follicular oocyte, the perivitelline space was present and a decrease in free SER and RER in the ooplasm occurred; finally, the nucleus migrated from an eccentric to a peripheral location. In conclusion, the growth of the yak oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte-specific structures such as the zona pellucida, desmosome-like connection and cortical granules. PMID:20059745

  13. Phonological Bases for L2 Morphological Learning

    ERIC Educational Resources Information Center

    Hu, Chieh-Fang

    2010-01-01

    Two experiments examined the hypothesis that L1 phonological awareness plays a role in children's ability to extract morphological patterns of English as L2 from the auditory input. In Experiment 1, 84 Chinese-speaking third graders were tested on whether they extracted the alternation pattern between the base and the derived form (e.g.,…

  14. Morphological Sensitivity in Deaf Readers of Dutch

    ERIC Educational Resources Information Center

    Van Hoogmoed, Anne H.; Verhoeven, Ludo; Schreuder, Robert; Knoors, Harry

    2011-01-01

    Deaf children experience difficulties with reading comprehension. These difficulties are not completely explained by their difficulties with the reading of single short words. Whether deaf children and adults lag behind in the morphological processing of longer words is therefore examined in two experiments in which the processing of prefixes by…

  15. Children's Early Productivity with Verbal Morphology

    ERIC Educational Resources Information Center

    Wagner, Laura; Swensen, Lauren D.; Naigles, Letitia R.

    2009-01-01

    Three studies using the intermodal preferential looking paradigm examined onset of productive comprehension of tense/aspect morphology in English. When can toddlers understand these forms with novel verbs and novel events? The first study used familiar verbs and showed that 26-36-month olds correctly matched a past/perfective form ("-ed" or…

  16. Computation of Semantic Number from Morphological Information

    ERIC Educational Resources Information Center

    Berent, Iris; Pinker, Steven; Tzelgov, Joseph; Bibi, Uri; Goldfarb, Liat

    2005-01-01

    The distinction between singular and plural enters into linguistic phenomena such as morphology, lexical semantics, and agreement and also must interface with perceptual and conceptual systems that assess numerosity in the world. Three experiments examine the computation of semantic number for singulars and plurals from the morphological…

  17. Morphology engineering - Osmolality and its effect on Aspergillus niger morphology and productivity

    PubMed Central

    2011-01-01

    Background The filamentous fungus Aspergillus niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most intriguing and often uncontrollable characteristics of this filamentous organism is its complex morphology, ranging from dense spherical pellets to viscous mycelia depending on culture conditions. Optimal productivity correlates strongly with a specific morphological form, thus making high demands on process control. Results In about 50 2L stirred tank cultivations the influence of osmolality on A. niger morphology and productivity was investigated. The specific productivity of fructofuranosidase producing strain A. niger SKAn 1015 could be increased notably from 0.5 to 9 U mg-1 h-1 around eighteen fold, by increasing the culture broth osmolality by addition of sodium chloride. The specific productivity of glucoamylase producing strain A. niger AB1.13, could be elevated using the same procedure. An optimal producing osmolality was shown to exist well over the standard osmolality at about 3.2 osmol kg-1 depending on the strain. Fungal morphology of all cultivations was examined by microscope and characterized by digital image analysis. Particle shape parameters were combined to a dimensionless Morphology number, which enabled a comprehensive characterization of fungal morphology correlating closely with productivity. A novel method for determination of germination time in submerged cultivations by laser diffraction, introduced in this study, revealed a decelerated germination process with increasing osmolality. Conclusions Through the introduction of the versatile Morphology number, this study provides the means for a desirable characterization of fungal morphology and demonstrates its relation to productivity. Furthermore, osmolality as a fairly new parameter in process engineering is introduced and found to affect fungal morphology and productivity. Osmolality might provide an auspicious and

  18. Balamuthia mandrillaris: Further morphological observations of trophozoites by light, scanning and transmission electron microscopy.

    PubMed

    González-Robles, Arturo; Lares-Villa, Fernando; Lares-Jiménez, Luis Fernando; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Martínez-Palomo, Adolfo

    2015-10-01

    Additional morphological features of Balamuthia mandrillaris observed by light and electron microscopy are reported. Trophozoites were extremely pleomorphic: their cell shapes ranged from rounded to elongated and sometimes they appeared exceptionally stretched out and branched. By transmission electron microscopy it was possible to observe two different cytoplasmic areas, the ectoplasm and the endoplasm and often sections of rough endoplasmic reticulum were found in the transition zone. The cytoplasm was very fibrogranular and most of the organelles typically found in eukaryotic cells were observed. A particular finding was the presence of numerous mitochondria with a different structure from those of other free-living amoebae. The observations reported here may reinforce the morphological knowledge of this amoeba and provide a background for further analyses. PMID:26297955

  19. Morphological Awareness and Children's Writing: Accuracy, Error, and Invention

    ERIC Educational Resources Information Center

    McCutchen, Deborah; Stull, Sara

    2015-01-01

    This study examined the relationship between children's morphological awareness and their ability to produce accurate morphological derivations in writing. Fifth-grade US students (n = 175) completed two writing tasks that invited or required morphological manipulation of words. We examined both accuracy and error, specifically errors in…

  20. Differential Regulation of Genes Coding for Organelle and Cytosolic ClpATPases under Biotic and Abiotic Stresses in Wheat

    PubMed Central

    Muthusamy, Senthilkumar K.; Dalal, Monika; Chinnusamy, Viswanathan; Bansal, Kailash C.

    2016-01-01

    A sub-group of class I Caseinolytic proteases (Clps) function as molecular chaperone and confer thermotolerance to plants. We identified class I Clp family consisting of five ClpB/HSP100, two ClpC, and two ClpD genes from bread wheat. Phylogenetic analysis showed that these genes were highly conserved across grass genomes. Subcellular localization prediction revealed that TaClpC and TaClpD subgroup proteins and TaClpB1 proteins are potentially targeted to chloroplast, while TaClpB5 to mitochondria, and TaClpB2, TaClpB3, and TaClpB4 to cytoplasm. Spatio-temporal expression pattern analysis revealed that four TaClpB and TaClpD2 genes are expressed in majority of all tissues and developmental stages of wheat. Real-time RT-PCR analysis of expression levels of Clp genes in seven wheat genotypes under different abiotic stresses revealed that genes coding for the cytosolic Clps namely TaClpB2 and TaClpB3 were upregulated under heat, salt and oxidative stress but were downregulated by cold stress in most genotypes. In contrast, genes coding for the chloroplastic Clps TaClpC1, TaClpC2, and TaClpD1 genes were significantly upregulated by mainly by cold stress in most genotypes, while TaClpD2 gene was upregulated >2 fold by salt stress in DBW16. The TaClpB5 gene coding for mitochondrial Clp was upregulated in all genotypes under heat, salt and oxidative stresses. In addition, we found that biotic stresses also upregulated TaClpB4 and TaClpD1. Among biotic stresses, Tilletia caries induced TaClpB2, TaClpB3, TaClpC1, and TaClpD1. Differential expression pattern under different abiotic and biotic stresses and predicted differential cellular localization of Clps suggest their non-redundant organelle and stress-specific roles. Our results also suggest the potential role of Clps in cold, salt and biotic stress responses in addition to the previously established role in thermotolerance of wheat. PMID:27446158

  1. Differential Regulation of Genes Coding for Organelle and Cytosolic ClpATPases under Biotic and Abiotic Stresses in Wheat.

    PubMed

    Muthusamy, Senthilkumar K; Dalal, Monika; Chinnusamy, Viswanathan; Bansal, Kailash C

    2016-01-01

    A sub-group of class I Caseinolytic proteases (Clps) function as molecular chaperone and confer thermotolerance to plants. We identified class I Clp family consisting of five ClpB/HSP100, two ClpC, and two ClpD genes from bread wheat. Phylogenetic analysis showed that these genes were highly conserved across grass genomes. Subcellular localization prediction revealed that TaClpC and TaClpD subgroup proteins and TaClpB1 proteins are potentially targeted to chloroplast, while TaClpB5 to mitochondria, and TaClpB2, TaClpB3, and TaClpB4 to cytoplasm. Spatio-temporal expression pattern analysis revealed that four TaClpB and TaClpD2 genes are expressed in majority of all tissues and developmental stages of wheat. Real-time RT-PCR analysis of expression levels of Clp genes in seven wheat genotypes under different abiotic stresses revealed that genes coding for the cytosolic Clps namely TaClpB2 and TaClpB3 were upregulated under heat, salt and oxidative stress but were downregulated by cold stress in most genotypes. In contrast, genes coding for the chloroplastic Clps TaClpC1, TaClpC2, and TaClpD1 genes were significantly upregulated by mainly by cold stress in most genotypes, while TaClpD2 gene was upregulated >2 fold by salt stress in DBW16. The TaClpB5 gene coding for mitochondrial Clp was upregulated in all genotypes under heat, salt and oxidative stresses. In addition, we found that biotic stresses also upregulated TaClpB4 and TaClpD1. Among biotic stresses, Tilletia caries induced TaClpB2, TaClpB3, TaClpC1, and TaClpD1. Differential expression pattern under different abiotic and biotic stresses and predicted differential cellular localization of Clps suggest their non-redundant organelle and stress-specific roles. Our results also suggest the potential role of Clps in cold, salt and biotic stress responses in addition to the previously established role in thermotolerance of wheat. PMID:27446158

  2. Examination Management and Examination Malpractice: The Nexus

    ERIC Educational Resources Information Center

    Ogunji, James A.

    2011-01-01

    Examination malpractice or cheating has become a global phenomenon. In different countries of the world today, developed and developing, academic dishonesty especially cheating in examinations has heightened and taken frightening dimension. In many countries of the world this phenomenon has become a serious matter of concern that has left many…

  3. FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

    PubMed

    Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

    2015-04-01

    Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM. PMID:25786736

  4. Outline of Polish Morphology.

    ERIC Educational Resources Information Center

    Bidwell, Charles E.

    This volume, one of a series of concise but relatively exhaustive descriptions of the grammatical structures of the principal standard Slavic languages, contains an outline of Polish morphology. The four major sections are morphophonemics, nominal inflection, the Polish verb (Part 1--stem alternation and conjugation, and the Polish verb (Part…

  5. Eutectic-Alloy Morphology

    NASA Technical Reports Server (NTRS)

    Pirich, R. G.; Poit, W. J.

    1985-01-01

    Deviation in controlled-rod eutectic morphology anticipated for diffusion only crystal growth characterized at low solidification velocities. Naturally induced, gravity-related convective instabilities result in nonalined irregularly dispersed fibers or platelets. Lower solidification limit for controlled growth Bi/Mn alloys is 1 centimeter/ hour.

  6. OUTLINE OF UKRAINIAN MORPHOLOGY.

    ERIC Educational Resources Information Center

    BIDWELL, CHARLES E.

    SIMILAR IN FORMAT TO THE AUTHOR'S "OUTLINE OF BIELORUSSIAN MORPHOLOGY," THIS STRUCTURALLY-ORIENTED TREATMENT OF UKRAINIAN INCLUDES SECTIONS ON --(1) PHONOLOGY, (2) MORPHOPHONEMIC ALTERNATIONS, (3) THE NOUN, (4) THE ADJECTIVE, (5) PRONOUNS, (6) NUMERALS, AND (7) THE VERB. THE AUTHOR BASES HIS FINDINGS ON PUBLISHED HANDBOOKS AND GRAMMARS AS WELL AS…

  7. Patient Eye Examinations - Adults

    MedlinePlus

    ... Examinations, Adults Patient Eye Examinations, Children Refractive Errors Scientists in the Laboratory Visual Acuity Testing Patient Eye Examinations, Adults × Warning message Automatic fallback to the cURL connection method kicked in to handle the request. Result code ...

  8. Morphological Analysis in Learning to Read Pseudowords in Hebrew

    ERIC Educational Resources Information Center

    Bar-On, Amalia; Ravid, Dorit

    2011-01-01

    This paper examines the role of morphology in gradeschool children's learning to read nonpointed Hebrew. It presents two experiments testing the reading of morphologically based nonpointed pseudowords. One hundred seventy-one Hebrew-speaking children and adolescents in seven age/schooling groups (beginning and end of 2nd, 3rd, 4th, 7th, and 11th…

  9. The Syntax-Semantics Interface in Distributed Morphology

    ERIC Educational Resources Information Center

    Kelly, Justin Robert

    2013-01-01

    Distributed Morphology (DM; Halle & Marantz 1993; Marantz 1997) is founded on the premise that the syntax is the only computational component of the grammar. Much research focuses on how this premise is relevant to the syntax-morphology interface in DM. In this dissertation, I examine theory-internal issues related to the syntax-semantics…

  10. Interspeaker Variability in Hard Palate Morphology and Vowel Production

    ERIC Educational Resources Information Center

    Lammert, Adam; Proctor, Michael; Narayanan, Shrikanth

    2013-01-01

    Purpose: Differences in vocal tract morphology have the potential to explain interspeaker variability in speech production. The potential acoustic impact of hard palate shape was examined in simulation, in addition to the interplay among morphology, articulation, and acoustics in real vowel production data. Method: High-front vowel production from…

  11. Morphology in Malay-English Biliteracy Acquisition: An Intervention Study

    ERIC Educational Resources Information Center

    Zhang, Dongbo

    2016-01-01

    This intervention study examined the effect of English morphological instruction on the development of English as well as Malay morphological awareness and word reading abilities among Malay-English bilingual fourth graders in Singapore, where English is the medium of instruction. The intervention group experienced semester-long instruction in…

  12. Morphological Innovation in the Acquisition of American Sign Language.

    ERIC Educational Resources Information Center

    van Hoek, Karen; And Others

    A study examined aspects of the acquisition of spatialized morphology and syntax in American Sign Language (ASL) learned natively by deaf children of deaf parents. Children aged 2 to 8 were shown story books to elicit narratives, and the resulting use of verbs contained morphological forms not appearing in adult grammar. Analysis of the creative…

  13. External morphology of stable fly (Diptera: Muscidae) larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scanning electron microscopy was used to examine the external morphology of first-, second-, and third-instar stable flies (Stomoxys calcitrans (L.)). In the cephalic region, the antennae, labial lobe, and maxillary palpi are morphologically similar among instars. Antennae comprise a prominent ante...

  14. Contributions of Morphological Skill to Children's Essay Writing

    ERIC Educational Resources Information Center

    Northey, Mary; McCutchen, Deborah; Sanders, Elizabeth A.

    2016-01-01

    Morphological skills have previously been found to reliably predict reading skill, including word reading, vocabulary, and comprehension. However, less is known about how morphological skills might contribute to writing skill, aside from its well-documented role in the development of spelling. This correlational study examines whether…

  15. Grain Size and Morphological Variability

    NASA Astrophysics Data System (ADS)

    Macmahan, J.; Gallagher, E.; Reniers, A.; Thornton, E.

    2008-12-01

    Grain size on natural beaches has traditionally been assumed to be uniform and modeling efforts assume a single mean grain size for an entire beach environment. Many recent studies contradict this assumption and suggest that sediment grain size on a beach is not homogeneous and that variations in sediment size and supply are important in sediment transport and morphodynamics at all scales. Unfortunately, measuring grain size is difficult, tedious and time consuming. Therefore, in spite of the evidence pointing to the importance of grain size in sediment transport and morphodynamics, many previous studies have been based on only a few field samples. Rubin (2004) introduced a technique for measuring surface grain size in situ in rivers and deeper coastal waters, using a digital camera and auto-correlation of digital images. Using this technique, information about the surface grain size distribution can be obtained quickly and inexpensively. Following Rubin (2004), we have developed a mobile digital imaging system (DIS) for surveying grain size on beaches. The DIS was used during two experiments: RCEX, a rip current experiment in Monterey, CA in April 2007 and Truc Vert '08, a multi-institutional, international experiment, on the Atlantic coast of France in March 2008. Preliminary results suggest that grain size varies spatially with the morphology of beach features and temporally with changes in tide level, wave energy, and morphodynamics. These data are being used to examine the relationship between morphological, sedimentological and hydrodynamic variability.

  16. Grain Size and Morphological Variability

    NASA Astrophysics Data System (ADS)

    Gallagher, E. L.; Reniers, A. J.; Macmahan, J. H.; Thornton, E. B.

    2009-12-01

    Grain size on natural beaches has traditionally been assumed to be uniform and modeling efforts assume a single mean grain size for an entire beach environment. Many recent studies contradict this assumption and suggest that sediment grain size on a beach is not homogeneous and that variations in sediment size and supply are important in sediment transport and morphodynamics at all scales. Unfortunately, measuring grain size is tedious and time consuming. Therefore, in spite of the evidence pointing to the importance of grain size in sediment transport and morphodynamics, many previous studies have been based on only a few field samples. Rubin (2004) introduced a technique for measuring surface grain size in situ in rivers and deeper coastal waters, using a digital camera and auto-correlation of digital images. Using this technique, information about the surface grain size distribution can be obtained quickly and inexpensively. Following Rubin (2004), we have developed a mobile digital imaging system (DIS) for surveying grain size on beaches. The DIS was used during three experiments: RCEX and REX, both rip current experiments in Monterey, CA (April 2007 and April 2009) and Truc Vert ’08, a multi-institutional, international experiment, on the Atlantic coast of France in March 2008. In 2007 and 2008 spatial surveys of surface grain size were completed every few days over large (~500x500m) intertidal areas. The 2009 experiment focused on twice-daily sampling of two cross shore lines to examine temporal grain size variability of the intertidal beach. Preliminary results suggest that grain size varies spatially with the morphology of beach features (eg, rip channels and shoals) and temporally with changes in tide level, wave energy, and morphodynamics. These data are being used to examine the relationship between morphological, sedimentological and hydrodynamic variability.

  17. Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

    PubMed Central

    Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

    2013-01-01

    All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

  18. [Functional morphology of endonasal blood capillaries].

    PubMed

    Grevers, G

    1989-01-01

    Harvey (1628) was the first to describe the blood circulatory system. 50 years later, the capillary system was discovered by Marcello Malpighi as a link between arteries and veins. Since then, the microvascular network has been examined abundantly by means of various morphological and physiological techniques. During the last decades the development of the technical equipment enabled us to receive detailed informations on the structural and ultrastructural aspects of the vasculature and--particularly--the capillary endothelium. New electron microscopic investigations on the behaviour of this special cell system. In the present study we combined our own morphological results on the endonasal microcirculatory system--using different morphological techniques--to interpret these anatomical finding under functional aspects. PMID:2644502

  19. Volcanology and morphology

    NASA Technical Reports Server (NTRS)

    Bryan, W. B.

    1976-01-01

    Apollo 15 photographs of the southern parts of Serenitatis and Imbrium were used for a study of the morphology and distribution of wrinkle ridges. Volcanic and structural features along the south margin of Serenitatis were also studied, including the Dawes basalt cinder cones. Volcanic and structural features in crater Aitken were investigated as well. Study of crater Goclenius showed a close relationship between morphology of the impact crater and grabens which tend to parallel directions of the lunar grid. Similar trends were observed in the walls of crater Tsiolkovsky and other linear structures. Small craters of possible volcanic origin were also studied. Possible cinder cones were found associated with the Dawes basalt and in the floor of craters Aitken and Goclenius. Small pit craters were observed in the floors of these craters. Attempts were made to obtain contour maps of specific small features and to compare Orbiter and Apollo photographs to determine short term changes associated with other processes.

  20. Uterine morphology during diapause and early pregnancy in the tammar wallaby (Macropus eugenii).

    PubMed

    Laird, Melanie K; Hearn, Cyrma M; Shaw, Geoff; Renfree, Marilyn B

    2016-09-01

    In mammals, embryonic diapause, or suspension of embryonic development, occurs when embryos at the blastocyst stage are arrested in growth and metabolism. In the tammar wallaby (Macropus eugenii), there are two separate uteri, only one of which becomes gravid with the single conceptus at a post-partum oestrus, so changes during pregnancy can be compared between the gravid and non-gravid uterus within the same individual. Maintenance of the viable blastocyst and inhibition of further conceptus growth during diapause in the tammar is completely dependent on the uterine environment. Although the specific endocrine and seasonal signals are well established, much less is known about the cellular changes required to create this environment. Here we present the first detailed study of uterine morphology during diapause and early pregnancy of the tammar wallaby. We combined transmission electron microscopy and light microscopy to describe the histological and ultrastructural changes to luminal and glandular epithelial cells. At entry into diapause after the post-partum oestrus and formation of the new conceptus, there was an increase in abundance of organelles associated with respiration in the endometrial cells of the newly gravid uterus, particularly in the endoplasmic reticulum and mitochondria, as well as an increase in secretory activity. Organelle changes and active secretion then ceased in these cells as they became quiescent and remained so for the duration of diapause. In contrast, cells of the non-gravid, post-partum, contralateral uterus underwent sloughing and remodelling during this time and some organelle changes in glandular epithelial cells continued throughout diapause, suggesting these cells are not completely quiescent during diapause, although no active secretion occurred. These findings demonstrate that diapause, like pregnancy, is under unilateral endocrine control in the tammar, and that preparation for and maintenance of diapause requires

  1. Examination Involving Students as Peer Examiners

    ERIC Educational Resources Information Center

    Ljungman, Anders G.; Silen, Charlotte

    2008-01-01

    The main interest in this article is students' involvement in assessment as a part of growth towards self-directedness in learning. In order to enhance students' development of autonomy in learning, a project involving "older" students as peer examiners for "younger" students was designed and carried out. Students in the sixth semester in a…

  2. Authigenic illite morphology: appearances can be deceiving

    SciTech Connect

    Cocker, J.D.

    1986-05-01

    Authigenic filamentous illite can severely reduce permeability despite relatively high porosities in sandstones. The effect of illite on rock properties is dependent on the illite's morphology. By combining careful core preservation, critical-point drying, and scanning electron microscopy examination, it can be shown that illite has various morphologies, both natural and artifactual . The native-state growth form of illite is filamentous into open, brine-filled pore space. If the illite grows as relatively thick filaments, its morphology is stable, unaltered by changes in liquid saturations or core drying. However, thin illite is delicate, and its filamentous form can be modified naturally and artificially, by the passage of interfaces between fluids. Hydrocarbons entering a reservoir modify the fiber distribution, giving rise to various illite textures. In a North Sea Brent reservoir, filaments are preserved in the water zone and in isolated water-saturated sandstones high in the oil zone, but in oil-saturated samples the illite is matted into flake and honeycomb forms. Many textures in the oil zone are similar to those of conventionally air-dried samples, which have artifactual illite morphologies. Without appropriate core preservation and critical-point drying, interpreting the textures as natural or artifactual is difficult - for example, some air-dried illite forms resemble those of smectite or detrital clays. Understanding the various illite morphologies is important for interpreting clay origins, core analysis, and potential productivity or injectivity of a formation.

  3. Rod examination gauge

    SciTech Connect

    Bacvinskas, W.S.; Bayer, J.E.; Davis, W.W.; Fodor, G.; Kikta, T.J.; Matchett, R.L.; Nilsen, R.J.; Wilczynski, R.

    1991-12-31

    The present invention is directed to a semi-automatic rod examination gauge for performing a large number of exacting measurements on radioactive fuel rods. The rod examination gauge performs various measurements underwater with remote controlled machinery of high reliability. The rod examination gauge includes instruments and a closed circuit television camera for measuring fuel rod length, free hanging bow measurement, diameter measurement, oxide thickness measurement, cladding defect examination, rod ovality measurement, wear mark depth and volume measurement, as well as visual examination. A control system is provided including a programmable logic controller and a computer for providing a programmed sequence of operations for the rod examination and collection of data.

  4. A Ribbon-like Structure in the Ejective Organelle of the Green Microalga Pyramimonas parkeae (Prasinophyceae) Consists of Core Histones and Polymers Containing N-acetyl-glucosamine.

    PubMed

    Yamagishi, Takahiro; Kurihara, Akira; Kawai, Hiroshi

    2015-11-01

    The green microalga, Pyramimonas parkeae (Prasinophyceae) has an ejective organelle containing a coiled ribbon structure resembling the ejectisome in Cryptophyta. This structure is discharged from the cell by a stimulus and extends to form a tube-like structure, but the molecular components of the structure have not been identified. Tricine-SDS-PAGE analysis indicated that the ribbon-like structure of P. parkeae contains some proteins and low molecular acidic polymers. Edman degradation, LC/MS/MS analyses and immunological studies demonstrated that their proteins are core histones (H3, H2A, H2B and H4). In addition, monosaccharide composition analysis of the ribbon-like structures and degradation by lysozyme strongly indicated that the ribbon-like structure consist of β (1-4) linked polymers containing N-acetyl-glucosamine. Purified polymers and recombinant histones formed glob-like or filamentous structures. Therefore we conclude that the ribbon-like structure of P. parkeae mainly consists of a complex of core histones (H3, H2A, H2B and H4) and polymers containing N-acetyl-glucosamine, and suggest to name the ejective organelle in P. parkeae the "histrosome" to distinguish it from the ejectisome in Cryptophyta. PMID:26398336

  5. Mitochondrial-Nuclear DNA Interactions Contribute to the Regulation of Nuclear Transcript Levels as Part of the Inter-Organelle Communication System

    PubMed Central

    Rodley, Chris D. M.; Grand, Ralph S.; Gehlen, Lutz R.; Greyling, Gary; Jones, M. Beatrix; O'Sullivan, Justin M.

    2012-01-01

    Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions. The interactions that form between mitochondrial and nuclear loci are dependent on the metabolic state of the yeast. Moreover, the frequency of specific mitochondrial - nuclear interactions (i.e. COX1-MSY1 and Q0182-RSM7) showed significant reductions in the absence of mitochondrial encoded reverse transcriptase machinery. Furthermore, these reductions correlated with increases in the transcript levels of the nuclear loci (MSY1 and RSM7). We propose that these interactions represent an inter-organelle DNA mediated communication system and that reverse transcription of mitochondrial RNA plays a role in this process. PMID:22292080

  6. A set of SNARE proteins in the contractile vacuole complex of Paramecium regulates cellular calcium tolerance and also contributes to organelle biogenesis.

    PubMed

    Schönemann, Barbara; Bledowski, Alexander; Sehring, Ivonne M; Plattner, Helmut

    2013-03-01

    The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca(2+). No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca(2+) after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca(2+) stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca(2+) in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca(2+) tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis. PMID:23280185

  7. Flash imaging of fine structures of cellular organelles by contact x-ray microscopy with a high intensity laser plasma x-ray source

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Ishino, Masahiko; Kishimoto, Maki; Tamotsu, Satoshi; Yasuda, Keiko; Kinjo, Yasuhito; Shinohara, Kunio

    2011-09-01

    X-ray flash imaging by contact microscopy with a highly intense laser-plasma x-ray source was achieved for the observation of wet biological cells. The exposure time to obtain a single x-ray image was about 600 ps as determined by the pulse duration of the driving laser pulse. The x-ray flash imaging makes it possible to capture an x-ray image of living biological cells without any artificial treatment such as staining, fixation, freezing, and so on. The biological cells were cultivated directly on the surface of the silicon nitride membranes, which are used for the x-ray microscope. Before exposing the cells to x-rays they were observed by a conventional fluorescent microscope as reference, since the fluorescent microscopes can visualize specific organelles stained with fluorescent dye. Comparing the x-ray images with the fluorescent images of the exact same cells, each cellular organelle observed in the x-ray images was identified one by one and actin filaments and mitochondria were clearly identified in the x-ray images.

  8. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    PubMed

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region. PMID:26626407

  9. Minding Morphology: How Morphological Awareness Relates to Reading for English Language Learners

    ERIC Educational Resources Information Center

    Goodwin, Amanda P.; Huggins, A. Corinne; Carlo, Maria S.; August, Diane; Calderon, Margarita

    2013-01-01

    This study explored subprocesses of reading for 157 fifth grade Spanish-speaking English language learners (ELLs) by examining whether morphological awareness made a unique contribution to reading comprehension beyond a strong covariate-phonological decoding. The role of word reading and reading vocabulary as mediators of this relationship was…

  10. Wood's lamp examination

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003386.htm Wood's lamp examination To use the sharing features on this page, please enable JavaScript. A Wood's lamp examination is a test that uses ultraviolet ( ...

  11. Wood's lamp examination

    MedlinePlus

    A Wood's lamp examination is a test that uses ultraviolet (UV) light to look at the skin closely. ... Gebhard RE. Wood's light examination. In: Pfenninger JL, Fowler GC, eds. ... Care . 3rd ed. Philadelphia, PA: Elsevier Mosby; 2010:chap ...

  12. The neurological examination.

    PubMed

    April, R S

    1995-06-01

    This chapter describes methods of clinical history taking and examination of the PLDD candidate with lumbar radicular symptoms. It stresses features of the classical neurological examination of the back and lower extremities in a concise, systematic fashion. PMID:10150641

  13. Wood's Lamp Examination

    MedlinePlus

    ... dermatologists to assist in the diagnosis of various pigment and infectious disorders. The examination is performed in ... lamp. If a fungal or bacterial infection or pigment disorder is present, Wood's lamp examination can strengthen ...

  14. Physics 300 Provincial Examination.

    ERIC Educational Resources Information Center

    Manitoba Dept. of Education and Training, Winnipeg.

    This document consists of the physics 300 provincial examination (English version), a separate "provincial summary report" on the results of giving the test, and a separate French language version of the examination. This physics examination contains a 53-item multiple choice section and an 12 item free response section. Subsections of the test…

  15. Vesta's shape and morphology.

    PubMed

    Jaumann, R; Williams, D A; Buczkowski, D L; Yingst, R A; Preusker, F; Hiesinger, H; Schmedemann, N; Kneissl, T; Vincent, J B; Blewett, D T; Buratti, B J; Carsenty, U; Denevi, B W; De Sanctis, M C; Garry, W B; Keller, H U; Kersten, E; Krohn, K; Li, J-Y; Marchi, S; Matz, K D; McCord, T B; McSween, H Y; Mest, S C; Mittlefehldt, D W; Mottola, S; Nathues, A; Neukum, G; O'Brien, D P; Pieters, C M; Prettyman, T H; Raymond, C A; Roatsch, T; Russell, C T; Schenk, P; Schmidt, B E; Scholten, F; Stephan, K; Sykes, M V; Tricarico, P; Wagner, R; Zuber, M T; Sierks, H

    2012-05-11

    Vesta's surface is characterized by abundant impact craters, some with preserved ejecta blankets, large troughs extending around the equatorial region, enigmatic dark material, and widespread mass wasting, but as yet an absence of volcanic features. Abundant steep slopes indicate that impact-generated surface regolith is underlain by bedrock. Dawn observations confirm the large impact basin (Rheasilvia) at Vesta's south pole and reveal evidence for an earlier, underlying large basin (Veneneia). Vesta's geology displays morphological features characteristic of the Moon and terrestrial planets as well as those of other asteroids, underscoring Vesta's unique role as a transitional solar system body. PMID:22582254

  16. Cluster Morphology Analysis

    PubMed Central

    Jacquez, Geoffrey M.

    2009-01-01

    Most disease clustering methods assume specific shapes and do not evaluate statistical power using the applicable geography, at-risk population, and covariates. Cluster Morphology Analysis (CMA) conducts power analyses of alternative techniques assuming clusters of different relative risks and shapes. Results are ranked by statistical power and false positives, under the rationale that surveillance should (1) find true clusters while (2) avoiding false clusters. CMA then synthesizes results of the most powerful methods. CMA was evaluated in simulation studies and applied to pancreatic cancer mortality in Michigan, and finds clusters of flexible shape while routinely evaluating statistical power. PMID:20234799

  17. Electron precipitation patterns and substorm morphology.

    NASA Technical Reports Server (NTRS)

    Hoffman, R. A.; Burch, J. L.

    1973-01-01

    Statistical analysis of data from the auroral particles experiment aboard OGO 4, performed in a statistical framework interpretable in terms of magnetospheric substorm morphology, both spatial and temporal. Patterns of low-energy electron precipitation observed by polar satellites are examined as functions of substorm phase. The implications of the precipitation boundaries identifiable at the low-latitude edge of polar cusp electron precipitation and at the poleward edge of precipitation in the premidnight sector are discussed.

  18. Morphological evolution of galaxies

    NASA Astrophysics Data System (ADS)

    Gardner, Jonathan P.; Heap, Sara R.; Malumuth, Eliot M.; Hill, Robert S.; Smith, Eric P.

    1997-05-01

    Recent studies of the Hubble Deep Field (Abraham et al. 1996) [1] and Medium Deep Survey (Driver, Windhorst & Griffiths 1995) [6] find that the frequency of irregular/peculiar/merger systems rises with increasing redshift. However, this finding must be carefully interpreted in light of UV images of low-redshift galaxies obtained by the Ultraviolet Imaging Telescope (Stecher et al. 1992) [9]. These UV images imply that K-correction effects may be at least partially responsible for the apparent increase in Irr galaxies with redshift. To assess the degree to which there is an overabundance of Irregular galaxies (relative to the present epoch), we must understand the degree to which the K-correction biases morphological studies. We demonstrate the importance of the morphological K-correction to the classification schemes used in the HDF. We find that high-redshift spiral galaxies are misclassified as Irr galaxies, while Elliptical/S0 galaxies, should not be affected substantially. We have been granted 40 orbits in Cycle 7 with STIS to place these conclusions on a statistical basis.

  19. Morphology of urethral tissues

    NASA Astrophysics Data System (ADS)

    Müller, Bert; Schulz, Georg; Herzen, Julia; Mushkolaj, Shpend; Bormann, Therese; Beckmann, Felix; Püschel, Klaus

    2010-09-01

    Micro computed tomography has been developed to a powerful technique for the characterization of hard and soft human and animal tissues. Soft tissues including the urethra, however, are difficult to be analyzed, since the microstructures of interest exhibit X-ray absorption values very similar to the surroundings. Selective staining using highly absorbing species is a widely used approach, but associated with significant tissue modification. Alternatively, one can suitably embed the soft tissue, which requires the exchange of water. Therefore, the more recently developed phase contrast modes providing much better contrast of low X-ray absorbing species are especially accommodating in soft tissue characterization. The present communication deals with the morphological characterization of sheep, pig and human urethras on the micrometer scale taking advantage of micro computed tomography in absorption and phase contrast modes. The performance of grating-based tomography is demonstrated for freshly explanted male and female urethras in saline solution. The micro-morphology of the urethra is important to understand how the muscles close the urethra to reach continence. As the number of incontinent patients is steadily increasing, the function under static and, more important, under stress conditions has to be uncovered for the realization of artificial urinary sphincters, which needs sophisticated, biologically inspired concepts to become nature analogue.

  20. How Specific Is the Connection between Morphological Awareness and Spelling? A Study of French Children

    ERIC Educational Resources Information Center

    Casalis, S.; Deacon, S. H.; Pacton, S.

    2011-01-01

    This study examined the relationship between morphological awareness and spelling. We show that French children in Grades 3 and 4 appear to use morphological information in spelling; spelling of sounds for which there are several alternatives was more accurate in derived than in nonderived words. The link between morphological awareness and…