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Sample records for organelle morphology examination

  1. Morphology and function of membrane-bound organelles.

    PubMed

    Heald, Rebecca; Cohen-Fix, Orna

    2014-02-01

    The cell interior is a busy and crowded place. A large fraction of the cell volume is taken up by organelles that come in a variety of shapes and sizes. These organelles are surrounded by membrane that not only acts as a diffusion barrier, but also provides each organelle with its unique morphology that contributes to its function, often in ways that are poorly understood. Here we discuss recent discoveries on the relationship between organelle structure and function. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Morphological Features of Organelles during Apoptosis: An Overview

    PubMed Central

    Bottone, Maria Grazia; Santin, Giada; Aredia, Francesca; Bernocchi, Graziella; Pellicciari, Carlo; Scovassi, Anna Ivana

    2013-01-01

    An apoptotic program leading to controlled cell dismantling implies perturbations of nuclear dynamics, as well as changes affecting the organelle structure and distribution. In human cancer cells driven to apoptosis by different stimuli, we have recently investigated the morphological properties of several organelles, including mitochondria, lysosomes, endoplasmic reticulum and Golgi apparatus. In this review, we will discuss the body of evidence in the literature suggesting that organelles are generally relocated and/or degraded during apoptosis, irrespectively of the apoptogenic stimulus and cell type. PMID:24709702

  3. Following mitochondria dynamism: confocal analysis of the organelle morphology.

    PubMed

    Mariotti, Francesca R; Corrado, Mauro; Campello, Silvia

    2015-01-01

    Mitochondria are highly dynamic organelles, whose morphology can vary from an elongated and interconnected network to fragmented units. In recent years, outstanding discoveries have linked mitochondrial morphology to the regulation of an increasing number of biological processes, such as biosynthetic pathways, oxidative phosphorylation and ATP production, calcium buffering, and cell death. Here we describe two of the main methods used to analyze the mitochondrial length in fixed cells and the mitochondrial fusion rate in live cells. Moreover, we focus one of the protocols on T cells, as an example of non-adherent cells, which present some particularities and difficulties in the analysis of mitochondrial shape. We also discuss the main mouse models carrying a mitochondrial targeted fluorescent protein, an invaluable tool to deeply investigate in vivo mitochondrial morphology.

  4. Plant peroxisomes: recent discoveries in functional complexity, organelle homeostasis, and morphological dynamics.

    PubMed

    Reumann, Sigrun; Bartel, Bonnie

    2016-12-01

    Peroxisomes are essential for life in plants. These organelles house a variety of metabolic processes that generate and inactivate reactive oxygen species. Our knowledge of pathways and mechanisms that depend on peroxisomes and their constituent enzymes continues to grow, and in this review we highlight recent advances in understanding the identity and biological functions of peroxisomal enzymes and metabolic processes. We also review how peroxisomal matrix and membrane proteins enter the organelle from their sites of synthesis. Peroxisome homeostasis is regulated by specific degradation mechanisms, and we discuss the contributions of specialized autophagy and a peroxisomal protease to the degradation of entire peroxisomes and peroxisomal enzymes that are damaged or superfluous. Finally, we review how peroxisomes can flexibly change their morphology to facilitate inter-organellar contacts. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. A novel organelle map framework for high-content cell morphology analysis in high throughput.

    PubMed

    Schauer, Kristine; Grossier, Jean-Philippe; Duong, Tarn; Chapuis, Violaine; Degot, Sébastien; Lescure, Aurianne; Del Nery, Elaine; Goud, Bruno

    2014-02-01

    A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box statistical testing. Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape and distribution of intracellular compartments avoiding strong cell-to-cell variation that is a major limitation of classical culture conditions. To detect changes in cell morphology induced by compound treatment, fluorescently labeled intracellular structures from several tens of micropatterned cells were transformed into probabilistic density maps. Then, the similarity or difference between two given density maps was quantified using statistical testing that evaluates differences directly from the data without additional analysis or any subjective decision. The versatility of this organelle mapping approach for different magnifications and its performance for different cell shapes has been assessed. Density-based analysis detected changes in cell morphology due to compound treatment in a small-scale proof-of-principle screen demonstrating its compatibility with high-throughput screening. This novel tool for high-content and high-throughput cellular phenotyping can potentially be used for a wide range of applications from drug screening to careful characterization of cellular processes.

  6. 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.

  7. 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

  8. 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.

  9. Effects of Fcj1-Mos1 and mitochondrial division on aggregation of mitochondrial DNA nucleoids and organelle morphology.

    PubMed

    Itoh, Kie; Tamura, Yasushi; Iijima, Miho; Sesaki, Hiromi

    2013-06-01

    Mitochondrial DNA (mtDNA) is packaged into DNA-protein complexes called nucleoids, which are distributed as many small foci in mitochondria. Nucleoids are crucial for the biogenesis and function of mtDNA. Here, using a yeast genetic screen for components that control nucleoid distribution and size, we identify Fcj1 and Mos1, two evolutionarily conserved mitochondrial proteins that maintain the connection between the cristae and boundary membranes. These two proteins are also important for establishing tubular morphology of mitochondria, as mitochondria lacking Fcj1 and Mos1 form lamellar sheets. We find that nucleoids aggregate, increase in size, and decrease in number in fcj1 and mos1 cells. In addition, Fcj1 form punctate structures and localized adjacent to nucleoids. Moreover, connecting mitochondria by deleting the DNM1 gene required for organelle division enhances aggregation of mtDNA nucleoids in fcj1 and mos1 cells, whereas single deletion of DNM1 does not affect nucleoids. Conversely, deleting F1Fo-ATP synthase dimerization factors generates concentric ring-like cristae, restores tubular mitochondrial morphology, and suppresses nucleoid aggregation in these mutants. Our findings suggest an unexpected role of Fcj1-Mos1 and organelle division in maintaining the distribution and size of mtDNA nucleoids.

  10. 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

  11. Tomographic reconstruction reveals the morphology of a unique cellular organelle, the aggregated macrotubules (Macrotubuli aggregati) of human retinal horizontal cells.

    PubMed

    Jastrow, Holger; Yarwood, Andrew; Majorovits, Endre; Harris, J Robin

    2015-04-01

    Horizontal cells of the human retina contain unique tubular organelles that have a diameter which is about 10 times larger than that of microtubules (~230 nm). These macrotubuli in most cases form regular aggregates. Therefore we propose to introduce them as Macrotubuli aggregati in the Terminologia histologica. Tomographic investigation of the structures revealed that the walls of the tubules most probably consist of intermediate filaments running nearly parallel to each other and show somewhat regularly attached ribosomes on their inner and also outer surface. About 2% of the organelles exhibit double- to multiple layered walls and less than 1% resemble large scrolls. The tubules may extend 10 to over 20 μm in the cytoplasm and are also encountered in soma-near processes extending into the outer plexiform layer. It remains unclear why these structures are only present in humans and few other species and why almost only in horizontal cells. Speculations on possible functions are discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Novel organelles in primate retinal epithelium.

    PubMed

    Biesemeier, A; Gouras, P

    2016-10-01

    We are investigating age-related changes in organelles in monkey retinal epithelium using transmission and analytic electron microscopy. We previously described a circular organelle in retinal epithelium with a diameter of about 0.5μm. The organelle is unique in containing a single, round vacuole within an otherwise electron dense interior. We suggested that the organelle might be a melanosome with lysosomal properties. We now find that there are two similar organelles with such a single vacuole but which differ in their chemical composition, electron density, cell location and according to age. Epon embedded sections from the macular epithelium of seven monkeys, ranging from 1 to 35 years of age, were examined by transmission electron microscopy. A seven year old monkey was processed for analytic electron microscopy to determine the chemical composition of the organelles. The number and location of the organelles in the retinal epithelium were determined. The chemical composition of these two organelles was different. One of the organelles contained high mole fractions of oxygen and nitrogen and little phosphorous characteristic of melanin; the other had little oxygen and nitrogen and higher mole fractions of phosphorous uncharacteristic of melanin, but more common with lysosomal organelles. The latter had an electron dense rim around the vacuole, a less electron dense interior than the melanin containing organelle and also contained iron. The melanin containing organelle was more common in young monkeys and in the middle third of the cell. The organelle without melanin was more common in old monkeys and localized in the basal third of the cell. Two similarly vacuolated organelles, not identified before in retinal epithelium, differ in their chemical composition. One contains melanin; the other does not. The former is more common in young and the latter more common in old monkeys. This suggests reorganization and or degradation of melanin-containing organelles

  13. Mitochondrion-related organelles in eukaryotic protists.

    PubMed

    Shiflett, April M; Johnson, Patricia J

    2010-01-01

    The discovery of mitochondrion-type genes in organisms thought to lack mitochondria led to the demonstration that hydrogenosomes share a common ancestry with mitochondria, as well as the discovery of mitosomes in multiple eukaryotic lineages. No examples of examined eukaryotes lacking a mitochondrion-related organelle exist, implying that the endosymbiont that gave rise to the mitochondrion was present in the first eukaryote. These organelles, known as hydrogenosomes, mitosomes, or mitochondrion-like organelles, are typically reduced, both structurally and biochemically, relative to classical mitochondria. However, despite their diversification and adaptation to different niches, all appear to play a role in Fe-S cluster assembly, as observed for mitochondria. Although evidence supports the use of common protein targeting mechanisms in the biogenesis of these diverse organelles, divergent features are also apparent. This review examines the metabolism and biogenesis of these organelles in divergent unicellular microbes, with a focus on parasitic protists.

  14. 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.

  15. Fluorescent proteins in cellular organelles: serious pitfalls and some solutions.

    PubMed

    Costantini, Lindsey M; Snapp, Erik Lee

    2013-11-01

    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.

  16. 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…

  17. 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…

  18. Mitochondrion-related Organelles in Parasitic Eukaryotes

    PubMed Central

    Shiflett, April; Johnson, Patricia J.

    2011-01-01

    The discovery of mitochondrial-type genes in organisms thought to lack mitochondria led to the demonstration that hydrogenosomes share a common ancestry with mitochondria, as well as the discovery of mitosomes in multiple eukaryotic lineages. No examples of examined eukaryotes lacking a mitochondrion-related organelle exist, implying that the endosymbiont that gave rise to the mitochondrion was present in the first eukaryote. These organelles, known as hydrogenosomes, mitosomes or mitochondrion-like organelles (MLO), are typically reduced, both structurally and biochemically, relative to classical mitochondria. However, despite diversification and adaptation to different niches, all appear to play a role in Fe-S cluster assembly, as observed for mitochondria. Although evidence supports the use of common protein targeting mechanisms in the biogenesis of these diverse organelles, divergent features are also apparent. The metabolism and biogenesis of these organelles in parasitic protists is discussed here. PMID:20528687

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

    PubMed Central

    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 assessments. Study 2 extended these results by giving 90 eighth-grade students expanded measures of vocabulary and morphology that assessed (a) definitional knowledge, (b) usage, (c) relational knowledge, and (d) knowledge of morphological variants, with each potential aspect of knowledge assessed using an identical set of 23 words to control for differential knowledge of specific vocabulary items. Results indicated that a single-factor model that encompassed morphological and vocabulary knowledge provided the best fit to the data. Finally, explanatory item response modeling was used to investigate sources of variance in the vocabulary and morphological awareness tasks we administered. Implications for assessment and instruction are discussed. PMID:26273128

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

    PubMed

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

    2015-09-01

    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 assessments. Study 2 extended these results by giving 90 eighth-grade students expanded measures of vocabulary and morphology that assessed (a) definitional knowledge, (b) usage, (c) relational knowledge, and (d) knowledge of morphological variants, with each potential aspect of knowledge assessed using an identical set of 23 words to control for differential knowledge of specific vocabulary items. Results indicated that a single-factor model that encompassed morphological and vocabulary knowledge provided the best fit to the data. Finally, explanatory item response modeling was used to investigate sources of variance in the vocabulary and morphological awareness tasks we administered. Implications for assessment and instruction are discussed.

  1. Dynamic regulation of endosymbiotic organelles by ubiquitination.

    PubMed

    Ling, Qihua; Jarvis, Paul

    2013-08-01

    Recent work has revealed that mitochondria and chloroplasts are subject to direct control by the ubiquitin-proteasome system (UPS). Ubiquitin E3 ligases are present at the outer membrane of both organelles where they mediate ubiquitination and turnover of other organellar proteins. Both organelles exhibit remarkable structural dynamism and UPS control is particularly concerned with these properties. In mitochondria, the UPS targets factors involved in organellar fission and fusion, with significant impacts upon organellar morphology, mitophagy, and apoptosis. In chloroplasts (and other plastids), the UPS targets components of the protein import machinery, facilitating reorganization of the organellar proteome to determine organellar development and functions. Acquisition of such regulatory control during evolution is perhaps linked to the dynamic characteristics of the two organelles, which are not paralleled in their prokaryotic relatives. Here we discuss our current understanding of the role of the UPS in the regulation of endosymbiotic organelles. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. 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.

  3. Mitochondrial fission factor Drp1 maintains oocyte quality via dynamic rearrangement of multiple organelles.

    PubMed

    Udagawa, Osamu; Ishihara, Takaya; Maeda, Maki; Matsunaga, Yui; Tsukamoto, Satoshi; Kawano, Natsuko; Miyado, Kenji; Shitara, Hiroshi; Yokota, Sadaki; Nomura, Masatoshi; Mihara, Katsuyoshi; Mizushima, Noboru; Ishihara, Naotada

    2014-10-20

    Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1, as well as analyzing a human patient harboring a point mutation in Drp1, showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically, and their organelle aggregation is related to germ cell formation and epigenetic regulation. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca(2+) signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement.

  4. Evolving a photosynthetic organelle.

    PubMed

    Nakayama, Takuro; Archibald, John M

    2012-04-24

    The evolution of plastids from cyanobacteria is believed to represent a singularity in the history of life. The enigmatic amoeba Paulinella and its 'recently' acquired photosynthetic inclusions provide a fascinating system through which to gain fresh insight into how endosymbionts become organelles.The plastids, or chloroplasts, of algae and plants evolved from cyanobacteria by endosymbiosis. This landmark event conferred on eukaryotes the benefits of photosynthesis--the conversion of solar energy into chemical energy--and in so doing had a huge impact on the course of evolution and the climate of Earth 1. From the present state of plastids, however, it is difficult to trace the evolutionary steps involved in this momentous development, because all modern-day plastids have fully integrated into their hosts. Paulinella chromatophora is a unicellular eukaryote that bears photosynthetic entities called chromatophores that are derived from cyanobacteria and has thus received much attention as a possible example of an organism in the early stages of organellogenesis. Recent studies have unlocked the genomic secrets of its chromatophore 23 and provided concrete evidence that the Paulinella chromatophore is a bona fide photosynthetic organelle 4. The question is how Paulinella can help us to understand the process by which an endosymbiont is converted into an organelle.

  5. Proteomics of regulated secretory organelles.

    PubMed

    Brunner, Yannick; Schvartz, Domitille; Couté, Yohann; Sanchez, Jean-Charles

    2009-01-01

    Regulated secretory organelles are important subcellular structures of living cells that allow the release in the extracellular space of crucial compounds, such as hormones and neurotransmitters. Therefore, the regulation of biogenesis, trafficking, and exocytosis of regulated secretory organelles has been intensively studied during the last 30 years. However, due to the large number of different regulated secretory organelles, only a few of them have been specifically characterized. New insights into regulated secretory organelles open crucial perspectives for a better comprehension of the mechanisms that govern cell secretion. The combination of subcellular fractionation, protein separation, and mass spectrometry is also possible to study regulated secretory organelles at the proteome level. In this review, we present different strategies used to isolate regulated secretory organelles, separate their protein content, and identify the proteins by mass spectrometry. The biological significance of regulated secretory organelles-proteomic analysis is discussed as well. Copyright 2009 Wiley Periodicals, Inc.

  6. Actin-Based Motility of Isolated Axoplasmic Organelles

    PubMed Central

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

    2010-01-01

    We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315–384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560–988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 ± .03 μm/s and an instantaneous velocity of 1.63 ± 0.29 μm/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity. PMID:8635200

  7. The Plant Organelles Database 2 (PODB2): an updated resource containing movie data of plant organelle dynamics.

    PubMed

    Mano, Shoji; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nishimura, Mikio

    2011-02-01

    The Plant Organelles Database (PODB) was launched in 2006 and provides imaging data of plant organelles, protocols for plant organelle research and external links to relevant websites. To provide comprehensive information on plant organelle dynamics and accommodate movie files that contain time-lapse images and 3D structure rotations, PODB was updated to the next version, PODB2 (http://podb.nibb.ac.jp/Organellome). PODB2 contains movie data submitted directly by plant researchers and can be freely downloaded. Through this organelle movie database, users can examine the dynamics of organelles of interest, including their movement, division, subcellular positioning and behavior, in response to external stimuli. In addition, the user interface for access and submission has been enhanced. PODB2 contains all of the information included in PODB, and the volume of data and protocols deposited in the PODB2 continues to grow steadily. Moreover, a new website, Plant Organelles World (http://podb.nibb.ac.jp/Organellome/PODBworld/en/index.html), which is based on PODB2, was recently launched as an educational tool to engage members of the non-scientific community such as students and school teachers. Plant Organelles World is written in layman's terms, and technical terms were avoided where possible. We would appreciate contributions of data from all plant researchers to enhance the usefulness of PODB2 and Plant Organelles World.

  8. The Plant Organelles Database 2 (PODB2): An Updated Resource Containing Movie Data of Plant Organelle Dynamics

    PubMed Central

    Mano, Shoji; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nishimura, Mikio

    2011-01-01

    The Plant Organelles Database (PODB) was launched in 2006 and provides imaging data of plant organelles, protocols for plant organelle research and external links to relevant websites. To provide comprehensive information on plant organelle dynamics and accommodate movie files that contain time-lapse images and 3D structure rotations, PODB was updated to the next version, PODB2 (http://podb.nibb.ac.jp/Organellome). PODB2 contains movie data submitted directly by plant researchers and can be freely downloaded. Through this organelle movie database, users can examine the dynamics of organelles of interest, including their movement, division, subcellular positioning and behavior, in response to external stimuli. In addition, the user interface for access and submission has been enhanced. PODB2 contains all of the information included in PODB, and the volume of data and protocols deposited in the PODB2 continues to grow steadily. Moreover, a new website, Plant Organelles World (http://podb.nibb.ac.jp/Organellome/PODBworld/en/index.html), which is based on PODB2, was recently launched as an educational tool to engage members of the non-scientific community such as students and school teachers. Plant Organelles World is written in layman's terms, and technical terms were avoided where possible. We would appreciate contributions of data from all plant researchers to enhance the usefulness of PODB2 and Plant Organelles World. PMID:21115470

  9. Application of proteomic marker ensembles to subcellular organelle identification.

    PubMed

    Andreyev, Alexander Y; Shen, Zhouxin; Guan, Ziqiang; Ryan, Andrea; Fahy, Eoin; Subramaniam, Shankar; Raetz, Christian R H; Briggs, Steven; Dennis, Edward A

    2010-02-01

    systematically redefine originally morphologically defined organelles as biochemical entities.

  10. 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

  11. Visualization of lipid droplet composition by direct organelle mass spectrometry.

    PubMed

    Horn, Patrick J; Ledbetter, Nicole R; James, Christopher N; Hoffman, William D; Case, Charlene R; Verbeck, Guido F; Chapman, Kent D

    2011-02-04

    An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules.

  12. 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.

  13. Mechanisms of organelle biogenesis govern stochastic fluctuations in organelle abundance.

    PubMed

    Mukherji, Shankar; O'Shea, Erin K

    2014-06-10

    Fluctuations in organelle abundance can profoundly limit the precision of cell biological processes from secretion to metabolism. We modeled the dynamics of organelle biogenesis and predicted that organelle abundance fluctuations depend strongly on the specific mechanisms that increase or decrease the number of a given organelle. Our model exactly predicts the size of experimentally measured Golgi apparatus and vacuole abundance fluctuations, suggesting that cells tolerate the maximum level of variability generated by the Golgi and vacuole biogenesis pathways. We observe large increases in peroxisome abundance fluctuations when cells are transferred from glucose-rich to fatty acid-rich environments. These increased fluctuations are significantly diminished in mutants lacking peroxisome fission factors, leading us to infer that peroxisome biogenesis switches from de novo synthesis to primarily fission. Our work provides a general framework for exploring stochastic organelle biogenesis and using fluctuations to quantitatively unravel the biophysical pathways that control the abundance of subcellular structures.DOI: http://dx.doi.org/10.7554/eLife.02678.001. Copyright © 2014, Mukherji and O'Shea.

  14. Mechanisms of organelle biogenesis govern stochastic fluctuations in organelle abundance

    PubMed Central

    Mukherji, Shankar; O'Shea, Erin K

    2014-01-01

    Fluctuations in organelle abundance can profoundly limit the precision of cell biological processes from secretion to metabolism. We modeled the dynamics of organelle biogenesis and predicted that organelle abundance fluctuations depend strongly on the specific mechanisms that increase or decrease the number of a given organelle. Our model exactly predicts the size of experimentally measured Golgi apparatus and vacuole abundance fluctuations, suggesting that cells tolerate the maximum level of variability generated by the Golgi and vacuole biogenesis pathways. We observe large increases in peroxisome abundance fluctuations when cells are transferred from glucose-rich to fatty acid-rich environments. These increased fluctuations are significantly diminished in mutants lacking peroxisome fission factors, leading us to infer that peroxisome biogenesis switches from de novo synthesis to primarily fission. Our work provides a general framework for exploring stochastic organelle biogenesis and using fluctuations to quantitatively unravel the biophysical pathways that control the abundance of subcellular structures. DOI: http://dx.doi.org/10.7554/eLife.02678.001 PMID:24916159

  15. Nonperturbative Chemical Imaging of Organelle Transport in Living Cells with Coherent Anti-Stokes Raman Scattering Microscopy

    PubMed Central

    Nan, Xiaolin; Potma, Eric O.; Xie, X. Sunney

    2006-01-01

    Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed subdiffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells. PMID:16632501

  16. Organelles are transported on sliding microtubules in Reticulomyxa.

    PubMed

    Orokos, D D; Cole, R W; Travis, J L

    2000-12-01

    Organelles and plasma membrane domains appear to be transported along Reticulomyxa's microtubule cytoskeleton. Previously we demonstrated that organelle and cell surface transport share the same enzymatic properties and suggested that both are powered by the same cytoplasmic dynein. Motility analysis in Reticulomyxa is complicated by the fact that the microtubules also are motile and appear to "slide" bidirectionally throughout the network. We have utilized laser ablation to address this frame-of-reference problem as to how each transport component (microtubule sliding vs. organelle translocations) contributes to reactivated bidirectional translocation of organelles along the microtubule cytoskeleton. Laser ablation was used to cut microtubule bundles from lysed networks into 4-15-microm segments. After examining these reactivated cut fragments, it appears that the majority of organelles did not move relative to microtubule fragments, but remained attached to microtubules and moved as the microtubules slid. Microtubule sliding stops after 1-2 min and cannot be reactivated even when perfused with fresh ATP. Furthermore, once sliding stops, organelle transport also stops. Our findings indicate that the majority of Reticulomyxa pseudopodial organelles do not move along the surface of the microtubules, rather it is the sliding of the microtubules to which they are attached that moves them.

  17. 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

  18. Cell biology of prokaryotic organelles.

    PubMed

    Murat, Dorothee; Byrne, Meghan; Komeili, Arash

    2010-10-01

    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.

  19. Basal Organelles of Bacterial Flagella

    PubMed Central

    Cohen-Bazire, Germaine; London, Jack

    1967-01-01

    Liberated by enzymatic lysis of the cells, the flagella of Rhodospirillum rubrum, R. molischianum, and R. fulvum all have a similar structure. The hook at the base of the flagellum is connected by a short, narrow collar to a paired disc in the basal organelle. This paired disc is in turn connected to a second paired disc. The disposition of flagella to which fragments of the cell membrane still adhere suggests that the narrow collar at the base of the hook traverses both the wall and the membrane, and that the upper pair of discs in the basal organelle lies just beneath the surface of the membrane. Images PMID:6039362

  20. Morphological variability in second language learners: An examination of electrophysiological and production data.

    PubMed

    Alemán Bañón, José; Miller, David; Rothman, Jason

    2017-10-01

    We examined sources of morphological variability in second language (L2) learners of Spanish whose native language (L1) is English, with a focus on L1-L2 similarity, morphological markedness, and knowledge type (receptive vs. expressive). Experiment 1 uses event-related potentials to examine noun-adjective number (present in L1) and gender agreement (absent in L1) in online sentence comprehension (receptive knowledge). For each feature, markedness was manipulated, such that half of the critical noun-adjective combinations were feminine (marked) and the other half were masculine; half were used in the plural (marked) and the other half were used in the singular. With this setup, we examined learners' potential overreliance on unmarked forms or "defaults" (singular/masculine). Experiment 2 examines similar dependencies in spoken sentence production (expressive knowledge). Learners (n = 22) performed better with number than gender overall, but their brain responses to both features were qualitatively native-like (i.e., P600), even though gender was probed with nouns that do not provide strong distributional cues to gender. In addition, variability with gender agreement was better accounted for by lexical (as opposed to syntactic) aspects. Learners showed no advantage for comprehension over production, and no systematic evidence of reliance on morphological defaults, although their online processing was sensitive to markedness in a native-like manner. Overall, these results suggest that there is facilitation for L2 properties that exist in the L1 and that markedness impacts L2 processing, but in a native-like manner. These results also speak against proposals arguing that adult L2ers have deficits at the level of the morphology or the syntax. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  1. Evolution of organelle-associated protein profiling.

    PubMed

    Yan, Wei; Aebersold, Ruedi; Raines, Elaine W

    2009-02-15

    Identification of the protein constituents of cell organelles forms the basis for studies to define the roles of specific proteins in organelle structure and functions. Over the past decade, the use of mass spectrometry-based proteomics has dissected various organelles and allowed the association of many novel proteins with particular organelles. This review chronicles the evolution of organelle proteomics technology, and discusses how many limitations, such as organelle heterogeneity and purity, can be avoided with recently developed quantitative profiling approaches. Although many challenges remain, quantitative profiling of organelles holds the promise to begin to address the complex and dynamic shuttling of proteins among organelles that will be critical for application of this advanced technology to disease-based changes in organelle function.

  2. Organelle extensions in plant cells.

    PubMed

    Mathur, Jaideep; Mammone, Alena; Barton, Kiah A

    2012-11-01

    Cell walls lock each cell in a specific position within the supra-organization of a plant. Despite its fixed location, each cell must be able to sense alterations in its immediate environment and respond rapidly to ensure the optimal functioning, continued growth and development, and eventual long-term survival of the plant. The ultra-structural detail that underlies our present understanding of the plant cell has largely been acquired from fixed and processed material that does not allow an appreciation of the dynamic nature of sub-cellular events in the cell. In recent years, fluorescent protein-aided imaging of living plant cells has added to our understanding of the dynamic nature of the plant cell. One of the major outcomes of live imaging of plant cells is the growing appreciation that organelle shapes are not fixed, and many organelles extend their surface transiently in rapid response to environmental stimuli. In many cases, the extensions appear as tubules extending from the main organelle. Specific terms such as stromules from plastids, matrixules from mitochondria, and peroxules from peroxisomes have been coined to describe the extensions. Here, we review our present understanding of organelle extensions and discuss how they may play potential roles in maintaining cellular homeostasis in plant cells. © 2012 Institute of Botany, Chinese Academy of Sciences.

  3. Cleaning House: Selective Autophagy of Organelles.

    PubMed

    Anding, Allyson L; Baehrecke, Eric H

    2017-04-10

    The selective clearance of organelles by autophagy is critical for the regulation of cellular homeostasis in organisms from yeast to humans. Removal of damaged organelles clears the cell of potentially toxic byproducts and enables reuse of organelle components for bioenergetics. Thus, defects in organelle clearance may be detrimental to the health of the cells, contributing to cancer, neurodegeneration, and inflammatory diseases. Organelle-specific autophagy can clear mitochondria, peroxisomes, lysosomes, ER, chloroplasts, and the nucleus. Here, we review our understanding of the mechanisms that regulate the clearance of organelles by autophagy and highlight gaps in our knowledge of these processes. Copyright © 2017 Elsevier Inc. All rights reserved.

  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 Role of Oocyte Organelles in Determining Developmental Competence.

    PubMed

    Reader, Karen L; Stanton, Jo-Ann L; Juengel, Jennifer L

    2017-09-18

    The ability of an oocyte to undergo successful cytoplasmic and nuclear maturation, fertilization and embryo development is referred to as the oocyte's quality or developmental competence. Quality is dependent on the accumulation of organelles, metabolites and maternal RNAs during the growth and maturation of the oocyte. Various models of good and poor oocyte quality have been used to understand the essential contributors to developmental success. This review covers the current knowledge of how oocyte organelle quantity, distribution and morphology differ between good and poor quality oocytes. The models of oocyte quality are also described and their usefulness for studying the intrinsic quality of an oocyte discussed. Understanding the key critical features of cytoplasmic organelles and metabolites driving oocyte quality will lead to methods for identifying high quality oocytes and improving oocyte competence, both in vitro and in vivo.

  6. Apoptotic death sensor: an organelle's alter ego?

    PubMed

    Bratton, S B; Cohen, G M

    2001-06-01

    Caspases are intracellular cysteine proteases that are primarily responsible for the stereotypic morphological and biochemical changes that are associated with apoptosis. Caspases are often activated by the apoptotic protease-activating factor 1 (APAF-1) apoptosome, a complex that is formed following mitochondrial release of cytochrome c in response to many death-inducing stimuli. Both pro- and anti-apoptotic BCL-2 family members regulate apoptosis, primarily by their effects on mitochondria, whereas many inhibitor of apoptosis proteins (IAPs) regulate apoptosis by directly inhibiting distinct caspases. Exposure of cells to chemicals and radiation, as well as loss of trophic stimuli, perturb cellular homeostasis and, depending on the type of cellular stress, particular or multiple organelles appear to 'sense' the damage and signal the cell to undergo apoptosis by stimulating the formation of unique and/or common caspase-activating complexes.

  7. Hybrid pigment organelles in an invertebrate.

    PubMed

    Schliwa, M; Euteneuer, U

    1979-02-28

    Observations of a number of vertebrate chromatophores have revealed the presence of more than one type of pigment organelles, suggesting that the different types are all derived from an equipotential organelle able to differentiate into any of the major pigment-containing organelles (Bagnara, 1972). Observations are presented concerning the occurrence of hybrid pigment inclusions, i.e., all kinds of intergrades between melanosomes, pterinosomes, and reflecting platelets in pigment cells of the daddy-long-legs. It therefore seems possible that pigment organelles in some invertebrates may also be derived from a common pluripotential primordial organelle.

  8. Routine blood examinations combined with morphological analysis for the diagnosis of myelodysplastic/myeloproliferative neoplasms

    PubMed Central

    Wu, Huanling; Sun, Hui; Zhang, Zhifen; Li, Xiangli; Li, Yuantang; Li, Li; Xu, Rui; Wang, Zie; Tian, Wenjun

    2016-01-01

    In 2008, the World Health Organization (WHO) introduced a new hematological neoplasm category; myelodysplastic/myeloproliferative neoplasms (MDS/MPN), which included four main subcategories. This disease is often misdiagnosed, which delays effective therapy. The present study evaluated the role of routine blood examinations and morphological analysis of peripheral blood cells in the reliable diagnosis of MDS/MPN. In total, 236 adult MDS/MPN patients were analyzed. The analysis included 10 routine blood parameters measured using a Sysmex XE-2100™, 3 differential percentage parameters and 7 morphological features of peripheral blood cells which were analyzed by optical microscopy, and 3 differential absolute count numbers obtained based on the corresponding differential percentages and absolute count of blood cells. The parameters were compared among the subcategories and a value of P<0.05 was considered to indicate a statistically significant difference. The median white blood cell and hemoglobin counts of the patients were 18.0×109/l and 88 g/l, respectively. The proportion of monocytes increased to 8% (1.82×109/l), the proportion of blast cells increased to 1% (0.5×109/l) and that of neutrophil precursors increased to 10% (1.98×109/l). A total of 87% of all patients presented with hypogranulation and 71% presented with abnormal condensed nuclear chromatin in granulocytes. Atypical monocytes were observed in 73% of all patients and Pseudo-Pelger cells were observed in 60%. Significant differences were detected among the subcategories. The present study demonstrated that combining blood routine parameters and the morphological analysis of peripheral blood cells have an essential role in the reliable diagnosis of MDS/MPN based on WHO categories. PMID:27895799

  9. 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.

  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.

  11. Reconstitution of microtubule-dependent organelle transport.

    PubMed

    Barak, Pradeep; Rai, Ashim; Dubey, Alok Kumar; Rai, Priyanka; Mallik, Roop

    2014-01-01

    Microtubule (MT)-based motor proteins transport many cellular factors to their functionally relevant locations within cells, and defects in transport are linked to human disease. Understanding the mechanism and regulation of this transport process in living cells is difficult because of the complex in vivo environment and limited means to manipulate the system. On the other hand, in vitro motility assays using purified motors attached to beads does not recapitulate the full complexity of cargo transport in vivo. Assaying motility of organelles in cell extracts is therefore attractive, as natural cargoes are being examined, but in an environment that is more amenable to manipulation. Here, we describe the purification and in vitro MT-based motility of phagosomes from Dictyostelium and lipid droplets from rat liver. These assays have the potential to address diverse questions related to endosome/phagosome maturation, fatty acid regulation, and could also serve as a starting point for reconstituting the motility of other types of organelles. © 2014 Elsevier Inc. All rights reserved.

  12. 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

  13. Evolution of apicomplexan secretory organelles

    PubMed Central

    Gubbels, Marc-Jan; Duraisingh, Manoj T.

    2013-01-01

    The alveolate superphylum includes many free-living and parasitic organisms, which are united by the presence of alveolar sacs lying proximal to the plasma membrane, providing cell structure. All species comprising the apicomplexan group of alveolates are parasites and have adapted to the unique requirements of the parasitic lifestyle. Here the evolution of apicomplexan secretory organelles that are involved in the critical process of egress from one cell and invasion of another is explored. The variations within the Apicomplexa and how these relate to species-specific biology will be discussed. In addition, recent studies have identified specific calcium-sensitive molecules that coordinate the various events and regulate the release of these secretory organelles within apicomplexan parasites. Some aspects of this machinery are conserved outside the Apicomplexa, and are beginning to elucidate the conserved nature of the machinery. Briefly, the relationship of this secretion machinery within the Apicomplexa will be discussed, compared with free-living and predatory alveolates, and how these might have evolved from a common ancestor. PMID:23068912

  14. 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

  15. Mechanisms of Polarized Organelle Distribution in Neurons.

    PubMed

    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.

  16. Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons.

    PubMed

    Kaasik, Allen; Safiulina, Dzhamilja; Choubey, Vinay; Kuum, Malle; Zharkovsky, Alexander; Veksler, Vladimir

    2007-11-09

    Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.

  17. The plant organelles database (PODB): a collection of visualized plant organelles and protocols for plant organelle research.

    PubMed

    Mano, Shoji; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nishimura, Mikio

    2008-01-01

    The plant organelles database (PODB; http://podb.nibb.ac.jp/Organellome) was built to promote a comprehensive understanding of organelle dynamics, including organelle function, biogenesis, differentiation, movement and interactions with other organelles. This database consists of three individual parts, the organellome database, the functional analysis database and external links to other databases and homepages. The organellome database provides images of various plant organelles that were visualized with fluorescent and nonfluorescent probes in various tissues of several plant species at different developmental stages. The functional analysis database is a collection of protocols for plant organelle research. External links give access primarily to other databases and Web pages with information on transcriptomes and proteomes. All the data and protocols in the organellome database and the functional analysis database are populated by direct submission of experimentally determined data from plant researchers and can be freely downloaded. Our database promotes the exchange of information between plant organelle researchers for the comprehensive study of the organelle dynamics that support integrated functions in higher plants. We would also appreciate contributions of data and protocols from all plant researchers to maximize the usefulness of the database.

  18. 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. Copyright © 2015 Elsevier GmbH. All rights reserved.

  19. 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.

  20. Organelle acidification: an ancient cellular leak detector.

    PubMed

    Thattai, Mukund

    2017-06-26

    Intracellular membrane-bounded organelles of eukaryotic cells transiently contact the extracellular environment during endocytosis and secretion. Such contacts must be precisely timed to prevent leakage of cargo. I argue that early eukaryotes evolved organelle acidification as a way to detect and prevent leakage.

  1. 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.

  2. Why are most organelle genomes transmitted maternally?

    PubMed

    Greiner, Stephan; Sobanski, Johanna; Bock, Ralph

    2015-01-01

    Why the DNA-containing organelles, chloroplasts, and mitochondria, are inherited maternally is a long standing and unsolved question. However, recent years have seen a paradigm shift, in that the absoluteness of uniparental inheritance is increasingly questioned. Here, we review the field and propose a unifying model for organelle inheritance. We argue that the predominance of the maternal mode is a result of higher mutational load in the paternal gamete. Uniparental inheritance evolved from relaxed organelle inheritance patterns because it avoids the spread of selfish cytoplasmic elements. However, on evolutionary timescales, uniparentally inherited organelles are susceptible to mutational meltdown (Muller's ratchet). To prevent this, fall-back to relaxed inheritance patterns occurs, allowing low levels of sexual organelle recombination. Since sexual organelle recombination is insufficient to mitigate the effects of selfish cytoplasmic elements, various mechanisms for uniparental inheritance then evolve again independently. Organelle inheritance must therefore be seen as an evolutionary unstable trait, with a strong general bias to the uniparental, maternal, mode. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

  3. Why are most organelle genomes transmitted maternally?

    PubMed Central

    Greiner, Stephan; Sobanski, Johanna; Bock, Ralph

    2015-01-01

    Why the DNA-containing organelles, chloroplasts, and mitochondria, are inherited maternally is a long standing and unsolved question. However, recent years have seen a paradigm shift, in that the absoluteness of uniparental inheritance is increasingly questioned. Here, we review the field and propose a unifying model for organelle inheritance. We argue that the predominance of the maternal mode is a result of higher mutational load in the paternal gamete. Uniparental inheritance evolved from relaxed organelle inheritance patterns because it avoids the spread of selfish cytoplasmic elements. However, on evolutionary timescales, uniparentally inherited organelles are susceptible to mutational meltdown (Muller's ratchet). To prevent this, fall-back to relaxed inheritance patterns occurs, allowing low levels of sexual organelle recombination. Since sexual organelle recombination is insufficient to mitigate the effects of selfish cytoplasmic elements, various mechanisms for uniparental inheritance then evolve again independently. Organelle inheritance must therefore be seen as an evolutionary unstable trait, with a strong general bias to the uniparental, maternal, mode. PMID:25302405

  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. Motors, anchors, and connectors: orchestrators of organelle inheritance.

    PubMed

    Knoblach, Barbara; Rachubinski, Richard A

    2015-01-01

    Organelle inheritance is a process whereby organelles are actively distributed between dividing cells at cytokinesis. Much valuable insight into the molecular mechanisms of organelle inheritance has come from the analysis of asymmetrically dividing cells, which transport a portion of their organelles to the bud while retaining another portion in the mother cell. Common principles apply to the inheritance of all organelles, although individual organelles use specific factors for their partitioning. Inheritance factors can be classified as motors, which are required for organelle transport; anchors, which immobilize organelles at distinct cell structures; or connectors, which mediate the attachment of organelles to motors and anchors. Here, we provide an overview of recent advances in the field of organelle inheritance and highlight how motor, anchor, and connector molecules choreograph the segregation of a multicopy organelle, the peroxisome. We also discuss the role of organelle population control in the generation of cellular diversity.

  6. The Upsides and Downsides of Organelle Interconnectivity.

    PubMed

    Gottschling, Daniel E; Nyström, Thomas

    2017-03-23

    Interconnectivity and feedback control are hallmarks of biological systems. This includes communication between organelles, which allows them to function and adapt to changing cellular environments. While the specific mechanisms for all communications remain opaque, unraveling the wiring of organelle networks is critical to understand how biological systems are built and why they might collapse, as occurs in aging. A comprehensive understanding of all the routes involved in inter-organelle communication is still lacking, but important themes are beginning to emerge, primarily in budding yeast. These routes are reviewed here in the context of sub-system proteostasis and complex adaptive systems theory. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. 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.

  8. Implications of mutation of organelle genomes for organelle function and evolution.

    PubMed

    Raven, John A

    2015-09-01

    Organelle genomes undergo more variation, including that resulting from damage, than eukaryotic nuclear genomes, or bacterial genomes, under the same conditions. Recent advances in characterizing the changes to genomes of chloroplasts and mitochondria of Zea mays should, when applied more widely, help our understanding of how damage to organelle genomes relates to how organelle function is maintained through the life of individuals and in succeeding generations. Understanding of the degree of variation in the changes to organelle DNA and its repair among photosynthetic organisms might help to explain the variations in the rate of nucleotide substitution among organelle genomes. Further studies of organelle DNA variation, including that due to damage and its repair might also help us to understand why the extent of DNA turnover in the organelles is so much greater than that in their bacterial (cyanobacteria for chloroplasts, proteobacteria for mitochondria) relatives with similar rates of production of DNA-damaging reactive oxygen species. Finally, from the available data, even the longest-lived organelle-encoded proteins, and the RNAs needed for their synthesis, are unlikely to maintain organelle function for much more than a week after the complete loss of organelle DNA. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  9. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. Morphological examination of the intraorbital muscles (musculi bulbi) in Persian cats in the perinatal period.

    PubMed

    Klećkowska, J; Pospieszny, N

    2005-02-01

    The study involved 34 fetuses of Persian cats from eight uteruses, gestational day 58. The intraorbital muscles were analysed morphologically in respect of the location of the recti muscles, oblique muscles and retractor bulbi muscle, their length and breadth, the distance of the muscle tendon insertions from the corneal limbus and the line of insertions.

  12. 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.

  13. Modelling organelle transport after traumatic axonal injury.

    PubMed

    Kuznetsov, I A; Kuznetsov, A V

    2015-01-01

    This paper is motivated by recent experimental research (Tang-Schomer et al. 2012) on the formation of periodic varicosities in axons after traumatic brain injury (TBI). TBI leads to the formation of undulated distortions in the axons due to their dynamic deformation. These distortions result in the breakage of some microtubules (MTs) near the peaks of undulations. The breakage is followed by catastrophic MT depolymerisation around the broken ends. Although after relaxation axons regain their straight geometry, the structure of the axon after TBI is characterised by the presence of periodic regions where the density of MTs has been decreased due to depolymerisation. We modelled organelle transport in an axon segment with such a damaged MT structure and investigated how this structure affects the distributions of organelle concentrations and fluxes. The modelling results suggest that organelles accumulate at the boundaries of the region where the density of MTs has been decreased by depolymerisation. According to the model, the presence of such damaged regions decreases the organelle flux by only about 12%. This provides evidence that axon degradation after TBI may be caused by organelle accumulation rather than by starvation due to insufficient organelle flux.

  14. Optogenetic control of organelle transport and positioning

    PubMed Central

    Hoogenraad, Casper C.; Kapitein, Lukas C.

    2016-01-01

    Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signaling, polarization, and growth1–8. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here, we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhances growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to directly explore site-specific organelle functions in different model systems. PMID:25561173

  15. 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

  16. 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.

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

    DOE PAGES

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

  18. 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

  19. Organelle membranes from germinating castro bean endosperm

    SciTech Connect

    Donaldson, R.P.; Tully, R.E.; Young, O.A.; Beevers, H.

    1981-01-01

    Glyoxysome ghosts were isolated from germinating castor bean endosperms using established methods. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. Two cytochrome reductases and phosphorylcholine glyceride transferase co-sedimented with the alkaline lipase, a known component of the glyoxysome membrane, in sucrose gradient centrifugation of osmotically shocked glyoxysomes. The activities of these enzymes in the glyoxysome membranes were compared to those in the endoplasmic reticulum relative to phospholipid content. On this basis, the phosphorylcholine glyceride transferase was 10-fold more active in the endoplasmic reticulum, whereas the lipase was 50-fold more active in the glyoxysome membrane. The cytochrome reductases were only 2-fold more active in the endoplasmic reticulum, indicating that they are components of the two membranes. Difference spectroscopy of the glyoxysome membrane suspension revealed the presence of a b5-type cytochrome similar to that found in the endoplasmic reticulum. Since the glyoxysome membrane is apparently derived from the endoplasmic reticulum, components of the endoplasmic reticulum such as these are likely to be incorporated into the glyoxysome membrane during biogenesis. Enzyme activites involving the cofactors NADH or CoA were measurable in broken, but not in intact, glyoxysomes. Thus, it appears that cofactors for enzymes within the organelle cannot pass through the membrane.

  20. 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

  1. [The morphological examination in the differential diagnosis of tuberculosis and sarcoidosis].

    PubMed

    Dvorkovskaia, I V; Maĭskaia, M Iu; Nasyrov, R A; Baranova, O P; Ariel', B M

    2014-01-01

    The paper considers the results of histological and bacterioscopic examinations of biopsy specimens from the lung and mediastinal lymph nodes of 120 patients with an unclear diagnosis of tuberculosis and sarcoidosis and with problem of their differential diagnosis. One hundred and five of these patients were microscopically diagnosed as having either tuberculosis or sarcoidosis. Additional examination of differently stained sections and that using immunohistochemical M. tuberculosis detection were conducted in 15 cases, which could diagnose tuberculosis and sarcoidosis in 7 and 8 patients, respectively. Bacterioscopic examination, in terms of extreme variability the phenotypic properties of mycobacteria, is of decisive importance in the differential diagnosis of tuberculosis and sarcoidosis.

  2. Morphological Examination and Transcriptomic Profiling To Identify Prednisolone Treatment in Beef Cattle.

    PubMed

    Cannizzo, Francesca T; Pegolo, Sara; Pregel, Paola; Manuali, Elisabetta; Salamida, Sonia; Divari, Sara; Scaglione, Frine E; Bollo, Enrico; Biolatti, Bartolomeo; Bargelloni, Luca

    2016-11-09

    In livestock production corticosteroids are licensed only for therapy; nevertheless, they are often illegally used as growth promoters. The aim of this study was to identify morphological or biomolecular alterations induced by prednisolone (PDN) in experimentally treated beef cattle, because PDN and its metabolites are no longer detectable by LC-MS/MS methods in biological fluids. Moreover, PDN does not induce any histological alterations in the thymus, different from dexamethasone treatments. Therefore, a marker of illicit treatment for this growth promoter could be useful. Eight male Italian Friesian beef cattle were administered prednisolone acetate 30 mg day(-1) per os for 35 days, and seven beef cattle represented the control group. Six days after drug withdrawal, the animals were slaughtered. Morphological and morphometric modifications were evaluated in the epididymis and testis, whereas transcriptomic changes induced by PDN administration were investigated in peripheral blood mononuclear cells (PBMCs) at different sampling times and in skeletal muscle and testis sampled at slaughtering. In the epididymis, spermatozoa number decreased in PDN-treated animals, and in some cases they were totally absent. Correspondingly, in the testis of treated animals, down-regulation for serine/threonine kinase 11 (STK11) gene expression was detected (p < 0.01). DNA microarray analysis revealed a total of 133 differentially expressed genes in skeletal muscle and testis, and 907 and 1416 in PBMCs after 33 days of treatment and at slaughtering, respectively. Histological investigations on epididymal content could represent a promising marker for PDN treatment in beef cattle and could be used as a screening method to identify animals worthy of further investigation with official methods. Moreover, the clear transcriptomic signature of PDN treatment evidenced in PBMCs supported the possibility of using this matrix to monitor the illicit treatment in vivo during ranching.

  3. Probing and tracking organelles in living plant cells.

    PubMed

    Chen, Tong; Wang, Xiaohua; von Wangenheim, Daniel; Zheng, Maozhong; Šamaj, Jozef; Ji, Wanquan; Lin, Jinxing

    2012-06-01

    Intracellular organelle movements and positioning play pivotal roles in enabling plants to proliferate life efficiently and to survive diverse environmental stresses. The elaborate dissection of organelle dynamics and their underlying mechanisms (e.g., the role of the cytoskeleton in organelle movements) largely depends on the advancement and efficiency of organelle tracking systems. Here, we provide an overview of some recently developed tools for labeling and tracking organelle dynamics in living plant cells.

  4. Calcium regulation in endosymbiotic organelles of plants.

    PubMed

    Bussemer, Johanna; Vothknecht, Ute C; Chigri, Fatima

    2009-09-01

    In plant cells calcium-dependent signaling pathways are involved in a large array of biological processes in response to hormones, biotic/abiotic stress signals and a variety of developmental cues. This is generally achieved through binding of calcium to diverse calcium-sensing proteins, which subsequently control downstream events by activating or inhibiting biochemical reactions. Regulation by calcium is considered as a eukaryotic trait and has not been described for prokaryotes. Nevertheless, there is increasing evidence indicating that organelles of prokaryotic origin, such as chloroplasts and mitochondria, are integrated into the calcium-signaling network of the cell. An important transducer of calcium in these organelles appears to be calmodulin. In this review we want to give an overview over present data showing that endosymbiotic organelles harbour calcium-dependent biological processes with a focus on calmodulin-regulation.

  5. On the move: organelle dynamics during mitosis.

    PubMed

    Jongsma, Marlieke L M; Berlin, Ilana; Neefjes, Jacques

    2015-03-01

    A cell constitutes the minimal self-replicating unit of all organisms, programmed to propagate its genome as it proceeds through mitotic cell division. The molecular processes entrusted with ensuring high fidelity of DNA replication and subsequent segregation of chromosomes between daughter cells have therefore been studied extensively. However, to process the information encoded in its genome a cell must also pass on its non-genomic identity to future generations. To achieve productive sharing of intracellular organelles, cells have evolved complex mechanisms of organelle inheritance. Many membranous compartments undergo vast spatiotemporal rearrangements throughout mitosis. These controlled organizational changes are crucial to enabling completion of the division cycle and ensuring successful progeny. Herein we review current understanding of intracellular organelle segregation during mitotic division in mammalian cells, with a focus on compartment organization and integrity throughout the inheritance process. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Harnessing yeast organelles for metabolic engineering.

    PubMed

    Hammer, Sarah K; Avalos, José L

    2017-08-01

    Each subcellular compartment in yeast offers a unique physiochemical environment and metabolite, enzyme, and cofactor composition. While yeast metabolic engineering has focused on assembling pathways in the cell cytosol, there is growing interest in embracing subcellular compartmentalization. Beyond harnessing distinct organelle properties, physical separation of organelles from the cytosol has the potential to eliminate metabolic crosstalk and enhance compartmentalized pathway efficiency. In this Perspective we review the state of the art in yeast subcellular engineering, highlighting the benefits of targeting biosynthetic pathways to subcellular compartments, including mitochondria, peroxisomes, the ER and/or Golgi, vacuoles, and the cell wall, in different yeast species. We compare the performances of strains developed with subcellular engineering to those of native producers or yeast strains previously engineered with cytosolic pathways. We also identify important challenges that lie ahead, which need to be addressed for organelle engineering to become as mainstream as cytosolic engineering in academia and industry.

  7. Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

    PubMed Central

    Cho, Moon-Il; Holt, Stanley C.; Iacono, Vincent J.; Pollock, Jerry J.

    1982-01-01

    The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short

  8. Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

    PubMed Central

    1987-01-01

    We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Peroxisome morphology in pathology.

    PubMed

    Ribeiro, D; Castro, I; Fahimi, H D; Schrader, M

    2012-06-01

    Peroxisomes are remarkably dynamic and versatile organelles that are essential for human health and development. They respond to physiological changes in the cellular environment by adapting their morphology, number, enzyme content and metabolic functions accordingly. With the discovery of the first key peroxisomal morphology proteins, the investigation of peroxisomal shape, distribution and dynamics has become an exciting new field in cell biology and biomedical sciences because of its relation to organelle functionality and its impact on developmental and physiological processes. In this review, we summarize recent findings on peroxisome biology, dynamics and the modulation of peroxisome morphology, especially in mammals. Furthermore, we discuss the roles of peroxisome dynamics and morphology in cell pathology and present recent examples for alterations in peroxisome morphology under disease conditions. Besides defects in the peroxisomal morphology machinery, we also address peroxisome biogenesis disorders, alterations of peroxisome number during carcinogenesis and liver cirrhosis, and morphological alterations of peroxisomes during viral infection.

  14. Autophagy, apoptosis and organelle features during cell exposure to cadmium.

    PubMed

    Vergilio, Cristiane dos Santos; de Melo, Edésio José Tenório

    2013-08-01

    Cadmium (Cd) induces several effects in different tissues, but our knowledge of the toxic effects on organelles is insufficient. To observe the progression of Cd effects on organelle structure and function, HuH-7 cells (human hepatic carcinoma cell line) were exposed to CdCl2 in increasing concentrations (1 microM - 20 microM) and exposure times (2 h - 24 h). During Cd treatment, the cells exhibited a progressive decrease in viability that was both time- and dose-dependent. Cd treated cells displayed progressive morphological changes that included cytoplasm retraction and nuclear condensation preceding a total loss of cell adhesion. Treatment with 10 microM for 12 h led to irreversible damages. Before these drastic and irreparable damages, treated cells (5 microM for 12 h) presented a progressive loss of mitochondrial function and cytoplasm acidification as well as dysfunction and disorganization of microfilaments and endoplasmic reticulum. These damages led to the induction of apoptotic events and an increase in autophagic bodies in the cytoplasm. These results revealed that Cd affects multiple intra-cellular targets that induce alterations in the mitochondria, cytoskeleton, endoplasmic reticulum and acidic compartments, ultimately culminating in cell death via apoptotic and autophagic pathways.

  15. Plant organelle proteomics: collaborating for optimal cell function.

    PubMed

    Agrawal, Ganesh Kumar; Bourguignon, Jacques; Rolland, Norbert; Ephritikhine, Geneviève; Ferro, Myriam; Jaquinod, Michel; Alexiou, Konstantinos G; Chardot, Thierry; Chakraborty, Niranjan; Jolivet, Pascale; Doonan, John H; Rakwal, Randeep

    2011-01-01

    Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear-encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter-plant and inter-tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in-depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in

  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. A workflow for the automatic segmentation of organelles in electron microscopy image stacks

    PubMed Central

    Perez, Alex J.; Seyedhosseini, Mojtaba; Deerinck, Thomas J.; Bushong, Eric A.; Panda, Satchidananda; Tasdizen, Tolga; Ellisman, Mark H.

    2014-01-01

    Electron microscopy (EM) facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson's and Alzheimer's diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM). Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime. PMID:25426032

  18. Organelles - understanding noise and heterogeneity in cell biology at an intermediate scale.

    PubMed

    Chang, Amy Y; Marshall, Wallace F

    2017-03-01

    Many studies over the years have shown that non-genetic mechanisms for producing cell-to-cell variation can lead to highly variable behaviors across genetically identical populations of cells. Most work to date has focused on gene expression noise as the primary source of phenotypic heterogeneity, yet other sources may also contribute. In this Commentary, we explore organelle-level heterogeneity as a potential secondary source of cellular 'noise' that contributes to phenotypic heterogeneity. We explore mechanisms for generating organelle heterogeneity and present evidence of functional links between organelle morphology and cellular behavior. Given the many instances in which molecular-level heterogeneity has been linked to phenotypic heterogeneity, we posit that organelle heterogeneity may similarly contribute to overall phenotypic heterogeneity and underline the importance of studying organelle heterogeneity to develop a more comprehensive understanding of phenotypic heterogeneity. Finally, we conclude with a discussion of the medical challenges associated with phenotypic heterogeneity and outline how improved methods for characterizing and controlling this heterogeneity may lead to improved therapeutic strategies and outcomes for patients. © 2017. Published by The Company of Biologists Ltd.

  19. Review on recent advances in the analysis of isolated organelles.

    PubMed

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

    2012-11-13

    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. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. 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

  1. How cells know the size of their organelles.

    PubMed

    Chan, Yee-Hung M; Marshall, Wallace F

    2012-09-07

    Cells have developed ways to sense and control the size of their organelles. Size-sensing mechanisms range from direct measurements provided by dedicated reporters to indirect functional readouts, and they are used to modify organelle size under both normal and stress conditions. Organelle size can also be controlled in the absence of an identifiable size sensor. Studies on flagella have dissected principles of size sensing and control, and it will be exciting to see how these principles apply to other organelles.

  2. Electron tomography for organelles, cells, and tissues.

    PubMed

    He, Wanzhong; He, Yongning

    2014-01-01

    Electron tomography (ET) is an emerging electron microscopy (EM) technique for three-dimensional (3D) visualization of molecular arrangements and ultrastructural architectures in organelles, cells, and tissues at 2-10 nm resolution. The 3D tomogram is reconstructed from a series of 2D EM images taken from a single specimen at different projecting orientations. The specimen for ET must be specially prepared to meet the ET imaging requirements, i.e., ultrastructural preservation, specimen thickness, tolerance of electron dose and vacuum, and image contrast. In this chapter, the strategies of specimen preparation of organelles, cells, and tissues and the corresponding EM imaging requirements for ET will be described in detail. In addition, the general procedures tomographic reconstruction and tomogram interpretation will be described.

  3. The peroxisome: still a mysterious organelle

    PubMed Central

    Fahimi, H. Dariush

    2008-01-01

    More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed. PMID:18274771

  4. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

    Kustatscher, Georg; Hégarat, Nadia; Wills, Karen L H; Furlan, Cristina; Bukowski-Wills, Jimi-Carlo; Hochegger, Helfrid; Rappsilber, Juri

    2014-01-01

    Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. PMID:24534090

  5. 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.

  6. Organelle-specific Hsp90 inhibitors.

    PubMed

    Seo, Young Ho

    2015-09-01

    Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is involved in the folding, activation, and stabilization of numerous oncogenic proteins. It has become an attractive therapeutic target, especially for eradicating malignant cancers and overcoming chemotherapy resistance. The Hsp90 family in mammalian cells is composed of four major homologs: Hsp90α, Hsp90β, 94-kDa glucose-regulated protein (Grp94), and TNF receptor-associated protein 1 (Trap1). Hsp90α and Hsp90β are mainly localized in the cytoplasm, while Grp94 and Trap1 reside in the endoplasmic reticulum and the mitochondria, respectively. Additionally, some Hsp90 s are secreted from the cytoplasm, commonly called extracellular Hsp90. Interestingly, each Hsp90 isoform is localized in a particular organelle, possesses a unique biological function, and participates in various physiological and pathological processes. To inhibit the organelle-specific Hsp90 chaperone function, there have been significant efforts to accumulate Hsp90 inhibitors in particular cellular compartments. This review introduces current studies regarding the delivery of Hsp90 inhibitors to subcellular organelles, particularly to the extracellular matrix and the mitochondria, and discusses their biological insights and therapeutic implications.

  7. Organelle size equalization by a constitutive process.

    PubMed

    Ludington, William B; Shi, Linda Z; Zhu, Qingyuan; Berns, Michael W; Marshall, Wallace F

    2012-11-20

    How cells control organelle size is an elusive problem. Two predominant models for size control can be distinguished: (1) induced control, where organelle genesis, maintenance, and disassembly are three separate programs that are activated in response to size change, and (2) constitutive control, where stable size results from the balance between continuous organelle assembly and disassembly. The problem has been studied in Chlamydomonas reinhardtii because the flagella are easy to measure, their size changes only in the length dimension, and the genetics are comparable to yeast. Length dynamics in Chlamydomonas flagella are quite robust: they maintain a length of about 12 μm and recover from amputation in about 90 min with a growth rate that decreases smoothly to zero as the length approaches 12 μm. Despite a wealth of experimental studies, existing data are consistent with both induced and constitutive control models for flagella. Here we developed novel microfluidic trapping and laser microsurgery techniques in Chlamydomonas to distinguish between length control models by measuring the two flagella on a single cell as they equilibrate after amputation of a single flagellum. The results suggest that cells equalize flagellar length by constitutive control.

  8. Morphology of the lumbar transversospinal muscles examined in a mouse bearing a muscle fiber-specific nuclear marker.

    PubMed

    Cornwall, Jon; Deries, Marianne; Duxson, Marilyn

    2010-12-01

    Although the morphology of human lumbar transversospinal (TSP) muscles has been studied, little is known about the structure of these muscles in the mouse (Mus musculus). Such information is relevant given mice are often used as a "normal" phenotype for studies modeling human development. This study describes the gross morphology, muscle fiber arrangement, and innervation pattern of the mouse lumbar TSP muscles. A unique feature of the study is the use of a transgenic mouse line bearing a muscle-specific nuclear marker that allows clear delineation of muscle fiber and connective tissue boundaries. The lumbar TSP muscles of five mice were examined bilaterally; at each spinal level muscles attached to the caudal edge of the spinous process and passed caudally as a single complex unit. Fibers progressively terminated over the four vertebral segments caudad, with multiple points of muscle fiber attachment on each vertebra. Motor endplates, defined with acetylcholinesterase histochemistry, were consistently located half way along each muscle fiber, regardless of length, with all muscle fibers arranged in-parallel rather than in-series. These results provide information relevant to interpretation of developmental and functional studies involving this muscle group in the mouse and show mouse lumbar TSP muscles are different in form to descriptions of equivalent muscles in humans and horses.

  9. Examination of the morphology of bacteria adhering to peritoneal dialysis catheters by scanning and transmission electron microscopy.

    PubMed Central

    Marrie, T J; Noble, M A; Costerton, J W

    1983-01-01

    We examined Tenckhoff peritoneal catheters by scanning and transmission electron microscopy to study the morphology of bacterial adherence. Two catheters were removed from uninfected patients, three from patients with exit site infections, four from patients with peritonitis, and one from a patient with both exit site infection and peritonitis. Infecting organisms included three of Staphylococcus aureus and one each of Enterobacter sp., Staphylococcus epidermidis, Achromobacter xylosoxidans, Serratia sp., Klebsiella sp., and Candida albicans. Considerable morphological variation in adherence to the peritoneal dialysis apparatus occurred. No inflammatory cells were ever seen in association with infected cuffs, only two of the five patients with peritonitis had inflammatory cells associated with their catheters. In both instances, these cells tended to occur in clumps and demonstrated no flattening when in contact with the surface. Colonization of the catheter was uneven--bacteria tended to occur in clusters. Extensive matrix formation was evident in several instances, and condensation of this matrix onto the bacteria during the dehydration process rendered clumps of bacterial cells amorphous at times. Bacteria were adherent to the subcutaneous cuff in those patients with exit site infections. Gram-negative bacteria attached to individual dacron fibers of the cuff, often several layers deep. Gram-positive bacteria tended to adhere in clusters. Images PMID:6228562

  10. Morphological examination of highly porous polylactic acid/Bioglass(®) scaffolds produced via nonsolvent induced phase separation.

    PubMed

    Rezabeigi, Ehsan; Wood-Adams, Paula M; Drew, Robin A L

    2016-09-19

    In this study, we produce highly porous (up to ∼91%) composite scaffolds of polylactic acid (PLA) containing 2 wt % sol-gel-derived 45S5 Bioglass(®) particles via nonsolvent induced phase separation at -23°C with no sacrificial phases involved. Before the incorporation of the bioglass with PLA, the particles are surface modified with a silane coupling agent which effectively diminishes agglomeration between them leading to a better dispersion of bioactive particles throughout the scaffold. Interestingly, the incorporation route (via solvent dichloromethane or nonsolvent hexane) of the surface modified particles in the foaming process has the greatest impact on porosity, crystallinity, and morphology of the scaffolds. The composite scaffolds with a morphology consisting of both mesopores and large macropores, which is potentially beneficial for bone regeneration applications, are examined further. SEM images show that the surface modified bioglass particles take-up a unique configuration within the mesoporous structure of these scaffolds ensuring that the particles are well interlocked but not completely covered by PLA such that they can be in contact with physiological fluids. The results of preliminary in vitro tests confirm that this PLA/bioglass configuration promotes the interaction of the bioactive phase with physiological fluids. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

  11. 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.

  12. Renaissance of morphological studies: the examination of functional structures in living animal organs using the in vivo cryotechnique.

    PubMed

    Ohno, Shinichi; Saitoh, Yurika; Ohno, Nobuhiko; Terada, Nobuo

    2017-01-01

    Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living animal organs, as well as the locations of their molecular components. Recently, the live imaging of animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living animal organs are compatible with those obtained from live images of the same organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living animal organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components-such as rapidly changing signaling molecules, membrane channels, or receptors-in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.

  13. Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells.

    PubMed

    Tutak, Wojtek; Jyotsnendu, Giri; Bajcsy, Peter; Simon, Carl G

    2017-07-01

    Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl

  14. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Organelle segregation during mitosis: lessons from asymmetrically dividing cells.

    PubMed

    Ouellet, Jimmy; Barral, Yves

    2012-02-06

    Studies on cell division traditionally focus on the mechanisms of chromosome segregation and cytokinesis, yet we know comparatively little about how organelles segregate. Analysis of organelle partitioning in asymmetrically dividing cells has provided insights into the mechanisms through which cells control organelle distribution. Interestingly, these studies have revealed that segregation mechanisms frequently link organelle distribution to organelle growth and formation. Furthermore, in many cases, cells use organelles, such as the endoplasmic reticulum and P granules, as vectors for the segregation of information. Together, these emerging data suggest that the coordination between organelle growth, division, and segregation plays an important role in the control of cell fate inheritance, cellular aging, and rejuvenation, i.e., the resetting of age in immortal lineages. © 2012 Ouellet and Barral

  16. Dynamic shape changes of cytoplasmic organelles translocating along microtubules

    PubMed Central

    1987-01-01

    Transient shape changes of organelles translocating along microtubules are directly visualized in thinly spread cytoplasmic processes of the marine foraminifer. Allogromia laticollaris, by a combination of high- resolution video-enhanced microscopy and fast-freezing electron microscopy. The interacting side of the organelle flattens upon binding to a microtubule, as if to maximize contact with it. Organelles typically assume a teardrop shape while moving, as if they were dragged through a viscous medium. Associated microtubules bend around attachments of the teardrop-shaped organelles, suggesting that they too are acted on by the forces deforming the organelles. An 18-nm gap between the organelles and the microtubules is periodically bridged by 10-nm-thick cross-bridge structures that may be responsible for the binding and motive forces deforming organelles and microtubules. PMID:3654751

  17. Dynamic shape changes of cytoplasmic organelles translocating along microtubules.

    PubMed

    Kachar, B; Bridgman, P C; Reese, T S

    1987-09-01

    Transient shape changes of organelles translocating along microtubules are directly visualized in thinly spread cytoplasmic processes of the marine foraminifer. Allogromia laticollaris, by a combination of high-resolution video-enhanced microscopy and fast-freezing electron microscopy. The interacting side of the organelle flattens upon binding to a microtubule, as if to maximize contact with it. Organelles typically assume a teardrop shape while moving, as if they were dragged through a viscous medium. Associated microtubules bend around attachments of the teardrop-shaped organelles, suggesting that they too are acted on by the forces deforming the organelles. An 18-nm gap between the organelles and the microtubules is periodically bridged by 10-nm-thick cross-bridge structures that may be responsible for the binding and motive forces deforming organelles and microtubules.

  18. The cytoskeleton maintains organelle partitioning required for single-cell C4 photosynthesis in Chenopodiaceae species.

    PubMed

    Chuong, Simon D X; Franceschi, Vincent R; Edwards, Gerald E

    2006-09-01

    Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C(4) photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein-tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm.

  19. Clinal variation in a brown lemur (Eulemur spp.) hybrid zone: combining morphological, genetic and climatic data to examine stability.

    PubMed

    Delmore, K E; Brenneman, R A; Lei, R; Bailey, C A; Brelsford, A; Louis, E E; Johnson, S E

    2013-08-01

    Studies of hybrid zones can inform our understanding of reproductive isolation and speciation. Two species of brown lemur (Eulemur rufifrons and E. cinereiceps) form an apparently stable hybrid zone in the Andringitra region of southeastern Madagascar. The aim of this study was to identify factors that contribute to this stability. We sampled animals at 11 sites along a 90-km transect through the hybrid zone and examined variation in 26 microsatellites, the D-loop region of mitochondrial DNA, six pelage and nine morphological traits; we also included samples collected in more distant allopatric sites. Clines in these traits were noncoincident, and there was no increase in either inbreeding coefficients or linkage disequilibrium at the centre of the zone. These results could suggest that the hybrid zone is maintained by weak selection against hybrids, conforming to either the tension zone or geographical selection-gradient model. However, a closer examination of clines in pelage and microsatellites indicates that these clines are not sigmoid or stepped in shape but instead plateau at their centre. Sites within the hybrid zone also occur in a distinct habitat, characterized by greater seasonality in precipitation and lower seasonality in temperature. Together, these findings suggest that the hybrid zone may follow the bounded superiority model, with exogenous selection favouring hybrids within the transitional zone. These findings are noteworthy, as examples supporting the bounded superiority model are rare and may indicate a process of ecologically driven speciation without geographical isolation.

  20. The Influence of Reserpine and Ethylenediaminetetraacetic Acid (EDTA) on Serotonin Storage Organelles of Blood Platelets

    PubMed Central

    Gerrard, Jonathan M.; Rao, Gundu H. R.; White, James G.

    1977-01-01

    The present investigation has evaluated the influence of reserpine on the serotonin-rich organelles bodies) in platelets from dogs, rabbits, and humans. Reserpine markedly depresses the levels of stored serotonin in human and animal platelets, accompanied by a small decrease in platelet ATP but no change in platelet ADP content. Thin sections of human platelets showed no change in the number or morphology of serotonin storage organelles during reserpine therapy, whereas a profound decrease in the size and number of dense bodies occurred in platelets from rabbits treated with reserpine. Dog platelets also showed a decrease in the number and density of serotonin storage organelles after reserpine therapy. The basis for the difference between rabbit and human platelets was explored by fixing platelets in glutaraldehyde and osmium in the presence or absence of the chelating agent ethylenediaminetetraacetic acid (EDTA). Most of the dense bodies in fixed human platelets were removed by EDTA while rabbit platelet dense bodies remained essentially intact. The results suggested that the opacity of rabbit platelet dense bodies following fixation with glutaraldehyde and osmium relate primarily to their serotonin content, while the electron density of human serotonin storage organelles in fixed cells is due primarily to their calcium content. Further confirmation of this concept came from studies of platelets using the whole mount technique. Rabbit platelet serotonin storage organelles were found to lack the inherent opacity of the human dense bodies, a finding consistent with the lower concentration of calcium in the rabbit organelles. ImagesFigures 1A-DFigure 2Figure 3Figure 4Figures 5 and 6Figure 7Figure 8 PMID:405872

  1. Prokaryotic cells: structural organisation of the cytoskeleton and organelles.

    PubMed

    Souza, Wanderley de

    2012-05-01

    For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

  2. 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

  3. Sharing the cell's bounty - organelle inheritance in yeast.

    PubMed

    Knoblach, Barbara; Rachubinski, Richard A

    2015-02-15

    Eukaryotic cells replicate and partition their organelles between the mother cell and the daughter cell at cytokinesis. Polarized cells, notably the budding yeast Saccharomyces cerevisiae, are well suited for the study of organelle inheritance, as they facilitate an experimental dissection of organelle transport and retention processes. Much progress has been made in defining the molecular players involved in organelle partitioning in yeast. Each organelle uses a distinct set of factors - motor, anchor and adaptor proteins - that ensures its inheritance by future generations of cells. We propose that all organelles, regardless of origin or copy number, are partitioned by the same fundamental mechanism involving division and segregation. Thus, the mother cell keeps, and the daughter cell receives, their fair and equitable share of organelles. This mechanism of partitioning moreover facilitates the segregation of organelle fragments that are not functionally equivalent. In this Commentary, we describe how this principle of organelle population control affects peroxisomes and other organelles, and outline its implications for yeast life span and rejuvenation. © 2015. Published by The Company of Biologists Ltd.

  4. Fluorescence spectral imaging of organelle interaction

    NASA Astrophysics Data System (ADS)

    Kohen, Elli; Hirschberg, Joseph G.; Kohen, Cahide; Schachtschabel, Dietrich O.; Monti, Marco; Stanikunaite, Rita

    2000-04-01

    In cell biology, one of the great mysteries, which has bene only superficially 8investigate,d is the integration of cytoplasmic and nuclear organelles as part of the intracellular regulatory mechanism. The methodology used for the exploration of such intracellular processes is the pixel-by-pixel scanning by means of fluorescence spectral imaging and excitation emission fluorescence spectroscopy. While several of the steps required by this method are still in the process of implementation, the Michelson interferometer, the Sagnac interferometer and the related 'pentaferometer' are possible components of the instrumental design. One of the illustrative experimental models to begin the study of intracellular integrative processes is based on the hypothesis of a 'nuclear pump' in conjunction with cell treatment by chemotherapeutic agents such as adriamycin. Preliminary observations initiated in cultured fibroblasts, and to be pursued in Cloudman's melanoma cells, suggest that this cytotoxic agent first moves into the nucleus, form which it is subsequently ejected to be incorporated into the lysosomes and Golgi apparatus, possibly prior to exclusion via the multiple drug resistance pathway. The timetable of such a process is under investigation. This subject has obvious implications for diagnostic, prognostic and therapeutic studies of organelles integration.

  5. Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules

    PubMed Central

    Valach, Matus; Burger, Gertraud; Gray, Michael W.; Lang, B. Franz

    2014-01-01

    5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator. PMID:25429974

  6. Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules.

    PubMed

    Valach, Matus; Burger, Gertraud; Gray, Michael W; Lang, B Franz

    2014-12-16

    5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.

    PubMed

    Nelson, Brook K; Cai, Xue; Nebenführ, Andreas

    2007-09-01

    Genome sequencing has resulted in the identification of a large number of uncharacterized genes with unknown functions. It is widely recognized that determination of the intracellular localization of the encoded proteins may aid in identifying their functions. To facilitate these localization experiments, we have generated a series of fluorescent organelle markers based on well-established targeting sequences that can be used for co-localization studies. In particular, this organelle marker set contains indicators for the endoplasmic reticulum, the Golgi apparatus, the tonoplast, peroxisomes, mitochondria, plastids and the plasma membrane. All markers were generated with four different fluorescent proteins (FP) (green, cyan, yellow or red FPs) in two different binary plasmids for kanamycin or glufosinate selection, respectively, to allow for flexible combinations. The labeled organelles displayed characteristic morphologies consistent with previous descriptions that could be used for their positive identification. Determination of the intracellular distribution of three previously uncharacterized proteins demonstrated the usefulness of the markers in testing predicted subcellular localizations. This organelle marker set should be a valuable resource for the plant community for such co-localization studies. In addition, the Arabidopsis organelle marker lines can also be employed in plant cell biology teaching labs to demonstrate the distribution and dynamics of these organelles.

  8. 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…

  9. 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…

  10. Cell-size-dependent control of organelle sizes during development.

    PubMed

    Hara, Yuki; Kimura, Akatsuki

    2011-01-01

    During development, cells differentiate into diverse cell types with different sizes. The size of intracellular organelles often correlates with the size of the cell, which may be important for cell homeostasis. The nucleus is a well-known example of an organelle whose size correlates with cell size. However, the mechanical basis of the correlation is unknown. The lengths of the mitotic spindle and contractile ring are emerging as model system to investigate the cell-size-dependent control mechanisms of organelle size. Mechanistic models are proposed for the cell-size-dependent control of these organelles. Understanding the cell-size dependency of organelle sizes is expected to impact not only on the morphogenesis of the individual organelle, but also on cell homeostasis, cell cycle progression, and cell differentiation.

  11. Right Time, Right Place: Probing the Functions of Organelle Positioning.

    PubMed

    van Bergeijk, Petra; Hoogenraad, Casper C; Kapitein, Lukas C

    2016-02-01

    The proper spatial arrangement of organelles underlies many cellular processes including signaling, polarization, and growth. Despite the importance of local positioning, the precise connection between subcellular localization and organelle function is often not fully understood. To address this, recent studies have developed and employed different strategies to directly manipulate organelle distributions, such as the use of (light-sensitive) heterodimerization to control the interaction between selected organelles and specific motor proteins, adaptor molecules, or anchoring factors. We review here the importance of subcellular localization as well as tools to control local organelle positioning. Because these approaches allow spatiotemporal control of organelle distribution, they will be invaluable tools to unravel local functioning and the mechanisms that control positioning. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. 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.

  13. Isolation of endocitic organelles by density gradient centrifugation.

    PubMed

    de Araùjo, Mariana Eça Guimarães; Huber, Lukas Alfons; Stasyk, Taras

    2008-01-01

    Advanced prefractionation strategies, in combination with highly sensitive and accurate mass spectrometers provide powerful means to detect and analyze low abundant proteins on the subcellular and organelle-specific level. Among enrichment techniques, subcellular fractionation has become the most commonly used. Its application gives access to less complex subproteomes and organelle constituents, facilitating downstream analysis. Furthermore, subcellular fractionation allows the identification of proteins that shuttle between different subcellular compartments in a stimulus dependent manner. As a paradigm of subcellular organelle isolation, we describe here endosomal purification protocols, based on differential centrifugation in continuous and discontinuous sucrose gradients. Described methods can be easily modified to isolate other organelles and are compatible with subsequent organelle- and functional organelle proteome analyses by, e.g., two-dimensional gel electrophoresis.

  14. The inheritance of organelle genes and genomes: patterns and mechanisms.

    PubMed

    Xu, Jianping

    2005-12-01

    Unlike nuclear genes and genomes, the inheritance of organelle genes and genomes does not follow Mendel's laws. In this mini-review, I summarize recent research progress on the patterns and mechanisms of the inheritance of organelle genes and genomes. While most sexual eukaryotes show uniparental inheritance of organelle genes and genomes in some progeny at least part of the time, increasing evidence indicates that strictly uniparental inheritance is rare and that organelle inheritance patterns are very diverse and complex. In contrast with the predominance of uniparental inheritance in multicellular organisms, organelle genes in eukaryotic microorganisms, such as protists, algae, and fungi, typically show a greater diversity of inheritance patterns, with sex-determining loci playing significant roles. The diverse patterns of inheritance are matched by the rich variety of potential mechanisms. Indeed, many factors, both deterministic and stochastic, can influence observed patterns of organelle inheritance. Interestingly, in multicellular organisms, progeny from interspecific crosses seem to exhibit more frequent paternal leakage and biparental organelle genome inheritance than those from intraspecific crosses. The recent observation of a sex-determining gene in the basidiomycete yeast Cryptococcus neoformans, which controls mitochondrial DNA inheritance, has opened up potentially exciting research opportunities for identifying specific molecular genetic pathways that control organelle inheritance, as well as for testing evolutionary hypotheses regarding the prevalence of uniparental inheritance of organelle genes and genomes.

  15. Mitochondria-organelle contact sites: the plot thickens.

    PubMed

    Elbaz-Alon, Yael

    2017-04-15

    Membrane contact sites (MCSs) are areas of close apposition between the membranes of two different organelles that enable non-vesicular transfer of ions and lipids. Recent studies reveal that mitochondria maintain contact sites with organelles other than the endoplasmic reticulum such as the vacuole, plasma membrane and peroxisomes. This review focuses on novel findings achieved mainly in yeast regarding tethers, function and regulation of mitochondria-organelle contact sites. The emerging network of MCSs linking virtually all cellular organelles is highly dynamic and integrated with cellular metabolism.

  16. 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.

  17. The peroxisome as a cell signaling organelle.

    PubMed

    Tripathi, Durga Nand; Walker, Cheryl Lyn

    2016-04-01

    Peroxisomes participate in lipid metabolism, and are a major source of ROS in the cell. Their importance in cellular energy balance and redox homeostasis is well-established, as is the need to maintain peroxisome homeostasis to prevent pathologies associated with too few, or too many, of these organelles. How cells regulate peroxisome number has remained somewhat elusive. Recently, the tumor suppressors ATM and TSC, which regulate mTORC1 signaling, have been localized to peroxisomes. When activated by peroxisomal ROS, ATM signals to TSC to repress mTORC1 signaling and increase autophagic flux in cells, and also phosphorylates the peroxisomal protein PEX 5 to target peroxisomes for selective autophagy (pexophagy), providing a mechanism for regulation of peroxisomal homeostasis using ROS as a rheostat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. The mitochondrial UPR - protecting organelle protein homeostasis.

    PubMed

    Haynes, Cole M; Ron, David

    2010-11-15

    Mitochondria are required for numerous essential metabolic processes including the regulation of apoptosis; therefore, proper maintenance of the mitochondrial proteome is crucial. The protein-folding environment in mitochondria is challenged by organelle architecture, the presence of reactive oxygen species and the difficulties associated with assembly of the electron transport chain, which consists of components encoded by both the mitochondrial and the nuclear genomes. Mitochondria have dedicated molecular chaperones and proteases that promote proper protein folding, complex assembly and quality control. Work in cultured mammalian cells and Caenorhabditis elegans has yielded clues to the mechanisms linking perturbations in the protein-folding environment in the mitochondrial matrix to the expression of nuclear genes encoding mitochondrial proteins. Here, we review the current knowledge of this mitochondrial unfolded protein response (UPR(mt)), compare it with the better understood UPR of the endoplasmic reticulum and highlight its potential impact on development and disease.

  19. Dynamic lipid landscape of picornavirus replication organelles.

    PubMed

    Belov, George A

    2016-08-01

    Picornavirus infection induces rapid reorganization of the cellular membrane architecture and appearance of novel membranous structures associated with the viral RNA replication and virion assembly-replication organelles. Recent studies significantly advanced our understanding of their lipid composition and cellular mechanisms involved in their development. Picornaviruses activate synthesis of both structural and signaling lipids and reroute cellular cholesterol trafficking pathways to create unique membranous domains favoring viral replication. Rapidly replicating picornaviruses rely on posttranslational activation and/or specific recruitment of cellular proteins rather than on modulation of expression of cellular genes to create favorable membrane microenvironment. At the same time picornaviruses demonstrate remarkable adaptability to changes in the lipid landscape which should be taken into account when developing novel antiviral strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Degradation of organelles or specific organelle components via selective autophagy in plant cells.

    PubMed

    Michaeli, Simon; Galili, Gad

    2014-05-05

    Macroautophagy (hereafter referred to as autophagy) is a cellular mechanism dedicated to the degradation and recycling of unnecessary cytosolic components by their removal to the lytic compartment of the cell (the vacuole in plants). Autophagy is generally induced by stresses causing energy deprivation and its operation occurs by special vesicles, termed autophagosomes. Autophagy also operates in a selective manner, recycling specific components, such as organelles, protein aggregates or even specific proteins, and selective autophagy is implicated in both cellular housekeeping and response to stresses. In plants, selective autophagy has recently been shown to degrade mitochondria, plastids and peroxisomes, or organelle components such as the endoplasmic-reticulum (ER) membrane and chloroplast-derived proteins such as Rubisco. This ability places selective-autophagy as a major factor in cellular steady-state maintenance, both under stress and favorable environmental conditions. Here we review the recent advances documented in plants for this cellular process and further discuss its impact on plant physiology.

  1. The effect of elevated intraocular oxygen on organelle degradation in the embryonic chicken lens.

    PubMed

    Bassnett, Steven; McNulty, Richard

    2003-12-01

    In the vertebrate lens, nuclei and other cytoplasmic organelles are degraded in fiber cells situated in the center of the tissue. This is believed to ensure the transparency of the tissue. The mechanism that triggers this process is unknown. We hypothesized that standing gradients of oxygen generated within the tissue may serve as a spatial cue for organelle degradation. To examine this possibility, we incubated fertilized chicken eggs under hyperoxic (50% O(2)) or normoxic (21% O(2)) conditions. Hyperoxic treatment was initiated on the seventh day of embryonic development (E7), five days before organelle degradation normally commences in the lens core. Hyperoxia was maintained until E17. Under normoxic conditions, the partial pressure of oxygen (P(O)) within the vitreous compartment was low. Direct measurement of P(O) using an optode oxygen sensor indicated values of 1.3 kPa and 0.4 kPa for the mid- and anterior vitreous, respectively. Similarly, treatment with pimonidazole, a bio-reductive hypoxia marker, led to the formation of immuno-positive protein adducts within the lens, suggesting that the embryonic lens is chronically hypoxic in situ. Following hyperoxic treatment, vitreous P(O) significantly increased, although pimonidazole staining in the lens was not markedly affected. Confocal microscopy of slices prepared from hyperoxic lenses revealed a significant increase in the size of the lens relative to age-matched normoxic controls. By E13, an organelle-free zone (OFZ) was present in the center of normoxic and hyperoxic lenses. However, in hyperoxic lenses, the OFZ was consistently smaller, and the distance from the lens surface to the border of the OFZ significantly larger, than in normoxic controls. These observations suggest that hyperoxia delays organelle breakdown and are consistent with a model in which hypoxia in the deep cortical layers of the normal lens serves as a trigger for the organelle loss process.

  2. Curvature of Double-Membrane Organelles Generated by Changes in Membrane Size and Composition

    PubMed Central

    Knorr, Roland L.; Dimova, Rumiana; Lipowsky, Reinhard

    2012-01-01

    Transient double-membrane organelles are key players in cellular processes such as autophagy, reproduction, and viral infection. These organelles are formed by the bending and closure of flat, double-membrane sheets. Proteins are believed to be important in these morphological transitions but the underlying mechanism of curvature generation is poorly understood. Here, we describe a novel mechanism for this curvature generation which depends primarily on three membrane properties: the lateral size of the double-membrane sheets, the molecular composition of their highly curved rims, and a possible asymmetry between the two flat faces of the sheets. This mechanism is evolutionary advantageous since it does not require active processes and is readily available even when resources within the cell are restricted as during starvation, which can induce autophagy and sporulation. We identify pathways for protein-assisted regulation of curvature generation, organelle size, direction of bending, and morphology. Our theory also provides a mechanism for the stabilization of large double-membrane sheet-like structures found in the endoplasmic reticulum and in the Golgi cisternae. PMID:22427874

  3. Multicompartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells.

    PubMed

    Godoy-Gallardo, Maria; Labay, Cédric; Trikalitis, Vasileios D; Kempen, Paul J; Larsen, Jannik B; Andresen, Thomas L; Hosta-Rigau, Leticia

    2017-02-13

    Cell organelles are subcellular structures entrapping a set of enzymes to achieve a specific functionality. The incorporation of artificial organelles into cells is a novel medical paradigm which might contribute to the treatment of various cell disorders by replacing malfunctioning organelles. In particular, artificial organelles are expected to be a powerful solution in the context of enzyme replacement therapy since enzymatic malfunction is the primary cause of organelle dysfunction. Although several attempts have been made to encapsulate enzymes within a carrier vehicle, only few intracellularly active artificial organelles have been reported to date and they all consist of single-compartment carriers. However, it is noted that biological organelles consist of multicompartment architectures where enzymatic reactions are executed within distinct subcompartments. Compartmentalization allows for multiple processes to take place in close vicinity and in a parallel manner without the risk of interference or degradation. Here, we report on a subcompartmentalized and intracellularly active carrier, a crucial step for advancing artificial organelles. In particular, we develop and characterize a novel capsosome system, which consists of multiple liposomes and fluorescent gold nanoclusters embedded within a polymer carrier capsule. We subsequently demonstrate that encapsulated enzymes preserve their activity intracellularly, allowing for controlled enzymatic cascade reaction within a host cell.

  4. Organelle growth control through limiting pools of cytoplasmic components.

    PubMed

    Goehring, Nathan W; Hyman, Anthony A

    2012-05-08

    The critical importance of controlling the size and number of intracellular organelles has led to a variety of mechanisms for regulating the formation and growth of cellular structures. In this review, we explore a class of mechanisms for organelle growth control that rely primarily on the cytoplasm as a 'limiting pool' of available material. These mechanisms are based on the idea that, as organelles grow, they incorporate subunits from the cytoplasm. If this subunit pool is limited, organelle growth will lead to depletion of subunits from the cytoplasm. Free subunit concentration therefore provides a measure of the number of incorporated subunits and thus the current size of the organelle. Because organelle growth rates are typically a function of subunit concentration, cytoplasmic depletion links organelle size, free subunit concentration, and growth rates, ensuring that as the organelle grows, its rate of growth slows. Thus, a limiting cytoplasmic pool provides a powerful mechanism for size-dependent regulation of growth without recourse to active mechanisms to measure size or modulate growth rates. Variations of this general idea allow not only for size control, but also cell-size-dependent scaling of cellular structures, coordination of growth between similar structures within a cell, and the enforcement of singularity in structure formation, when only a single copy of a structure is desired. Here, we review several examples of such mechanisms in cellular processes as diverse as centriole duplication, centrosome and nuclear size control, cell polarity, and growth of flagella. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Emergent complexity in Myosin V-based organelle inheritance.

    PubMed

    Mast, Fred D; Rachubinski, Richard A; Dacks, Joel B

    2012-03-01

    How is adaptability generated in a system composed of interacting cellular machineries, each with a separate and functionally critical job to perform? The machinery for organelle inheritance is precisely one such system, requiring coordination between robust and ancient cellular modules, including the cell cycle, cytoskeleton, and organelle biogenesis/identity. Budding yeasts have emerged as powerful models to study these processes, which are critical for cellular survival, propagation, and differentiation, as organelles must compete for access to myosin V motors that travel along polarized actin cables to vectorially deliver bound cargo to the bud. Under the direction of the cell cycle, myosin V motors are recruited to organelles by specific interactions between their carboxyl-terminal globular tail domains and organelle-specific receptors. We used comparative genomics, phylogenetics, and secondary structure modeling to characterize the evolutionary history of these organelle-specific receptors. We find that while some receptors are retained widely across the animals and fungi, others are limited primarily to the Saccharomycetaceae family of budding yeast, with the emergent pattern of a conserved biogenic and inheritance factor often paired with an evolutionarily novel inheritance adaptor. We propose an evolutionary model whereby the emergence of myosin V-based organelle inheritance has utilized mechanisms of paralogy, mutation, and the appearance of pliable evolutionarily novel adaptor proteins. Our findings suggest an overarching evolutionary mechanism for how diverse cargoes compete for a single myosin V motor in organelle transport and detail one system's solution to obtaining evolutionary adaptability amongst constrained cellular modules.

  6. Effects of Cisplatin in Neuroblastoma Rat Cells: Damage to Cellular Organelles

    PubMed Central

    Santin, Giada; Scietti, Luigi; Veneroni, Paola; Barni, Sergio; Bernocchi, Graziella; Bottone, Maria Grazia

    2012-01-01

    Cisplatin (cisPt) is a chemotherapy agent used as a treatment for several types of cancer. The main cytotoxic effect of cisplatin is generally accepted to be DNA damage. Recently, the mechanism by which cisPt generates the cascade of events involved in the apoptotic process has been demonstrated. In particular it has been shown that some organelles are cisPt target and are involved in cell death. This paper aims to describe the morphological and functional changes of the Golgi apparatus and lysosomes during apoptosis induced in neuronal rat cells (B50) by cisplatin. The results obtained show that the cellular organelles are the target of cisPt, so their damage can induce cell death. PMID:22505928

  7. 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

  8. Engineering carbon fixation with artificial protein organelles.

    PubMed

    Giessen, Tobias W; Silver, Pamela A

    2017-08-01

    Based on projections for global population growth, current techniques for improving agricultural yields will not be able to address future demands for major food crops. Improving photosynthetic efficiency by engineering carbon fixation has been identified as one of the most important approaches for increasing agricultural output. Recent studies indicate that introducing cyanobacterial-like carbon concentrating mechanisms (CCMs) into plant chloroplasts represents a promising strategy for enhancing plant photosynthesis. Here, we give a general outline for transferring CCMs to plants. The proposed trajectory includes introducing bicarbonate transporters and CO2-fixing organelles into plant chloroplasts as well as minimizing stromal carbonic anhydrase (CA) activity. We focus on different approaches for constructing compartments that co-localize the CO2-fixing enzyme d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and CA, aimed at increasing RuBisCO turnover and decreasing wasteful photorespiration. We consider strategies based on cyanobacterial carboxysomes and on other protein-based compartments, specifically encapsulin nanocompartments. Finally, recent advances in expressing catalytic and structural carboxysomal components in plants will be highlighted. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable.

    PubMed

    Schliwa, M; Shimizu, T; Vale, R D; Euteneuer, U

    1991-03-01

    Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.

  10. Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.

    PubMed

    Niclas, J; Allan, V J; Vale, R D

    1996-05-01

    Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.

  11. Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable

    PubMed Central

    1991-01-01

    Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement. PMID:1825662

  12. [Persistent thrombocytopenia in a child: morphological examination of blood platelets established the diagnosis of Wiskott-Aldrich syndrome].

    PubMed

    Latger-Cannard, V; Lacroix, F; Devignes, J; Salignac, S; Bensoussan, D; Salmon, A; Mansuy, L; Bordigoni, P; Lecompte, T

    2008-01-01

    Thrombocytopenia frequently occurs in laboratory practice. The present work illustrates, through the presentation of a case report of Wiskott-Aldrich syndrome, the difficulties encountered to identify and characterize thrombocytopenia. The clinicobiological validation of a low platelet count involves both the biologist, who must assume the validation of numeration while mentioning the morphological characteristics of the platelets and other blood cells, as well as the physician who has to interpret these data according to the clinical context.

  13. 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.

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

    PubMed

    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.

  15. Peroxisomes as dynamic organelles: peroxisome abundance in yeast.

    PubMed

    Saraya, Ruchi; Veenhuis, Marten; van der Klei, Ida J

    2010-08-01

    Peroxisomes are cell organelles that are present in almost all eukaryotic cells and involved in a large range of metabolic pathways. The organelles are highly dynamic in nature: their number and enzyme content is highly variable and continuously adapts to prevailing environmental conditions. This review summarizes recent relevant developments in research on processes that are involved in the regulation of peroxisome abundance and maintenance. These processes include fission of the organelles, formation of new peroxisomes from the endoplasmic reticulum, autophagic degradation and segregation/inheritance during cell division.

  16. The biogenesis of lysosomes and lysosome-related organelles.

    PubMed

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

    2014-09-02

    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. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  17. 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

  18. 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

    2015-07-27

    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.

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

    PubMed

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

    2011-10-01

    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 be compatible with microscale 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 the pH of each magnetically retained individual endocytic organelle. 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.

  20. 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

  1. Metabolic Capacity of Mitochondrion-related Organelles in the Free-living Anaerobic Stramenopile Cantina marsupialis.

    PubMed

    Noguchi, Fumiya; Shimamura, Shigeru; Nakayama, Takuro; Yazaki, Euki; Yabuki, Akinori; Hashimoto, Tetsuo; Inagaki, Yuji; Fujikura, Katsunori; Takishita, Kiyotaka

    2015-11-01

    Functionally and morphologically degenerate mitochondria, so-called mitochondrion-related organelles (MROs), are frequently found in eukaryotes inhabiting hypoxic or anoxic environments. In the last decade, MROs have been discovered from a phylogenetically broad range of eukaryotic lineages and these organelles have been revealed to possess diverse metabolic capacities. In this study, the biochemical characteristics of an MRO in the free-living anaerobic protist Cantina marsupialis, which represents an independent lineage in stramenopiles, were inferred based on RNA-seq data. We found transcripts for proteins known to function in one form of MROs, the hydrogenosome, such as pyruvate:ferredoxin oxidoreductase, iron-hydrogenase, acetate:succinate CoA-transferase, and succinyl-CoA synthase, along with transcripts for acetyl-CoA synthetase (ADP-forming). These proteins possess putative mitochondrial targeting signals at their N-termini, suggesting dual ATP generation systems through anaerobic pyruvate metabolism in Cantina MROs. In addition, MROs in Cantina were also shown to share several features with canonical mitochondria, including amino acid metabolism and an "incomplete" tricarboxylic acid cycle. Transcripts for all four subunits of complex II (CII) of the electron transport chain were detected, while there was no evidence for the presence of complexes I, III, IV, or F1Fo ATPase. Cantina MRO biochemistry challenges the categories of mitochondrial organelles recently proposed.

  2. Organization of organelles and VAMP-associated vesicular transport systems in differentiating skeletal muscle cells.

    PubMed

    Tajika, Yuki; Takahashi, Maiko; Ueno, Hitoshi; Murakami, Tohru; Yorifuji, Hiroshi

    2015-01-01

    Vesicular transport plays an important role in the regulation of cellular function and differentiation of the cell, and intracellular vesicles play a role in the delivery of membrane components and in sorting membrane proteins to appropriate domains in organelles and the plasma membrane. Research on vesicular transport in differentiating cells has mostly focused on neurons and epithelial cells, and few such studies have been carried out on skeletal muscle cells. Skeletal muscle cells have specialized organelles and plasma membrane domains, including T-tubules, sarcoplasmic reticulum, neuromuscular junctions, and myotendinous junctions. The differentiation of skeletal muscle cells is achieved by multiple steps, i.e., proliferation of myoblasts, formation of myotubes by cell-cell fusion, and maturation of myotubes into myofibers. Systematic vesicular transport is expected to play a role in the maintenance and development of skeletal muscle cells. Here, we review a map of the vesicular transport system during the differentiation of skeletal muscle cells. The characteristics of organelle arrangement in myotubes are described according to morphological studies. Vesicular transport in myotubes is explained by the expression profiles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins.

  3. The conserved AAA-ATPase Msp1 confers organelle specificity to tail-anchored proteins.

    PubMed

    Okreglak, Voytek; Walter, Peter

    2014-06-03

    The accuracy of tail-anchored (TA) protein targeting to the endoplasmic reticulum (ER) depends on the Guided Entry of Tail-Anchored (Get) protein targeting machinery. The fate of TA proteins that become inappropriately inserted into other organelles, such as mitochondria, is unknown. Here, we identify Msp1, a conserved, membrane-anchored AAA-ATPase (ATPase associated with a variety of cellular activities) that localizes to mitochondria and peroxisomes, as a critical factor in a quality control pathway that senses and degrades TA proteins mistargeted to the outer mitochondrial membrane (OMM). Pex15 is normally targeted by the Get pathway to the ER, from where it travels to peroxisomes. Loss of Msp1 or loss of the Get pathway results in the redistribution of Pex15 to mitochondria. Cells lacking both a functional Get pathway and Msp1 accumulate increased amounts of Pex15 on the OMM and display severely dysfunctional mitochondrial morphology. In addition, Msp1 binds and promotes the turnover of a Pex15 mutant that is misdirected to the OMM. Our data suggest that Msp1 functions in local organelle surveillance by extracting mistargeted proteins, ensuring the fidelity of organelle specific-localization of TA proteins.

  4. Ultrastructural relationship of the phagophore with surrounding organelles

    PubMed Central

    Biazik, Joanna; Ylä-Anttila, Päivi; Vihinen, Helena; Jokitalo, Eija; Eskelinen, Eeva-Liisa

    2015-01-01

    Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation. PMID:25714487

  5. Ultrastructural relationship of the phagophore with surrounding organelles.

    PubMed

    Biazik, Joanna; Ylä-Anttila, Päivi; Vihinen, Helena; Jokitalo, Eija; Eskelinen, Eeva-Liisa

    2015-01-01

    Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.

  6. Fungal evo-devo: organelles and multicellular complexity.

    PubMed

    Jedd, Gregory

    2011-01-01

    Peroxisome-derived Woronin bodies of the Ascomycota phyla, and the endoplasmic reticulum (ER)-derived septal pore cap (SPC) of the Basidiomycota, are both fungal organelles that prevent cytoplasmic bleeding when multicellular hyphal filaments are wounded. Analysis of Woronin body constituent proteins suggests that these organelles evolved in part through gene duplication and co-opting of non-essential genes for new functions, indicating that new organelles can arise through typical evolutionary mechanisms. Interestingly, clades possessing the Woronin body and SPC also produce the largest and most complex multicellular fungal reproductive structures. Certain Woronin body and SPC mutants have defects in growth and development, suggesting functions beyond cellular wound healing. I argue that studying these specialized systems will help to reveal the basis for fungal diversity and provide general principles for co-evolution of organelles and multicellular complexity. Copyright © 2010 Elsevier Ltd. All rights reserved.

  7. 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

  8. 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.

  9. 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

  10. Optogenetic control of molecular motors and organelle distributions in cells.

    PubMed

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

    2015-05-21

    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 exposure to blue light, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven toward the cell periphery upon light-induced recruitment of kinesin, or toward 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The Cytoskeleton Maintains Organelle Partitioning Required for Single-Cell C4 Photosynthesis in Chenopodiaceae Species[W

    PubMed Central

    Chuong, Simon D.X.; Franceschi, Vincent R.; Edwards, Gerald E.

    2006-01-01

    Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C4 photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein–tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm. PMID:16905659

  12. Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells.

    PubMed

    Tanji, Takahiro; Nishikori, Kenji; Haga, Syoko; Kanno, Yuki; Kobayashi, Yusuke; Takaya, Mai; Gengyo-Ando, Keiko; Mitani, Shohei; Shiraishi, Hirohisa; Ohashi-Kobayashi, Ayako

    2016-01-27

    The intestinal cells of Caenorhabditis elegans are filled with heterogeneous granular organelles that are associated with specific organ functions. The best studied of these organelles are lipid droplets and acidified gut granules associated with GLO-1, a homolog of the small GTPase Rab38. In this study, we characterized a subset of the intestinal granules in which HAF-4 and HAF-9 localize on the membrane. HAF-4 and HAF-9 are ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing-like, ABCB9). Using transgenic worms expressing fluorescent protein-tagged marker proteins, we demonstrated that the HAF-4- and HAF-9-localizing organelles are not lipid droplets and do not participate in yolk protein transport. They were also ruled out as GLO-1-positive acidified gut granules. Furthermore, we clarified that the late endosomal protein RAB-7 localizes to the HAF-4- and HAF-9-localizing organelles and is required for their biogenesis. Our results indicate that the HAF-4- and HAF-9-localizing organelles are distinct intestinal organelles associated with the endocytic pathway.

  13. In vivo examination of the morphology of the tendinous inscription of the human semitendinosus muscle: gender and joint position effects.

    PubMed

    Kellis, Eleftherios; Balidou, Anna

    2014-01-01

    A tendinous inscription divides the semitendinosus muscle in two parts and it may have an effect on its function. The purpose of this study was to determine the effects of joint position and gender on the tendinous inscription morphology. Ultrasonography scans were taken from 76 young males and females at rest, in nine combinations of hip and knee joint angles. The length of the tendinous inscription arms and the angles formed by the two arms (apex angle), the tendinous inscription with the superficial (surface angle), and deep (deep angle) aponeurosis were determined. The tendinous inscription was clearly visible in 70 (out of 76) subjects. Analysis of variance designs showed that increasing hip flexion angle from 0 to 90° increased the long arm and muscle thickness but decreased the short tendinous inscription arm (P < 0.05). Changing knee flexion angle from 0 to 90° was accompanied by a longer tendinous inscription arm and an increased apex angle (P < 0.05). Long arm length and muscle thickness significantly increased from the shortest (hip 0° - knee 90°) to the longest muscle lengths (hip 0° - knee 90°). Males had a significantly higher surface, apex, and deep angle and a lower normalized tendinous inscription long arm than females (P < 0.05). These results indicate that the effect of the tendinous inscription (if any) on semitendinous muscle function depends on hip and knee joint angle while it may be gender dependent.

  14. 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…

  15. 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…

  16. 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

  17. Microbial arms race: Ballistic "nematocysts" in dinoflagellates represent a new extreme in organelle complexity.

    PubMed

    Gavelis, Gregory S; Wakeman, Kevin C; Tillmann, Urban; Ripken, Christina; Mitarai, Satoshi; Herranz, Maria; Özbek, Suat; Holstein, Thomas; Keeling, Patrick J; Leander, Brian S

    2017-03-01

    We examine the origin of harpoon-like secretory organelles (nematocysts) in dinoflagellate protists. These ballistic organelles have been hypothesized to be homologous to similarly complex structures in animals (cnidarians); but we show, using structural, functional, and phylogenomic data, that nematocysts evolved independently in both lineages. We also recorded the first high-resolution videos of nematocyst discharge in dinoflagellates. Unexpectedly, our data suggest that different types of dinoflagellate nematocysts use two fundamentally different types of ballistic mechanisms: one type relies on a single pressurized capsule for propulsion, whereas the other type launches 11 to 15 projectiles from an arrangement similar to a Gatling gun. Despite their radical structural differences, these nematocysts share a single origin within dinoflagellates and both potentially use a contraction-based mechanism to generate ballistic force. The diversity of traits in dinoflagellate nematocysts demonstrates a stepwise route by which simple secretory structures diversified to yield elaborate subcellular weaponry.

  18. Rab GTPases and myosin motors in organelle motility.

    PubMed

    Seabra, Miguel C; Coudrier, Evelyne

    2004-06-01

    The actin cytoskeleton is essential to ensure the proper location of, and communication between, intracellular organelles. Some actin-based myosin motors have been implicated in this process, particularly members of the class V myosins. We discuss here the emerging role of the Ras-like GTPases of the Rab family as regulators of myosin function in organelle transport. Evidence from yeast secretory vesicles and mitochondria, and mammalian melanosomes and endosomes suggests that Rab GTPases are crucial components of the myosin organelle receptor machinery. Better understood is the case of the melanosome where Rab27a recruits a specific effector called melanophilin, which in turn binds myosin Va. The presence of a linker protein between a Rab and a myosin may represent a general mechanism. We argue that Rabs are ideally suited to perform this role as they are exquisite organelle markers. Furthermore, the molecular switch property of Rabs may enable them to regulate the timing of the myosin association with the target organelle.

  19. Systematic Structural Analyses of Attachment Organelle in Mycoplasma pneumoniae.

    PubMed

    Nakane, Daisuke; Kenri, Tsuyoshi; Matsuo, Lisa; Miyata, Makoto

    2015-12-01

    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.

  20. 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

  1. Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

    PubMed Central

    Kato, Naohiro; Reynolds, Dexter; Brown, Matthew L; Boisdore, Marietta; Fujikawa, Yukichi; Morales, Andrea; Meisel, Lee A

    2008-01-01

    Background The isolation of green fluorescent protein (GFP) and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types. Results We generated a transgenic Arabidopsis plant that contains four spectrally different fluorescent proteins. Nuclei, plastids, mitochondria and plasma membranes were genetically tagged with cyan, red, yellow and green fluorescent proteins, respectively. In addition, methods to track nuclei, mitochondria and chloroplasts and quantify the interaction between these organelles at a submicron resolution were developed. These analyzes revealed that N-ethylmaleimide disrupts nuclear-mitochondrial but not nuclear-plastids interactions in root epidermal cells of live Arabidopsis seedlings. Conclusion We developed a tool and associated methods for analyzing the complex dynamic of organelle-organelle interactions in real time in planta. Homozygous transgenic Arabidopsis (Kaleidocell) is available through Arabidopsis Biological Resource Center. PMID:18489765

  2. The dynamic behavior of storage organelles in developing cereal seeds and its impact on the production of recombinant proteins

    PubMed Central

    Arcalis, Elsa; Ibl, Verena; Peters, Jenny; Melnik, Stanislav; Stoger, Eva

    2014-01-01

    Cereal endosperm is a highly differentiated tissue containing specialized organelles for the accumulation of storage proteins, which are ultimately deposited either within protein bodies derived from the endoplasmic reticulum, or in protein storage vacuoles (PSVs). During seed maturation endosperm cells undergo a rapid sequence of developmental changes, including extensive reorganization and rearrangement of the endomembrane system and protein transport via several developmentally regulated trafficking routes. Storage organelles have been characterized in great detail by the histochemical analysis of fixed immature tissue samples. More recently, in vivo imaging and the use of tonoplast markers and fluorescent organelle tracers have provided further insight into the dynamic morphology of PSVs in different cell layers of the developing endosperm. This is relevant for biotechnological applications in the area of molecular farming because seed storage organelles in different cereal crops offer alternative subcellular destinations for the deposition of recombinant proteins that can reduce proteolytic degradation, allow control over glycan structures and increase the efficacy of oral delivery. We discuss how the specialized architecture and developmental changes of the endomembrane system in endosperm cells may influence the subcellular fate and post-translational modification of recombinant glycoproteins in different cereal species. PMID:25232360

  3. Lysosome-Related Organelles in Intestinal Cells Are a Zinc Storage Site in C. elegans

    PubMed Central

    Roh, Hyun Cheol; Collier, Sara; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

    2014-01-01

    SUMMARY Zinc is an essential trace element involved in many biological processes and human diseases. Because zinc deficiency and excess are deleterious, animals require homeostatic mechanisms to maintain zinc levels in response to dietary fluctuations. Here we demonstrate that lysosome-related organelles in intestinal cells of C. elegans, called gut granules, function as the major site of zinc storage. Zinc storage in gut granules promotes detoxification and subsequent mobilization, linking cellular and organismal zinc metabolism. The cation diffusion facilitator protein CDF-2 plays a critical role in this process by transporting zinc into gut granules. In response to high dietary zinc, gut granules displayed structural changes characterized by a bilobed morphology with asymmetric distributions of zinc and molecular markers. We defined a genetic pathway that mediates the formation of bilobed morphology. These findings elucidate mechanisms of zinc storage, detoxification and mobilization in C. elegans and may be relevant to other animals. PMID:22225878

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

    SciTech Connect

    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 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. In conclusion, 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.

  5. 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.

  6. 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.

  7. Poles apart: prokaryotic polar organelles and their spatial regulation.

    PubMed

    Kirkpatrick, Clare L; Viollier, Patrick H

    2011-03-01

    While polar organelles hold the key to understanding the fundamentals of cell polarity and cell biological principles in general, they have served in the past merely for taxonomical purposes. Here, we highlight recent efforts in unraveling the molecular basis of polar organelle positioning in bacterial cells. Specifically, we detail the role of members of the Ras-like GTPase superfamily and coiled-coil-rich scaffolding proteins in modulating bacterial cell polarity and in recruiting effector proteins to polar sites. Such roles are well established for eukaryotic cells, but not for bacterial cells that are generally considered diffusion-limited. Studies on spatial regulation of protein positioning in bacterial cells, though still in their infancy, will undoubtedly experience a surge of interest, as comprehensive localization screens have yielded an extensive list of (polarly) localized proteins, potentially reflecting subcellular sites of functional specialization predicted for organelles.

  8. 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

  9. 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.

  10. Spatiotemporal analysis of organelle and macromolecular complex inheritance

    PubMed Central

    Menendez-Benito, Victoria; van Deventer, Sjoerd J.; Jimenez-Garcia, Victor; Roy-Luzarraga, Marina; van Leeuwen, Fred; Neefjes, Jacques

    2013-01-01

    Following mitosis, daughter cells must inherit a functional set of essential proteins and organelles. We applied a genetic tool to simultaneously monitor the kinetics and distribution of old and new proteins marking all intracellular compartments in budding yeasts. Most organelles followed a general pattern whereby preexisting proteins are symmetrically partitioned followed by template-based incorporation of new proteins. Peroxisomes belong to this group, supporting a model of biogenesis by growth and division from preexisting peroxisomes. We detected two exceptions: the nuclear pore complex (NPC) and the spindle pole body (SPB). Old NPCs are stably inherited during successive generations but remained separated from new NPCs, which are incorporated de novo in mother and daughter cells. Only the SPB displayed asymmetrical distribution, with old components primarily inherited by daughter cells and new proteins equally incorporated in both cells. Our analysis resolves conflicting models (peroxisomes, NPC) and reveals unique patterns (NPC, SPB) of organelle inheritance. PMID:23248297

  11. Lam6 Regulates the Extent of Contacts between Organelles

    PubMed Central

    Elbaz-Alon, Yael; Eisenberg-Bord, Michal; Shinder, Vera; Stiller, Sebastian Berthold; Shimoni, Eyal; Wiedemann, Nils; Geiger, Tamar; Schuldiner, Maya

    2015-01-01

    Summary Communication between organelles is crucial for eukaryotic cells to function as one coherent unit. An important means of communication is through membrane contact sites, where two organelles come into close proximity allowing the transport of lipids and small solutes between them. Contact sites are dynamic in size and can change in response to environmental or cellular stimuli; however, how this is regulated has been unclear. Here, we show that Saccharomyces cerevisiae Lam6 resides in several central contact sites: ERMES (ER/mitochondria encounter structure), vCLAMP (vacuole and mitochondria patch), and NVJ (nuclear vacuolar junction). We show that Lam6 is sufficient for expansion of contact sites under physiological conditions and necessary for coordination of contact site size. Given that Lam6 is part of a large protein family and is conserved in vertebrates, our work opens avenues for investigating the underlying principles of organelle communication. PMID:26119743

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

    PubMed

    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.

  13. (+)RNA viruses rewire cellular pathways to build replication organelles.

    PubMed

    Belov, George A; van Kuppeveld, Frank J M

    2012-12-01

    Positive-strand RNA [(+)RNA] viruses show a significant degree of conservation of their mechanisms of replication. The universal requirement of (+)RNA viruses for cellular membranes for genome replication, and the formation of membranous replication organelles with similar architecture, suggest that they target essential control mechanisms of membrane metabolism conserved among eukaryotes. Recently, significant progress has been made in understanding the role of key host factors and pathways that are hijacked for the development of replication organelles. In addition, electron tomography studies have shed new light on their ultrastructure. Collectively, these studies reveal an unexpected complexity of the spatial organization of the replication membranes and suggest that (+)RNA viruses actively change cellular membrane composition to build their replication organelles. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Biogenesis of a bacterial organelle: the carboxysome assembly pathway.

    PubMed

    Cameron, Jeffrey C; Wilson, Steven C; Bernstein, Susan L; Kerfeld, Cheryl A

    2013-11-21

    The carboxysome is a protein-based organelle for carbon fixation in cyanobacteria, keystone organisms in the global carbon cycle. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase. Here, we describe the stages of carboxysome assembly and the requisite gene products necessary for progression through each. Our results demonstrate that, unlike membrane-bound organelles of eukaryotes, in carboxysomes the interior of the compartment forms first, at a distinct site within the cell. Subsequently, shell proteins encapsulate this procarboxysome, inducing budding and distribution of functional organelles within the cell. We propose that the principles of carboxysome assembly that we have uncovered extend to diverse bacterial microcompartments.

  15. Cancer therapy: Targeting mitochondria and other sub-cellular organelles.

    PubMed

    Ubah, Obinna C; Wallace, Heather M

    2014-01-01

    Tumour cell death is required for the clearance of malignant cells and is a vital part of the mechanism of natural tumour suppression. Cancer cells, having acquired multiple deregulated pathways involving several cellular oragenelles, are capable of disrupting these normally finely tuned processes thereby evading both physiological and therapeutic intervention. Although current available data indicate the dependence of successful tumour cell clearance on classical apoptotic pathways (intrinsic and/or extrinsic pathways), there is now evidence suggesting that alternative apoptotic and non-apoptotic pathways may effectively contribute to tumour cell death. The mitochondria, proteasomes, endoplasmic reticulum, Golgi apparatus, lysosomes and lysosome-related organelles of tumour cells exhibit a number of deregulations which have been identified as potential druggable targets for successful rational drug design and therapy. In this review, we summarise the roles of these cellular organelles in tumour initiation and establishment as well as current trends in development of agents that target deregulations in these organelles.

  16. Organelle-localized potassium transport systems in plants.

    PubMed

    Hamamoto, Shin; Uozumi, Nobuyuki

    2014-05-15

    Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K(+) transport systems allowing K(+) to move across the membrane. K(+) transport systems in plant organelles act coordinately with the plasma membrane intrinsic K(+) transport systems to maintain cytosolic K(+) concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K(+) channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K(+) homeostasis of the cytoplasm. The initial electrophysiological measurements of K(+) transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K(+) transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K(+) transport system has been isolated from cyanobacteria, which may add to our understanding of K(+) flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K(+) transport proteins. Copyright © 2014 Elsevier GmbH. All rights reserved.

  17. AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS(1).

    PubMed

    Kato, Syou; Misawa, Kazuharu; Takahashi, Fumio; Sakayama, Hidetoshi; Sano, Satomi; Kosuge, Keiko; Kasai, Fumie; Watanabe, Makoto M; Tanaka, Jiro; Nozaki, Hisayoshi

    2011-10-01

    Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat-based dimorphism of chloroplast DNA (cpDNA). Here, we examined the "shallow" and "deep" subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima's D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both "shallow" and "deep" haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat-based dimorphism of cpDNA from field-collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes.

  18. The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure.

    PubMed

    Otulak, Katarzyna; Chouda, Marcin; Bujarski, Józef; Garbaczewska, Grażyna

    2015-03-01

    Tobraviruses, like other (+) stranded RNA viruses of plants, replicate their genome in cytoplasm and use such usual membranous structures like endoplasmic reticulum. Based on the ultrastructural examination of Tobacco rattle virus (TRV)-infected potato and tobacco leaf tissues, in this work we provide evidence of the participation of not only the membranous and vesicular ER structures but also other cell organelles during the viral infection cycle. Non-capsidated TRV PSG particles (potato isolate from the Netherlands) (long and short forms) were observed inside the nucleus while the presence of TRV capsid protein (CP) was detected in the nucleus caryolymph and within the nucleolus area. Both capsidated and non-capsidated viral particles were localized inside the strongly disorganized chloroplasts and mitochondria. The electron-dense TRV particles were connected with vesicular structures of mitochondria as well as with chloroplasts in both potato and tobacco tissues. At 15-30 days after infection, vesicles filled with TRV short particles were visible in mitochondria revealing the expanded cristae structures. Immunodetection analysis revealed the TRV PSG CP epitope inside chloroplast with disorganized thylakoids structure as well as in mitochondria of different tobacco and potato tissues. The ultrastructural analysis demonstrated high dynamics of the main cell organelles during the TRV PSG-Solanaceous plants interactions. Moreover, our results suggest a relationship between organelle changes and different stages of virus infection cycle and/or particle formation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

    PubMed Central

    Vigil, Eugene L.

    1970-01-01

    Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix. PMID:4121486

  20. Melanosomes – dark organelles enlighten endosomal membrane transport

    PubMed Central

    Raposo, Graça; Marks, Michael S.

    2009-01-01

    Melanosomes are tissue-specific “lysosome-related” organelles of pigment cells in which melanins are synthesized and stored. Analyses of the trafficking and fate of melanosomal components are beginning to reveal how melanosomes are formed through novel pathways from early endosomal intermediates. These studies unveil generalized structural and functional modifications of the endosomal system in specialized cells, and provide unexpected insights into the biogenesis of multivesicular bodies and how compartmentalization regulates protein refolding. Moreover, genetic disorders that affect the biogenesis of melanosomes and other lysosome-related organelles have shed light into the molecular machinery that controls specialized endosomal sorting events. PMID:17878918

  1. The contribution of specific organelles to side scatter

    NASA Astrophysics Data System (ADS)

    Mourant, Judith R.; Marina, Oana C.; Sanders, Claire K.

    2013-02-01

    Knowledge of which cellular structures scatter light is needed to fully utilize the information available from light scattering measurements of cells and tissues. To determine how specific organelles contribute to light scattering, wide angle side scattering was imaged simultaneously with fluorescence from specific organelles for thousands of cells using flow cytometry. Images were obtained with different depth of field conditions and analyzed with different assumptions. Both sets of data demonstrated that mitochondria and lysosomes, contribute similarly to side scatter. The nucleus contributes as much or more light scatter than either the mitochondria or the lysosomes.

  2. METABOLIC REGULATION OF ORGANELLE HOMEOSTASIS IN LUPUS T CELLS

    PubMed Central

    Caza, Tiffany N; Talaber, Gergely; Perl, Andras

    2012-01-01

    Abnormal T-cell signaling and activation is a characteristic feature in systemic lupus erythematosus (SLE). Lupus T cells are shifted towards an over-activated state, important signaling pathways are rewired, and signaling molecules are replaced. Disturbances in metabolic and organelle homeostasis, importantly within the mitochondrial, endosomal, and autophagosomal compartments, underlie the changes in signal transduction. Mitochondrial hyperpolarization, enhanced endosomal recycling, and dysregulated autophagy are hallmarks of pathologic organelle homeostasis in SLE. This review is focused on the metabolic checkpoints of endosomal traffic that control immunological synapse formation and mitophagy and may thus serve as targets for treatment in SLE. PMID:22836085

  3. Morphological examinations of hard tissues of periodontium and evaluation of selected processes of lipid peroxidation in blood serum of rats in the course of experimental periodontitis.

    PubMed

    Sobaniec, H; Sobaniec-Lotowska, M E

    2000-01-01

    The problem of teeth loss as a result of periodontitis is growing continuously. In the study we aimed to show the correlation between the disease and lipid metabolism disorders. We performed morphological examinations of hard tissues of rats' periodontium in the course of experimental ligature-induced periodontitis and we demonstrated the destruction of alveolodental ligament. The following changes were observed: degenerative changes including necrosis within periodontium, progressive destruction of bone mass of alveolar process of the mandible in the region of inflammatory infiltration. Simultaneously, biochemical examinations of blood serum were performed revealing decrease of basic antioxidant enzymes activities: SOD, GSH-Px, GSH-R with simultaneous increase of MDA--the final product of lipid peroxidation.

  4. Fresh-frozen, optimal cutting temperature (OCT) compound-embedded bone marrow aspirates: a reliable resource for morphological, immunohistochemical and molecular examinations.

    PubMed

    Lim, J; Kim, Y; Lee, W; Kim, M; Lee, E J; Kang, C S; Han, K

    2010-02-01

    The usefulness of fresh-frozen, optimal cutting temperature (OCT) compound-embedded (FFOE) bone marrow (BM) aspirates was evaluated as a reliable resource for morphological, immunohistochemical and molecular examinations. One hundred BM aspirates were collected in polypropylene tubes and immediately frozen for 2 h in a deep freezer. Frozen BM was transferred to a cryomold filled with OCT compound and the prepared samples were stored in a deep freezer. Histological examination and immunohistochemical staining, polymerase chain reaction (PCR), sequencing and reverse transcription (RT)-PCR were performed to evaluate the quality of the FFOE BM sections in 10% of randomly selected samples. FFOE BM sections revealed better morphologies than paraffin-embedded clot sections in haematoxylin and eosin staining because mature erythrocytes were removed during the staining process in frozen BM sections. Immunohistochemical staining for CD34 revealed excellent staining quality and oil red O staining showed that fat vacuoles in cells were well preserved. The quality of genomic DNA in FFOE BM sections was suitable for obtaining about 2000 bp PCR product for the human leucocyte antigen-A locus followed by direct sequencing of the sample, and the quality of total RNA was suitable for detection of BCR-ABL fusion transcript. FFOE BM aspirates are a reliable resource for various laboratory tests of diagnostic and research arenas.

  5. 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.

  6. 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

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

    PubMed

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

    2010-02-17

    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

  8. Plant cell organelle proteomics in response to abiotic stress.

    PubMed

    Hossain, Zahed; Nouri, Mohammad-Zaman; Komatsu, Setsuko

    2012-01-01

    Proteomics is one of the finest molecular techniques extensively being used for the study of protein profiling of a given plant species experiencing stressed conditions. Plants respond to a stress by alteration in the pattern of protein expression, either by up-regulating of the existing protein pool or by the synthesizing novel proteins primarily associated with plants antioxidative defense mechanism. Improved protein extraction protocols and advance techniques for identification of novel proteins have been standardized in different plant species at both cellular and whole plant level for better understanding of abiotic stress sensing and intracellular stress signal transduction mechanisms. In contrast, an in-depth proteome study of subcellular organelles could generate much detail information about the intrinsic mechanism of stress response as it correlates the possible relationship between the protein abundance and plant stress tolerance. Although a wealth of reviews devoted to plant proteomics are available, review articles dedicated to plant cell organelle proteins response under abiotic stress are very scanty. In the present review, an attempt has been made to summarize all significant contributions related to abiotic stresses and their impacts on organelle proteomes for better understanding of plants abiotic stress tolerance mechanism at protein level. This review will not only provide new insights into the plants stress response mechanisms, which are necessary for future development of genetically engineered stress tolerant crop plants for the benefit of humankind, but will also highlight the importance of studying changes in protein abundance within the cell organelles in response to abiotic stress.

  9. More than a locomotive organelle: flagella in Escherichia coli.

    PubMed

    Zhou, Mingxu; Yang, Yang; Chen, Panlin; Hu, Huijie; Hardwidge, Philip R; Zhu, Guoqiang

    2015-11-01

    The flagellum is a locomotive organelle that allows bacteria to respond to chemical gradients. This review summarizes the current knowledge regarding Escherichia coli flagellin variants and the role of flagella in bacterial functions other than motility, including the relationship between flagella and bacterial virulence.

  10. 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.

  11. Neurovascular Events After Subarachnoid Hemorrhage: Focusing on Subcellular Organelles

    PubMed Central

    Chen, Sheng; Wu, Haijian; Tang, Jiping; Zhang, Jianmin; Zhang, John H.

    2015-01-01

    Subarachnoid hemorrhage (SAH) is a devastating condition with high morbidity and mortality rates due to the lack of effective therapy. Early brain injury (EBI) and cerebral vasospasm (CVS) are the two most important pathophysiological mechanisms for brain injury and poor outcomes for patients with SAH. CVS has traditionally been considered the sole cause of delayed ischemic neurological deficits after SAH. However, the failure of antivasospastic therapy in patients with SAH supported changing the research target from CVS to other mechanisms. Currently, more attention has been focused on global brain injury within 3 days after ictus, designated as EBI. The dysfunction of subcellular organelles, such as endoplasmic reticulum stress, mitochondrial failure, and autophagy–lysosomal system activation, has developed during EBI and delayed brain injury after SAH. To our knowledge, there is a lack of review articles addressing the direction of organelle dysfunction after SAH. In this review, we discuss the roles of organelle dysfunction in the pathogenesis of SAH and present the opportunity to develop novel therapeutic strategies of SAH via modulating the functions of organelles. PMID:25366597

  12. Neurovascular events after subarachnoid hemorrhage: focusing on subcellular organelles.

    PubMed

    Chen, Sheng; Wu, Haijian; Tang, Jiping; Zhang, Jianmin; Zhang, John H

    2015-01-01

    Subarachnoid hemorrhage (SAH) is a devastating condition with high morbidity and mortality rates due to the lack of effective therapy. Early brain injury (EBI) and cerebral vasospasm (CVS) are the two most important pathophysiological mechanisms for brain injury and poor outcomes for patients with SAH. CVS has traditionally been considered the sole cause of delayed ischemic neurological deficits after SAH. However, the failure of antivasospastic therapy in patients with SAH supported changing the research target from CVS to other mechanisms. Currently, more attention has been focused on global brain injury within 3 days after ictus, designated as EBI. The dysfunction of subcellular organelles, such as endoplasmic reticulum stress, mitochondrial failure, and autophagy-lysosomal system activation, has developed during EBI and delayed brain injury after SAH. To our knowledge, there is a lack of review articles addressing the direction of organelle dysfunction after SAH. In this review, we discuss the roles of organelle dysfunction in the pathogenesis of SAH and present the opportunity to develop novel therapeutic strategies of SAH via modulating the functions of organelles.

  13. 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

  14. Off to the organelles - killing cancer cells with targeted gold nanoparticles.

    PubMed

    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.

  15. Behaviour of cytoplasmic organelles and cytoskeleton during oocyte maturation.

    PubMed

    Mao, Luna; Lou, Hangying; Lou, Yiyun; Wang, Ning; Jin, Fan

    2014-03-01

    Assisted reproduction technology (ART) has become an attractive option for infertility treatment and holds tremendous promise. However, at present, there is still room for improvement in its success rates. Oocyte maturation is a process by which the oocyte becomes competent for fertilization and subsequent embryo development. To better understand the mechanism underlying oocyte maturation and for the future improvement of assisted reproduction technology, this review focuses on the complex processes of cytoplasmic organelles and the dynamic alterations of the cytoskeleton that occur during oocyte maturation. Ovarian stimulation and in-vitro maturation are the major techniques used in assisted reproduction technology and their influence on the organelles of oocytes is also discussed. Since the first birth by assisted reproduction treatment was achieved in 1978, numerous techniques involved in assisted reproduction have been developed and have become attractive options for infertility treatment. However, the unsatisfactory success rate remains as a main challenge. Oocyte maturation is a process by which the oocyte becomes competent for fertilization and subsequent embryo development. Oocyte maturation includes both nuclear and cytoplasmic maturation. Nuclear maturation primarily involves chromosomal segregation, which has been well studied, whereas cytoplasmic maturation involves a series of complicated processes, and there are still many parts of this process that remain controversial. Ovarian stimulation and in-vitro maturation (IVM) are the major techniques of assisted reproduction. The effect of ovarian stimulation or IVM on the behaviour of cell organelles of the oocyte has been postulated as the reason for the reduced developmental potential of in-vitro-produced embryos. To further understanding of the mechanism of oocyte maturation and future improvement of assisted reproduction treatment, the complex events of cytoplasmic organelles and the cytoskeleton that

  16. Differentiation of sympatric populations of the band-rumped storm-petrel in the Galapagos Islands: an examination of genetics, morphology, and vocalizations.

    PubMed

    Smith, A L; Friesen, V L

    2007-04-01

    In each of at least two locations within the Galapagos Islands, breeding band-rumped storm-petrels (Oceanodroma castro) form two distinct populations that use the same colony site at separate times of the year for reproduction. Temporal segregation of these populations raises the possibility that they are reproductively isolated and represent cryptic species. We examined variation in mitochondrial DNA, morphology, and vocalizations of storm-petrel populations nesting 6 months apart on the islet of Plaza Norte in the Galapagos. Seasonal populations displayed low but significant levels of differentiation in the mitochondrial control region, five morphological variables, and one feature of male vocalizations. Breeding populations appear to have been separated for approximately 1700 years. Given the recent divergence date and relatively high effective population sizes (4000-5600 females each), seasonal populations are unlikely to be in genetic equilibrium. As a result, the low divergence estimate probably reflects historical association and not contemporary genetic exchange. These populations are not sufficiently differentiated to be considered cryptic species. However, they are probably in the early stages of divergence. Consequently, we recommend that cool- and hot season populations on Plaza Norte be recognized as separate management units.

  17. Cytoplasmic inheritance of organelles in brown algae.

    PubMed

    Motomura, Taizo; Nagasato, Chikako; Kimura, Kei

    2010-03-01

    Brown algae, together with diatoms and chrysophytes, are a member of the heterokonts. They have either a characteristic life cycle of diplohaplontic alternation of gametophytic and sporophytic generations that are isomorphic or heteromorphic, or a diplontic life cycle. Isogamy, anisogamy and oogamy have been recognized as the mode of sexual reproduction. Brown algae are the characteristic group having elaborated multicellular organization within the heterokonts. In this study, cytoplasmic inheritance of chloroplasts, mitochondria and centrioles was examined, with special focus on sexual reproduction and subsequent zygote development. In oogamy, chloroplasts and mitochondria are inherited maternally. In isogamy, chloroplasts in sporophyte cells are inherited biparentally (maternal or paternal); however, mitochondria (or mitochondrial DNA) derived from the female gamete only remained during zygote development after fertilization. Centrioles in zygotes are definitely derived from the male gamete, irrespective of the sexual reproduction pattern. Female centrioles in zygotes are selectively broken down within 1-2 h after fertilization. The remaining male centrioles play a crucial role as a part of the centrosome for microtubule organization, mitosis, determination of the cytokinetic plane and cytokinesis, as well as for maintaining multicellularity and regular morphogenesis in brown algae.

  18. 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

  19. Cadmium stress disrupts the endomembrane organelles and endocytosis during Picea wilsonii pollen germination and tube growth.

    PubMed

    Wang, Xiaoxia; Gao, Yuan; 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.

  20. Mitochondrial dynamics controlled by mitofusins define organelle positioning and movement during mouse oocyte maturation.

    PubMed

    Wakai, Takuya; Harada, Yuichirou; Miyado, Kenji; Kono, Tomohiro

    2014-11-01

    Mitochondria are abundant in fully grown mammalian oocytes with a unique spherical morphology, but the mechanisms controlling mitochondria behavior are not well understood. Here we describe for the first time the control of mitochondrial behavior in mouse oocytes by a fusion/fission mechanism. Mitofusins (Mfn1 and Mfn2) and OPA1 proteins are required for outer and inner mitochondrial membrane fusion, respectively, whereas Drp1 is the key regulator of mitochondrial fission. We show that mouse oocytes express the Mfn1, Mfn2, Opa1 and Drp1 proteins, both in immature and mature oocytes at similar levels. Overexpression of Mfn1 or Mfn2 causes marked mitochondrial aggregation, particularly in the perinuclear region during meiotic progression. Tracking of mitochondria with chromosomes or endoplasmic reticulum (ER) throughout oocyte maturation demonstrates that Mfn1 and Mfn2-promoted mitochondrial aggregation disturbs the spatiotemporal dynamic of the chromosomes and ER, respectively. Our findings suggest that organelle dynamics are co-ordinately controlled during meiotic division, and an imbalance of mitochondrial fusion/fission leads to disorganization of the organelle compartments. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. The border-to-border distribution method for analysis of cytoplasmic particles and organelles.

    PubMed

    Yacovone, Shalane K; Ornelles, David A; Lyles, Douglas S

    2016-02-01

    Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.

  2. Multi-detector row CT as a "one-stop" examination in the preoperative evaluation of the morphology and function of living renal donors: preliminary study.

    PubMed

    Su, Chen; Yan, Chaogui; Guo, Yan; Zhou, Xuhui; Chen, Yaqing; Liu, Mingjuan; Wang, Wenjuan; Zhang, Xiaoling

    2011-02-01

    We designed to investigate the feasibility of multi-detector row computerized tomography (CT) as a "one-stop" examination for the simultaneous preoperative evaluation of the morphology and function of living renal donors. 21 living renal donors were examined by 64-slice spiral CT with a three-phase enhancement CT scan and two inserted dynamic scans. The maximum intensity projection (MIP), multi-planar reformation (MPR), and volume reconstruction (VR) procedures were performed to compare the renal parenchyma, renal vessels, and collecting system with operational findings. The known Patlak equation was used to calculate the glomerular filtration rate (GFR); exact GFR information was acquired by single photon emission computed tomography (SPECT). Our results as following, there were 3 cases of artery variation and 3 cases of vein variation. CT findings all corresponded with the operation, and the sensitivity, positive predictive value, specialty, and negative predictive value of CT were all 100%. The r of the GFR values estimated from CT is 0.894 (left) (P < 0.001) and 0.881 (right) (P < 0.001). In conclusions, our findings demonstrate that 64-slice spiral CT may offer a "one-stop" examination to replace SPECT in the preoperative evaluation of living renal donors to simultaneously provide information regarding both anatomy and the GFR of living renal donors.

  3. When size does matter: organelle size influences the properties of transport mediated by molecular motors.

    PubMed

    De Rossi, María Cecilia; Bruno, Luciana; Wolosiuk, Alejandro; Despósito, Marcelo A; Levi, Valeria

    2013-11-01

    Organelle transport is driven by the action of molecular motors. In this work, we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment. We used single particle tracking to obtain trajectories of melanosomes (pigmented organelles in Xenopus laevis melanophores). In response to certain hormones, melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors. Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent (α) provided information regarding the trajectories' topography and thus of the processes causing it. During aggregation, the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments (IF). Depolymerization of IF significantly reduced α values of small organelles during aggregation but slightly affect their directionality during dispersion. Our results could be interpreted considering that the number of copies of active motors increases with organelle size. Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors. Also, we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment. The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs. dynein at the single molecule level. © 2013 Elsevier B.V. All rights reserved.

  4. The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes

    PubMed Central

    Wilson, Nedra F.; Foglesong, Mary J.; Snell, William J.

    1997-01-01

    In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ∼3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 106 fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes. PMID:9199169

  5. Clinical significance of the low normal sperm morphology value as proposed in the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen

    PubMed Central

    Menkveld, Roelof

    2010-01-01

    The very low cut-off value for sperm morphology of 4% morphologically normal spermatozoa, as proposed in the new edition of the World Health Organization (WHO) manual on semen analysis, is in agreement with recently published values and reflects the trend of a decline in reported mean values for normal sperm morphology. The reduced value for morphologically normal spermatozoa over the years may be due to several factors. The first is the introduction of strict criteria for the evaluation of sperm morphology. Other reasons may include the introduction of additional criteria for sperm morphology abnormalities and the suggested decrease in semen parameters because of increasing negative environmental influences. Although on its own the newly proposed very low normal value may not provide the strong predictive value for a males' fertility potential, as originally reported for sperm morphology evaluated according to strict criteria, a good predictive value can still be obtained if the holistic, strict approach for sperm morphology evaluation is followed together with additional sperm morphology parameters now available, because certain morphology patterns and sperm abnormalities are now known to be of strong prognostic value. In addition, better international standardization of the technical methodology, consensus on the interpretation of sperm morphology evaluation criteria and standardized international external quality control (EQC) schemes, are of utmost importance to maintain the good predictive value of sperm morphology. PMID:20111081

  6. Clinical significance of the low normal sperm morphology value as proposed in the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen.

    PubMed

    Menkveld, Roelof

    2010-01-01

    The very low cut-off value for sperm morphology of 4% morphologically normal spermatozoa, as proposed in the new edition of the World Health Organization (WHO) manual on semen analysis, is in agreement with recently published values and reflects the trend of a decline in reported mean values for normal sperm morphology. The reduced value for morphologically normal spermatozoa over the years may be due to several factors. The first is the introduction of strict criteria for the evaluation of sperm morphology. Other reasons may include the introduction of additional criteria for sperm morphology abnormalities and the suggested decrease in semen parameters because of increasing negative environmental influences. Although on its own the newly proposed very low normal value may not provide the strong predictive value for a males' fertility potential, as originally reported for sperm morphology evaluated according to strict criteria, a good predictive value can still be obtained if the holistic, strict approach for sperm morphology evaluation is followed together with additional sperm morphology parameters now available, because certain morphology patterns and sperm abnormalities are now known to be of strong prognostic value. In addition, better international standardization of the technical methodology, consensus on the interpretation of sperm morphology evaluation criteria and standardized international external quality control (EQC) schemes, are of utmost importance to maintain the good predictive value of sperm morphology.

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

    PubMed

    Capetanaki, Yassemi; 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.

  8. 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.

  9. A nonproteolytic proteasome activity controls organelle fission in yeast.

    PubMed

    Hofmann, Line; Saunier, Rémy; Cossard, Raynald; Esposito, Michela; Rinaldi, Teresa; Delahodde, Agnès

    2009-10-15

    To understand the processes underlying organelle function, dynamics and inheritance, it is necessary to identify and characterize the regulatory components involved. Recently in yeast and mammals, proteins of the membrane fission machinery (Dnm1-Mdv1-Caf4-Fis1 in yeast and DLP1-FIS1 in human) have been shown to have a dual localization on mitochondria and peroxisomes, where they control mitochondrial fission and peroxisome division. Here, we show that whereas vacuole fusion is regulated by the proteasome degradation function, mitochondrial fission and peroxisomal division are not controlled by the proteasome activity but rather depend on a new function of the proteasomal lid subunit Rpn11. Rpn11 was found to regulate the Fis1-dependent fission machinery of both organelles. These findings indicate a unique role of the Rpn11 protein in mitochondrial fission and peroxisomal proliferation that is independent of its role in proteasome-associated deubiquitylation.

  10. 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

  11. 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.

  12. 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.

  13. Effects of mutual interaction of Laccaria laccata with Trichoderma harzianum and T. virens on the morphology of microtubules and mitochondria.

    PubMed

    Zadworny, M; Tuszyńska, S; Samardakiewicz, S; Werner, A

    2007-01-01

    Organelles are known to respond to challenges caused by many stress factors. The morphology of the microtubular cytoskeleton and mitochondria during mutual interaction in coculture of Laccaria laccata with Trichoderma harzianum and T. virens were examined. Hyphae from the interaction region were sampled between 4 and 12 days of growth. Microtubules were labelled with a specific antibody and mitochondria with 3,3'-dihexyloxacarbocyanine iodide, and the organelles were examined microscopically. The morphology of microtubules and mitochondria were similar in all three fungi. Microtubules were arranged in long arrays parallel to the hyphal axis and mitochondria formed an interconnected network. In hyphae growing within the interaction zone, microtubules became wavy and eventually fragmented or depolymerised, and mitochondria also became fragmented. The effects were time-dependent. In general, the organelles of all three fungi were affected during the interaction, but L. laccata was affected the least and to the same extent by each of the saprotrophic fungi. The saprotrophic fungi were affected by L. laccata to a similar extent at 4 and 8 days of interaction. Our results suggest that the studied fungi antagonistically affect each other at the cellular level, although the mechanisms involved remain to be elucidated.

  14. Quantitative optical trapping on single cellular organelles in cell extract

    PubMed Central

    Barak, Pradeep; Rai, Ashim; Rai, Priyanka; Mallik, Roop

    2012-01-01

    We develop optical trapping methodology to precisely measure the force generated by motor-proteins on single organelles of unknown size in cell extract. Native motor-complexes can now be interrogated functionally, overcoming limitations of assays with purified motors coated on artificial beads. Forces, number and activity of kinesin-1 is measured on motile lipid droplets isolated from liver of normal and fasted rats to detect a correlation between metabolic state and kinesin-1 activity. PMID:23241632

  15. Genomes at the interface between bacteria and organelles.

    PubMed Central

    Douglas, Angela E; Raven, John A

    2003-01-01

    The topic of the transition of the genome of a free-living bacterial organism to that of an organelle is addressed by considering three cases. Two of these are relatively clear-cut as involving respectively organisms (cyanobacteria) and organelles (plastids). Cyanobacteria are usually free-living but some are involved in symbioses with a range of eukaryotes in which the cyanobacterial partner contributes photosynthesis, nitrogen fixation, or both of these. In several of these symbioses the cyanobacterium is vertically transmitted, and in a few instances, sufficient unsuccessful attempts have been made to culture the cyanobiont independently for the association to be considered obligate for the cyanobacterium. Plastids clearly had a cyanobacterial ancestor but cannot grow independently of the host eukaryote. Plastid genomes have at most 15% of the number of genes encoded by the cyanobacterium with the smallest number of genes; more genes than are retained in the plastid genome have been transferred to the eukaryote nuclear genome, while the rest of the cyanobacterial genes have been lost. Even the most cyanobacteria-like plastids, for example the "cyanelles" of glaucocystophyte algae, are functionally and genetically very similar to other plastids and give little help in indicating intermediates in the evolution of plastids. The third case considered is the vertically transmitted intracellular bacterial symbionts of insects where the symbiosis is usually obligate for both partners. The number of genes encoded by the genomes of these obligate symbionts is intermediate between that of organelles and that of free-living bacteria, and the genomes of the insect symbionts also show rapid rates of sequence evolution and AT (adenine, thymine) bias. Genetically and functionally, these insect symbionts show considerable similarity to organelles. PMID:12594915

  16. Kinesin-3 is an organelle motor in the squid giant axon.

    PubMed

    DeGiorgis, Joseph A; Petukhova, Tatyana A; Evans, Teresa A; Reese, Thomas S

    2008-11-01

    Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the squid giant axon, where it is thought to move organelles on microtubules. In this study, we identify a second squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the axon for unidirectional movement of organelles along microtubules.

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

    DOE PAGES

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; ...

    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 Cd (whichmore » 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. In conclusion, 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

  18. Protein localization and dynamics within a bacterial organelle

    PubMed Central

    Hughes, H. Velocity; Huitema, Edgar; Pritchard, Sean; Keiler, Kenneth C.; Brun, Yves V.; Viollier, Patrick H.

    2010-01-01

    Protein localization mechanisms dictate the functional and structural specialization of cells. Of the four polar surface organelles featured by the dimorphic bacterium Caulobacter crescentus, the stalk, a cylindrical extension of all cell envelope layers, is the least well characterized at the molecular level. Here we apply a powerful experimental scheme that integrates genetics with high-throughput localization to discover StpX, an uncharacterized bitopic membrane protein that modulates stalk elongation and is sequestered to the stalk. In stalkless mutants StpX is dispersed. Two populations of StpX were discernible within the stalk with different mobilities: an immobile one near the stalk base and a mobile one near the stalk tip. Molecular anatomy provides evidence that (i) the StpX transmembrane domain enables access to the stalk organelle, (ii) the N-terminal periplasmic domain mediates retention in the stalk, and (iii) the C-terminal cytoplasmic domain enhances diffusion within the stalk. Moreover, the accumulation of StpX and an N-terminally truncated isoform is differentially coordinated with the cell cycle. Thus, at the submicron scale the localization and the mobility of a protein are precisely regulated in space and time and are important for the correct organization of a subcellular compartment or organelle such as the stalk. PMID:20212131

  19. 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.

  20. Cell-penetrating peptides with intracellular organelle targeting.

    PubMed

    Cerrato, Carmine Pasquale; Künnapuu, Kadri; Langel, Ülo

    2017-02-01

    One of the major limiting steps in order to have an effective drug is the passage through one or more cell membranes to reach its site of action. To reach the action-site, the specific macromolecules are required to be delivered specifically to the cell compartment/organelle in their (pre)active form. Areas covered: In this review, we will discuss cell-penetrating peptides (CPPs) developed in the last decade to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into specific sites of the cell. The article describes CPPs in complex with cargo molecules that target specific intracellular organelles and their potential for pharmacological or clinical use. Expert opinion: Organelle targeting is the ultimate goal to ensure selective delivery to the site of action in the cells. CPP technologies represent an important strategy to address drug delivery to specific intracellular compartments by covalent conjugation to targeting sequences, potentially enabling strategies to combat genomic diseases as well as infections, cancer, neurodegenerative and hereditary diseases. They have proven to be successful in delivering various therapeutic agents into cells however, further in vivo experiments and clinical trials are required to demonstrate the efficacy of this technology.

  1. Autophagy and mitophagy participate in ocular lens organelle degradation.

    PubMed

    Costello, M Joseph; Brennan, Lisa A; Basu, Subharsee; Chauss, Daniel; Mohamed, Ashik; Gilliland, Kurt O; Johnsen, Sönke; Menko, Sue; Kantorow, Marc

    2013-11-01

    The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Connection of Protein Transport and Organelle Contact Sites in Mitochondria.

    PubMed

    Ellenrieder, Lars; Rampelt, Heike; Becker, Thomas

    2017-07-07

    Mitochondrial biogenesis and function depend on the intensive exchange of molecules with other cellular compartments. The mitochondrial outer membrane plays a central role in this communication process. It is equipped with a number of specific protein machineries that enable the transport of proteins and metabolites. Furthermore, the outer membrane forms molecular contact sites with other cell organelles like the endoplasmic reticulum (ER), thus integrating mitochondrial function in cellular physiology. The best-studied mitochondrial organelle contact site, the ER-mitochondria encounter structure (ERMES) has been linked to many vital processes including mitochondrial division, inheritance, mitophagy, and phospholipid transport. Strikingly, ER-mitochondria contact sites are closely connected to outer membrane protein translocases. The translocase of the outer mitochondrial membrane (TOM) represents the general mitochondrial entry gate for precursor proteins that are synthesized on cytosolic ribosomes. The outer membrane also harbors the sorting and assembly machinery (SAM) that mediates membrane insertion of β-barrel proteins. Both of these essential protein translocases are functionally linked to ER-mitochondria contact sites. First, the SAM complex associates with an ERMES core component to promote assembly of the TOM complex. Second, several TOM components have been co-opted as ER-mitochondria tethers. We propose that protein import and organelle contact sites are linked to coordinate processes important for mitochondrial biogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Organelle DNA accumulation in the recently evolved papaya sex chromosomes.

    PubMed

    VanBuren, Robert; Ming, Ray

    2013-06-01

    Sex chromosomes are a pair of specialized chromosomes containing a sex determination region that is suppressed for recombination. Without recombination, Y chromosomes are thought to accumulate repetitive DNA sequences which contribute to their degeneration. A pair of primitive sex chromosomes controls sex type in papaya with male and hermaphrodite determined by the slightly different male-specific region of the Y (MSY) and hermaphrodite-specific region of Y(h) (HSY) chromosomes, respectively. Here, we show that the papaya HSY and MSY in the absence of recombination have accumulated nearly 12 times the amount of chloroplast-derived DNA than the corresponding region of the X chromosome and 4 times the papaya genome-wide average. Furthermore, a chloroplast genome fragment containing the rsp15 gene has been amplified 23 times in the HSY, evidence of retrotransposon-mediated duplication. Surprisingly, mitochondria-derived sequences are less abundant in the X and HSY compared to the whole genome. Shared organelle integrations are sparse between X and HSY, with only 11 % of chloroplast and 12 % of mitochondria fragments conserved, respectively, suggesting that the accelerated accumulation of organelle DNA occurred after the HSY was suppressed for recombination. Most of the organelle-derived sequences have divergence times of <7 MYA, reinforcing this notion. The accumulated chloroplast DNA is evidence of the slow degeneration of the HSY.

  4. Intracellular delivery of nanocarriers and targeting to subcellular organelles.

    PubMed

    Jhaveri, Aditi; Torchilin, Vladimir

    2016-01-01

    Recent trends in drug delivery indicate a steady increase in the use of targeted therapeutics to enhance the specific delivery of biologically active payloads to diseased tissues while avoiding their off-target effects. However, in most cases, the distribution of therapeutics inside cells and their targeting to intracellular targets still presents a formidable challenge. The main barrier to intracellular delivery is the translocation of therapeutic molecules across the cell membrane, and ultimately through the membrane of their intracellular target organelles. Another prerequisite for an efficient intracellular localization of active molecules is their escape from the endocytic pathway. Pharmaceutical nanocarriers have demonstrated substantial advantages for the delivery of therapeutics and offer elegant platforms for intracellular delivery. They can be engineered with both intracellular and organelle-specific targeting moieties to deliver encapsulated or conjugated cargoes to specific sub-cellular targets. In this review, we discuss important aspects of intracellular drug targeting and delivery with a focus on nanocarriers modified with various ligands to specifically target intracellular organelles. Intracellular delivery affords selective localization of molecules to their target site, thus maximizing their efficacy and safety. The advent of novel nanocarriers and targeting ligands as well as exploration of alternate routes for the intracellular delivery and targeting has prompted extensive research, and promises an exciting future for this field.

  5. Gregarina niphandrodes may lack both a plastid genome and organelle.

    PubMed

    Toso, Marc A; Omoto, Charlotte K

    2007-01-01

    Gregarines are early diverging apicomplexans that appear to be closely related to Cryptosporidium. Most apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess both plastids and corresponding plastid genomes. Cryptosporidium lacks both the organelle and the genome. To investigate the evolutionary history of plastids in the Apicomplexa, we tried to determine whether gregarines possess a plastid and/or its genome. We used PCR and dot-blot hybridization to determine whether the gregarine Gregarina niphandrodes possesses a plastid genome. We used an inhibitor of plastid function for any reduction in gregarine infection, and transmission electron microscopy to search for plastid ultrastructure. Despite an extensive search, an organelle of the appropriate ultrastructure in transmission electron microscopy, was not observed. Triclosan, an inhibitor of the plastid-specific enoyl-acyl carrier reductase enzyme, did not reduce host infection by G. niphandrodes. Plastid-specific primers produced amplicons with the DNA of Babesia equi, Plasmodium falciparum, and Toxoplasma gondii as templates, but not with G. niphandrodes DNA. Plastid-specific DNA probes, which hybridized to Babesia equi, failed to hybridize to G. niphandrodes DNA. This evidence indicates that G. niphandrodes is not likely to possess either a plastid organelle or its genome. This raises the possibility that the plastid was lost in the Apicomplexan following the divergence of gregarines and Cryptosporidium.

  6. Reorganization of axoplasmic organelles following beta, beta'- iminodipropionitrile administration

    PubMed Central

    1981-01-01

    beta, beta'-Iminodipropionitrile (IDPN), a synthetic compound that selectively impairs slow axonal transport, produced a rearrangement of the axonal cytoskeleton, smooth endoplasmic reticulum, and mitochondria. Immunoperoxidase staining using an antiserum to the 68,000-dalton neurofilament subunit demonstrated a displacement of neurofilaments toward the periphery of the axons of IDPN-treated rats. This change occurred simultaneously along the entire length of the sciatic nerve. Ultrastructural morphometry of the axonal organelles confirmed the peripheral relocation of neurofilaments and also showed a displacement of microtubules, smooth endoplasmic reticulum, and mitochondria to the center of the axons. The overall density of axonal mitochondria was increased, whereas those of other organelles were not significantly changed. Axons were reduced in size by 10--24%, the large axons being more affected than the small ones. The observed rearrangement of axonal organelles may be due to an effect of IDPN on microtubule-neurofilament interactions, which could in turn explain the impairment of the slow transport. Axons in IDPN intoxication are a useful model to study the organization of the axoplasm and the mechanism of axonal transport. PMID:7199048

  7. Guanine Nucleotide Exchange Factors (GEFs) Have a Critical but Not Exclusive Role in Organelle Localization of Rab GTPases*

    PubMed Central

    Cabrera, Margarita; Ungermann, Christian

    2013-01-01

    Membrane fusion at eukaryotic organelles is initiated by Rab GTPases and tethering factors. Rabs in their GDP-bound form are kept soluble in the cytoplasm by the GDP dissociation inhibitor (GDI) chaperone. Guanine nucleotide exchange factors (GEFs) are found at organelles and are critical for Rab function. Here, we surveyed the overall role of GEFs in Rab localization. We show that GEFs, but none of the proposed GDI displacement factors, are essential for the correct membrane localization of yeast Rabs. In the absence of the GEF, Rabs lost their primary localization to the target organelle. Several Rabs, such as vacuolar Ypt7, were found at the endoplasmic reticulum and thus were still membrane-bound. Surprisingly, a Ypt7 mutant that undergoes facilitated nucleotide exchange localized to vacuoles independently of its GEF Mon1-Ccz1 and rescued vacuole morphology. In contrast, wild-type Ypt7 required its GEF for localization and to counteract the extraction by GDI. Our data agree with the emerging model that GEFs are critical for Rab localization but raise the possibility that additional factors can contribute to this process. PMID:23979137

  8. Reduction of Cr (VI) into Cr (III) by organelles of Chlorella vulgaris in aqueous solution: An organelle-level attempt.

    PubMed

    Chen, Zunwei; Song, Shufang; Wen, Yuezhong

    2016-12-01

    The priority pollutant chromium (Cr) was ubiquitous and great efforts have been made to reduce Cr (VI) into less-toxic Cr (III) by alga for the convenient availability and low expense. However, the functional role of organelle inside the algal cell in Cr (VI) reduction was poorly understood. In this study, organelles in green algae Chlorella vulgaris were extracted and further decorated for Cr (VI) reduction tests. Results showed that the chloroplast exhibited not only adsorption ability of total Cr (21.18% comparing to control) but also reduction potential of Cr (VI) (almost 70% comparing to control), whose most suitable working concentration was at 17μg/mL. Furtherly, the isolated thylakoid membrane (ITM) showed better Cr (VI) reduction potential with the presence of sodium alginate (SA), even though the Hill reaction activity (HRA) was inhibited. As for photosystem II (PSII), the addition of mesoporous silica SBA-15 enhanced the reduction ability through improving the light-harvesting complex (LHC) II efficiency and electron transport rate. On the whole, the reduction ability order of the three kinds of materials based on chloroplast in C. vulgaris was PSII@SBA-15>Chloroplast>ITM@SA. The attempt made in this study to reduce the Cr (VI) with C. vulgaris organelles might not only offer basement to detect the potential action mechanism of Cr (VI) reduction by C. vulgaris but also provide a new sight for the scavenge of heavy metal with biological materials. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. 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

    2014-08-04

    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.

  10. Neospora caninum Recruits Host Cell Structures to Its Parasitophorous Vacuole and Salvages Lipids from Organelles.

    PubMed

    Nolan, Sabrina J; Romano, Julia D; Luechtefeld, Thomas; Coppens, Isabelle

    2015-05-01

    Toxoplasma gondii and Neospora caninum, which cause the diseases toxoplasmosis and neosporosis, respectively, are two closely related apicomplexan parasites. They have similar heteroxenous life cycles and conserved genomes and share many metabolic features. Despite these similarities, T. gondii and N. caninum differ in their transmission strategies and zoonotic potential. Comparative analyses of the two parasites are important to identify the unique biological features that underlie the basis of host preference and pathogenicity. T. gondii and N. caninum are obligate intravacuolar parasites; in contrast to T. gondii, events that occur during N. caninum infection remain largely uncharacterized. We examined the capability of N. caninum (Liverpool isolate) to interact with host organelles and scavenge nutrients in comparison to that of T. gondii (RH strain). N. caninum reorganizes the host microtubular cytoskeleton and attracts endoplasmic reticulum (ER), mitochondria, lysosomes, multivesicular bodies, and Golgi vesicles to its vacuole though with some notable differences from T. gondii. For example, the host ER gathers around the N. caninum parasitophorous vacuole (PV) but does not physically associate with the vacuolar membrane; the host Golgi apparatus surrounds the N. caninum PV but does not fragment into ministacks. N. caninum relies on plasma lipoproteins and scavenges cholesterol from NPC1-containing endocytic organelles. This parasite salvages sphingolipids from host Golgi Rab14 vesicles that it sequesters into its vacuole. Our data highlight a remarkable degree of conservation in the intracellular infection program of N. caninum and T. gondii. The minor differences between the two parasites related to the recruitment and rearrangement of host organelles around their vacuoles likely reflect divergent evolutionary paths.

  11. Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles

    PubMed Central

    1994-01-01

    Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein. PMID:7528220

  12. Neospora caninum Recruits Host Cell Structures to Its Parasitophorous Vacuole and Salvages Lipids from Organelles

    PubMed Central

    Nolan, Sabrina J.; Luechtefeld, Thomas

    2015-01-01

    Toxoplasma gondii and Neospora caninum, which cause the diseases toxoplasmosis and neosporosis, respectively, are two closely related apicomplexan parasites. They have similar heteroxenous life cycles and conserved genomes and share many metabolic features. Despite these similarities, T. gondii and N. caninum differ in their transmission strategies and zoonotic potential. Comparative analyses of the two parasites are important to identify the unique biological features that underlie the basis of host preference and pathogenicity. T. gondii and N. caninum are obligate intravacuolar parasites; in contrast to T. gondii, events that occur during N. caninum infection remain largely uncharacterized. We examined the capability of N. caninum (Liverpool isolate) to interact with host organelles and scavenge nutrients in comparison to that of T. gondii (RH strain). N. caninum reorganizes the host microtubular cytoskeleton and attracts endoplasmic reticulum (ER), mitochondria, lysosomes, multivesicular bodies, and Golgi vesicles to its vacuole though with some notable differences from T. gondii. For example, the host ER gathers around the N. caninum parasitophorous vacuole (PV) but does not physically associate with the vacuolar membrane; the host Golgi apparatus surrounds the N. caninum PV but does not fragment into ministacks. N. caninum relies on plasma lipoproteins and scavenges cholesterol from NPC1-containing endocytic organelles. This parasite salvages sphingolipids from host Golgi Rab14 vesicles that it sequesters into its vacuole. Our data highlight a remarkable degree of conservation in the intracellular infection program of N. caninum and T. gondii. The minor differences between the two parasites related to the recruitment and rearrangement of host organelles around their vacuoles likely reflect divergent evolutionary paths. PMID:25750213

  13. Myosin-Va and Dynamic Actin Oppose Microtubules to Drive Long-Range Organelle Transport

    PubMed Central

    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.

    2014-01-01

    Summary 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 [1–8]. 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 [2]. 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 [5, 9]. 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 [10, 11]. PMID:25065759

  14. 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'.

  15. Highly divergent mitochondrion-related organelles in anaerobic parasitic protozoa.

    PubMed

    Makiuchi, Takashi; Nozaki, Tomoyoshi

    2014-05-01

    The mitochondria have arisen as a consequence of endosymbiosis of an ancestral α-proteobacterium with a methane-producing archae. The main function of the canonical aerobic mitochondria include ATP generation via oxidative phosphorylation, heme and phospholipid synthesis, calcium homeostasis, programmed cell death, and the formation of iron-sulfur clusters. Under oxygen-restricted conditions, the mitochondrion has often undergone remarkable reductive alterations of its content and function, leading to the generation of mitochondrion-related organelles (MROs), such as mitosomes, hydrogenosomes, and mithochondrion-like organelles, which are found in a wide range of anaerobic/microaerophilic eukaryotes that include several medically important parasitic protists such as Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Cryptosporidium parvum, Blastocystis hominis, and Encephalitozoon cuniculi, as well as free-living protists such as Sawyeria marylandensis, Neocallimastix patriciarum, and Mastigamoeba balamuthi. The transformation from canonical aerobic mitochondria to MROs apparently have occurred in independent lineages, and resulted in the diversity of their components and functions. Due to medical and veterinary importance of the MRO-possessing human- and animal-pathogenic protozoa, their genomic, transcriptomic, proteomic, and biochemical evidence has been accumulated. Detailed analyses of the constituents and functions of the MROs in such anaerobic pathogenic protozoa, which reside oxygen-deprived or oxygen-poor environments such as the mammalian intestine and the genital organs, should illuminate the current evolutionary status of the MROs in these organisms, and give insight to environmental constraints that drive the evolution of eukaryotes and their organelles. In this review, we summarize and discuss the diverse metabolic functions and protein transport systems of the MROs from anaerobic parasitic protozoa.

  16. 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.

  17. Organelle biogenesis and intracellular lipid transport in eukaryotes.

    PubMed Central

    Voelker, D R

    1991-01-01

    The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those

  18. 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

  19. Organelle evolution, fragmented rRNAs, and Carl.

    PubMed

    Gray, Michael W

    2014-01-01

    I am honored to have been asked to contribute to this memorial issue, although I cannot claim to have known Carl Woese well. Carl's insights and the discoveries that his research group made over the years certainly stimulated my own research program, and at several points early on, interactions with him were pivotal in my career. Here I comment on these personal dealings with Carl and emphasize his influence in two areas of long-standing interest in my lab: organelle evolution and rRNA evolution.

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

    PubMed

    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

    2015-02-02

    Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, create vaccines, and 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 to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. This unit describes the methods for generating and screening the iPhage display system, and explains how to select and validate candidate internalizing homing peptide.

  1. Sequence-Specific Polyampholyte Phase Separation in Membraneless Organelles

    NASA Astrophysics Data System (ADS)

    Lin, Yi-Hsuan; Forman-Kay, Julie D.; Chan, Hue Sun

    2016-10-01

    Liquid-liquid phase separation of charge- and/or aromatic-enriched intrinsically disordered proteins (IDPs) is critical in the biological function of membraneless organelles. Much of the physics of this recent discovery remains to be elucidated. Here, we present a theory in the random phase approximation to account for electrostatic effects in polyampholyte phase separations, yielding predictions consistent with recent experiments on the IDP Ddx4. The theory is applicable to any charge pattern and thus provides a general analytical framework for studying sequence dependence of IDP phase separation.

  2. Organelle evolution, fragmented rRNAs, and Carl

    PubMed Central

    Gray, Michael W

    2014-01-01

    I am honored to have been asked to contribute to this memorial issue, although I cannot claim to have known Carl Woese well. Carl’s insights and the discoveries that his research group made over the years certainly stimulated my own research program, and at several points early on, interactions with him were pivotal in my career. Here I comment on these personal dealings with Carl and emphasize his influence in two areas of long-standing interest in my lab: organelle evolution and rRNA evolution. PMID:24572720

  3. 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

  4. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Inheritance of organelle DNA sequences in a citrus-poncirus intergeneric cross.

    PubMed

    Moreira, C D; Gmitter, F G; Grosser, J W; Huang, S; Ortega, V M; Chase, C D

    2002-01-01

    Many land plants deviate from the maternal pattern of organelle inheritance. In this study, heterologous mitochondrial and chloroplast probes were used to investigate the inheritance of organelle genomes in the progeny of an intergeneric cross. The seed parent was LB 1-18 (a hybrid of Citrus reticulata Blanco cv. Clementine x C. paradisi Macf. cv. Duncan) and the pollen parent was the cross-compatible species Poncirus trifoliata (L.) Raf. All 26 progeny examined exhibited maternal inheritance of plastid petA and petD loci. However, 17 of the 26 progeny exhibited an apparent biparental inheritance of mitochondrial atpA, cob, coxII, and coxIII restriction fragment length polymorphisms (RFLPs) and maternal inheritance of mitochondrial rrn26 and coxI RFLPs. The remaining nine progeny inherited only maternal mitochondrial DNA (mtDNA) configurations. Investigations of plant mitochondrial genome inheritance are complicated by the multipartite structure of this genome, nuclear gene control over mitochondrial genome organization, and transfer of mitochondrial sequences to the nucleus. In this study, paternal mtDNA configurations were not detected in purified mtDNA of progeny plants, but were present in progeny DNA preparations enriched for nuclear genome sequences. MtDNA sequences in the nuclear genome therefore produced an inheritance pattern that mimics biparental inheritance of mtDNA.

  6. 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.

  7. A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles.

    PubMed

    Matthiesen, S H; Shenoy, S M; Kim, K; Singer, R H; Satir, B H

    2001-12-01

    We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.

  8. 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

  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 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.

  11. The effect of organelle discovery upon sub-cellular protein localisation.

    PubMed

    Breckels, L M; Gatto, L; Christoforou, A; Groen, A J; Lilley, K S; Trotter, M W B

    2013-08-02

    Prediction of protein sub-cellular localisation by employing quantitative mass spectrometry experiments is an expanding field. Several methods have led to the assignment of proteins to specific subcellular localisations by partial separation of organelles across a fractionation scheme coupled with computational analysis. Methods developed to analyse organelle data have largely employed supervised machine learning algorithms to map unannotated abundance profiles to known protein-organelle associations. Such approaches are likely to make association errors if organelle-related groupings present in experimental output are not included in data used to create a protein-organelle classifier. Currently, there is no automated way to detect organelle-specific clusters within such datasets. In order to address the above issues we adapted a phenotype discovery algorithm, originally created to filter image-based output for RNAi screens, to identify putative subcellular groupings in organelle proteomics experiments. We were able to mine datasets to a deeper level and extract interesting phenotype clusters for more comprehensive evaluation in an unbiased fashion upon application of this approach. Organelle-related protein clusters were identified beyond those sufficiently annotated for use as training data. Furthermore, we propose avenues for the incorporation of observations made into general practice for the classification of protein-organelle membership from quantitative MS experiments. Protein sub-cellular localisation plays an important role in molecular interactions, signalling and transport mechanisms. The prediction of protein localisation by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavour in improving protein annotation. Several such approaches use gradient-based separation of cellular organelle content to measure relative protein abundance across distinct gradient fractions. The distribution profiles are commonly mapped in

  12. 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

  13. Characterization of a Planctomycetal Organelle: a Novel Bacterial Microcompartment for the Aerobic Degradation of Plant Saccharides

    PubMed Central

    Erbilgin, Onur; McDonald, Kent L.

    2014-01-01

    Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae. PMID:24487526

  14. 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.

  15. 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

  16. Genetic Regulation of Caenorhabditis elegans Lysosome Related Organelle Function

    PubMed Central

    Soukas, Alexander A.; Carr, Christopher E.; Ruvkun, Gary

    2013-01-01

    Lysosomes are membrane-bound organelles that contain acid hydrolases that degrade cellular proteins, lipids, nucleic acids, and oligosaccharides, and are important for cellular maintenance and protection against age-related decline. Lysosome related organelles (LROs) are specialized lysosomes found in organisms from humans to worms, and share many of the features of classic lysosomes. Defective LROs are associated with human immune disorders and neurological disease. Caenorhabditis elegans LROs are the site of concentration of vital dyes such as Nile red as well as age-associated autofluorescence. Even though certain short-lived mutants have high LRO Nile red and high autofluorescence, and other long-lived mutants have low LRO Nile red and low autofluorescence, these two biologies are distinct. We identified a genetic pathway that modulates aging-related LRO phenotypes via serotonin signaling and the gene kat-1, which encodes a mitochondrial ketothiolase. Regulation of LRO phenotypes by serotonin and kat-1 in turn depend on the proton-coupled, transmembrane transporter SKAT-1. skat-1 loss of function mutations strongly suppress the high LRO Nile red accumulation phenotype of kat-1 mutation. Using a systems approach, we further analyzed the role of 571 genes in LRO biology. These results highlight a gene network that modulates LRO biology in a manner dependent upon the conserved protein kinase TOR complex 2. The results implicate new genetic pathways involved in LRO biology, aging related physiology, and potentially human diseases of the LRO. PMID:24204312

  17. Anesthesia-Induced Developmental Neurodegeneration: The Role of Neuronal Organelles

    PubMed Central

    Jevtovic-Todorovic, Vesna; Boscolo, A.; Sanchez, V.; Lunardi, N.

    2012-01-01

    Exposure to general anesthetics (GAs) and antiepileptics during critical stages of brain development causes significant neurotoxicity to immature neurons. Many animal, and emerging human studies have shown long-term functional sequelae manifested as behavioral deficits and cognitive impairments. Since GAs and antiepileptic drugs are a necessity, current research is focused on deciphering the mechanisms responsible for anesthesia-induced developmental neurotoxicity so that protective strategies can be devised. These agents promote massive and wide-spread neuroapoptosis that is caused by the impairment of integrity and function of neuronal organelles. Mitochondria and endoplasmic reticulum are particularly vulnerable. By promoting significant release of intracellular calcium from the endoplasmic reticulum, anesthetics cause an increase in mitochondrial calcium load resulting in the loss of their integrity, release of pro-apoptotic factors, functional impairment of ATP synthesis, and enhanced accumulation of reactive oxygen species. The possibility that GAs may have direct damaging effects on mitochondria, resulting in the impairment of their morphogenesis, also has been proposed. This review will present evidence that neuronal organelles are critical and early targets of anesthesia-induced developmental neurotoxicity. PMID:23087668

  18. Selective Lysosomal Transporter Degradation by Organelle Membrane Fusion.

    PubMed

    McNally, Erin Kate; Karim, Mahmoud Abdul; Brett, Christopher Leonard

    2017-01-23

    Lysosomes rely on their resident transporter proteins to return products of catabolism to the cell for reuse and for cellular signaling, metal storage, and maintaining the lumenal environment. Despite their importance, little is known about the lifetime of these transporters or how they are regulated. Using Saccharomyces cerevisiae as a model, we discovered a new pathway intrinsic to homotypic lysosome membrane fusion that is responsible for their degradation. Transporter proteins are selectively sorted by the docking machinery into an area between apposing lysosome membranes, which is internalized and degraded by lumenal hydrolases upon organelle fusion. These proteins have diverse lifetimes that are regulated in response to protein misfolding, changing substrate levels, or TOR activation. Analogous to endocytosis for controlling surface protein levels, the "intralumenal fragment pathway" is critical for lysosome membrane remodeling required for organelle function in the context of cellular protein quality control, ion homeostasis, and metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

    Gordon, Natalie K; Gordon, Richard

    2016-03-10

    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).

  20. Unraveling the complexity of lipid body organelles in human eosinophils

    PubMed Central

    Melo, Rossana C. N.; Weller, Peter F.

    2014-01-01

    Lipid-rich organelles are common in many cell types. In cells, such as adipocytes, these organelles are termed LDs, whereas in other cells, such as leukocytes, they are called LBs. The study of leukocyte LBs has attracted attention as a result of their association with human diseases. In leukocytes, such as eosinophils, LB accumulation has been documented extensively during inflammatory conditions. In these cells, LBs are linked to the regulation of immune responses by compartmentalization of several proteins and lipids involved in the control and biosynthesis of inflammatory mediators (eicosanoids). However, it has been unclear how diverse proteins, including membrane-associated enzymes involved in eicosanoid formation, incorporate into LBs, especially if the internal content of LBs is assumed to consist solely of stores of neutral lipids, as present within adipocyte LDs. Studies of the formation, function, and ultrastructure of LBs in eosinophils have been providing insights pertinent to LBs in other leukocytes. Here, we review current knowledge of the composition and function of leukocyte LBs as provided by studies of human eosinophil LBs, including recognitions of the internal architecture of eosinophil LBs based on 3D electron tomographic analyses. PMID:25210147

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

    PubMed

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

    2012-12-01

    Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multiprotein 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. Copyright © 2012 John Wiley & Sons, Ltd.

  2. Organelles in developing neurons: essential regulators of neuronal morphogenesis and function.

    PubMed

    Sekine, Sayaka; Miura, Masayuki; Chihara, Takahiro

    2009-01-01

    Eukaryotic cells contain multiple intracellular organelles which are structurally and functionally distinct membrane-delimited compartments. Organelles play vital roles in many cellular events in essentially all eukaryotic cells. Although the canonical roles of organelles are well described by classical in vitro studies, little is known about the specific physiological roles of organelles in neurons, which possess extremely polarized cellular structures and have a massive cellular volume compared with most eukaryotic cells. Studies that make use of recently developed genetic and microscopic techniques are currently elucidating the unexpectedly specialized roles of intracellular, membrane-delimited organelles in neuronal morphogenesis and function, and in human disease. Here we review recent advances in understanding the roles of organelles (the ER-Golgi secretory pathway, endosomes and mitochondria) in developing neurons.

  3. Seeing is believing: on the use of image databases for visually exploring plant organelle dynamics.

    PubMed

    Mano, Shoji; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nishimura, Mikio

    2009-12-01

    Organelle dynamics vary dramatically depending on cell type, developmental stage and environmental stimuli, so that various parameters, such as size, number and behavior, are required for the description of the dynamics of each organelle. Imaging techniques are superior to other techniques for describing organelle dynamics because these parameters are visually exhibited. Therefore, as the results can be seen immediately, investigators can more easily grasp organelle dynamics. At present, imaging techniques are emerging as fundamental tools in plant organelle research, and the development of new methodologies to visualize organelles and the improvement of analytical tools and equipment have allowed the large-scale generation of image and movie data. Accordingly, image databases that accumulate information on organelle dynamics are an increasingly indispensable part of modern plant organelle research. In addition, image databases are potentially rich data sources for computational analyses, as image and movie data reposited in the databases contain valuable and significant information, such as size, number, length and velocity. Computational analytical tools support image-based data mining, such as segmentation, quantification and statistical analyses, to extract biologically meaningful information from each database and combine them to construct models. In this review, we outline the image databases that are dedicated to plant organelle research and present their potential as resources for image-based computational analyses.

  4. Characteristics in Sliding Motions of Small Organelles in a Nitella Internodal Cell

    NASA Astrophysics Data System (ADS)

    Uchida, Go; Nemoto, Tomomi; Tsuchiya, Yoshimi

    1995-12-01

    Steady velocities of small organelles smoothly moving on chloroplasts in a Nitella internodal cell have been investigated at various temperatures. It has been found that variance in the velocities of the organelles changes in proportion to their average velocity, which has been first elucidated from the temperature dependence of the organelle's velocity. This result suggests that the generation process of the force due to the actin-myosin is a Poisson like stochastic one. Thus, we have discussed a stochastic model for the motion of the organelle with many myosin-like molecules and estimated the force to be 4.2×10-12 N.

  5. Organelle DB: a cross-species database of protein localization and function.

    PubMed

    Wiwatwattana, Nuwee; Kumar, Anuj

    2005-01-01

    To efficiently utilize the growing body of available protein localization data, we have developed Organelle DB, a web-accessible database cataloging more than 25,000 proteins from nearly 60 organelles, subcellular structures and protein complexes in 154 organisms spanning the eukaryotic kingdom. Organelle DB is the first on-line resource devoted to the identification and presentation of eukaryotic proteins localized to organelles and subcellular structures. As such, Organelle DB is a strong resource of data from the human proteome as well as from the major model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans and Mus musculus. In particular, Organelle DB is a central repository of yeast data, incorporating results--and actual fluorescent imagesfrom ongoing large-scale studies of protein localization in S.cerevisiae. Each protein in Organelle DB is presented with its sequence and, as available, a detailed description of its function; functions were extracted from relevant model organism databases, and links to these databases are provided within Organelle DB. To facilitate data interoperability, we have annotated all protein localizations using vocabulary from the Gene Ontology consortium. We also welcome new data for inclusion in Organelle DB, which may be freely accessed at http://organelledb.lsi.umich.edu.

  6. Structure and function of ER membrane contact sites with other organelles.

    PubMed

    Phillips, Melissa J; Voeltz, Gia K

    2016-02-01

    The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

  7. Modeling transport of a pulse of radiolabeled organelles in a Drosophila unipolar motor neuron.

    PubMed

    Kuznetsov, A V

    2013-01-01

    Based on published experimental evidence, this paper develops a model for the transport of a pulse of radiolabeled organelles in a unipolar Drosophila motor neuron. In particular, since published data indicate that no microtubules (MTs) travel from the primary neurite into the dendrite, it is investigated how organelles are transported into the dendrite. Analytical solutions describing concentrations of kinesin- and dynein-driven organelles in the primary neurite, axon, and dendrite are obtained. The effects of increasing the width of the pulse and increasing the rate of organelle transition rate from the kinesin-driven to the dynein-driven state are investigated.

  8. Mechanisms of organelle transport and capture along proplatelets during platelet production

    PubMed Central

    Richardson, Jennifer L.; Shivdasani, Ramesh A.; Boers, Chad; Hartwig, John H.; Italiano, Joseph E.

    2005-01-01

    Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. Platelet packaging and release concludes at the tips of each proplatelet. Essential in this process is the distribution of organelles and platelet-specific granules into the nascent platelets. To investigate the mechanism of delivery of organelles into putative platelets, the distribution and dynamics of organelles/granules was monitored. Individual organelles are sent from the cell body to the proplatelets where they move bidirectionally until they are captured at proplatelet ends. Movement occurs at approximately 0.2 μm/min, but pauses and changes in direction are frequent. At any given time, approximately 30% of organelles/granules are in motion. Actin poisons do not diminish organelle motion, and vesicular structures are intimately associated with the microtubules. Therefore, movement appears to involve microtubule-based forces. Bidirectional organelle movement is conveyed by the bipolar organization of microtubules within the proplatelet, as kinesin-coated beads move bidirectionally on the microtubule arrays of permeabilized proplatelets. Movement of organelles along proplatelets involves 2 mechanisms: organelles travel along microtubules, and the linked microtubules move relative to each other. These studies demonstrate that the components that form platelets are delivered to and assembled de novo along proplatelets. PMID:16118320

  9. Structure and function of ER membrane contact sites with other organelles

    PubMed Central

    Phillips, Melissa J.; Voeltz, Gia K.

    2016-01-01

    The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes. PMID:26627931

  10. 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. Copyright © 2011 Elsevier GmbH. All rights reserved.

  11. 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.

  12. Organelle targeting during bacterial infection: insights from Listeria.

    PubMed

    Lebreton, Alice; Stavru, Fabrizia; Cossart, Pascale

    2015-06-01

    Listeria monocytogenes, a facultative intracellular bacterium responsible for severe foodborne infections, is now recognized as a multifaceted model in infection biology. Comprehensive studies of the molecular and cellular basis of the infection have unraveled how the bacterium crosses the intestinal and feto-placental barriers, invades several cell types in which it multiplies and moves, and spreads from cell to cell. Interestingly, although Listeria does not actively invade host cell organelles, it can interfere with their function. We discuss the effect of Listeria on the endoplasmic reticulum (ER) and the mechanisms leading to the fragmentation of the mitochondrial network and its consequences, and review the strategies used by Listeria to subvert nuclear functions, more precisely to control host gene expression at the chromatin level. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Cilia in the CNS: the Quiet Organelle Claims Center Stage

    PubMed Central

    Louvi, Angeliki; Grove, Elizabeth A.

    2011-01-01

    Summary The primary cilium is a cellular organelle that is almost ubiquitous in eukaryotes, yet its functions in vertebrates have been slow to emerge. The last fifteen years have been marked by accelerating insight into the biology of primary cilia, arising from the synergy of three major lines of research. These research programs describe a specialized mode of protein trafficking in cilia, reveal that genetic disruptions of primary cilia cause complex human disease syndromes, and establish that Sonic hedgehog (Shh) signal transduction requires the primary cilium. New lines of research have branched off to investigate the role of primary cilia in neuronal signaling, adult neurogenesis, and brain tumor formation. We review a fast expanding literature to determine what we now know about the primary cilium in the developing and adult CNS, and what new directions should lead to further clarity. PMID:21435552

  14. Physiological role of taurine--from organism to organelle.

    PubMed

    Lambert, I H; Kristensen, D M; Holm, J B; Mortensen, O H

    2015-01-01

    Taurine is often referred to as a semi-essential amino acid as newborn mammals have a limited ability to synthesize taurine and have to rely on dietary supply. Taurine is not thought to be incorporated into proteins as no aminoacyl tRNA synthetase has yet been identified and is not oxidized in mammalian cells. However, taurine contributes significantly to the cellular pool of organic osmolytes and has accordingly been acknowledged for its role in cell volume restoration following osmotic perturbation. This review describes taurine homeostasis in cells and organelles with emphasis on taurine biophysics/membrane dynamics, regulation of transport proteins involved in active taurine uptake and passive taurine release as well as physiological processes, for example, development, lung function, mitochondrial function, antioxidative defence and apoptosis which seem to be affected by a shift in the expression of the taurine transporters and/or the cellular taurine content. © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  15. Transport Selectivity of Nuclear Pores, Phase Separation, and Membraneless Organelles.

    PubMed

    Schmidt, H Broder; Görlich, Dirk

    2016-01-01

    Nuclear pore complexes (NPCs) provide a selective passageway for receptor-mediated active transport between nucleus and cytoplasm, while maintaining the distinct molecular compositions of both compartments at large. In this review we discuss how NPCs gain a remarkable sorting selectivity from non-globular FG domains and their phase separation into dense polymer meshworks. The resulting sieve-like FG hydrogels are effective barriers to normal macromolecules but are at the same time highly permeable to shuttling nuclear transport receptors, which bind to FG motifs as well as to their designated cargoes. Phase separation driven by disordered protein domains was recently also recognized as being pivotal to the formation of membraneless organelles, making it an important emerging principle in cell biology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Sizing Subcellular Organelles and Nanoparticles Confined within Aqueous Droplets

    PubMed Central

    Gadd, Jennifer C.; Kuyper, Christopher L.; Fujimoto, Bryant S.; Allen, Richard W.; Chiu, Daniel T.

    2009-01-01

    This paper describes two complementary techniques, single-particle tracking and correlation spectroscopy, for accurately sizing nanoparticles confined within picoliter-volume aqueous droplets. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged, and we demonstrated this approach for sizing single fluorescent beads. Fluorescence correlation spectroscopy detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules; we demonstrated FCS for sizing synaptic vesicles confined in aqueous droplets. In combination with recent advances in droplet manipulations and analysis, we anticipate this capability to size single nanoparticles and molecules in free solution will complement existing tools for probing cellular systems, subcellular organelles, and nanoparticles. PMID:18363409

  17. Quantum dots targeted to the assigned organelle in living cells.

    PubMed

    Hoshino, Akiyoshi; Fujioka, Kouki; Oku, Taisuke; Nakamura, Shun; Suga, Masakazu; Yamaguchi, Yukio; Suzuki, Kazuo; Yasuhara, Masato; Yamamoto, Kenji

    2004-01-01

    Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear- and mitochondria-targeting ligands. When QDs with nuclear-localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.

  18. Proteomic changes in bovine heart mitochondria with age: using a novel technique for organelle separation and enrichment.

    PubMed

    Kiri, Ajay N; Tran, Hung-Cuong; Drahos, Kate L; Lan, Wenkui; McRorie, Donald K; Horn, Marcus J

    2005-12-01

    Separation and enrichment of organelles from complex biological mixtures are important for proteomic analysis. Two widely used current standard techniques to isolate individual organelles include differential and density-gradient centrifugation. Although these techniques have proven useful for processing small volumes of sample, multiple rounds of centrifugation are required when performing a large-scale purification. In this report, we have introduced a novel technique: continuous-flow ultracentrifugation using a sucrose gradient to separate, accumulate, and highly enrich bovine heart mitochondria in one step. To demonstrate the advantage of the technique, mitochondrial proteins from two different bovine hearts (3-8 mo and 18-30 mo old) were examined. For each age group, 100 g of bovine heart tissue were homogenized by a blending procedure. After removal of the nuclei, the entire remaining homogenate was loaded onto a proteomics continuous-flow ultracentrifuge to separate and enrich the organelles. Fractions were collected and mitochondria-enriched fractions were identified by Western blot analysis. To study the protein profile changes with aging in the mitochondrial proteome, the mitochondria-enriched fractions were applied to two-dimensional gel electrophoresis. The resulting two-dimensional PAGE gels were subsequently analyzed by image analysis software to identify proteins unique to each age group and proteins with at least twofold differences in protein expression. These proteins were then digested with trypsin and identified by mass spectrometer. Significant differences in the protein profiles of the two differently aged mitochondria preparations were found. The continuous-flow ultracentrifugation technique was demonstrated to be a powerful tool for separation and enrichment of organelles and their sub-types.

  19. 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

  20. 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

  1. An intracellular nanotrap redirects proteins and organelles in live bacteria.

    PubMed

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

    2015-01-13

    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. 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 building synthetic cellular structures

  2. 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

  3. 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…

  4. 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…

  5. Investigating the AGN-Galaxy Interaction Relationship by Examining the Color and Morphology Measurements of Real and Simulated AGN Host Galaxies

    NASA Astrophysics Data System (ADS)

    Pierce, Christina M.

    2009-01-01

    UV-optical colors provide a clear distinction between quiescent galaxies and those undergoing star formation. Galaxy morphology measurements, such as the Gini coefficient, M20, concentration, asymmetry, and the Sersic index, allow identification of interacting galaxies and separation of non-interacting galaxies into bulge or disk-dominated systems. Thus, one can use the colors and morphologies of AGN host galaxies to probe the predicted relationship between galaxy interactions and significant black hole growth (an AGN stage). However, due to the UV excess observed in many AGNs (particularly quasars and Seyfert 1 galaxies) and the potentially significant optical contribution from AGNs that are not heavily obscured, one must exercise caution when interpreting the results from color and morphology measurements of AGN host galaxies. With this in mind, we created a set of simulated AGNs to test the reliability of color and morphology measurements of AGN host galaxies. The results were compared to observations of AGN host galaxies at z 1 from the All-wavelength Extended Groth Strip International Survey (AEGIS). Our observed results reveal a population of X-ray luminous AGN hosts that appear to have green UV-optical colors, indicative of recent star-formation, and a largely disk-dominated profile, suggesting a bulge that is not yet fully developed. Comparison with results from our simulated AGNs suggest that at least some of the observational results are not likely to be due to color or morphological contamination from the presence of an AGN. Therefore, the observed AGN hosts seem to represent a real population that may be going through a transition phase, during which significant star-formation has recently ceased, but for which the black hole remains quite luminous.

  6. Describing autophagy via analysis of individual organelles by capillary electrophoresis with laser induced fluorescence detection.

    PubMed

    Satori, Chad P; Arriaga, Edgar A

    2013-12-03

    Autophagy is a cellular process responsible for the degradation of intracellular cargo. Its dynamic nature and the multiple types of autophagy organelles present at a given time make current measurements, such as those done by Western blotting, insufficient to understand autophagy and its roles in aging and disease. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) has been used previously to count and determine properties of individual organelles, but has never been used on autophagy organelles or for determination of changes of such properties. Here we used autophagy organelles isolated from L6 cells expressing GFP-LC3, which is an autophagy marker, to develop a CE-LIF method for the determination of the number of autophagy organelles, their individual GFP-LC3 fluorescence intensities, and their individual electrophoretic mobilities. These properties were compared under basal and rapamycin-driven autophagy, which showed differences in the number of detected organelles and electrophoretic mobility distributions of autophagy organelles. Vinblastine treatment was also used to halt autophagy and further characterize changes and provide additional insight on the nature of autophagy organelles. This approach revealed dramatic and opposite directions in changes of organelle numbers, GFP-LC3 contents, and electrophoretic mobilities during the duration of the vinblastine treatment. These trends suggested the identity of organelle types being detected. These observations demonstrate that individual organelle analysis by CE-LIF is a powerful technology to investigate the complexity and nature of autophagy, a process that plays critical roles in response to drug treatments, aging, and disease.

  7. Describing Autophagy via Analysis of Individual Organelles by Capillary Electrophoresis with Laser Induced Fluorescence Detection

    PubMed Central

    Satori, Chad P.

    2013-01-01

    Autophagy is a cellular process responsible for the degradation of intracellular cargo. Its dynamic nature and the multiple types of autophagy organelles present at a given time make current measurements, such as those done by Western blotting, insufficient to understand autophagy and its roles in aging and disease. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) has been used previously to count and determine properties of individual organelles, but has never been used on autophagy organelles or for determination of changes of such properties. Here we used autophagy organelles isolated from L6 cells expressing GFP-LC3, which is an autophagy marker, to develop a CE-LIF method for the determination of the number of autophagy organelles, their individual GFP-LC3 fluorescence intensities, and their individual electrophoretic mobilities. These properties were compared under basal and rapamycin-driven autophagy, which showed differences in the number of detected organelles and electrophoretic mobility distributions of autophagy organelles. Vinblastine treatment was also used to halt autophagy and further characterize changes and provide additional insight on the nature of autophagy organelles. This approach revealed dramatic and opposite directions in changes of organelle numbers, GFP-LC3 contents, and electrophoretic mobilities during the duration of the vinblastine treatment. These trends suggested the identity of organelle types being detected. These observations demonstrate that individual organelle analysis by CE-LIF is a powerful technology to investigate the complexity and nature of autophagy, a process that plays critical roles in response to drug treatments, aging, and disease. PMID:24164243

  8. Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists.

    PubMed

    Sanitá Lima, Matheus; Smith, David Roy

    2017-09-21

    Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing (NGS) technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq) data. Little is known about organelle transcription in non-model species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or non-coding content. For every species analyzed, transcripts covered at least 85% of the mitochondrial and/or plastid genomes (all of which were ≤ 105 kb), indicating that most of the organelle DNA-coding and non-coding-is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that non-coding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb) across a broader range of eukaryotes, this could indicate that non-coding organelle RNAs are regulating fundamental processes within eukaryotic cells. Copyright © 2017, G3: Genes, Genomes, Genetics.

  9. Morphological changes in human spermatozoa as examined under scanning electron microscope after in vitro exposure to saponins isolated from Sapindus mukorossi.

    PubMed

    Dhar, J D; Bajpai, V K; Setty, B S; Kamboj, V P

    1989-05-01

    Saponins isolated from Sapindus mukorossi have potent spermicidal activity. Morphological changes in human ejaculated spermatozoa after exposure to these saponins were evaluated under Scanning Electron Microscope. The minimum effective concentration (0.05% in spot test) did not affect the surface topography after exposure for 1 minute. However, incubation of spermatozoa for 10 minutes resulted in extensive vesiculation and disruption of plasma membrane in the head region. Higher concentrations (0.1%, 1.25%, 2.5% and 5.0%) caused more or less similar changes which included vesiculation, vacuolation, disruption or erosion of membranes in the head region. These findings suggest that the morphological changes observed are due to alterations in the glycoproteins associated with the lipid bilayer of plasma membrane of spermatozoa.

  10. Recognising moulting behaviour in trilobites by examining morphology, development and preservation: Comment on Błażejowski et al. 2015

    PubMed Central

    Daley, Allison C.

    2016-01-01

    A 365 million year‐old trilobite moult‐carcass assemblage was described by Błażejowski et al. (2015) as the oldest direct evidence of moulting in the arthropod fossil record. Unfortunately, their suppositions are insufficiently supported by the data provided. Instead, the morphology, configuration and preservational context of the highly fossiliferous locality (Kowala Quarry, Poland) suggest that the specimen consists of two overlapping, queued carcasses. The wider fossil record of moulting actually extends back 520 million years, providing an unparalleled opportunity to study behaviour, ecology and development in early animals. Taking cues from modern analogues, it is possible to quantify precise details about moulting behaviour to determine broad‐scale evolutionary trends, ontogenetic sequences and morphological selection pressures. In this review, we argue that this rich source of data has been underused in evolutionary studies, though has great potential for investigating the life history and evolution of arthropods in deep time. PMID:27545417

  11. 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.

  12. 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.

  13. Myosin IIA is critical for organelle distribution and F-actin organization in megakaryocytes and platelets.

    PubMed

    Pertuy, Fabien; Eckly, Anita; Weber, Josiane; Proamer, Fabienne; Rinckel, Jean-Yves; Lanza, François; Gachet, Christian; Léon, Catherine

    2014-02-20

    During proplatelet formation, a relatively homogeneous content of organelles is transported from the megakaryocyte (MK) to the nascent platelets along microtubule tracks. We found that platelets from Myh9(-/-) mice and a MYH9-RD patient were heterogeneous in their organelle content (granules and mitochondria). In addition, Myh9(-/-) MKs have an abnormal cytoplasmic clustering of organelles, suggesting that the platelet defect originates in the MKs. Myosin is not involved in the latest stage of organelle traffic along microtubular tracks in the proplatelet shafts as shown by confocal observations of proplatelet buds. By contrast, it is required for the earlier distribution of organelles within the large MK preplatelet fragments shed into the sinusoid circulation before terminal proplatelet remodeling. We show here that F-actin is abnormally clustered in the cytoplasm of Myh9(-/-) MKs and actin polymerization is impaired in platelets. Myosin IIA is required for normal granule motility and positioning within MKs, mechanisms that may be dependent on organelle traveling and tethering onto F-actin cytoskeleton tracks. Altogether, our results indicate that the distribution of organelles within platelets critically depends on a homogeneous organelle distribution within MKs and preplatelet fragments, which requires myosin IIA.

  14. Protein import into the photosynthetic organelles of Paulinella chromatophora and its implications for primary plastid endosymbiosis.

    PubMed

    Mackiewicz, Paweł; Bodył, Andrzej; Gagat, Przemysław

    2012-12-01

    The rhizarian amoeba Paulinella chromatophora harbors two photosynthetically active organelles of cyanobacterial origin that have been acquired independently of classic primary plastids. Because their acquisition did take place relatively recently, they are expected to provide new insight into the ancient cyanobacterial primary endosymbiosis. During the process of Paulinella endosymbiont-to-organelle transformation, more than 30 genes have been transferred from the organelle to the host nuclear genome via endosymbiotic gene transfer (EGT). The article discusses step-by-step protein import of EGT-derived proteins into Paulinella photosynthetic organelles with the emphasis on the nature of their targeting signals and the final passage of proteins through the inner organelle membrane. The latter most probably involves a simplified Tic translocon composed of Tic21- and Tic32-like proteins as well as a Hsp70-based motor responsible for pulling of imported proteins into the organelle matrix. Our results indicate that although protein translocation across the inner membrane of Paulinella photosynthetic organelles seems to resemble the one in classic primary plastids, the transport through the outer membrane does not. The differences could result from distinct integration pathways of Paulinella photosynthetic organelles and primary plastids with their respective host cells.

  15. Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

    PubMed Central

    Davis, Lianne C.; Wagner, Siegfried K.T.Y.; Lewis, Alexander M.; Parrington, John; Churchill, Grant C.

    2013-01-01

    The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca2+ stores that release Ca2+ in response to the second messengers IP3 and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca2+ released from acidic organelles by NAADP subsequently recruits IP3 or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca2+ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca2+ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca2+-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca2+ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca2+ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca2+ oscillations and waves. PMID:23479744

  16. Characteristics of weak base-induced vacuoles formed around individual acidic organelles.

    PubMed

    Hiruma, Hiromi; Kawakami, Tadashi

    2011-01-01

    We have previously found that the weak base 4-aminopyridine induces Brownian motion of acidic organelles around which vacuoles are formed, causing organelle traffic disorder in neurons. Our present study investigated the characteristics of vacuoles induced by weak bases (NH(4)Cl, aminopyridines, and chloroquine) using mouse cells. Individual vacuoles included acidic organelles identified by fluorescent protein expression. Mitochondria and actin filaments were extruded outside the vacuoles, composing the vacuole rim. Staining with amine-reactive fluorescence showed no protein/amino acid content in vacuoles. Thus, serous vacuolar contents are probably partitioned by viscous cytosol, other organelles, and cytoskeletons, but not membrane. The weak base (chloroquine) was immunochemically detected in intravacuolar organelles, but not in vacuoles. Early vacuolization was reversible, but long-term vacuolization caused cell death. The vacuolization and cell death were blocked by the vacuolar H(+)-ATPase inhibitor and Cl--free medium. Staining with LysoTracker or LysoSensor indicated that intravacuolar organelles were strongly acidic and vacuoles were slightly acidic. This suggests that vacuolization is caused by accumulation of weak base and H(+) in acidic organelles, driven by vacuolar H(+)-ATPase associated with Cl(-) entering, and probably by subsequent extrusion of H(+) and water from organelles to the surrounding cytoplasm.

  17. Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes.

    PubMed

    Stechmann, Alexandra; Hamblin, Karleigh; Pérez-Brocal, Vicente; Gaston, Daniel; Richmond, Gregory S; van der Giezen, Mark; Clark, C Graham; Roger, Andrew J

    2008-04-22

    Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.

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

    PubMed

    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.

  19. Organelles contribute differentially to reactive oxygen species-related events during extended darkness.

    PubMed

    Rosenwasser, Shilo; Rot, Ilona; Sollner, Evelyn; Meyer, Andreas J; Smith, Yoav; Leviatan, Noam; Fluhr, Robert; Friedman, Haya

    2011-05-01

    Treatment of Arabidopsis (Arabidopsis thaliana) leaves by extended darkness generates a genetically activated senescence program that culminates in cell death. The transcriptome of leaves subjected to extended darkness was found to contain a variety of reactive oxygen species (ROS)-specific signatures. The levels of transcripts constituting the transcriptome footprints of chloroplasts and cytoplasm ROS stresses decreased in leaves, as early as the second day of darkness. In contrast, an increase was detected in transcripts associated with mitochondrial and peroxisomal ROS stresses. The sequential changes in the redox state of the organelles during darkness were examined by redox-sensitive green fluorescent protein probes (roGFP) that were targeted to specific organelles. In plastids, roGFP showed a decreased level of oxidation as early as the first day of darkness, followed by a gradual increase to starting levels. However, in mitochondria, the level of oxidation of roGFP rapidly increased as early as the first day of darkness, followed by an increase in the peroxisomal level of oxidation of roGFP on the second day. No changes in the probe oxidation were observed in the cytoplasm until the third day. The increase in mitochondrial roGFP degree of oxidation was abolished by sucrose treatment, implying that oxidation is caused by energy deprivation. The dynamic redox state visualized by roGFP probes and the analysis of microarray results are consistent with a scenario in which ROS stresses emanating from the mitochondria and peroxisomes occur early during darkness at a presymptomatic stage and jointly contribute to the senescence program.

  20. Predicting the clinical outcome of ICSI by sperm head vacuole examination.

    PubMed

    Pocate-Cheriet, Khaled; Heilikman, Ilan; Porcher, Raphael; Barraud-Lange, Virginie; Sermondade, Nathalie; Herbemont, Charlene; Wolf, Jean Philippe; Sifer, Christophe

    2017-02-01

    To assess whether high magnification sperm head vacuole examination (SHVE) and/or standard sperm morphology assessment can predict ICSI outcomes in terms of fertilization, embryo quality, and delivery rates, a prospective observational bicentric study was conducted in two publicly funded assisted reproductive technology (ART) units in France between January and July of 2012. A total of 111 ICSI cycles for exclusively male infertility factors were included. A Spearman's correlation test was performed to validate SHVE reproducibility between the ART units. The normal morphology rate and SHVE performed on selected spermatozoa were respectively determined according to David's and Vanderzwalmen's classifications used for motile sperm organelle morphology examination (MSOME) on the day of the ICSI. Receiver Operating Characteristic (ROC) curve analysis was performed to determine thresholds associated with the occurrence of a delivery. There was an excellent correlation between the two operators (r=0.98), thus validating the study's SHVE data. Percentages of normal morphology grade spermatozoa using the standard classification and first-best morphology grade spermatozoa determined by SHVE were not significantly associated with (i) delivery (p=0.58; 0.90 /area under curve (AUC) =0.532; 0.507), (ii) fertilization (p=0.88; 0.90), (iii) top-quality embryos (p=0.27; 0.98), and (iv) good quality embryo rates (p=0.73; 0.98), respectively. In conclusion, high magnification SHVE and standard sperm morphology assessment cannot predict clinical or biological ICSI outcomes.

  1. Wiring through tunneling nanotubes--from electrical signals to organelle transfer.

    PubMed

    Abounit, Saïda; Zurzolo, Chiara

    2012-03-01

    Tunneling nanotubes (TNTs) represent a subset of F-actin-based transient tubular connections that allow direct communication between distant cells. Recent studies have provided new insights into the existence of TNTs in vivo, and this novel mechanism of intercellular communication is implicated in various essential processes, such as development, immunity, tissue regeneration and transmission of electrical signals. TNTs are versatile structures known to facilitate the transfer of various cargos, such as organelles, plasma membrane components, pathogens and Ca(2+). Recently, a new function of TNTs in the long-range transfer of electrical signals that involves gap junctions has been suggested. This indicates that different types of TNTs might exist, and supports the notion that TNTs might not be just passive open conduits but rather are regulated by gating mechanisms. Furthermore, TNTs have been found in different cell lines and are characterized by their diversity in terms of morphology. Here we discuss these novel findings in the context of the two models that have been proposed for TNT formation, and focus on putative proteins that could represent TNT specific markers. We also shed some light on the molecular mechanisms used by TNTs to transfer cargos, as well as chemical and electrical signals.

  2. High-throughput quantitation of intracellular trafficking and organelle disruption by flow cytometry.

    PubMed

    Chia, Pei Zhi Cheryl; Ramdzan, Yasmin M; Houghton, Fiona J; Hatters, Danny M; Gleeson, Paul A

    2014-05-01

    Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry-based method that circumvents these limitations. The method utilizes fluorescent pulse-width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse-width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso-endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane-to-endosomes-to-Golgi, by decreasing pulse-width values. A block in transport upon RNAi-mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse-width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. 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

  4. Live cell imaging of cytoskeletal and organelle dynamics in gravity-sensing cells in plant gravitropism.

    PubMed

    Nakamura, Moritaka; Toyota, Masatsugu; Tasaka, Masao; Morita, Miyo Terao

    2015-01-01

    Plants sense gravity and change their morphology/growth direction accordingly (gravitropism). The early process of gravitropism, gravity sensing, is supposed to be triggered by sedimentation of starch-filled plastids (amyloplasts) in statocytes such as root columella cells and shoot endodermal cells. For several decades, many scientists have focused on characterizing the role of the amyloplasts and observed their intracellular sedimentation in various plants. Recently, it has been discovered that the complex sedimentary movements of the amyloplasts are created not only by gravity but also by cytoskeletal/organelle dynamics, such as those of actin filaments and the vacuolar membrane. Thus, to understand how plants sense gravity, we need to analyze both amyloplast movements and their regulatory systems in statocytes. We have developed a vertical-stage confocal microscope that allows multicolor fluorescence imaging of amyloplasts, actin filaments and vacuolar membranes in vertically oriented plant tissues. We also developed a centrifuge microscope that allows bright-field imaging of amyloplasts during centrifugation. These microscope systems provide new insights into gravity-sensing mechanisms in Arabidopsis.

  5. Granulovacuolar Degeneration Bodies of Alzheimer’s Disease Resemble Late-stage Autophagic Organelles

    PubMed Central

    Funk, Kristen E.; Mrak, Robert E.; Kuret, Jeff

    2010-01-01

    Aims Granulovacuolar degeneration involves the accumulation of large, double membrane-bound bodies within certain neurons during the course of Alzheimer’s disease and other adultonset dementias. Because of the two-layer membrane morphology, it has been proposed that the bodies are related to autophagic organelles. The aim of this study was to test this hypothesis, and determine the approximate stage at which the pathway stalled in Alzheimer’s disease. Methods Spatial colocalization of autophagic and endocytic markers with casein kinase 1 delta, a marker for GVD bodies, was evaluated in hippocampal sections prepared from postmortem Braak stage IV and V Alzheimer’s disease cases using double-label confocal fluorescence microscopy. Results GVD bodies colocalized weakly with early-stage autophagy markers LC3 and p62, but strongly with late-stage marker LAMP1 (lysosome-associated membrane protein 1), which decorated their surrounding membranes. GVD bodies also colocalized strongly with CHMP2B (charged multivesicular body protein 2B), which colocalized with the core granule, but less strongly with lysosomal marker cathepsin D. Conclusions The resultant immunohistochemical signature suggests that GVD bodies contain late-stage autophagic markers, and accumulate at the nexus of autophagic and endocytic pathways. . The data further suggest that failure to complete autolysosome formation may be an important correlate of GVD body accumulation. PMID:20946470

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

    PubMed

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

    2016-03-22

    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 μm(2)). 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.

  7. Differential expression of nuclear- and organelle-encoded genes during tomato fruit development.

    PubMed

    Piechulla, B

    1988-12-01

    Steady-state mRNA levels of nuclear-and organelle-encoded genes were determined during fruit development and ripening. Transcripts specific for subunits of the mitochondrial and chloroplast ATPase complexes appear simultaneously and reach high levels two to three weeks after anthesis, but follow a different expression pattern during the ripening period. While the chloroplast-specific mRNA levels continuously decrease to low levels in ripe tomato fruits, the transcripts specific for two mitochondrial ATPase subunits continue to be present at relative high levels in red fruits. Transcript levels for the fructose-1,6-bisphosphate aldolase increase significantly during ripening. Structural proteins such as the alpha-subunit of tubulin and the hydroxyproline-rich glycoprotein extensin are expressed during maximal fruit growth. In addition, comparisons of mRNA levels of different genes in several plant organs (leaf, fruit, stem, and root) show characteristic differences. The results presented in this paper demonstrate that changes at the transcriptional or post-transcriptional level during fruit development can be correlated with morphological and physiological alterations.

  8. 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. Copyright © 2016, American Association for the Advancement of Science.

  9. Mechanisms for the Intracellular Manipulation of Organelles by Conventional Electroporation

    PubMed Central

    Esser, Axel T.; Smith, Kyle C.; Gowrishankar, T.R.; Vasilkoski, Zlatko; Weaver, James C.

    2010-01-01

    Abstract Conventional electroporation (EP) changes both the conductance and molecular permeability of the plasma membrane (PM) of cells and is a standard method for delivering both biologically active and probe molecules of a wide range of sizes into cells. However, the underlying mechanisms at the molecular and cellular levels remain controversial. Here we introduce a mathematical cell model that contains representative organelles (nucleus, endoplasmic reticulum, mitochondria) and includes a dynamic EP model, which describes formation, expansion, contraction, and destruction for the plasma and all organelle membranes. We show that conventional EP provides transient electrical pathways into the cell, sufficient to create significant intracellular fields. This emerging intracellular electrical field is a secondary effect due to EP and can cause transmembrane voltages at the organelles, which are large enough and long enough to gate organelle channels, and even sufficient, at some field strengths, for the poration of organelle membranes. This suggests an alternative to nanosecond pulsed electric fields for intracellular manipulations. PMID:20513394

  10. Positioning of large organelles by a membrane- associated cytoskeleton in Plasmodium sporozoites.

    PubMed

    Kudryashev, Mikhail; Lepper, Simone; Stanway, Rebecca; Bohn, Stefan; Baumeister, Wolfgang; Cyrklaff, Marek; Frischknecht, Friedrich

    2010-03-01

    Cellular organelles are usually linked to the cytoskeleton, which often provides a scaffold for organelle function. In malaria parasites, no link between the cytoskeleton and the major organelles is known. Here we show that during fast, stop-and-go motion of Plasmodium sporozoites, all organelles stay largely fixed in respect to the moving parasite. Cryogenic electron tomography reveals that the nucleus, mitochondrion, apicoplast and the microtubules of Plasmodium sporozoites are linked to the parasite pellicle via long tethering proteins. These tethers originate from the inner membrane complex and are arranged in a periodic fashion following a 32 nm repeat. The tethers pass through a subpellicular structure that encompasses the entire parasite, probably as a network of membrane-associated filaments. While the spatial organization of the large parasite organelles appears dependent on their linkage to the cortex, the specialized secretory vesicles are mostly not linked to microtubules or other cellular structures that could provide support for movement.

  11. A Conceptual Mathematical Model of the Dynamic Self-Organisation of Distinct Cellular Organelles

    PubMed Central

    Binder, Bernd; Goede, Andrean; Berndt, Nikolaus; Holzhütter, Hermann-Georg

    2009-01-01

    Formation, degradation and renewal of cellular organelles is a dynamic process based on permanent budding, fusion and inter-organelle traffic of vesicles. These processes include many regulatory proteins such as SNAREs, Rabs and coats. Given this complex machinery, a controversially debated issue is the definition of a minimal set of generic mechanisms necessary to enable the self-organization of organelles differing in number, size and chemical composition. We present a conceptual mathematical model of dynamic organelle formation based on interacting vesicles which carry different types of fusogenic proteins (FP) playing the role of characteristic marker proteins. Our simulations (ODEs) show that a de novo formation of non-identical organelles, each accumulating a different type of FP, requires a certain degree of disproportionation of FPs during budding. More importantly however, the fusion kinetics must indispensably exhibit positive cooperativity among these FPs, particularly for the formation of larger organelles. We compared different types of cooperativity: sequential alignment of corresponding FPs on opposite vesicle/organelles during fusion and pre-formation of FP-aggregates (equivalent, e.g., to SNARE clusters) prior to fusion described by Hill kinetics. This showed that the average organelle size in the system is much more sensitive to the disproportionation strength of FPs during budding if the vesicular transport system gets along with a fusion mechanism based on sequential alignments of FPs. Therefore, pre-formation of FP aggregates within the membranes prior to fusion introduce robustness with respect to organelle size. Our findings provide a plausible explanation for the evolution of a relatively large number of molecules to confer specificity on the fusion machinery compared to the relatively small number involved in the budding process. Moreover, we could speculate that a specific cooperativity which may be described by Hill kinetics (aggregates

  12. Applying systems-level spectral imaging and analysis to reveal the organelle interactome.

    PubMed

    Valm, Alex M; Cohen, Sarah; Legant, Wesley R; Melunis, Justin; Hershberg, Uri; Wait, Eric; Cohen, Andrew R; Davidson, Michael W; Betzig, Eric; Lippincott-Schwartz, Jennifer

    2017-06-01

    The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as

  13. A conceptual mathematical model of the dynamic self-organisation of distinct cellular organelles.

    PubMed

    Binder, Bernd; Goede, Andrean; Berndt, Nikolaus; Holzhütter, Hermann-Georg

    2009-12-30

    Formation, degradation and renewal of cellular organelles is a dynamic process based on permanent budding, fusion and inter-organelle traffic of vesicles. These processes include many regulatory proteins such as SNAREs, Rabs and coats. Given this complex machinery, a controversially debated issue is the definition of a minimal set of generic mechanisms necessary to enable the self-organization of organelles differing in number, size and chemical composition. We present a conceptual mathematical model of dynamic organelle formation based on interacting vesicles which carry different types of fusogenic proteins (FP) playing the role of characteristic marker proteins. Our simulations (ODEs) show that a de novo formation of non-identical organelles, each accumulating a different type of FP, requires a certain degree of disproportionation of FPs during budding. More importantly however, the fusion kinetics must indispensably exhibit positive cooperativity among these FPs, particularly for the formation of larger organelles. We compared different types of cooperativity: sequential alignment of corresponding FPs on opposite vesicle/organelles during fusion and pre-formation of FP-aggregates (equivalent, e.g., to SNARE clusters) prior to fusion described by Hill kinetics. This showed that the average organelle size in the system is much more sensitive to the disproportionation strength of FPs during budding if the vesicular transport system gets along with a fusion mechanism based on sequential alignments of FPs. Therefore, pre-formation of FP aggregates within the membranes prior to fusion introduce robustness with respect to organelle size. Our findings provide a plausible explanation for the evolution of a relatively large number of molecules to confer specificity on the fusion machinery compared to the relatively small number involved in the budding process. Moreover, we could speculate that a specific cooperativity which may be described by Hill kinetics (aggregates

  14. Cytonuclear interactions and relaxed selection accelerate sequence evolution in organelle ribosomes.

    PubMed

    Sloan, Daniel B; Triant, Deborah A; Wu, Martin; Taylor, Douglas R

    2014-03-01

    Many mitochondrial and plastid protein complexes contain subunits that are encoded in different genomes. In animals, nuclear-encoded mitochondrial proteins often exhibit rapid sequence evolution, which has been hypothesized to result from selection for mutations that compensate for changes in interacting subunits encoded in mutation-prone animal mitochondrial DNA. To test this hypothesis, we analyzed nuclear genes encoding cytosolic and organelle ribosomal proteins in flowering plants. The model angiosperm genus Arabidopsis exhibits low organelle mutation rates, typical of most plants. Nevertheless, we found that (nuclear-encoded) subunits of organelle ribosomes in Arabidopsis have higher amino acid sequence polymorphism and divergence than their counterparts in cytosolic ribosomes, suggesting that organelle ribosomes experience relaxed functional constraint. However, the observed difference between organelle and cytosolic ribosomes was smaller than in animals and could be partially attributed to rapid evolution in N-terminal organelle-targeting peptides that are not involved in ribosome function. To test the role of organelle mutation more directly, we used transcriptomic data from an angiosperm genus (Silene) with highly variable rates of organelle genome evolution. We found that Silene species with unusually fast-evolving mitochondrial and plastid DNA exhibited increased amino acid sequence divergence in ribosomal proteins targeted to the organelles but not in those that function in cytosolic ribosomes. Overall, these findings support the hypothesis that rapid organelle genome evolution has selected for compensatory mutations in nuclear-encoded proteins. We conclude that coevolution between interacting subunits encoded in different genomic compartments within the eukaryotic cell is an important determinant of variation in rates of protein sequence evolution.

  15. 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.

  16. 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

  17. Subcompartmentalized Nanoreactors as Artificial Organelle with Intracellular Activity.

    PubMed

    Thingholm, Bo; Schattling, Philipp; Zhang, Yan; Städler, Brigitte

    2016-04-06

    Cell mimicry is an approach which aims at substituting missing or lost activity. In this context, the goal of artificial organelles is to provide intracellularly active nanoreactors to affect the cellular performance. So far, only a handful of reports discuss concepts addressing this challenge based on single-component reactors. Here, the assembly of nanoreactors equipped with glucose oxidase (GOx)-loaded liposomal subunits coated with a poly(dopamine) polymer layer and RGD targeting units is reported. When comparing different surface modifications, the uptake of the nanoreactors by endothelial cells and macrophages with applied shear stress is confirmed without inherent cytotoxicity. Furthermore, the encapsulation and preserved activity of GOx within the nanoreactors is shown. The intracellular activity is demonstrated by exposing macrophages with internalized nanoreactors to glucose and assessment of the cell viability after 6 and 24 h. The macrophage viability is found to be reduced due to the intracellularly produced hydrogen peroxide by GOx. This report on the first intracellular active subcompartmentalized nanoreactors is a considerable step in therapeutic cell mimicry. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Proteome analysis of human embryonic stem cells organelles.

    PubMed

    Shekari, Faezeh; Nezari, Hossein; Larijani, Mehran Rezaei; Han, Chia-Li; Baharvand, Hossein; Chen, Yu-Ju; Salekdeh, Ghasem Hosseini

    2017-06-06

    As the functions of proteins are associated with their cellular localization, the comprehensive sub-cellular proteome knowledge of human embryonic stem cells (hESCs) is indispensable for ensuring a therapeutic effect. Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices. Among ten enriched signaling pathways highlighted in our results, non-canonical Wnt signaling were analyzed in greater detail. The functions of three novel hESC membrane proteins (ERBB4, GGT1 and ZDHHC13) have been assessed in terms of pluripotency. Our report is the most comprehensive for organellar proteomics of hESCs. Mass spectrometric identification of proteins using a TripleTOF 5600 from nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomal fractions highlighted the significance of the non-canonical Wnt signaling in human embryonic stem cells. Copyright © 2017. Published by Elsevier B.V.

  19. The perennial organelle: assembly and disassembly of the primary cilium

    PubMed Central

    Seeley, E. Scott; Nachury, Maxence V.

    2010-01-01

    Primary cilia contain signaling receptors of diverse classes, and ciliary dysfunction results in a variety of developmental defects. Thus, primary cilia are thought to have an important role in sensing and transducing cellular signals. Although there is clear evidence demonstrating that these organelles are assembled and disassembled dynamically as cells progress through the cell cycle, the mechanisms by which the cell cycle controls the assembly and disassembly of the primary cilium remain poorly understood. In this Commentary, we review the basic cellular mechanisms that underlie the early stages of cilium assembly and discuss how the cell cycle communicates with the ciliation program. A commonly held view is that ciliation occurs exclusively in cells that have exited the cell cycle and entered quiescence or differentiation. However, this concept is at odds with the finding that, during development, many actively proliferating cells require cilia-mediated signaling pathways to instruct their developmental fate. Here, we reassess the quiescence-centric view of ciliation by reviewing historic and current literature. We discuss ample evidence that cilia are in fact present on many proliferating cells, and that a transient peak of ciliation before the G1-S transition might be tightly coupled to entry into the DNA replication phase. Finally, we touch on the relationship between the ciliation and cell-division cycles and the tissue distribution of primary cilia in order to highlight potential roles for the primary cilium in restraining cells from the hyperproliferative state that contributes to cancer. PMID:20144999

  20. Lipid Composition of Organelles from Germinating Castor Bean Endosperm 1

    PubMed Central

    Donaldson, Robert P.; Beevers, Harry

    1977-01-01

    Glyoxysome, endoplasmic reticulum, mitochondria, and proplastid fractions were isolated from endosperm of castor beans (Ricinus communis) germinated for 5 days at 30 C. Samples from sucrose density gradients were diluted with 0.15 m KCI and the membranes pelleted. Lipid extracts of these membranes were analyzed for phosphoglyceride, acyl lipid, and sterol content. The endoplasmic reticulum contains 1.24 μmol of phosphoglyceride per mg of protein; the mitochondria, 0.65 μmol/mg; and the glyoxysome membranes, 0.55 μmol/mg. Phosphatidyl choline and phosphatidyl ethanolamine are the most abundant lipids in all membranes studied, accounting for 70% or more of the lipid phosphorus and 50% or more of the fatty acid. Glyoxysome membranes and endoplasmic reticulum also contain phosphatidyl inositol (respectively, 9 and 17% of the lipid phosphorus) and free fatty acids (13% of the total fatty acid in each). Compared with other organelles, mitochondrial membranes have more phosphatidyl ethanolamine relative to phosphatidyl choline and are characterized by the presence of cardiolipin, in which 80% of the fatty acid is linoleate. The relative amounts of linoleate, palmitate, oleate, stearate, and linolenate in each of the phosphotoglycerides are constant regardless of the membrane source. Stimasgasterol and β-sitosterol are present in the membranes (1-9 nmol each/mg protein). The data provide further evidence that glyoxysome membranes are derived from the endoplasmic reticulum but at the same time indicate some differentiation. PMID:16659829

  1. tRNA travels from the cytoplasm to organelles

    PubMed Central

    Rubio, Mary Anne T.; Hopper, Anita K.

    2011-01-01

    Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitchondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles. PMID:21976284

  2. 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.

  3. Organelle Redox of CF and CFTR-Corrected Airway Epithelia

    PubMed Central

    Schwarzer, Christian; Illek, Beate; Suh, Jung H.; Remington, S. James; Fischer, Horst; Machen, Terry E.

    2014-01-01

    In cystic fibrosis reduced CFTR function may alter redox properties of airway epithelial cells. Redox-sensitive GFP (roGFP1) and imaging microscopy were used to measure redox potentials of cytosol, ER, mitochondria and cell surface of cystic fibrosis nasal epithelial cells and CFTR-corrected cells. We also measured glutathione and cysteine thiol redox states in cell lysates and apical fluids to provide coverage over a range of redox potentials and environments that might be affected by CFTR. As measured with roGFP1, redox potentials at the cell surface (~ -207 ±8 mV) and in the ER (~ -217 ±1 mV) and rates of regulation of the apical fluid and ER lumen following DTT treatment were similar for CF and CFTR-corrected cells. CF and CFTR-corrected cells had similar redox potentials in mitochondria (-344 ±9 mV) and cytosol (-322 ±7 mV). Oxidation of carboxy-dichlorodihydrofluoresceindiacetate and of apical Amplex Red occurred at equal rates in CF and CFTR-corrected cells. Glutathione and cysteine redox couples in cell lysates and apical fluid were equal in CF and CFTR-corrected cells. These quantitative estimates of organelle redox potentials combined with apical and cell measurements using small molecule couples confirmed there were no differences in redox properties of CF and CFTR-corrected cells. PMID:17603939

  4. 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

  5. Shear bond strength to enamel of primary teeth irradiated with varying Er:YAG laser energies and SEM examination of the surface morphology: an in vitro study.

    PubMed

    Wanderley, Rosimeyri L; Monghini, Elisângela M; Pecora, Jesus D; Palma-Dibb, Regina G; Borsatto, Maria C

    2005-06-01

    This study aimed to assess in vitro the influence of Er:YAG laser energy on the shear bond strength of a total-etch adhesive system to lased enamel of primary teeth, and to observe by SEM the morphological appearance of laser-ablated enamel surfaces. For the SBS test, primary canines were assigned to four groups (n = 12): a control (G1) and three groups irradiated with different Er:YAG laser energies- 60 mJ/2 Hz (G2), 80 mJ/2 Hz (G3), and 100 mJ/2 Hz (G4). In all groups, enamel surfaces were acidetched, Single Bond was applied, and resin cylinders were fabricated from Z250 resin. Bond strength was tested in shear (0.5 mm/min). For morphological analysis, 21 specimens were irradiated using the same energies, with or without acid-etching, and observed by SEM. SBS means, in MPa, were: G1-14.28 (+/-3.24); G2-18.48 (+/-4.58); G3-17.82 (+/-4.38); G4-16.59 (+/-5.40). Overall, Er:YAG laser ablation of primary teeth enamel, prior to the adhesive protocol, influenced the shear bond strength. Bond strengths recorded after irradiation with energies of 60 and 80 mJ were statistically similar among them (p > 0.05), and both were superior to those yielded by the acid-etched control group (p < 0.05). No significant difference (p < 0.05) was found between bond strengths recorded for control specimens and those irradiated with 100 mJ. SEM analysis revealed that the increase of laser energy resulted in increasingly uneven and microroughened surfaces, regardless of acid-etching association. The favorable results of the present study suggest that Er:YAG laser irradiation may be a viable approach for the preparation and treatment of primary teeth enamel prior to the placement of adhesive restorative systems.

  6. 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

  7. Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient.

    PubMed

    Pretlow, T G; Kreisberg, J I; Fine, W D; Zieman, G A; Brattain, M G; Pretlow, T P

    1978-07-15

    Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient.

  8. Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient.

    PubMed Central

    Pretlow, T G; Kreisberg, J I; Fine, W D; Zieman, G A; Brattain, M G; Pretlow, T P

    1978-01-01

    Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient. Images Fig. 2. Fig. 3. Fig. 4. PMID:697757

  9. A method to rapidly induce organelle-specific molecular activities and membrane tethering.

    PubMed

    Komatsu, Toru; Inoue, Takanari

    2014-01-01

    In this chapter we describe a technique for rapid protein targeting to individual intracellular organelles. This method enables a real-time imaging-based study of cellular behavior in response to controlled induction of signaling events in a specifically targeted cellular compartment. We provide rationales and a step-by-step protocol for probe design and imaging of protein targeting along with two different applications of this technique. One application involves organelle-specific activation of small GTPases, while the other application involves membrane tethering of two different organelles. In the former case, we activate Rac1 and Ras at distinct intracellular locations in order to study compartmentalization of their signaling pathways, and in the latter example, we induce membrane tethering of the endoplasmic reticulum and mitochondria in order to study organelle-organelle communication. The described technique allows to rapidly perturb molecular activities and organelle-organelle communications at precise locations with specified timing and represents a powerful strategy to dissect spatiotemporally complex biological processes.

  10. The big and intricate dreams of little organelles: Embracing complexity in the study of membrane traffic.

    PubMed

    Liu, Allen P; Botelho, Roberto J; Antonescu, Costin N

    2017-09-01

    Compartmentalization of eukaryotic cells into dynamic organelles that exchange material through regulated membrane traffic governs virtually every aspect of cellular physiology including signal transduction, metabolism and transcription. Much has been revealed about the molecular mechanisms that control organelle dynamics and membrane traffic and how these processes are regulated by metabolic, physical and chemical cues. From this emerges the understanding of the integration of specific organellar phenomena within complex, multiscale and nonlinear regulatory networks. In this review, we discuss systematic approaches that revealed remarkable insight into the complexity of these phenomena, including the use of proximity-based proteomics, high-throughput imaging, transcriptomics and computational modeling. We discuss how these methods offer insights to further understand molecular versatility and organelle heterogeneity, phenomena that allow a single organelle population to serve a range of physiological functions. We also detail on how transcriptional circuits drive organelle adaptation, such that organelles may shift their function to better serve distinct differentiation and stress conditions. Thus, organelle dynamics and membrane traffic are functionally heterogeneous and adaptable processes that coordinate with higher-order system behavior to optimize cell function under a range of contexts. Obtaining a comprehensive understanding of organellar phenomena will increasingly require combined use of reductionist and system-based approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. 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

    2016-03-07

    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.

  12. A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii.

    PubMed

    Breinich, Manuela S; Ferguson, David J P; Foth, Bernardo J; van Dooren, Giel G; Lebrun, Maryse; Quon, Doris V; Striepen, Boris; Bradley, Peter J; Frischknecht, Friedrich; Carruthers, Vern B; Meissner, Markus

    2009-02-24

    Apicomplexans contain only a core set of factors involved in vesicular traffic. Yet these obligate intracellular parasites evolved a set of unique secretory organelles (micronemes, rhoptries, and dense granules) that are required for invasion and modulation of the host cell. Apicomplexa replicate by budding from or within a single mother cell, and secretory organelles are synthesized de novo at the final stage of division. To date, the molecular basis for their biogenesis is unknown. We demonstrate that the apicomplexan dynamin-related protein B (DrpB) belongs to an alveolate specific family of dynamins that is expanded in ciliates. DrpB accumulates in a cytoplasmic region close to the Golgi that breaks up during replication and reforms after assembly of the daughter cells. Conditional ablation of DrpB function results in mature daughter parasites that are devoid of micronemes and rhoptries. In the absence of these organelles, invasion-related secretory proteins are mistargeted to the constitutive secretory pathway. Mutant parasites are able to replicate but are unable to escape from or invade into host cells. DrpB is the essential mechanoenzyme for the biogenesis of secretory organelles in Apicomplexa. We suggest that DrpB is required during replication to generate vesicles for the regulated secretory pathway that form the unique secretory organelles. Our study supports a role of an alveolate-specific dynamin that was required for the evolution of novel, secretory organelles. In the case of Apicomplexa, these organelles further evolved to enable a parasitic lifestyle.

  13. Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles.

    PubMed

    de Longevialle, Andéol Falcon; Small, Ian D; Lurin, Claire

    2010-07-01

    Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleo-protein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

  14. An Organelle Gatekeeper Function for Caenorhabditis elegans UNC-16 (JIP3) at the Axon Initial Segment

    PubMed Central

    Edwards, Stacey L.; Yu, Szi-chieh; Hoover, Christopher M.; Phillips, Barret C.; Richmond, Janet E.; Miller, Kenneth G.

    2013-01-01

    Neurons must cope with extreme membrane trafficking demands to produce axons with organelle compositions that differ dramatically from those of the cell soma and dendrites; however, the mechanism by which they accomplish this is not understood. Here we use electron microscopy and quantitative imaging of tagged organelles to show that Caenorhabditis elegans axons lacking UNC-16 (JIP3/Sunday Driver) accumulate Golgi, endosomes, and lysosomes at levels up to 10-fold higher than wild type, while ER membranes are largely unaffected. Time lapse microscopy of tagged lysosomes in living animals and an analysis of lysosome distributions in various regions of unc-16 mutant axons revealed that UNC-16 inhibits organelles from escaping the axon initial segment (AIS) and moving to the distal synaptic part of the axon. Immunostaining of native UNC-16 in C. elegans neurons revealed a localized concentration of UNC-16 at the initial segment, although UNC-16 is also sparsely distributed in distal regions of axons, including the synaptic region. Organelles that escape the AIS in unc-16 mutants show bidirectional active transport within the axon commissure that occasionally deposits them in the synaptic region, where their mobility decreases and they accumulate. These results argue against the long-standing, untested hypothesis that JIP3/Sunday Driver promotes anterograde organelle transport in axons and instead suggest an organelle gatekeeper model in which UNC-16 (JIP3/Sunday Driver) selectively inhibits the escape of Golgi and endosomal organelles from the AIS. This is the first evidence for an organelle gatekeeper function at the AIS, which could provide a regulatory node for controlling axon organelle composition. PMID:23633144

  15. An organelle gatekeeper function for Caenorhabditis elegans UNC-16 (JIP3) at the axon initial segment.

    PubMed

    Edwards, Stacey L; Yu, Szi-chieh; Hoover, Christopher M; Phillips, Barret C; Richmond, Janet E; Miller, Kenneth G

    2013-05-01

    Neurons must cope with extreme membrane trafficking demands to produce axons with organelle compositions that differ dramatically from those of the cell soma and dendrites; however, the mechanism by which they accomplish this is not understood. Here we use electron microscopy and quantitative imaging of tagged organelles to show that Caenorhabditis elegans axons lacking UNC-16 (JIP3/Sunday Driver) accumulate Golgi, endosomes, and lysosomes at levels up to 10-fold higher than wild type, while ER membranes are largely unaffected. Time lapse microscopy of tagged lysosomes in living animals and an analysis of lysosome distributions in various regions of unc-16 mutant axons revealed that UNC-16 inhibits organelles from escaping the axon initial segment (AIS) and moving to the distal synaptic part of the axon. Immunostaining of native UNC-16 in C. elegans neurons revealed a localized concentration of UNC-16 at the initial segment, although UNC-16 is also sparsely distributed in distal regions of axons, including the synaptic region. Organelles that escape the AIS in unc-16 mutants show bidirectional active transport within the axon commissure that occasionally deposits them in the synaptic region, where their mobility decreases and they accumulate. These results argue against the long-standing, untested hypothesis that JIP3/Sunday Driver promotes anterograde organelle transport in axons and instead suggest an organelle gatekeeper model in which UNC-16 (JIP3/Sunday Driver) selectively inhibits the escape of Golgi and endosomal organelles from the AIS. This is the first evidence for an organelle gatekeeper function at the AIS, which could provide a regulatory node for controlling axon organelle composition.

  16. The CoRR hypothesis for genes in organelles.

    PubMed

    Allen, John F

    2017-04-11

    Chloroplasts and mitochondria perform energy transduction in photosynthesis and respiration. These processes can be described in physico-chemical terms with no obvious requirement for co-located genetic systems, separat from those of the rest of the cell. Accordingly, biochemists once tended to regard endosymbiosis as untestable evolutionary speculation. Lynn Sagan's seminal 1967 paper "On the Origin of Mitosing Cells" outlined the evolution of eukaryotic cells by endosymbiosis of prokaryotes. The endosymbiont hypothesis is consistent with presence of DNA in chloroplasts and mitochondria, but does not assign it a function. Biochemistry and molecular biology now show that Sagan's proposal has an explanatory reach far beyond that originally envisaged. Prokaryotic origins of photosynthetic and respiratory mechanisms are apparent in protein structural insights into energy coupling. Genome sequencing confirms the underlying, prokaryotic architecture of chloroplasts and mitochondria and illustrates the profound influence of the original mergers of their ancestors' genes and proteins with those of their host cells. Peter Mitchell's 1961 chemiosmotic hypothesis applied the concept of vectorial catalysis that underlies biological energy transduction and cell structure, function, and origins. Continuity of electrical charge separation and membrane sidedness requires compartments within compartments, together with intricate mechanisms for transport within and between them. I suggest that the reason for the persistence of distinct genetic systems within bioenergetic organelles is the selective advantage of subcellular co-location of specific genes with their gene products. Co-location for Redox Regulation - CoRR - provides for a dialogue between chemical reduction-oxidation and the action of genes encoding its protein catalysts. These genes and their protein products are in intimate contact, and cannot be isolated from each other without loss of an essential mechanism of

  17. Chemokinesis and necrotaxis of human granulocytes: the important cellular organelles.

    PubMed

    Gruler, H; de Boisfleury Chevance, A

    1987-01-01

    The directed and non-directed locomotion of human polymorphonuclear leukocytes on a glass surface was compared to Brownian and drift motion. The average track velocity was measured under different conditions. The track velocity of colchicine treated cells was the same as control cells. However, cytochalasin B treated cells and cytokineplasts had a reduced track velocity compared with the control cells. The non-directed locomotion was investigated by measuring the mean square displacement as a function of time. The diffusion constant, D, which quantitates the random walk process, and the characteristic time, tau, which governs the migration of the cell, was calculated. The value of the diffusion constant depended on the cell treatment: For control cells 261 micron2/min, for colchicine treated cells 145 micron2/min, for cytochalasin B treated cells 55 micron2/min, and for cytokineplasts 47 micron2/min. The characteristic time was about 40 s. The measurement showed that the nondirected locomotion can be described by the Brownian motion. The directed locomotion was investigated by a necrotactic assay and quantitated by the McCutcheon index. This index was for control cells 0.85 +/- 0.07, for colchicine treated cells 0.8 +/- 0.07, and for cytokineplasts 0.75 +/- 0.1. The measurement showed that the directed locomotion can be described by a process which is called drift mode. From this method of analysis it was determined that the important organelles of the cell for the directed and the non-directed locomotion are: (i) A part of the plasma membrane, (ii) the microfilaments, and (iii) an unstructurated part of the cytoplasme. The microtubules of the cell are only of minor importance for the directed and the non-directed locomotion.

  18. 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

  19. Effects of organelle shape on fluorescence recovery after photobleaching.

    PubMed

    Sbalzarini, Ivo F; Mezzacasa, Anna; Helenius, Ari; Koumoutsakos, Petros

    2005-09-01

    The determination of diffusion coefficients from fluorescence recovery data is often complicated by geometric constraints imposed by the complex shapes of intracellular compartments. To address this issue, diffusion of proteins in the lumen of the endoplasmic reticulum (ER) is studied using cell biological and computational methods. Fluorescence recovery after photobleaching (FRAP) experiments are performed in tissue culture cells expressing GFP-KDEL, a soluble, fluorescent protein, in the ER lumen. The three-dimensional (3D) shape of the ER is determined by confocal microscopy and computationally reconstructed. Within these ER geometries diffusion of solutes is simulated using the method of particle strength exchange. The simulations are compared to experimental FRAP curves of GFP-KDEL in the same ER region. Comparisons of simulations in the 3D ER shapes to simulations in open 3D space show that the constraints imposed by the spatial confinement result in two- to fourfold underestimation of the molecular diffusion constant in the ER if the geometry is not taken into account. Using the same molecular diffusion constant in different simulations, the observed speed of fluorescence recovery varies by a factor of 2.5, depending on the particular ER geometry and the location of the bleached area. Organelle shape considerably influences diffusive transport and must be taken into account when relating experimental photobleaching data to molecular diffusion coefficients. This novel methodology combines experimental FRAP curves with high accuracy computer simulations of diffusion in the same ER geometry to determine the molecular diffusion constant of the solute in the particular ER lumen.

  20. Effects of Organelle Shape on Fluorescence Recovery after Photobleaching

    PubMed Central

    Sbalzarini, Ivo F.; Mezzacasa, Anna; Helenius, Ari; Koumoutsakos, Petros

    2005-01-01

    The determination of diffusion coefficients from fluorescence recovery data is often complicated by geometric constraints imposed by the complex shapes of intracellular compartments. To address this issue, diffusion of proteins in the lumen of the endoplasmic reticulum (ER) is studied using cell biological and computational methods. Fluorescence recovery after photobleaching (FRAP) experiments are performed in tissue culture cells expressing GFP–KDEL, a soluble, fluorescent protein, in the ER lumen. The three-dimensional (3D) shape of the ER is determined by confocal microscopy and computationally reconstructed. Within these ER geometries diffusion of solutes is simulated using the method of particle strength exchange. The simulations are compared to experimental FRAP curves of GFP–KDEL in the same ER region. Comparisons of simulations in the 3D ER shapes to simulations in open 3D space show that the constraints imposed by the spatial confinement result in two- to fourfold underestimation of the molecular diffusion constant in the ER if the geometry is not taken into account. Using the same molecular diffusion constant in different simulations, the observed speed of fluorescence recovery varies by a factor of 2.5, depending on the particular ER geometry and the location of the bleached area. Organelle shape considerably influences diffusive transport and must be taken into account when relating experimental photobleaching data to molecular diffusion coefficients. This novel methodology combines experimental FRAP curves with high accuracy computer simulations of diffusion in the same ER geometry to determine the molecular diffusion constant of the solute in the particular ER lumen. PMID:15951382

  1. Bidirectional organelle transport can occur in cell processes that contain single microtubules

    PubMed Central

    1985-01-01

    Intracellular organelle transport was studied in a new model system, the giant freshwater ameba Reticulomyxa. The ameba extends a large reticulate network of cytoplasmic strands in which various phase-dense organelles can be seen to move at a rate of up to 25 microns/s. This combined light and high voltage electron microscopic study shows that organelles move bidirectionally in even the finest network strands that contain only a single microtubule. In terms of microtubule-associated intracellular transport, this observation defines a minimum set of conditions necessary for such movement. The implications of this finding for possible models of force generation are discussed. PMID:3965478

  2. Bidirectional organelle transport can occur in cell processes that contain single microtubules.

    PubMed

    Koonce, M P; Schliwa, M

    1985-01-01

    Intracellular organelle transport was studied in a new model system, the giant freshwater ameba Reticulomyxa. The ameba extends a large reticulate network of cytoplasmic strands in which various phase-dense organelles can be seen to move at a rate of up to 25 microns/s. This combined light and high voltage electron microscopic study shows that organelles move bidirectionally in even the finest network strands that contain only a single microtubule. In terms of microtubule-associated intracellular transport, this observation defines a minimum set of conditions necessary for such movement. The implications of this finding for possible models of force generation are discussed.

  3. Scanning electron microscopy and transmitted electron backscatter diffraction examination of asbestos standard reference materials, amphibole particles of differing morphology, and particle phase discrimination from talc ores.

    PubMed

    Bandli, Bryan R; Gunter, Mickey E

    2014-12-01

    Since 1972, when the US Occupational Health and Safety Administration established the first limits on occupational exposure to asbestos fibers, numerous analytical methods employing several microscopy techniques have been developed to identify a group of minerals defined by legislation as asbestos. While transmission electron microscopy (TEM) is implemented in standardized analytical methods, these methods specify the use of selected area electron diffraction. Because of this constraint, the diffraction data a TEM can provide are often underutilized due to challenges associated with collecting and interpreting individual diffraction patterns. It has been shown that transmission electron backscatter diffraction (tEBSD) produces diffraction patterns nearly identical to electron backscatter diffraction, but from smaller crystal domains. This paper explores the utility of tEBSD for characterization of asbestiform particles from reference asbestos materials, a suite of amphibole minerals of varying morphologies to determine if there is a correlation between mineral habit (i.e., crystal form), microscopic particle shape preferred orientation, and mineral specimens from an industrial talc deposit to provide a case study of the utility and limitations of the technique.

  4. A molecular and morphological re-examination of the generic limits of truffles in the tarzetta-geopyxis lineage - Densocarpa, Hydnocystis, and Paurocotylis.

    PubMed

    Kumar, Leticia M; Smith, Matthew E; Nouhra, Eduardo R; Orihara, Takamichi; Sandoval Leiva, Pablo; Pfister, Donald H; McLaughlin, David J; Trappe, James M; Healy, Rosanne A

    2017-03-01

    Truffle species within the /tarzetta-geopyxis lineage share smooth, globose, hyaline spores, but differ in the amount of convolution of hymenia in ascomata. The relationships among truffle species in this lineage have historically been confused. Phylogenetic analyses of the ITS and 28S nuclear ribosomal DNA from recently collected members of the /tarzetta-geopyxis lineage from Asia, Austral Asia, North America, and South America prompted a reinvestigation of species and generic limits in the truffle genera Hydnocystis, Paurocotylis, and Stephensia. Our analyses support emendations of Hydnocystis and Paurocotylis, abandonment of Stephensia and the resurrection of the genus Densocarpa. Nomenclatural changes include the transfer of Stephensia bombycina to Hydnocystis, the transfer of Hydnocystis singeri and Stephensia bynumii to Paurocotylis, the reinstatement of Densocarpa for Stephensia shanori and transfer of Stephensia crocea to Densocarpa. This is the first detection of the genus Paurocotylis in the Americas. We describe three new species, Hydnocystis transitoria from North America, Paurocotylis patagonica from South America, and Paurocotylis watlingii from Australia. Our work highlights the unexplored diversity, morphological plasticity, and remaining taxonomic problems among truffles in the /tarzetta-geopyxis lineage.

  5. Sperm fluorescence in situ hybridization study in nine men carrying a Robertsonian or a reciprocal translocation: relationship between segregation modes and high-magnification sperm morphology examination.

    PubMed

    Cassuto, Nino Guy; Le Foll, Nathalie; Chantot-Bastaraud, Sandra; Balet, Richard; Bouret, Dominique; Rouen, Alexandre; Bhouri, Rakia; Hyon, Capucine; Siffroi, Jean Pierre

    2011-10-01

    To evaluate whether observation of spermatozoa at × 6,100 magnification can distinguish between those with and without a balanced chromosomal content. Retrospective research study. Genetics laboratory of a university hospital and in vitro fertilization center. Six men carrying a reciprocal translocation and three men with a Robertsonian translocation. Sperm fluorescence in situ hybridization (FISH) with a specific set of three probes for each translocation for determining chromosomal content, performed on both unselected spermatozoa and on spermatozoa selected at × 6,100 magnification according to the Cassuto-Barak classification. Chromosomal content in unselected and selected spermatozoa. Chromosomal translocations lead to gametes carrying either a balanced or an unbalanced karyotype in offspring and consequently to changes in chromosome position within sperm nucleus and potentially in nuclear morphology. In the unselected spermatozoa, the rate of chromosomally balanced nuclei ranged from 37.1% to 52.6% and from 70% to 88.6% in reciprocal and Robertsonian translocations, respectively, which is in agreement with published data. In selected spermatozoa, there was no statistically significant difference between the rates of segregation modes when compared with their frequencies in unselected sperm cells. The observation of spermatozoa at high-magnification in translocation carriers cannot be used to select sperm cells with a balanced chromosomal content. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

    PubMed

    Dietrich, Denise; Seiler, Florian; Essmann, Frank; Dodt, Gabriele

    2013-12-01

    The attachment of organelles to the cytoskeleton and directed organelle transport is essential for cellular morphology and function. In contrast to other cell organelles like the endoplasmic reticulum or the Golgi apparatus, peroxisomes are evenly distributed in the cytoplasm, which is achieved by binding of peroxisomes to microtubules and their bidirectional transport by the microtubule motor proteins kinesin-1 (Kif5) and cytoplasmic dynein. KifC3, belonging to the group of C-terminal kinesins, has been identified to interact with the human peroxin PEX1 in a yeast two-hybrid screen. We investigated the potential involvement of KifC3 in peroxisomal transport. Interaction of KifC3 and the AAA-protein (ATPase associated with various cellular activities) PEX1 was confirmed by in vivo colocalization and by coimmunoprecipitation from cell lysates. Furthermore, knockdown of KifC3 using RNAi resulted in an increase of cells with perinuclear-clustered peroxisomes, indicating enhanced minus-end directed motility of peroxisomes. The occurrence of this peroxisomal phenotype was cell cycle phase independent, while microtubules were essential for phenotype formation. We conclude that KifC3 may play a regulatory role in minus-end directed peroxisomal transport for example by blocking the motor function of dynein at peroxisomes. Knockdown of KifC3 would then lead to increased minus-end directed peroxisomal transport and cause the observed peroxisomal clustering at the microtubule-organizing center. © 2013.

  7. Six new examples of the bipartite trapezoid bone: morphology, significant population variation, and an examination of pre-existing criteria to identify bipartition of individual carpal bones.

    PubMed

    Burnett, Scott E; Stojanowski, Christopher M; Mahakkanukrauh, Pasuk

    2015-03-01

    Carpal bone bipartition is a developmental variant resulting in the division of a normally singular carpal into two distinct segments. Cases involving the scaphoid are best known, though many other carpals can be affected, including the trapezoid. Six new examples of bipartite trapezoids, identified in African and Asian anatomical and archeological samples, are reported here and compared with the eight previously known. While the site of bipartition is consistent, the resulting segments exhibit variability in their articulations with neighboring carpals. Five of the six affected trapezoids were identified in African or African-derived samples, yielding a significantly higher frequency (0.323%) of bipartite trapezoid than seen in anatomical or archeological series of European origin. Bilateral bipartite trapezoids in archeological remains from the Mid Holocene site of Gobero (Niger) are potentially the oldest bipartite carpals yet identified in humans. Their discovery may indicate that trapezoid bipartition is a condition that has been present in African populations since prehistoric times, though more data are needed. Because bipartite carpals may be symptomatic and can occur as part of syndromes, the significant population variation in frequency identified here has potential utility in both anatomical and clinical contexts. However, a comparison of the morphological appearance of bipartite trapezoids with the suggested criteria for bipartite scaphoid diagnosis indicates that these criteria are not equally applicable to other carpals. Fortunately, due to the rarity of fracture, identification of the bipartite trapezoid and separating it from pathological conditions is considerably easier than diagnosing a bipartite scaphoid. Copyright © 2014 Elsevier GmbH. All rights reserved.

  8. Organelle genome complexity scales positively with organism size in volvocine green algae.

    PubMed

    Smith, David Roy; Hamaji, Takashi; Olson, Bradley J S C; Durand, Pierre M; Ferris, Patrick; Michod, Richard E; Featherston, Jonathan; Nozaki, Hisayoshi; Keeling, Patrick J

    2013-04-01

    It has been argued that for certain lineages, noncoding DNA expansion is a consequence of the increased random genetic drift associated with long-term escalations in organism size. But a lack of data has prevented the investigation of this hypothesis in most plastid-bearing protists. Here, using newly sequenced mitochondrial and plastid genomes, we explore the relationship between organelle DNA noncoding content and organism size within volvocine green algae. By looking at unicellular, colonial, and differentiated multicellular algae, we show that organelle DNA complexity scales positively with species size and cell number across the volvocine lineage. Moreover, silent-site genetic diversity data suggest that the volvocine species with the largest cell numbers and most bloated organelle genomes have the smallest effective population sizes. Together, these findings support the view that nonadaptive processes, like random genetic drift, promote the expansion of noncoding regions in organelle genomes.

  9. Integrated regulation of motor-driven organelle transport by scaffolding proteins.

    PubMed

    Fu, Meng-meng; Holzbaur, Erika L F

    2014-10-01

    Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Purification of yeast membranes and organelles by sucrose density gradient centrifugation.

    PubMed

    Chang, Jennifer; Ruiz, Victoria; Vancura, Ales

    2008-01-01

    Many experiments require isolation and purification of membranes and organelles from a cell-free lysate. A combination of differential and sucrose density gradient centrifugation provides adequate separation of most yeast organelles in a single experiment. Yeast cells are converted to spheroplasts and gently lysed under conditions that preserve the integrity of organelles. The total lysate is subjected to differential centrifugation and the resulting membrane pellets are fractionated on density gradients. The method is based on the fact that different membranes contain different ratios of lipid to protein, and thus exhibit different density, allowing them to migrate through the gradient until they reach isopycnic position. The fractionated gradients are analyzed by Western blotting with antibodies that recognize marker proteins specific for individual organelles.

  11. Food Analysis Using Organelle DNA and the Effects of Processing on Assays.

    PubMed

    Caldwell, Jane M

    2017-02-28

    Extrachromosomal DNA such as organelle DNA are increasingly targeted in molecular detection assays where samples have been degraded by physical or chemical means. Owing to multiple organelles per cell and greater copy numbers than nuclear genes, organelle gene targets provide a more robust signal in polymerase chain reaction (PCR), quantitative PCR (qPCR), and other emerging molecular technologies. Because of these advantages, direct analysis of organelle DNA in food matrices is used for detection of contaminants and identification and authentication of food ingredients and allergens. Non-nuclear DNA is also used as an assay normalizer for detection of genetically modified organisms (GMOs) in foods. This review describes these protocols plus the effects of processing on efficacy, with special emphasis on thermally produced DNA fragmentation. Future research may incorporate molecular techniques beyond detection, used instead as time-temperature indicators in thermal food processing or quality indicators in food fermentation or acidification.

  12. PAHs pass through the cell wall and partition into organelles of arbuscular mycorrhizal roots of ryegrass.

    PubMed

    Gao, Yanzheng; Cao, Xuezhang; Kang, Fuxing; Cheng, Zhaoxia

    2011-01-01

    The subcellular process and distribution of polycyclic aromatic hydrocarbons (PAHs) in arbuscular mycorrhizal plants remains to be elucidated. This work used a greenhouse experiment to show that, accompanied by the apoplastic and symplastic water movement through the root, acenaphthene (ACE) as a representative PAH passed through the cell-wall boundary, dissolved in the cell solution, and partition organelles in arbuscular mycorrhizal roots of ryegrass (Lolium multiflorum Lam.). The observed concentrations of ACE in organelles were 0.6 to 4.4 times higher than in the cell walls. The cell wall and organelles were the dominant storage domains for ACE in the root, and the distribution of ACE in cells of mycorrhizal ryegrass roots was, in descending order, cell organelles (40.8-70.8%) > cell wall (19.7-3.8%) cell solution (9.6-20.5%).

  13. Assessing light scattering of intracellular organelles in single intact living cells

    PubMed Central

    Kalashnikov, Maxim; Choi, Wonshik; Yu, Chung-Chieh; Sung, Yongjin; Dasari, Ramachandra R.; Badizadegan, Kamran; Feld, Michael S.

    2010-01-01

    This report presents a model-independent method of assessing contributions to the light scattering from individual organelles in single intact cells. We first measure the 3D index map of a living cell, and then modify the map in such a way so as to eliminate contrast due to a particular intracellular organelle. By calculating and comparing the light scattering distributions calculated from the original and modified index maps using the Rytov approximation, we extract the light scattering contribution from the particular organelle of interest. The relative contributions of the nucleus and nucleolus to the scattering of the entire cell are thus determined, and the applicability of the homogeneous spherical model to non-spherical and heterogeneous organelles in forward scattering is evaluated. PMID:19997187

  14. FtsZ and the division of prokaryotic cells and organelles.

    PubMed

    Margolin, William

    2005-11-01

    Binary fission of many prokaryotes as well as some eukaryotic organelles depends on the FtsZ protein, which self-assembles into a membrane-associated ring structure early in the division process. FtsZ is homologous to tubulin, the building block of the microtubule cytoskeleton in eukaryotes. Recent advances in genomics and cell-imaging techniques have paved the way for the remarkable progress in our understanding of fission in bacteria and organelles.

  15. Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly "Targeted" Drug Delivery Systems.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-04

    Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

  16. Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

    PubMed

    Neuman, Benjamin W

    2016-11-01

    Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains.

  17. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

    PubMed Central

    Puchner, Elias M.; Walter, Jessica M.; Kasper, Robert; Huang, Bo; Lim, Wendell A.

    2013-01-01

    Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes. PMID:24043832

  18. Novel mitochondrion-related organelles in the anaerobic amoeba Mastigamoeba balamuthi.

    PubMed

    Gill, Erin E; Diaz-Triviño, Sara; Barberà, Maria José; Silberman, Jeffrey D; Stechmann, Alexandra; Gaston, Daniel; Tamas, Ivica; Roger, Andrew J

    2007-12-01

    Unicellular eukaryotes that lack mitochondria typically contain related organelles such as hydrogenosomes or mitosomes. To characterize the evolutionary diversity of these organelles, we conducted an expressed sequence tag (EST) survey on the free-living amoeba Mastigamoeba balamuthi, a relative of the human parasite Entamoeba histolytica. From 19 182 ESTs, we identified 21 putative mitochondrial proteins implicated in protein import, amino acid interconversion and carbohydrate metabolism, two components of the iron-sulphur cluster (Fe-S) assembly apparatus as well as two enzymes characteristic of hydrogenosomes. By immunofluorescence microscopy and subcellular fractionation, we show that mitochondrial chaperonin 60 is targeted to small abundant organelles within Mastigamoeba. In transmission electron micrographs, we identified double-membraned compartments that likely correspond to these mitochondrion-derived organelles, The predicted organellar proteome of the Mastigamoeba organelle indicates a unique spectrum of functions that collectively have never been observed in mitochondrion-related organelles. However, like Entamoeba, the Fe-S cluster assembly proteins in Mastigamoeba were acquired by lateral gene transfer from epsilon-proteobacteria and do not possess obvious organellar targeting peptides. These data indicate that the loss of classical aerobic mitochondrial functions and acquisition of anaerobic enzymes and Fe-S cluster assembly proteins occurred in a free-living member of the eukaryote super-kingdom Amoebozoa.

  19. 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.

  20. 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.

  1. Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells.

    PubMed

    Roudeau, Stéphane; Carmona, Asuncion; Perrin, Laura; Ortega, Richard

    2014-11-01

    X-ray chemical element imaging has the potential to enable fundamental breakthroughs in the understanding of biological systems because chemical element interactions with organelles can be studied at the sub-cellular level. What is the distribution of trace metals in cells? Do some elements accumulate within sub-cellular organelles? What are the chemical species of the elements in these organelles? These are some of the fundamental questions that can be addressed by use of X-ray chemical element imaging with synchrotron radiation beams. For precise location of the distribution of the elements, identification of cellular organelles is required; this can be achieved, after appropriate labelling, by use of fluorescence microscopy. As will be discussed, this approach imposes some limitations on sample preparation. For example, standard immunolabelling procedures strongly modify the distribution of the elements in cells as a result of the chemical fixation and permeabilization steps. Organelle location can, however, be performed, by use of a variety of specific fluorescent dyes or fluorescent proteins, on living cells before cryogenic fixation, enabling preservation of element distribution. This article reviews the methods used for fluorescent organelle labelling and X-ray chemical element imaging and speciation of single cells. Selected cases from our work and from other research groups are presented to illustrate the potential of the combination of the two techniques.

  2. Mechanical properties of organelles driven by microtubule-dependent molecular motors in living cells.

    PubMed

    Bruno, Luciana; Salierno, Marcelo; Wetzler, Diana E; Despósito, Marcelo A; Levi, Valeria

    2011-04-01

    The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is ∼ one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules.

  3. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory.

    PubMed

    Puchner, Elias M; Walter, Jessica M; Kasper, Robert; Huang, Bo; Lim, Wendell A

    2013-10-01

    Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.

  4. A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

    PubMed Central

    Wu, Qindong; Sandrock, Tanya M.; Turgeon, B. Gillian; Yoder, Olen C.; Wirsel, Stefan G.; Aist, James R.

    1998-01-01

    A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology. PMID:9436993

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

    PubMed

    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. Graphical Abstract ᅟ.

  6. 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.

  7. Complex morphological and molecular genetic examination of amelogenesis imperfecta: a case presentation of two Czech siblings with a non-syndrome form of the disease.

    PubMed

    Kripnerova, Tereza; Krulisova, Veronika; Ptakova, Nikola; Macek, Milan; Dostalova, Tatjana

    2014-01-01

    Amelogenesis imperfecta (AI) is an overarching term for a group of rare inherited disorders of hard tooth tissues. It is characterized by various defects in proper enamel formation. AI is a severe disorder that affects both the aesthetics and function of the dentition, with affected teeth increasingly suffering from dental caries. Therefore, early diagnosis and lifelong stomatological interventions are important. Due to the complex nature of AI family history, stomatological, radiographic, and molecular genetic examinations should be part of the diagnostic portfolio. Additionally, we utilized new visualization methods for the assessment of teeth demineralization. We present a case report of two affected Czech sisters (6 and 8 years old) with clinically defined AI. These are the first Czech cases in which comprehensive clinical and genetic analysis had been carried out and reflect the complex clinical nature, positive treatment options, and limitations of candidate-gene molecular genetic testing.

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

    PubMed

    Furt, Fabienne; Lemoi, Kyle; Tüzel, Erkan; Vidali, Luis

    2012-05-17

    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. 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. To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution and dynamics of organelles

  9. ChloroMitoCU: Codon patterns across organelle genomes for functional genomics and evolutionary applications.

    PubMed

    Sablok, Gaurav; Chen, Ting-Wen; Lee, Chi-Ching; Yang, Chi; Gan, Ruei-Chi; Wegrzyn, Jill L; Porta, Nicola L; Nayak, Kinshuk C; Huang, Po-Jung; Varotto, Claudio; Tang, Petrus

    2017-06-01

    Organelle genomes are widely thought to have arisen from reduction events involving cyanobacterial and archaeal genomes, in the case of chloroplasts, or α-proteobacterial genomes, in the case of mitochondria. Heterogeneity in base composition and codon preference has long been the subject of investigation of topics ranging from phylogenetic distortion to the design of overexpression cassettes for transgenic expression. From the overexpression point of view, it is critical to systematically analyze the codon usage patterns of the organelle genomes. In light of the importance of codon usage patterns in the development of hyper-expression organelle transgenics, we present ChloroMitoCU, the first-ever curated, web-based reference catalog of the codon usage patterns in organelle genomes. ChloroMitoCU contains the pre-compiled codon usage patterns of 328 chloroplast genomes (29,960 CDS) and 3,502 mitochondrial genomes (49,066 CDS), enabling genome-wide exploration and comparative analysis of codon usage patterns across species. ChloroMitoCU allows the phylogenetic comparison of codon usage patterns across organelle genomes, the prediction of codon usage patterns based on user-submitted transcripts or assembled organelle genes, and comparative analysis with the pre-compiled patterns across species of interest. ChloroMitoCU can increase our understanding of the biased patterns of codon usage in organelle genomes across multiple clades. ChloroMitoCU can be accessed at: http://chloromitocu.cgu.edu.tw/. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  10. Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo

    PubMed Central

    Kimura, Akatsuki

    2010-01-01

    The centrosome is generally maintained at the center of the cell. In animal cells, centrosome centration is powered by the pulling force of microtubules, which is dependent on cytoplasmic dynein. However, it is unclear how dynein brings the centrosome to the cell center, i.e., which structure inside the cell functions as a substrate to anchor dynein. Here, we provide evidence that a population of dynein, which is located on intracellular organelles and is responsible for organelle transport toward the centrosome, generates the force required for centrosome centration in Caenorhabditis elegans embryos. By using the database of full-genome RNAi in C. elegans, we identified dyrb-1, a dynein light chain subunit, as a potential subunit involved in dynein anchoring for centrosome centration. DYRB-1 is required for organelle movement toward the minus end of the microtubules. The temporal correlation between centrosome centration and the net movement of organelle transport was found to be significant. Centrosome centration was impaired when Rab7 and RILP, which mediate the association between organelles and dynein in mammalian cells, were knocked down. These results indicate that minus end-directed transport of intracellular organelles along the microtubules is required for centrosome centration in C. elegans embryos. On the basis of this finding, we propose a model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. This is the first model, to our knowledge, providing a mechanical basis for cytoplasmic pulling force for centrosome centration. PMID:21173218

  11. 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.

  12. Intracellular Microreactors as Artificial Organelles to Conduct Multiple Enzymatic Reactions Simultaneously.

    PubMed

    Godoy-Gallardo, María; Labay, Cédric; Jansman, Michelle M T; Ek, Pramod K; Hosta-Rigau, Leticia

    2017-02-01

    The creation of artificial organelles is a new paradigm in medical therapy that aims to substitute for missing cellular function by replenishing a specific cellular task. Artificial organelles tackle the challenge of mimicking metabolism, which is the set of chemical reactions that occur within a cell, mainly catalyzed by enzymes. So far, the few reported carriers able to conduct enzymatic reactions intracellularly are based on single-compartment carriers. However, cell organelles outperform by conducting multiple reactions simultaneously within confined sub-compartments. Here, the field of artificial organelles is advanced by reporting the assembly of a microreactor consisting of polymer capsules entrapping gold nanoclusters (AuNCs) and liposomes as sub-compartments. The fluorescence properties of AuNCs are employed to monitor the microreactors uptake by macrophages. Encapsulation is demonstrated and functionality of microreactors with trypsin (TRP) and horseradish peroxidase (HRP)-loaded liposomes is preserved. Multiple enzymatic reactions taking place simultaneously is demonstrated by exposing macrophages with the internalized microreactors to bis-(benzyloxycarbonyl-Ile-Pro-Arg)-Rho-110 and Amplex Red substrates, which are specific for TRP and HRP, respectively. Conversion of the substrates into the respective fluorescent products is observed. This report on the first microreactor conducting multiple enzymatic reactions simultaneously inside a cell is a considerable step in the field of artificial organelles.

  13. Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

    PubMed Central

    Gelfand, Vladimir I.

    2013-01-01

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport. PMID:24300413

  14. New organelles by gene duplication in a biophysical model of eukaryote endomembrane evolution.

    PubMed

    Ramadas, Rohini; Thattai, Mukund

    2013-06-04

    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.

  15. 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

  16. Role of centrins 2 and 3 in organelle segregation and cytokinesis in Trypanosoma brucei.

    PubMed

    Selvapandiyan, Angamuthu; Kumar, Praveen; Salisbury, Jeffrey L; Wang, Ching C; Nakhasi, Hira L

    2012-01-01

    Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in Trypanosoma brucei (T. brucei) displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of T. brucei centrin2 (TbCen2) and T. brucei 3 (TbCen3) in the early events of T. brucei procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1-3). In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.

  17. 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.

  18. 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.

  19. Organelle transport in cultured Drosophila cells: S2 cell line and primary neurons.

    PubMed

    Lu, Wen; Del Castillo, Urko; Gelfand, Vladimir I

    2013-11-20

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

  20. The exception proves the rule? Dual targeting of nuclear-encoded proteins into endosymbiotic organelles.

    PubMed

    Baudisch, Bianca; Langner, Uwe; Garz, Ingo; Klösgen, Ralf Bernd

    2014-01-01

    Plant cells harbor two types of endosymbiotic organelle: mitochondria and chloroplasts. As a consequence of endosymbiotic gene transfer, the majority of their proteins are encoded in the nucleus and post-translationally 're'-imported into the respective target organelle. The corresponding transport signals are usually selective for a single organelle, but several proteins are transported into both the mitochondria and chloroplasts. To estimate the number of proteins with such dual targeting properties in Arabidopsis, we classified the proteins encoded by nuclear genes of endosymbiotic origin according to the respective targeting specificity of their N-terminal transport signals as predicted by the TargetP software package. Selected examples of the resulting protein classes were subsequently analyzed by transient transformation assays as well as by in organello protein transport experiments. It was found that most proteins with high prediction values for both organelles show dual targeting with both experimental approaches. Unexpectedly, however, dual targeting was even found among those proteins that are predicted to be localized solely in one of the two endosymbiotic organelles. In total, among the 16 candidate proteins analyzed, we identified 10 proteins with dual targeting properties. This unexpectedly high proportion suggests that such transport properties are much more abundant than anticipated.

  1. 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

  2. Functional diversity of Arabidopsis organelle-localized RNA-recognition motif-containing proteins.

    PubMed

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

    2017-09-01

    RNA-Binding Proteins (RBPs) play key roles in plant gene expression and regulation. RBPs contain a variety of RNA-binding motifs, the most abundant and most widespread one in eukaryotes is the RNA recognition motif (RRM). Many nucleus-encoded RRM-containing proteins are transported into chloroplasts and/or mitochondria, and participate in various RNA-related processes in plant organelles. Loss of these proteins can have a detrimental effect on some critical processes such as photosynthesis and respiration, sometimes leading to lethality. Progress has been made in the last few years in understanding the function of particular organelle-localized RRM-containing proteins. Members of the Organelle RRM protein (ORRM, some also characterized as Glycine-Rich RNA-Binding Proteins) family and the Chloroplast RiboNucleoProtein (cpRNP) family, are involved in various types of RNA metabolism, including RNA editing, RNA stability and RNA processing. Organelle-localized RRM proteins also function in plant development and stress responses, in some conditions acting as protein or RNA chaperones. There has been recent progress in characterizing the function of organelle-localized RRM proteins in RNA-related processes and how RRM proteins contribute to the normal growth and development of plants. WIREs RNA 2017, 8:e1420. doi: 10.1002/wrna.1420 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

  3. Can polymeric vesicles that confine enzymatic reactions act as simplified organelles?

    PubMed

    Tanner, Pascal; Egli, Stefan; Balasubramanian, Vimalkumar; Onaca, Ozana; Palivan, Cornelia G; Meier, Wolfgang

    2011-06-06

    In various pathological conditions an advantage may be gained by reinforcing an intrinsic organismal response. This can be achieved, for example, by enzyme replacement therapy, which can amplify specific, intrinsic activities of the organelles. In this respect, polymeric nanoreactors composed of vesicles that encapsulate an enzyme or a combination of enzymes in their cavities represent a novel approach in therapeutic applications because they behave like simplified organelles. As compartments, polymeric vesicles possess a membrane that is more stable than the corresponding lipid membrane of liposomes, with the dual role of protecting enzymes and simultaneously allowing them to act in situ. A complex scenario of requirements must be fulfilled by enzyme-containing polymeric nanoreactors if they are to function under biological conditions and serve to model organelles. Nanoreactors are described here in terms of the existing models and the challenges faced in developing artificial organelles for therapeutic applications. We will focus on describing how polymeric vesicles can be used to provide a protected compartment for enzymatic reactions, and serve as simplified organelles inside cells. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  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. Nucleotide specificity for the bidirectional transport of membrane-bounded organelles in isolated axoplasm.

    PubMed

    Leopold, P L; Snyder, R; Bloom, G S; Brady, S T

    1990-01-01

    Video microscopy of isolated axoplasm from the squid giant axon permits correlated quantitative analyses of membrane-bounded organelle transport both in the intact axoplasm and along individual microtubules. As a result, the effects of experimental manipulations on both anterograde and retrograde movements of membrane-bounded organelles can be evaluated under nearly physiological conditions. Since anterograde and retrograde fast axonal transport are similar but distinct cellular processes, a systematic biochemical analysis is important for a further understanding of the molecular mechanisms for each. In this series of experiments, we employed isolated axoplasm of the squid to define the nucleoside triphosphate specificity for bidirectional organelle motility in the axon. Perfusion of axoplasm with 2-20 mM ATP preserved optimal vesicle velocities in both the anterograde and retrograde directions. Organelle velocities decreased to less than 50% of optimal values when the axoplasm was perfused with 10-20 mM UTP, GTP, ITP, or CTP with simultaneous depletion of endogenous ATP with hexokinase. Under the same conditions, TTP and ATP-gamma-S were unable to support significant levels of transport. None of the NTPs tested had a differential effect on anterograde vs. retrograde movement of vesicles. Surprisingly, several inconsistencies were revealed when a comparison was made between these results and nucleoside triphosphate specificities that have been reported for putative organelle motors by using in vitro assays. These data may be used in conjunction with data from well-defined in vitro assays to develop models for the molecular mechanisms of axonal transport.

  7. 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.

  8. GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles.

    PubMed

    Rodriguez-Garcia, Arancha; Rojo-Ruiz, Jonathan; Navas-Navarro, Paloma; Aulestia, Francisco Javier; Gallego-Sandin, Sonia; Garcia-Sancho, Javier; Alonso, Maria Teresa

    2014-02-18

    Genetically encoded calcium indicators allow monitoring subcellular Ca(2+) signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca(2+) sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg(2+), tunable Ca(2+) affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca(2+) indicators for organelles and becomes a valuable tool for in vivo Ca(2+) imaging applications.

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

    PubMed Central

    Melchionda, Manuela; Pittman, Jon K.

    2016-01-01

    Increasing evidence implicates Ca2+ in the control of cell migration. However, the underlying mechanisms are incompletely understood. Acidic Ca2+ stores are fast emerging as signaling centers. But how Ca2+ is taken up by these organelles in metazoans and the physiological relevance for migration is unclear. Here, we identify a vertebrate Ca2+/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 Ca2+ signals, and regulates cell-matrix adhesion during migration. Our data provide new molecular insight into how Ca2+ is handled by acidic organelles and link this to migration, thereby underscoring the role of noncanonical Ca2+ stores in the control of Ca2+-dependent function. PMID:27002171

  10. Organelle DNA variation and systematic relationships in the genus Zea: Teosinte

    PubMed Central

    Timothy, D. H.; Levings, C. S.; Pring, D. R.; Conde, M. F.; Kermicle, J. L.

    1979-01-01

    Chloroplast and mitochondrial DNAs from six races of annual teosinte (Guatemala, Huehuetenango, Balsas, Central Plateau, Chalco, and Nobogame), perennial teosinte, and maize were compared and grouped by restriction endonuclease fragment analyses. Three groups of chloroplast DNAs were detected: (i) perennial teosinte and Guatemala; (ii) Balsas and Huehuetenango; and (iii) all other teosintes. Four groups of mitochondrial DNAs were separated: (i) perennial teosinte; (ii) Guatemala; (iii) Nobogame; and (iv) all other teosintes. Separation of the teosinte and maize organelle DNAs into five groups (Guatemala; perennial teosinte; Balsas and Huehuetenango; Central Plateau and Chalco; Nobogame and maize) approximated the biosystematic relationships of the taxa. It was suggested that the evolutions of the chloroplast and mitochondrial DNAs may be independent of each other, that variation of organelle DNA within a species complex of an organism may be the common condition, and that the DNAs of the organelle and nuclear systems evolve in reasonable harmony. Images PMID:16592708

  11. Organelle DNA variation and systematic relationships in the genus Zea: Teosinte.

    PubMed

    Timothy, D H; Levings, C S; Pring, D R; Conde, M F; Kermicle, J L

    1979-09-01

    Chloroplast and mitochondrial DNAs from six races of annual teosinte (Guatemala, Huehuetenango, Balsas, Central Plateau, Chalco, and Nobogame), perennial teosinte, and maize were compared and grouped by restriction endonuclease fragment analyses. Three groups of chloroplast DNAs were detected: (i) perennial teosinte and Guatemala; (ii) Balsas and Huehuetenango; and (iii) all other teosintes. Four groups of mitochondrial DNAs were separated: (i) perennial teosinte; (ii) Guatemala; (iii) Nobogame; and (iv) all other teosintes. Separation of the teosinte and maize organelle DNAs into five groups (Guatemala; perennial teosinte; Balsas and Huehuetenango; Central Plateau and Chalco; Nobogame and maize) approximated the biosystematic relationships of the taxa. It was suggested that the evolutions of the chloroplast and mitochondrial DNAs may be independent of each other, that variation of organelle DNA within a species complex of an organism may be the common condition, and that the DNAs of the organelle and nuclear systems evolve in reasonable harmony.

  12. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. 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.

  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. 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.

  16. Healthspan and longevity in mammals: a family game for cellular organelles?

    PubMed

    Nisoli, Enzo; Valerio, Alessandra

    2014-01-01

    Healthy mitochondria are essential generators of cellular energy, while senescent or damaged mitochondria are bioenergetically inefficient and are sources of reactive oxygen species. The mitochondrial life cycle, comprising biogenesis, fusion/fission events and mitophagic elimination, is carefully orchestrated, and age-related decay of the lifecycle contributes to chronic degenerative diseases. Mitochondria make contacts with other cellular organelles in the endomembrane system (endoplasmic reticulum, peroxisomes and lysosomes) whose dynamics are co-regulated and interactions finely tuned to meet the cell requirements and maintain the health of the organism. This review will consider the evidence that mitochondrial biogenesis and quality control, as well as the complex interplay among cellular organelles, may be affected by the aging process(es), with negative consequences for the well being of elderly individuals. Moreover, we propose that nutrients or drugs able to maintain organelle homeostasis may represent novel preventive and/or therapeutic approaches for chronic age-related diseases.

  17. 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. © 2016 Melchionda et al.

  18. GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

    PubMed Central

    Rodriguez-Garcia, Arancha; Rojo-Ruiz, Jonathan; Navas-Navarro, Paloma; Aulestia, Francisco Javier; Gallego-Sandin, Sonia; Garcia-Sancho, Javier; Alonso, Maria Teresa

    2014-01-01

    Genetically encoded calcium indicators allow monitoring subcellular Ca2+ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca2+ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg2+, tunable Ca2+ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca2+ indicators for organelles and becomes a valuable tool for in vivo Ca2+ imaging applications. PMID:24501126

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

    PubMed

    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.

  20. 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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. 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.

  2. Reduction of organelle motility by removal of potassium and other solutes

    PubMed Central

    Yin, David; Wolkoff, Allan W.

    2017-01-01

    There are surprisingly few studies that describe how the composition of cell culture medium may affect the trafficking of organelles. Here we utilize time lapse multi-channel fluorescent imaging to show that short term exposure of Huh-7 cells to medium lacking potassium, sodium, or chloride strongly reduces but does not eliminate the characteristic back and forth and cell-traversing movement of fluorescent EGF (FL-EGF) containing organelles. We focused on potassium because of its relatively low abundance in media and serum and its energy requiring accumulation into cells. Upon exposure to potassium free medium, organelle motility declined steadily through 90 min and then persisted at a low level. Reduced motility was confirmed in 5 independent cell lines and for organelles of the endocytic pathway (FL-EGF and Lysotracker), autophagosomes (LC3-GFP), and mitochondria (TMRE). As has been previously established, potassium free medium also inhibited endocytosis. We expected that diminished cellular metabolism would precede loss of organelle motility. However, extracellular flux analysis showed near normal mitochondrial oxygen consumption and only a small decrease in extracellular acidification, the latter suggesting decreased glycolysis or proton efflux. Other energy dependent activities such as the accumulation of Lysotracker, TMRE, DiBAC4(3), and the exclusion of propidium iodide remained intact, as did the microtubule cytoskeleton. We took advantage of cell free in vitro motility assays and found that removal of potassium or sodium from the reconstituted cytosolic medium decreased the movement of endosomes on purified microtubules. The results indicate that although changes in proton homeostasis and cell energetics under solute depletion are not fully understood, potassium as well as sodium appear to be directly required by the motile machinery of organelles for optimal trafficking. PMID:28922372

  3. The mitochondrion-like organelle of Trimastix pyriformis contains the complete glycine cleavage system.

    PubMed

    Zubáčová, Zuzana; Novák, Lukáš; Bublíková, Jitka; Vacek, Vojtěch; Fousek, Jan; Rídl, Jakub; Tachezy, Jan; Doležal, Pavel; Vlček, Cestmír; Hampl, Vladimír

    2013-01-01

    All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins - mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

  4. 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. Copyright © 2016 Elsevier Inc

  5. The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles.

    PubMed

    Johnson, Matthew D

    2011-01-01

    Many non-photosynthetic species of protists and metazoans are capable of hosting viable algal endosymbionts or their organelles through adaptations of phagocytic pathways. A form of mixotrophy combining phototrophy and heterotrophy, acquired phototrophy (AcPh) encompasses a suite of endosymbiotic and organelle retention interactions, that range from facultative to obligate. AcPh is a common phenomenon in aquatic ecosystems, with endosymbiotic associations generally more prevalent in nutrient poor environments, and organelle retention typically associated with more productive ones. All AcPhs benefit from enhanced growth due to access to photosynthetic products; however, the degree of metabolic integration and dependency in the host varies widely. AcPh is found in at least four of the major eukaryotic supergroups, and is the driving force in the evolution of secondary and tertiary plastid acquisitions. Mutualistic resource partitioning characterizes most algal endosymbiotic interactions, while organelle retention is a form of predation, characterized by nutrient flow (i.e., growth) in one direction. AcPh involves adaptations to recognize specific prey or endosymbionts and to house organelles or endosymbionts within the endomembrane system but free from digestion. In many cases, hosts depend upon AcPh for the production of essential nutrients, many of which remain obscure. The practice of AcPh has led to multiple independent secondary and tertiary plastid acquisition events among several eukaryote lineages, giving rise to the diverse array of algae found in modern aquatic ecosystems. This article highlights those AcPhs that are model research organisms for both metazoans and protists. Much of the basic biology of AcPhs remains enigmatic, particularly (1) which essential nutrients or factors make certain forms of AcPh obligatory, (2) how hosts regulate and manipulate endosymbionts or sequestered organelles, and (3) what genomic imprint, if any, AcPh leaves on non

  6. RNase P branches out from RNP to protein: organelle-triggered diversification?

    PubMed Central

    Goldfarb, Katherine C.; Borah, Sumit; Cech, Thomas R.

    2012-01-01

    RNase P is the enzyme that removes 5′ leader sequences from precursor tRNAs. Remarkably, in most organisms, RNase P is a ribonucleoprotein particle where the RNA component is responsible for catalysis. In this issue of Genes & Development, Gutmann and colleagues (pp. 1022–1027) report the first organism, Arabidopsis thaliana, to employ protein-only RNase P in both its nucleus and organelles. An intriguing possibility is that replacement of RNase P ribonucleoprotein particles (RNPs) by proteins may have been triggered by the acquisition of organelles. PMID:22588715

  7. Targeting of host organelles by pathogenic bacteria: a sophisticated subversion strategy.

    PubMed

    Escoll, Pedro; Mondino, Sonia; Rolando, Monica; Buchrieser, Carmen

    2016-01-01

    Many bacterial pathogens have evolved the ability to subvert and exploit host functions in order to enter and replicate in eukaryotic cells. For example, bacteria have developed specific mechanisms to target eukaryotic organelles such as the nucleus, the mitochondria, the endoplasmic reticulum and the Golgi apparatus. In this Review, we highlight the most recent advances in our understanding of the mechanisms that bacterial pathogens use to target these organelles. We also discuss how these strategies allow bacteria to manipulate host functions and to ultimately enable bacterial infection.

  8. Intracellular control of axial shape in non-uniform neurites: a serial electron microscopic analysis of organelles and microtubules in AI and AII retinal amacrine neurites

    PubMed Central

    1984-01-01

    AI and AII cat retinal amacrine cells have highly varicose non-uniform, neuritic processes. Processes of both types were reconstructed via a computer system using serial electron micrographs. These reconstructions were analyzed for (a) varicosity volume, surface area, and length, (b) "neck" volume, surface area, and length, (c) number of microtubules within the varicosity, (d) number of microtubules within the "neck," and (e) volume and surface area of mitochondria and smooth endoplasmic reticulum and large smooth vesicular bodies within the processes. Correlation of these parameters revealed a linear relationship between the number of microtubules in the necks and mean neck cross-sectional area (rs = 0.780, P less than 0.001), while microtubule number within the varicosities showed no correlation with varicosity volume (rs = 0.239, P greater than 0.2). Varicosity volume did, however, correlate strongly with the summed volume of mitochondria and smooth vesicular bodies contained within the varicosity for both cell types examined. The ratio between membranous organelle volume and varicosity volume for AI amacrine processes of 1:6.97 (rs = 0.927), differed from the ratio of 1:1.80 for the AII amacrine processes (rs = 0.987). Similar relationships were observed in other nonvaricose neurites such as optic tract axons. Membranous organelles appear to contribute an additional obligatory volume to the cytosol that can be as much as seven times the organelles' direct volume. These observations suggest that both the cytoskeletal components, and the membrane organelles play a direct role in determining neurite shape. PMID:6538879

  9. Using a Morphological Analyzer to Teach Theoretical Morphology.

    ERIC Educational Resources Information Center

    Klavans, Judith L.; Chodorow, Martin S.

    1991-01-01

    Discusses the use of an instructional morphological parser (IMP) in the teaching of courses in theoretical and computational morphology. Provides an overview of computational morphology. Outlines the two courses and speculates on computational and linguistic concepts that students learned. Examines problems encountered in teaching about recursion.…

  10. FINE STRUCTURE AND ORGANELLE ASSOCIATIONS IN BROWN ALGAE

    PubMed Central

    Bouck, G. Benjamin

    1965-01-01

    The structural interrelationships among several membrane systems in the cells of brown algae have been examined by electron microscopy. In the brown algae the chloroplasts are surrounded by two envelopes, the outer of which in some cases is continuous with the nuclear envelope. The pyrenoid, when present, protrudes from the chloroplast, is also surrounded by the two chloroplast envelopes, and, in addition, is capped by a third dilated envelope or "pyrenoid sac." The regular apposition of the membranes around the pyrenoid contrasts with their looser appearance over the remainder of the chloroplast. The Golgi apparatus is closely associated with the nuclear envelope in all brown algae examined, but in the Fucales this association may extend to portions of the cytoplasmic endoplasmic reticulum as well. Evidence is presented for the derivation of vesicles, characteristic of those found in the formative region of the Golgi apparatus, from portions of the underlying nuclear envelope. The possibility that a structural channeling system for carbohydrate reserves and secretory precursors may be present in brown algae is considered. Other features of the brown algal cell, such as crystal-containing bodies, the variety of darkly staining vacuoles, centrioles, and mitochondria, are examined briefly, and compared with similar structures in other plant cells. PMID:5865936

  11. In Vitro Assays Demonstrate That Pollen Tube Organelles Use Kinesin-Related Motor Proteins to Move along MicrotubulesW⃞

    PubMed Central

    Romagnoli, Silvia; Cai, Giampiero; Cresti, Mauro

    2003-01-01

    The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro–polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear

  12. Divide and Conquer: the Application of Organelle Proteomics to Heart Failure

    PubMed Central

    Agnetti, Giulio; Husberg, Cathrine; Van Eyk, Jennifer E.

    2013-01-01

    Chronic heart failure is a worldwide cause of mortality and morbidity and is the final outcome of a number of different etiologies. This reflects both the complexity of the disease and our incomplete understanding of its underlying molecular mechanisms. One experimental approach to address this is to study subcellular organelles and how their functions are activated and synchronized under physiological and pathological conditions. In this review, we discuss the application of proteomic technologies to organelles and how this has deepened our perception of the cellular proteome and its alterations with heart failure. The use of proteomics to monitor protein quantity and post-translational modifications (PTMs) has revealed a highly intricate and sophisticated level of protein regulation. PTMs have the potential to regulate organelle function and interplay most likely by targeting both structural and signaling proteins throughout the cell, ultimately coordinating their responses. The potentials and limitations of current proteomic technologies are also discussed emphasizing that the development of novel methods will enhance our ability to further investigate organelles and decode intracellular communication. PMID:21335433

  13. 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

  14. 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

  15. Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7.

    PubMed

    Lantsman, Yelena; Tan, Kevin S W; Morada, Mary; Yarlett, Nigel

    2008-09-01

    A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.

  16. Cold-induced organelle relocation in the liverwort Marchantia polymorpha L.

    PubMed

    Ogasawara, Yuka; Ishizaki, Kimitsune; Kohchi, Takayuki; Kodama, Yutaka

    2013-08-01

    Organelles change their subcellular positions in response to various environmental conditions. Recently, we reported that cold treatments alter the intracellular position of chloroplasts and nuclei (cold positioning) in the fern Adiantum capillus-veneris; chloroplasts and nuclei localized to the periclinal cell wall relocated to anticlinal cell wall after cold treatments. To further understand organelle positioning under cold conditions, we studied cold-induced organelle relocation in the liverwort Marchantia polymorpha L. When sporelings and gemmmalings were treated under low temperature (5 °C), chloroplast cold positioning response was successfully induced both in the sporelings and the gemmmalings of M. polymorpha. Using a genetic transformation, nuclei, mitochondria or peroxisomes were visualized with a fluorescent protein, and the transgenic gemmmalings were incubated under the cold condition. Nuclei and peroxisomes, but not mitochondria, clearly relocated from the periclinal cell wall to the anticlinal cell wall after cold treatments. Our findings suggest that several organelles concurrently change their positions in the liverwort cell to cope with cold temperature. © 2013 John Wiley & Sons Ltd.

  17. Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii.

    PubMed

    Stewart, Rebecca J; Ferguson, David J P; Whitehead, Lachlan; Bradin, Clare H; Wu, Hong J; Tonkin, Christopher J

    2016-02-01

    Upon infection, apicomplexan parasites quickly invade host cells and begin a replicative cycle rapidly increasing in number over a short period of time, leading to tissue lysis and disease. The secretory pathway of these highly polarized protozoan parasites tightly controls, in time and space, the biogenesis of specialized structures and organelles required for invasion and intracellular survival. In other systems, regulation of protein trafficking can occur by phosphorylation of vesicle fusion machinery. Previously, we have shown that Toxoplasma gondii αSNAP - a protein that controls the disassembly of cis-SNARE complexes--is phosphorylated. Here, we show that this post-translational modification is required for the correct function of αSNAP in controlling secretory traffic. We demonstrate that during intracellular development conditional expression of a non-phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, we show that the vestigial plastid (termed apicoplast), although reported not to be reliant on Golgi trafficking for biogenesis, is also affected upon overexpression of αSNAP and is much more sensitive to the levels of this protein than targeting to other organelles. This work highlights the importance of αSNAP and its phosphorylation in Toxoplasma organelle biogenesis and exposes a hereto fore-unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Calcium signaling in plant cell organelles delimited by a double membrane.

    PubMed

    Xiong, Tou-Cheu; Bourque, Stéphane; Lecourieux, David; Amelot, Nicolas; Grat, Sabine; Brière, Christian; Mazars, Christian; Pugin, Alain; Ranjeva, Raoul

    2006-11-01

    Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.

  19. Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation.

    PubMed

    Tovar, Jorge; León-Avila, Gloria; Sánchez, Lidya B; Sutak, Robert; Tachezy, Jan; van der Giezen, Mark; Hernández, Manuel; Müller, Miklós; Lucocq, John M

    2003-11-13

    Giardia intestinalis (syn. lamblia) is one of the most widespread intestinal protozoan pathogens worldwide, causing hundreds of thousands of cases of diarrhoea each year. Giardia is a member of the diplomonads, often described as an ancient protist group whose primitive nature is suggested by the lack of typical eukaryotic organelles (for example, mitochondria, peroxisomes), the presence of a poorly developed endomembrane system and by their early branching in a number of gene phylogenies. The discovery of nuclear genes of putative mitochondrial ancestry in Giardia and the recent identification of mitochondrial remnant organelles in amitochondrial protists such as Entamoeba histolytica and Trachipleistophora hominis suggest that the eukaryotic amitochondrial state is not a primitive condition but is rather the result of reductive evolution. Using an in vitro protein reconstitution assay and specific antibodies against IscS and IscU--two mitochondrial marker proteins involved in iron-sulphur cluster biosynthesis--here we demonstrate that Giardia contains mitochondrial remnant organelles (mitosomes) bounded by double membranes that function in iron-sulphur protein maturation. Our results indicate that Giardia is not primitively amitochondrial and that it has retained a functional organelle derived from the original mitochondrial endosymbiont.

  20. Role of plant myosins in motile organelles: is a direct interaction required?

    PubMed

    Buchnik, Limor; Abu-Abied, Mohamad; Sadot, Einat

    2015-01-01

    Plant organelles are highly motile, with speed values of 3-7 µm/s in cells of land plants and about 20-60 µm/s in characean algal cells. This movement is believed to be important for rapid distribution of materials around the cell, for the plant's ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins. Most plants express multiple members of two main classes: myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in all kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate. © 2014 Institute of Botany, Chinese Academy of Sciences.

  1. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

    PubMed

    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

    2015-03-05

    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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Changes in architecture of the Golgi complex and other subcellular organelles during myogenesis

    PubMed Central

    1993-01-01

    Myogenesis involves changes in both gene expression and cellular architecture. Little is known of the organization, in muscle in vivo, of the subcellular organelles involved in protein synthesis despite the potential importance of targeted protein synthesis for formation and maintenance of functional domains such as the neuromuscular junction. A panel of antibodies to markers of the ER, the Golgi complex, and the centrosome were used to localize these organelles by immunofluorescence in myoblasts and myotubes of the mouse muscle cell line C2 in vitro, and in intact single muscle fibers from the rat flexor digitorum brevis. Antibodies to the ER stained structures throughout the cytoplasm of both C2 myoblasts and myotubes. In contrast, the spatial relationship between nucleus, centrosome, and Golgi complex was dramatically altered. These changes could also be observed in a low- calcium medium that allowed differentiation while preventing myoblast fusion. Muscle fibers in vivo resembled myotubes except that the ER occupied a smaller volume of cytoplasm and no staining was found for one of the Golgi complex markers, the enzyme alpha-mannosidase II. Electron microscopy, however, clearly showed the presence of stacks of Golgi cisternae in both junctional and extrajunctional regions of muscle fibers. The perinuclear distribution of the Golgi complex was also observed in live muscle fibers stained with a fluorescent lipid. Thus, the distribution of subcellular organelles of the secretory pathway was found to be similar in myotubes and muscle fibers, and all organelles were found in both junctional and extrajunctional areas of muscle. PMID:7678420

  3. 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

  4. 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

  5. Organelle-Specific Sensors for Monitoring Ca2+ Dynamics in Neurons

    PubMed Central

    Kwon, Seok-Kyu; Hirabayashi, Yusuke; Polleux, Franck

    2016-01-01

    Calcium (Ca2+) plays innumerable critical functions in neurons ranging from regulation of neurotransmitter release and synaptic plasticity to activity-dependent transcription. Therefore, more than any other cell types, neurons are critically dependent on spatially and temporally controlled Ca2+ dynamics. This is achieved through an exquisite level of compartmentalization of Ca2+ storage and release from various organelles. The function of these organelles in the regulation of Ca2+ dynamics has been studied for decades using electrophysiological and optical methods combined with pharmacological and genetic alterations. Mitochondria and the endoplasmic reticulum (ER) are among the organelles playing the most critical roles in Ca2+ dynamics in neurons. At presynaptic boutons, Ca2+ triggers neurotransmitter release and synaptic plasticity, and postsynaptically, Ca2+ mobilization mediates long-term synaptic plasticity. To explore Ca2+ dynamics in live cells and intact animals, various synthetic and genetically encoded fluorescent Ca2+ sensors were developed, and recently, many groups actively increased the sensitivity and diversity of genetically encoded Ca2+ indicators (GECIs). Following conjugation with various signal peptides, these improved GECIs can be targeted to specific subcellular compartments, allowing monitoring of organelle-specific Ca2+ dynamics. Here, we review recent findings unraveling novel roles for mitochondria- and ER-dependent Ca2+ dynamics in neurons and at synapses. PMID:27695411

  6. Sperm morphological abnormalities visualised at high magnification predict embryonic development, from fertilisation to the blastocyst stage, in couples undergoing ICSI.

    PubMed

    Setti, Amanda Souza; Braga, Daniela Paes de Almeida Ferreira; Vingris, Livia; Serzedello, Thais; Figueira, Rita de Cássia Sávio; Iaconelli, Assumpto; Borges, Edson

    2014-11-01

    To investigate the predictive value of the motile sperm organelle morphology examination (MSOME) on embryo morphology. The morphologies of 540 embryos obtained from 60 couples undergoing ICSI were evaluated from days 1 to 5 of development and were examined for associations with the percentages of morphologically normal paternal sperm and of the paternal sperm with large nuclear vacuoles (LNVs) as determined by MSOME. An increased percentage of LNV sperm was associated with increased odds of a zygote presenting with pronuclear abnormalities. It was also associated with decreased odds of (i) normal cleavage on days 2 and 3 of development, (ii) the presence of a high-quality embryo on day 3, (iii) the development of an embryo to the blastocyst stage, and (iv) an embryo possessing a normal trophectoderm and inner cell mass. The calculated areas under the curves differed for the embryos that did and did not develop to the blastocyst stage and for the high- and low-quality blastocysts. The optimal cut-off value for the percentage of LNV sperm that maximised proper blastocyst formation was ≤24.5 %, and the cut-off value that maximised blastocyst quality was ≤19.5 %. These results suggest a very early onset of paternal influences on embryo development. The evaluation of the incidence of vacuoles by MSOME may significantly improve upon the prognostic information provided by conventional semen analyses.

  7. Mitochondrial Morphological Features Are Associated with Fission and Fusion Events

    PubMed Central

    Martin, Katie R.; Hlavacek, William S.; MacKeigan, Jeffrey P.

    2014-01-01

    Mitochondria are dynamic organelles that undergo constant remodeling through the regulation of two opposing processes, mitochondrial fission and fusion. Although several key regulators and physiological stimuli have been identified to control mitochondrial fission and fusion, the role of mitochondrial morphology in the two processes remains to be determined. To address this knowledge gap, we investigated whether morphological features extracted from time-lapse live-cell images of mitochondria could be used to predict mitochondrial fate. That is, we asked if we could predict whether a mitochondrion is likely to participate in a fission or fusion event based on its current shape and local environment. Using live-cell microscopy, image analysis software, and supervised machine learning, we characterized mitochondrial dynamics with single-organelle resolution to identify features of mitochondria that are predictive of fission and fusion events. A random forest (RF) model was trained to correctly classify mitochondria poised for either fission or fusion based on a series of morphological and positional features for each organelle. Of the features we evaluated, mitochondrial perimeter positively correlated with mitochondria about to undergo a fission event. Similarly mitochondrial solidity (compact shape) positively correlated with mitochondria about to undergo a fusion event. Our results indicate that fission and fusion are positively correlated with mitochondrial morphological features; and therefore, mitochondrial fission and fusion may be influenced by the mechanical properties of mitochondrial membranes. PMID:24733410

  8. Mitochondrial morphological features are associated with fission and fusion events.

    PubMed

    Westrate, Laura M; Drocco, Jeffrey A; Martin, Katie R; Hlavacek, William S; MacKeigan, Jeffrey P

    2014-01-01

    Mitochondria are dynamic organelles that undergo constant remodeling through the regulation of two opposing processes, mitochondrial fission and fusion. Although several key regulators and physiological stimuli have been identified to control mitochondrial fission and fusion, the role of mitochondrial morphology in the two processes remains to be determined. To address this knowledge gap, we investigated whether morphological features extracted from time-lapse live-cell images of mitochondria could be used to predict mitochondrial fate. That is, we asked if we could predict whether a mitochondrion is likely to participate in a fission or fusion event based on its current shape and local environment. Using live-cell microscopy, image analysis software, and supervised machine learning, we characterized mitochondrial dynamics with single-organelle resolution to identify features of mitochondria that are predictive of fission and fusion events. A random forest (RF) model was trained to correctly classify mitochondria poised for either fission or fusion based on a series of morphological and positional features for each organelle. Of the features we evaluated, mitochondrial perimeter positively correlated with mitochondria about to undergo a fission event. Similarly mitochondrial solidity (compact shape) positively correlated with mitochondria about to undergo a fusion event. Our results indicate that fission and fusion are positively correlated with mitochondrial morphological features; and therefore, mitochondrial fission and fusion may be influenced by the mechanical properties of mitochondrial membranes.

  9. 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

  10. Organelle Communication at Membrane Contact Sites (MCS): From Curiosity to Center Stage in Cell Biology and Biomedical Research.

    PubMed

    Simmen, Thomas; Tagaya, Mitsuo

    2017-01-01

    Cell biology has long recognized that organelles can communicate with each other. Initially, such communication was thought to occur primarily via vesicular trafficking between biochemically distinct organelles. However, studies starting in the 1970s on lipid metabolism have unearthed another way how organelles can communicate and have spawned the field of membrane contact sites (MCS). While, initially, MCS had been recognized as fluid entities that mediate lipid and ion transport in an ad hoc manner, more recently MCS have been found to depend on protein-protein interactions that control themselves a variety of MCS functions. As a result, the cell biological definition of an intracellular organelle as an isolated membrane compartment is now being revised. Accordingly, the organelle definition now describes organelles as dynamic membrane compartments that function in a milieu of coordinated contacts with other organelles. Through these mercurial functions, MCS dictate the function of organelles to a large extent but also play important roles in a number of diseases, including type 2 diabetes, neurodegenerative diseases, infections, and cancer. This book assembles reviews that describe our quickly evolving knowledge about organellar communication on MCS and the significance of MCS for disease.

  11. Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging.

    PubMed

    Wang, Hongliang; Tai, Andrew W

    2017-01-01

    Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication. In chronically infected cells, it is not known whether these viral replication organelles are being continually resupplied by newly synthesized viral proteins in situ, or whether they are generated de novo. Here we aimed to study temporal events in replication organelles formation and maturation. Here we use pulse-chase labeling in combination with confocal microscopy, correlative light electron microscopy and biochemical methods to identify temporally distinct populations of replication organelles in living cells and study the formation, morphogenesis as well as compositional and functional changes of replication organelles over time. We found that HCV replication organelles are continuously generated de novo at spatially distinct sites from preformed ones. This process is accompanied by accumulated intracellular membrane alteration, increased cholesterol delivery, NS5A phosphorylation, and positive-strand RNA content, and by eventual association with HCV core protein around lipid droplets. Generation of spatially segregated foci requires viral NS5A and the host factors phosphatidylinositol 4-kinase and oxysterol-binding protein, while association of foci with lipid droplets requires cholesterol. Our results reveal that HCV replication organelles are not static structures, but instead are continuously generated and dynamically change in composition and possibly also in function. Hepatitis C virus replication membrane structures are continuously generated at spatially distinct sites. New replication organelles are different in composition, and possibly also in function, compared to old replication organelles. Published by Elsevier B.V.

  12. Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution

    PubMed Central

    Dacks, Joel B.; Poon, Pak P.; Field, Mark C.

    2008-01-01

    The process by which some eukaryotic organelles, for example the endomembrane system, evolved without endosymbiotic input remains poorly understood. This problem largely arises because many major cellular systems predate the last common eukaryotic ancestor (LCEA) and thus do not provide examples of organellogenesis in progress. A model is emerging whereby gene duplication and divergence of multiple “specificity-” or “identity-” encoding proteins for the various endomembranous organelles produced the diversity of nonendosymbiotically derived cellular compartments present in modern eukaryotes. To address this possibility, we analyzed three molecular components of the endocytic membrane-trafficking machinery. Phylogenetic analyses of the endocytic syntaxins, Rab 5, and the β-adaptins each reveal a pattern of ancestral, undifferentiated endocytic homologues in the LCEA. Subsequently, these undifferentiated progenitors independently duplicated in widely divergent lineages, convergently producing components with similar endocytic roles, e.g., β1 and β2-adaptin. In contrast, β3, β4, and all other adaptin complex subunits, as well as paralogues of the syntaxins and Rabs specific for the other membrane-trafficking organelles, all evolved before the LCEA. Thus, the process giving rise to the differentiated organelles of the endocytic system appears to have been interrupted by the major speciation event that produced the extant eukaryotic lineages. These results suggest that although many endocytic components evolved before the LCEA, other major features evolved independently and convergently after diversification into the primary eukaryotic supergroups. This finding provides an example of a basic cellular system that was simpler in the LCEA than in many extant eukaryotes and yields insight into nonendosymbiotic organelle evolution. PMID:18182495

  13. Analysis of Organelle Targeting by DIL Domains of the Arabidopsis Myosin XI Family.

    PubMed

    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R

    2011-01-01

    The Arabidopsis thaliana genome encodes 13 myosin XI motor proteins. Previous insertional mutant analysis has implicated substantial redundancy of function of plant myosin XIs in transport of intracellular organelles. Considerable information is available about the interaction of cargo with the myosin XI-homologous yeast myosin V protein myo2p. We identified a region in each of 12 myosin XI sequences that correspond to the yeast myo2p secretory-vesicle binding domain (the "DIL" domain). Structural modeling of the myosin DIL domain region of plant myosin XIs revealed significant similarity to the yeast myo2p and myo4p DIL domains. Transient expression of YFP fusions with the Arabidopsis myosin XI DIL domain resulted in fluorescent labeling of a variety of organelles, including the endoplasmic reticulum, peroxisomes, Golgi, and nuclear envelope. With the exception of the YFP::MYA1 DIL fusion, expression of the DIL-YFP fusions resulted in loss of motility of labeled organelles, consistent with a dominant-negative effect. Certain fusions resulted in localization to the cytoplasm, plasma membrane, or to unidentified vesicles. The same YFP-domain fusion sometimes labeled more than one organelle. Expression of a YFP fusion to a yeast myo2p DIL domain resulted in labeling of plant peroxisomes. Fusions with some of the myosin XI domains resulted in labeling of known cargoes of the particular myosin XI; however, certain myosin XI YFP fusions labeled organelles that had not previously been found to be detectably affected by mutations nor by expression of dominant-negative constructs.

  14. ATP-dependent uptake of anti-neoplastic agents by acidic organelles.

    PubMed

    Moriyama, Y; Manabe, T; Yoshimori, T; Tashiro, Y; Futai, M

    1994-02-01

    Daunomycin, an anti-neoplastic agent, is known to be sequestered by acidic organelles in normal and multidrug-resistant cells [Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesman, M.M., & Pastan, I. (1986) Cancer Res. 46, 5941-5946]. We studied the mechanism of accumulation of daunomycin into acidic organelles using chromaffin granule vesicles and proteoliposomes reconstituted with purified F-type H(+)-ATPase as model systems. Radiolabeled daunomycin was taken up by chromaffin vesicles upon addition of ATP. Its ATP-dependent uptake was stimulated about 1.4- to 1.8-fold by valinomycin plus K+, but was inhibited by ammonium chloride (10 mM) and nigericin plus K+. Quinidine (5 microM), verapamil (5 microM), or vanadate (0.5 mM), inhibitors of P-glycoprotein, had no effect on its uptake. Daunomycin was also taken up by liposomes reconstituted with F-type H(+)-ATPase. Furthermore, doxorubicin and vinblastine were taken up by these vesicles, whereas colchicine and rhodamine 123 were not. The accumulations of daunomycin and doxorubicin in acidic organelles of cultured cells were decreased by inhibiting vacuolar ATPase by addition of bafilomycin A1 or concanamycin A, or by increasing the internal pH by addition of nigericin. Melittin and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide dissipated the delta pH and inhibited accumulation of daunomycin in the membrane vesicles and acidic organelles in cultured cells. These results indicate that the delta pH established by vacuolar-type ATPase drives the uptake of daunomycin, doxorubicin or vinblastine into acidic organelles, and that no specific transporters are involved in their uptakes.

  15. 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

    2016-05-09

    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.

  16. Analysis of Organelle Targeting by DIL Domains of the Arabidopsis Myosin XI Family

    PubMed Central

    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R.

    2011-01-01

    The Arabidopsis thaliana genome encodes 13 myosin XI motor proteins. Previous insertional mutant analysis has implicated substantial redundancy of function of plant myosin XIs in transport of intracellular organelles. Considerable information is available about the interaction of cargo with the myosin XI-homologous yeast myosin V protein myo2p. We identified a region in each of 12 myosin XI sequences that correspond to the yeast myo2p secretory-vesicle binding domain (the “DIL” domain). Structural modeling of the myosin DIL domain region of plant myosin XIs revealed significant similarity to the yeast myo2p and myo4p DIL domains. Transient expression of YFP fusions with the Arabidopsis myosin XI DIL domain resulted in fluorescent labeling of a variety of organelles, including the endoplasmic reticulum, peroxisomes, Golgi, and nuclear envelope. With the exception of the YFP::MYA1 DIL fusion, expression of the DIL–YFP fusions resulted in loss of motility of labeled organelles, consistent with a dominant-negative effect. Certain fusions resulted in localization to the cytoplasm, plasma membrane, or to unidentified vesicles. The same YFP-domain fusion sometimes labeled more than one organelle. Expression of a YFP fusion to a yeast myo2p DIL domain resulted in labeling of plant peroxisomes. Fusions with some of the myosin XI domains resulted in labeling of known cargoes of the particular myosin XI; however, certain myosin XI YFP fusions labeled organelles that had not previously been found to be detectably affected by mutations nor by expression of dominant-negative constructs. PMID:22645548

  17. 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.

  18. Phylogeny of endocytic components yields insight into the process of nonendosymbiotic organelle evolution.

    PubMed

    Dacks, Joel B; Poon, Pak P; Field, Mark C

    2008-01-15

    The process by which some eukaryotic organelles, for example the endomembrane system, evolved without endosymbiotic input remains poorly understood. This problem largely arises because many major cellular systems predate the last common eukaryotic ancestor (LCEA) and thus do not provide examples of organellogenesis in progress. A model is emerging whereby gene duplication and divergence of multiple "specificity-" or "identity-" encoding proteins for the various endomembranous organelles produced the diversity of nonendosymbiotically derived cellular compartments present in modern eukaryotes. To address this possibility, we analyzed three molecular components of the endocytic membrane-trafficking machinery. Phylogenetic analyses of the endocytic syntaxins, Rab 5, and the beta-adaptins each reveal a pattern of ancestral, undifferentiated endocytic homologues in the LCEA. Subsequently, these undifferentiated progenitors independently duplicated in widely divergent lineages, convergently producing components with similar endocytic roles, e.g., beta1 and beta2-adaptin. In contrast, beta3, beta4, and all other adaptin complex subunits, as well as paralogues of the syntaxins and Rabs specific for the other membrane-trafficking organelles, all evolved before the LCEA. Thus, the process giving rise to the differentiated organelles of the endocytic system appears to have been interrupted by the major speciation event that produced the extant eukaryotic lineages. These results suggest that although many endocytic components evolved before the LCEA, other major features evolved independently and convergently after diversification into the primary eukaryotic supergroups. This finding provides an example of a basic cellular system that was simpler in the LCEA than in many extant eukaryotes and yields insight into nonendosymbiotic organelle evolution.

  19. Organelles Contribute Differentially to Reactive Oxygen Species-Related Events during Extended Darkness1[C][W][OA

    PubMed Central

    Rosenwasser, Shilo; Rot, Ilona; Sollner, Evelyn; Meyer, Andreas J.; Smith, Yoav; Leviatan, Noam; Fluhr, Robert; Friedman, Haya

    2011-01-01

    Treatment of Arabidopsis (Arabidopsis thaliana) leaves by extended darkness generates a genetically activated senescence program that culminates in cell death. The transcriptome of leaves subjected to extended darkness was found to contain a variety of reactive oxygen species (ROS)-specific signatures. The levels of transcripts constituting the transcriptome footprints of chloroplasts and cytoplasm ROS stresses decreased in leaves, as early as the second day of darkness. In contrast, an increase was detected in transcripts associated with mitochondrial and peroxisomal ROS stresses. The sequential changes in the redox state of the organelles during darkness were examined by redox-sensitive green fluorescent protein probes (roGFP) that were targeted to specific organelles. In plastids, roGFP showed a decreased level of oxidation as early as the first day of darkness, followed by a gradual increase to starting levels. However, in mitochondria, the level of oxidation of roGFP rapidly increased as early as the first day of darkness, followed by an increase in the peroxisomal level of oxidation of roGFP on the second day. No changes in the probe oxidation were observed in the cytoplasm until the third day. The increase in mitochondrial roGFP degree of oxidation was abolished by sucrose treatment, implying that oxidation is caused by energy deprivation. The dynamic redox state visualized by roGFP probes and the analysis of microarray results are consistent with a scenario in which ROS stresses emanating from the mitochondria and peroxisomes occur early during darkness at a presymptomatic stage and jointly contribute to the senescence program. PMID:21372201

  20. How transient alterations of organelles in mammalian cells submitted to electric field may explain some aspects of gene electrotransfer process.

    PubMed

    Phez, Emilie; Gibot, Laure; Rols, Marie-Pierre

    2016-12-01

    Electric pulses can be used to transiently permeabilize the cell plasma membrane. This method is nowadays employed as a safe and efficient means to deliver therapeutic molecules into target cells and tissues. Despite the large bulk of literature on this topic, there is a lack of knowledge about the mechanism(s) of molecule delivery. The behavior of the cells both while the field is on and after its application is indeed not well described. Questions about cell organelle alterations remain unanswered. We report here evidence for a number of ultrastructural alterations in mammalian cells exposed to electric pulses. Specifically, CHO cells were subjected to trains of 10 pulses lasting 5ms using an electric field of 800V/cm, i.e. under conditions leading both to membrane permeabilization, gene transfer and expression. Cells were observed to undergo morphological alterations of the mitochondria and nucleus. These modifications, detected in the minutes following pulse delivery, were transient. They may have direct consequences on molecule delivery and therefore may explain various aspects of the mechanisms of DNA electrotransfer. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. 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

  2. Comparison of ciliature microtubule organelles in three hypotrichous ciliate species

    NASA Astrophysics Data System (ADS)

    Li, Yisong; Shi, Lei; Gu, Fukang

    2010-05-01

    We examined the structure and spatial organization of ciliature base-associated microtubules (BAM) in three hypotrichous ciliates ( Stylonychia mytilus, Pseudourostyla cristata, Euplotes woodruffi) in fluorescence microscopy. The results revealed that BAM, including the anterior (ALM), posterior longitudinal microtubule (PLM) and the transverse microtubule (TM) bands, are composed of tubulin. The respective microtubular bands have cytoplasmic polarization patterns that are significantly asymmetric. The BAM of the midventral files in P. cristata appear cord-shaped compared with the ALM bands of transverse cirri in both S. mytilus and E. woodruffi, which extend to the left anterior side of the cell before converging. The TM bands of the left marginal cirri (MC) in S. mytilus extend to the right side of the cell, while those of the right MC bands extend to the left. Our observations suggest that BAM traits are common in hypotrichous ciliates even though different species possess different microtubule arrangements related to the conserved cirral morphogenetic patterns in the respective species. The differing development of BAM in the three ciliate suggests that the microtubules may be conserved in different hypotrichs. We have also demonstrated that the BAM, which appear polar and asymmetric, are localized in specific cytoskeletal positions and extend in different orientations within the cortex to connect with other ciliature-associated structures and, thus, strengthen the cortex. These BAM features indicate that they are directly associated with cell motion.

  3. Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia.

    PubMed

    Touz, María C; Kulakova, Liudmila; Nash, Theodore E

    2004-07-01

    Giardia lamblia is an early branching protist that possesses peripheral vacuoles (PVs) with characteristics of lysosome-like organelles, located underneath the plasma membrane. In more evolved cells, lysosomal protein trafficking is achieved by cargo recognition involving adaptor protein (AP) complexes that recognize specific amino acid sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins. Previously, we reported that Giardia has a tyrosine-based sorting system, which mediates the targeting of a membrane-associated cysteine protease (encystation-specific cysteine protease, ESCP) to the PVs. Here, we show that Giardia AP1 mediates the transport of ESCP and the soluble acid phosphatase (AcPh) to the PVs. By using the yeast two-hybrid assay we found that the ESCP tyrosine-based motif interacts specifically with the medium subunit of AP1 (Gimicroa). Hemagglutinin-tagged Gimicroa colocalizes with ESCP and AcPh and coimmunoprecipitates with clathrin, suggesting that protein trafficking toward the PVs is clathrin-adaptin dependent. Targeted disruption of Gimicroa results in mislocalization of ESCP and AcPh but not of variant-specific surface proteins. Our results suggest that, unlike mammalian cells, only AP1 is involved in anterograde protein trafficking to the PVs in Giardia. Moreover, even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, the presence of a clathrin-adaptor system suggests that this parasite possess a primitive secretory organelle capable of sorting proteins similar to that of more evolved cells.

  4. Neuronal transport of acid hydrolases and peroxidase within the lysosomal system or organelles: involvement of agranular reticulum-like cisterns.

    PubMed

    Broadwell, R D; Oliver, C; Brightman, M W

    1980-04-01

    Neurosecretory neurons of the hyperosmotically stressed hypothalamo-neurohypophysial system have been a useful model with which to demonstrate interrelationships among perikaryal lysosomes, agranular reticulum-like cisterns, endocytotic vacuoles, and the axoplasmic transport of acid hydrolases and horseradish peroxidase. Supraoptic neurons from normal mice and mice given 2% salt water to drink for 5--8 days have been studied using enzyme cytochemical techniques for peroxidase and lysosomal acid hydrolases. Peroxidase-labeling of these neurons was accomplished by intravenous injection or cerebral ventriculocisternal perfusion of the protein as previously reported (Broadwell and Brightman, '79). Compared to normal controls, supraoptic cell bodies from hyperosmotically stimulated mice contained elevated concentrations of peroxidase-labeled dense bodies demonstrated to be secondary lysosomes and acid hydrolase-positive and peroxidase-positive cisterns either attached or unattached to secondary lysosomes. These cisterns were smooth-surfaced and 400--1,000 A wide. Their morphology was similar to that of the agranular reticulum. Some of the cisterns contained both peroxidase and acid hydrolase activities. The cisterns probably represent an elongated form of lysosome and, therefore, are not elements of the agranular reticulum per se. By virtue of their direct connections with perikaryal secondary lysosomes, these cisterns may provide the route by which acid hydrolases and exogenous macromolecules can leave perikaryal secondary lysosomes for anterograde flow down the axon. Very few smooth-surfaced cisterns were involved in the retrograde transport of peroxidase within pituitary stalk axons from normal and salt-treated mice injected intravenously with peroxidase. Peroxidase undergoing retrograde transport was predominantly in endocytotic structures such as vacuoles and cup-shaped organelles, which deliver this exogenous macromolecule directly to secondary lysosomes for

  5. 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

  6. Sperm morphological normality under high magnification predicts laboratory and clinical outcomes in couples undergoing ICSI.

    PubMed

    Vingris, Livia; Setti, Amanda Souza; De Almeida Ferreira Braga, Daniela Paes; De Cassia Savio Figueira, Rita; Iaconelli, Assumpto; Borges, Edson

    2015-06-01

    The objective of this study was to evaluate whether 'motile sperm organelle morphology examination' (MSOME) is correlated with the outcome of ICSI. A total of 14400 spermatozoa from 72 couples undergoing ICSI were analysed by MSOME (x6600) and graded into four groups: grade I, normal form and no vacuoles; grade II, normal form and lesser than or equal to 2 small vacuoles; grade III, normal form greater than 2 small vacuoles or at least one large vacuole and grade IV, large vacuole and abnormal head shapes or other abnormalities. The correlations between the proportion of morphologically normal spermatozoa (grade I + II) and ICSI outcomes were assessed. The proportion of grade I+ II spermatozoa was lower in patients with oligoasthenoteratozoospermia (OAT) compared to patients with other types of semen alterations (10.6% vs. 17.0%, p = 0.001). The proportion of grade I+ II spermatozoa was positively correlated with blastocyst formation (S = 8.31, R(2):13.5%, p = 0.014) and implantation rates (S = 8.32, R(2): 7.9%, p = 0.030). The proportion of grade I + II spermatozoa was higher in patients with ongoing pregnancy in comparison with those who had a miscarriage (23.2% vs. 10.8%, p = 0.007). Sperm morphological normality was lower in oligoasthenoteratozoospermia patients but correlated with blastocyst formation, implantation and miscarriage rates in couples undergoing ICSI. MSOME may be valuable in predicting ICSI outcomes.

  7. Novel morphological features in the death of MCF-7 human breast cancer cells after exposure to anticancer drugs.

    PubMed

    Kugawa, F; Dalkhuren, S-O; Ueno, A; Yamashita, K

    2012-10-01

    Cell death of human breast cancer cell line MCF-7/pDsRed2-Mito, caused by independent- or multi-administration of three anticancer drugs, cyclophosphamide [CPA], doxorubicin [DXR], and 5-fluorouracil [5-FU], was studied using fluorescence and electron microscopy. In our previous study using cell viability assays, microscopic inspection of heterochromatin condensation, a DNA fragmentation assay, and flow cytometric analyses, the death of MCF-7 cells was classified into two groups. The cell death induced by CPA or 5-FU was classified as apoptotic, while the cell death induced by DXR treatment or a mixture of all three anticancer drugs was classified as non-apoptotic. Here, we examined the morphology of the whole cell and its organelles, including the mitochondria, using electron microscopy. Mitochondria are of particular interest because they are the key organelle for the molecular apoptotic-death cascade. To monitor mitochondrial morphology, we used our previously constructed MCF-7/pDsRed2-Mito line, generated by introducing the pDsRed2-Mito vector into MCF-7 cells. The mitochondria in these cells emit red fluorescence. We found that the administration of DXR alone or of all three anticancer drugs together resulted in the clumping of the red-fluorescent materials on both sides of the round dying cells, interrupted by the nucleus. Detailed electron microscopic observation revealed that the novel morphology of the dying MCF-7 cells might be owing, not to destruction of the mitochondrial membrane, but to the tight structure of the nuclear membrane. Other anticancer drugs showed different, characteristic features in electron microscopic images, which suggested that death induced by anti-cancer drugs in the human breast cancer cell line, MCF-7, may result from any of a number of diverse processes.

  8. Attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus.

    PubMed Central

    Ong, C J; Wong, M L; Smit, J

    1990-01-01

    Caulobacters attach to surfaces in the environment via their holdfasts, attachment organelles located at the base of the flagellum in swarmer cells and later at the end of the cellular stalk in the stalked cells which develop from the swarmer cells. There seems to be little specificity with respect to the types of surfaces to which holdfasts adhere. A notable exception is that the holdfast of one cell does not adhere to the cell surface of another caulobacter, except by joining holdfasts, typically forming "rosettes" of stalked cells. Thus, the localized adhesion of the holdfasts to the cells is in some way a specialized attachment. We investigated this holdfast-cell attachment by developing an adhesion screening assay and analyzing several mutants of Caulobacter crescentus CB2A selected to be defective in adhesion. One class of mutants made a normal holdfast by all available criteria, yet the attachment to the cell was very weak, such that the holdfast was readily shed. Another class of mutants made no holdfast at all, but when mixed with a wild-type strain, a mutant of this class participated in rosette formation. The mutant could also attach to the discarded holdfast produced by a shedding mutant. In addition, when rosettes composed of holdfast-defective and wild-type cells were examined, an increase in the number of holdfast-defective cells was correlated with a decrease in the ability of the holdfast material at the center of the rosette to bind colloidal gold particles. Gold particles are one type of surface to which holdfasts adhere well, suggesting that the stalk end and the colloidal gold particles occupy the same sites on the holdfast substance. Taken together, the data support the interpretation that there is a specialized attachment site for the holdfast at the base of the flagellum which later becomes the end of the stalk, but not a specialized region of the holdfast for attachment to this site. Also, attachment to the cell is accomplished by bond

  9. Immunological studies of isolated particulates of Paramecium aurella. I. Antigenic relationships between cytoplasmic organelles and evidence for mitochondrial variations as demonstrated by gel diffusion.

    PubMed

    FINGER, I; KABACK, M; KITTNER, P; HELLER, C

    1960-12-01

    Mitochondria and other particulates-cilia, trichocysts, and "small granules"-have been isolated from several stocks of Paramecium aurelia, syngen 2. Antisera against these particles and against breis have been used to characterize the fractions by diffusion in gel. Evidence is presented for the relationship of particles, as demonstrated by immunologic cross-reactivity of the soluble antigens extracted from them. Although some antigens are unique for a fraction, cross-reacting antigens in two or more fractions, as determined by "spur" formation in agar, suggest a relationship between morphologically diverse particles. A procedure for studying cross-reactions in gels is described using the specific immobilization antigens as a model. The localization of these antigens within cilia, and perhaps trichocysts, has been confirmed. Other organelles, specifically mitochondria and "small granules," appear to alter their specificity spontaneously and reversibly during cell reproduction, a pattern reminiscent of the immobilization serotypes which can transform to one another during clonal growth.

  10. 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.

  11. Maternal inheritance of mitochondrial DNA: degradation of paternal mitochondria by allogeneic organelle autophagy, allophagy.

    PubMed

    Sato, Miyuki; Sato, Ken

    2012-03-01

    Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) or