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Sample records for organelle morphology examination

  1. Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME).

    PubMed

    Perdrix, A; Saïdi, R; Ménard, J F; Gruel, E; Milazzo, J P; Macé, B; Rives, N

    2012-08-01

    With the motile sperm organelle morphology examination (MSOME), spermatozoa morphology may be assessed directly on motile spermatozoa at high magnification (up to 6600×). This procedure describes more precisely spermatozoa abnormalities, especially head vacuoles. However, no consensus has been established concerning normal or abnormal MSOME criteria. The aim of our study was to define MSOME vacuole criteria assessed objectively with a digital imaging system software to establish a potential relationship between conventional semen parameters. A total of 440 semen samples were obtained from males consulting in Rouen University Hospital Reproductive Biology Laboratory. Conventional semen analysis (volume, sperm concentration, progressive motility, vitality and morphology) and MSOME assessment {sperm head length, width and area as well as vacuole number, vacuole area and relative vacuole area to sperm head [RVA (%) = [vacuole area (μm(2))/head area (μm(2))] × 100)]} were performed for each semen sample. Among our 440 males, 109 presented normal conventional semen parameters and 331 abnormal ones. Sperm head vacuoles were significantly larger in abnormal semen samples (p < 0.0001). RVA was the most discriminative MSOME criterion between normal and abnormal semen samples according to ROC curves analysis, and was negatively correlated with poor sperm morphology (r = -0.53, p < 0.0001). We concluded to (i) the normal occurrence of vacuoles in sperm head whatever the normality or abnormality of semen parameters, (ii) the discriminative function of the RVA to distinguish semen samples with normal and abnormal parameters, and (iii) the strong correlation between high RVA and poor sperm morphology.

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

    PubMed

    Perdrix, Anne; Rives, Nathalie

    2013-01-01

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

  3. The effects of male age on sperm analysis by motile sperm organelle morphology examination (MSOME)

    PubMed Central

    2012-01-01

    Background This study aimed to investigate the influence of age on sperm quality, as analysed by motile sperm organelle morphology examination (MSOME). Methods Semen samples were collected from 975 men undergoing evaluation or treatment for infertility. Sperm cells were evaluated at 8400× magnification using an inverted microscope equipped with Nomarski (differential interference contrast) optics. Two forms of spermatozoa were considered: normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV, defined as vacuoles occupying > 50% of the sperm nuclear area). At least 200 spermatozoa per sample were evaluated, and the percentages of normal and LNV spermatozoa were determined. The subjects were divided into three groups according to age: Group I, less than or equal to 35 years; Group II, 36-40 years; and Group III, greater than or equal to 41 years. Results There was no difference in the percentages of normal sperm between the two younger (I and II) groups (P >0.05). The percentage of normal sperm in the older group (III) was significantly lower than that in the younger (I and II) groups (P < 0.05). There was no difference in the percentage of LNV spermatozoa between the younger (I and II) groups (P >0.05). The percentage of LNV spermatozoa was significantly higher in the older group (III) than in the younger (I and II) groups (P < 0.05). Regression analysis demonstrated a significant decrease in the incidence of normal sperm with increasing age (P < 0.05; r = -0.10). However, there was a significant positive correlation between the percentage of spermatozoa with LNV and male age (P < 0.05, r = 0.10). Conclusion The results demonstrated a consistent decline in semen quality, as reflected by morphological evaluation by MSOME, with increased age. Considering the relationship between nuclear vacuoles and DNA damage, these age-related changes predict that increased paternal age should be associated with unsuccessful or abnormal pregnancy as a consequence of

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

    PubMed

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

    2015-11-01

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

  5. 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.

  6. 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

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

    PubMed

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

    2016-04-01

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

  8. 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

  9. Organelle transformation.

    PubMed

    Bhattacharya, Anjanabha; Kumar, Anish; Desai, Nirali; Parikh, Seema

    2012-01-01

    The source of genetic information in a plant cell is contained in nucleus, plastids, and mitochondria. Organelle transformation is getting a lot of attention nowadays because of its superior performance over the conventional and most commonly used nuclear transformation for obtaining transgenic lines. Absence of gene silencing, strong predictable transgene expression, and its application in molecular pharming, both in pharmaceutical and nutraceuticals, are some of many advantages. Other important benefits of utilizing this technology include the absence of transgene flow, as organelles are maternally inherited. This may increase the acceptability of organelle transformation technology in the development of transgenic crops in a wider scale all over the globe. As the need for crop productivity and therapeutic compounds increases, organelle transformation may be able to bridge the gap, thereby having a definite promise for the future.

  10. Organelle transformation.

    PubMed

    Bhattacharya, Anjanabha; Kumar, Anish; Desai, Nirali; Parikh, Seema

    2012-01-01

    The source of genetic information in a plant cell is contained in nucleus, plastids, and mitochondria. Organelle transformation is getting a lot of attention nowadays because of its superior performance over the conventional and most commonly used nuclear transformation for obtaining transgenic lines. Absence of gene silencing, strong predictable transgene expression, and its application in molecular pharming, both in pharmaceutical and nutraceuticals, are some of many advantages. Other important benefits of utilizing this technology include the absence of transgene flow, as organelles are maternally inherited. This may increase the acceptability of organelle transformation technology in the development of transgenic crops in a wider scale all over the globe. As the need for crop productivity and therapeutic compounds increases, organelle transformation may be able to bridge the gap, thereby having a definite promise for the future. PMID:22610643

  11. Droplet organelles?

    PubMed

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

    2016-08-01

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

  12. Droplet organelles?

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. 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.

  15. 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

  16. 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

  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. 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

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

    PubMed

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

    2015-11-01

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

  20. Organelle fission in eukaryotes.

    PubMed

    Osteryoung, K W

    2001-12-01

    The cellular machineries that power chloroplast and mitochondrial division in eukaryotes carry out the topologically challenging job of constricting and severing these double-membraned organelles. Consistent with their endosymbiotic origins, mitochondria in protists and chloroplasts in photosynthetic eukaryotes have evolved organelle-targeted forms of FtsZ, the prokaryotic ancestor of tubulin, as key components of their fission complexes. In fungi, animals and plants, mitochondria no longer utilize FtsZ for division, but several mitochondrial division proteins that localize to the outer membrane and intermembrane space, including two related to the filament-forming dynamins, have been identified in yeast and animals. Although the reactions that mediate organelle division are not yet understood, recent progress in uncovering the constituents of the organelle division machineries promises rapid advancement in our understanding of the biochemical mechanisms underlying the distinct but related processes of chloroplast and mitochondrial division in eukaryotes.

  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.

  2. Organelle morphogenesis by active membrane remodeling

    NASA Astrophysics Data System (ADS)

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

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

  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. PMID:25264261

  4. Cryptic organelles in parasitic protists and fungi.

    PubMed

    Williams, Bryony A P; Keeling, Patrick J

    2003-01-01

    A number of parasitic protists and fungi have adopted extremely specialised characteristics of morphology, biochemistry, and molecular biology, sometimes making it difficult to discern their evolutionary origins. One aspect of several parasitic groups that reflects this is their metabolic organelles, mitochondria and plastids. These organelles are derived from endosymbiosis with an alpha-proteobacterium and a cyanobacterium respectively, and are home to a variety of core metabolic processes. As parasites adapted, new demands, or perhaps a relaxation of demands, frequently led to significant changes in these organelles. At the extreme, the organelles are degenerated and transformed beyond recognition, and are referred to as "cryptic". Generally, there is no prior cytological evidence for a cryptic organelle, and its presence is only discovered through phylogenetic analysis of molecular relicts followed by their localisation to organelle-like structures. Since the organelles are derived from eubacteria, the genes for proteins and RNAs associated with them are generally easily recognisable, and since the metabolic activities retained in these organelles are prokaryotic, or at least very unusual, they often serve as an important target for therapeutics. Cryptic mitochondria are now known in several protist and fungal parasites. In some cases (e.g., Trichomonas), well characterised but evolutionarily enigmatic organelles called hydrogenosomes were shown to be derived from mitochondria. In other cases (e.g., Entamoeba and microsporidia), "amitochondriate" parasites have been shown to harbour a previously undetected mitochondrial organelle. Typically, little is known about the functions of these newly discovered organelles, but recent progress in several groups has revealed a number of potential functions. Cryptic plastids have now been found in a small number of parasites that were not previously suspected to have algal ancestors. One recent case is the discovery that

  5. Composition of the von Willebrand factor storage organelle (Weibel- Palade body) isolated from cultured human umbilical vein endothelial cells

    PubMed Central

    1987-01-01

    von Willebrand factor (VWF) is a large, adhesive glycoprotein that is biosynthesized and secreted by cultured endothelial cells (EC). Although these cells constitutively release VWF, they also contain a storage pool of this protein that can be rapidly mobilized. In this study, a dense organelle fraction was isolated from cultured umbilical vein endothelial cells by centrifugation on a self-generated Percoll gradient. Stimulation of EC by 4-phorbol 12-myristate 13-acetate (PMA) resulted in the disappearance of this organelle fraction and the synchronous loss of Weibel-Palade bodies as judged by immunoelectron microscopy. Electrophoretic and serologic analyses of biosynthetically labeled dense organelle fraction revealed that it is comprised almost exclusively of VWF and its cleaved pro sequence. These two polypeptides were similarly localized exclusively to Weibel-Palade bodies by ultrastructural immunocytochemistry. The identity of the dense organelle as the Weibel-Palade body was further established by direct morphological examination of the dense organelle fraction. The VWF derived from this organelle is distributed among unusually high molecular weight multimers composed of fully processed monomeric subunits and is rapidly and quantitatively secreted in unmodified form after PMA stimulation. These studies: establish that the Weibel-Palade body is the endothelial-specific storage organelle for regulated VWF secretion; demonstrate that in cultured EC, the VWF concentrated in secretory organelles is of unusually high molecular weight and that this material may be rapidly mobilized in unmodified form; imply that proteolytic processing of VWF involved in regulated secretion takes place after translocation to the secretory organelle; provide a basis for further studies of intracellular protein trafficking in EC. PMID:3494734

  6. Diverse Bacterial Microcompartment Organelles

    PubMed Central

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

    2014-01-01

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

  7. Scaling properties of cell and organelle size

    PubMed Central

    Marshall, Wallace F

    2010-01-01

    How size is controlled is a fundamental question in biology. In this review, we discuss the use of scaling relationships—for example, power-laws of the form y∝xα—to provide a framework for comparison and interpretation of size measurements. Such analysis can illustrate the biological and physical principles underlying observed trends, as has been proposed for the allometric dependence of metabolic rate or limb structure on organism mass. Techniques for measuring size at smaller length-scales continue to improve, leading to more data on the control of size in cells and organelles. Size scaling of these structures is expected to influence growth patterns, functional capacity and intracellular transport. Furthermore, organelles such as the nucleus, mitochondria and endoplasmic reticulum show widely varying morphologies that affect their scaling properties. We provide brief summaries of these issues for individual organelles, and conclude with a discussion on how to apply this concept to better understand the mechanisms of size control in the cellular environment. PMID:20885855

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

    PubMed

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

    2014-01-01

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

  9. 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.

  10. 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.

  11. Organelle positioning and cell polarity.

    PubMed

    Bornens, Michel

    2008-11-01

    In spite of conspicuous differences in their polarized architecture, swimming unicellular eukaryotes and migrating cells from metazoa display a conserved hierarchical interlocking of the main cellular compartments, in which the microtubule network has a dominant role. A microtubule array can organize the distribution of endomembranes owing to a cell-wide and polarized extension around a unique nucleus-associated structure. The nucleus-associated structure in animal cells contains a highly conserved organelle, the centriole or basal body. This organelle has a defined polarity that can be transmitted to the cell. Its conservative mode of duplication seems to be a core mechanism for the transmission of polarities through cell division.

  12. 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.

  13. Organelle biogenesis and interorganellar connections

    PubMed Central

    Daniele, Tiziana; Schiaffino, Maria Vittoria

    2014-01-01

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

  14. [Value of hemophagocytosis in the morphological examination of the bone marrow (author's transl)].

    PubMed

    Brugues, R M; Vives-Corrons, J L; Rozman, C; Jou, J M; Aguilar, J L; Cardellach, F; Urbano, A; Feliu, E

    1979-12-15

    The hemophagocytosis or the process of ingestion of blood cells by phagocytes and macrophages of the mononuclear phagocytic system (MFS), is a phenomenon that rarely could be observed in the morphological examination of the bone marrow aspirate. Occasionally it is present in certain pathologic conditions such as, malignant histiocitosis, autimmune hemolytic anemia, or some chronic inflammatory diseases. The capacity of ingestion is not an exlusive property of the phagocytes and macrophages of the MFS, so that different neoplastic cells can show it too. This study analyzes the presence of hemophagocytosis by neoplastic cells in a total of 552 bone marrow aspirates corresponding to a series of 130 patients with acute leukemia and 422 patients with diverse solid tumors. In the group of patients with acute leukemia, hemophagocytosis by blastic cells was observed in five cases with acute monocytic leukemia. In the other group with solid tumors, hemophagocytosis was present in three patients with oat-cell lung carcinomas and diffuse bone marrow metastases. The interest of the evaluation of hemophagocytosis by neoplastic cells in the morphological examination of the bone marrow is stressed, as well as it possible value in the cytological diagnosis of acute monocytic leukemia. However, in these circumstances a sdiffuse metastases by solid tumors should be always discarded, since their cytomorphological characteristics are in most cases superimposed to those of the leukemic bone marrow infiltrate.

  15. Fungal peroxisomes as biosynthetic organelles.

    PubMed

    Stehlik, Thorsten; Sandrock, Björn; Ast, Julia; Freitag, Johannes

    2014-12-01

    Peroxisomes are nearly ubiquitous single-membrane organelles harboring multiple metabolic pathways beside their prominent role in the β-oxidation of fatty acids. Here we review the diverse metabolic functions of peroxisomes in fungi. A variety of fungal metabolites are at least partially synthesized inside peroxisomes. These include the essential co-factor biotin but also different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters for example β-oxidation intermediates. In several ascomycetes a subtype of peroxisomes has been identified that is metabolically inactive but is required to plug the septal pores of wounded hyphae. Thus, peroxisomes are versatile organelles that can adapt their function to the life style of an organism. This remarkable variability suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive. Moreover, in fungi peroxisomes are non-essential under laboratory conditions making them attractive organelles for biotechnological approaches and the design of novel metabolic pathways in customized peroxisomes.

  16. 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.

  17. 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.

  18. 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

  19. 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

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

    PubMed

    Desfougères, Yann; Neumann, Heinz; Mayer, Andreas

    2016-07-15

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

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

    PubMed

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

    2016-01-01

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

  2. 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.

  3. 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.

  4. Laser Surgery: Organelles to Organs

    NASA Astrophysics Data System (ADS)

    Berns, Michael W. D.

    1998-03-01

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

  5. Endosymbiotic theory for organelle origins.

    PubMed

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

    2014-12-01

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

  6. 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

  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.

  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. 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.

  11. Organelle remodeling at membrane contact sites.

    PubMed

    Henne, W Mike

    2016-10-01

    Cellular organelles must execute sophisticated biological processes to persist, and often communicate with one another to exchange metabolites and information. Recent studies suggest inter-organelle membrane contact sites (MCSs) are hubs for this cellular cross-talk. MCSs also govern membrane remodeling, thus controlling aspects of organelle shape, identity, and function. Here, we summarize three emerging phenomena that MCSs appear to govern: 1) organelle identity via the non-vesicular exchange of lipids, 2) mitochondrial shape and division, and 3) endosomal migration in response to sterol trafficking. We also discuss the role for ER-endolysosomal contact sites in cholesterol metabolism, and the potential biomedical importance this holds. Indeed, the emerging field inter-organellar cross-talk promises substantial advances in the fields of lipid metabolism and cell signaling.

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

    DOE PAGES

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

    2015-12-30

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

  13. 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.

  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. Optogenetic control of organelle transport and positioning.

    PubMed

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

    2015-02-01

    Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. 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 rat hippocampal 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 enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.

  16. 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.

  17. 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

  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. 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.

  20. Morphological and biochemical examination of Cosmos 1887 rat heart tissue: Part I--Ultrastructure.

    PubMed

    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 (1). 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 (2-4).

  1. 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.

  2. 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.

  3. 4D Confocal Imaging of Yeast Organelles.

    PubMed

    Day, Kasey J; Papanikou, Effrosyni; Glick, Benjamin S

    2016-01-01

    Yeast cells are well suited to visualizing organelles by 4D confocal microscopy. Typically, one or more cellular compartments are labeled with a fluorescent protein or dye, and a stack of confocal sections spanning the entire cell volume is captured every few seconds. Under appropriate conditions, organelle dynamics can be observed for many minutes with only limited photobleaching. Images are captured at a relatively low signal-to-noise ratio and are subsequently processed to generate movies that can be analyzed and quantified. Here, we describe methods for acquiring and processing 4D data using conventional scanning confocal microscopy. PMID:27631997

  4. [Results of the morphological examination of stomach in the different terms after stitching of a perforation duodenal ulcer].

    PubMed

    Tumanidze, G Z; Khardzeishvili, O M; Chkhikvadze, T F; Khazaradze, D V

    2005-01-01

    Analyses of 184 cases of morphological examination of resected part of stomach in the different terms after stitching of a perforative duodenal ulcer is presented. The authors confirm, that ulcerative disease of duodenum and it's various complications including perforation is associated with antrum gastritis in all cases, which has superficial character. The authors are confirming that a gastric metaplasia of a mucosa of duodenum assists to the helicobacterial colonization, which represents one of the main factors of an ulcer formation along with a fast transition of acid gastric contents into the duodenum. The authors consider the gastric metaplasia as preulcerative pathological condition of duodenum.

  5. Morphological and cellular examinations of experimentally induced malocclusion in mice mandibular condyle.

    PubMed

    Jung, Jae-Kwang; Sohn, Wern-Joo; Lee, Youngkyun; Bae, Yong Chul; Choi, Jae-Kap; Kim, Jae-Young

    2014-02-01

    Occlusal alignment is known clinically to have a widespread influence on the stomatognathic system, including the temporomandibular joint and masticatory muscles. However, while occlusion is still an important determinant of most dental treatments, the exact effect of occlusal alignment is unclear because of a lack of conclusive scientific evidence. In this study, a malocclusion model system is used to examine the cellular and histologic alterations in the contralateral condyle of mice after a malocclusion was induced by a build-up of resin on the left maxillary molars. A significant decrease in the thickness of the condylar cartilage was found in the 1-week experimental group, together with increased apoptosis and decreased proliferation in the condylar head, which included cartilage and subchondral bone. Additionally, the number of TRAP-positive osteoclasts and MPO- and F4/80-positive inflammatory cells in the subchondral bone were significantly higher in the 1-week experimental group. Unbalanced malocclusion caused increased bone remodeling, as evidenced by increased osteoclastic activity and inflammatory responses (macrophages and neutrophils, respectively). However, these alterations in the 1-week experimental group were subsequently attenuated and restored almost to the baseline at 3 weeks after the induction of the malocclusion.

  6. Pareto optimality in organelle energy metabolism analysis.

    PubMed

    Angione, Claudio; Carapezza, Giovanni; Costanza, Jole; Lió, Pietro; Nicosia, Giuseppe

    2013-01-01

    In low and high eukaryotes, energy is collected or transformed in compartments, the organelles. The rich variety of size, characteristics, and density of the organelles makes it difficult to build a general picture. In this paper, we make use of the Pareto-front analysis to investigate the optimization of energy metabolism in mitochondria and chloroplasts. Using the Pareto optimality principle, we compare models of organelle metabolism on the basis of single- and multiobjective optimization, approximation techniques (the Bayesian Automatic Relevance Determination), robustness, and pathway sensitivity analysis. Finally, we report the first analysis of the metabolic model for the hydrogenosome of Trichomonas vaginalis, which is found in several protozoan parasites. Our analysis has shown the importance of the Pareto optimality for such comparison and for insights into the evolution of the metabolism from cytoplasmic to organelle bound, involving a model order reduction. We report that Pareto fronts represent an asymptotic analysis useful to describe the metabolism of an organism aimed at maximizing concurrently two or more metabolite concentrations.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. Morphologic examination of mesenchymal cells in healing wounds of normal and tight skin mice.

    PubMed Central

    Hembry, R. M.; Bernanke, D. H.; Hayashi, K.; Trelstad, R. L.; Ehrlich, H. P.

    1986-01-01

    The healing process of an open wound as effected by wound contraction is complete by 3 weeks in the normal mouse. In contrast, its onset is delayed by 3 weeks and complete healing requires 6 weeks in the tight skin mouse (TSM), a mutant mouse strain with the autosomal dominant gene for tight skin. Possible mechanisms for this delay were evaluated. The frequency and distribution of myofibroblasts were studied during the 3-week delay in wound contraction by actin staining and electron microscopy. It was determined, by electron microscopy and phalloidin staining, that myofibroblasts were found in high density in noncontracting TSM wounds. Electron microscopy showed, however, that these myofibroblasts were surrounded by a pericellular matrix that separated their surface from adjacent collagen fibers. No pericellular matrix was found around cells in granulation tissue of normal mice. At 3 weeks, as TSM wounds began to contract, the number and intensity of cells stained by phalloidin in this tissue was less than that seen earlier. The pericellular matrix was fragmented at this time, and cell surface and collagen fiber associations were apparent. Finally, at 5 weeks, when wound contraction was well developed in the TSM, only a small area in the center of the healing wound beneath the epidermis contained phalloidin-positive myofibroblasts. Electron-microscopic examination of the residual granulation tissue at this time revealed the complete absence of the pericellular matrix. It is postulated that during the 3-week delay in wound closure, the presence of a localized pericellular matrix prevents the interaction between cells and collagen fibers necessary for the reorganization of collagen. It is also thought that the tightly adherent uninjured skin surrounding the healing wound may cause delayed wound closure. There was no evidence that the absence of myofibroblasts is responsible for delayed wound contraction. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID

  10. 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

  11. 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.

  12. 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

  13. 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

  14. The Organelle Genome Database Project (GOBASE).

    PubMed Central

    Korab-Laskowska, M; Rioux, P; Brossard, N; Littlejohn, T G; Gray, M W; Lang, B F; Burger, G

    1998-01-01

    The taxonomically broad organelle genome database (GOBASE) organizes and integrates diverse data related to organelles (mitochondria and chloroplasts). The current version of GOBASE focuses on the mitochondrial subset of data and contains molecular sequences, RNA secondary structures and genetic maps, as well as taxonomic information for all eukaryotic species represented. The database has been designed so that complex biological queries, especially ones posed in a comparative genomics context, are supported. GOBASE has been implemented as a relational database with a web-based user interface (http://megasun.bch.umontreal.ca/gobase/gobas e.html ). Custom software tools have been written in house to assist in the population of the database, data validation, nomenclature standardization and front-end design. The database is fully operational and publicly accessible via the World Wide Web, allowing interactive browsing, sophisticated searching and easy downloading of data. PMID:9399818

  15. A Eukaryote without a Mitochondrial Organelle.

    PubMed

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

    2016-05-23

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

  16. 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

  17. A Eukaryote without a Mitochondrial Organelle.

    PubMed

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

    2016-05-23

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

  18. 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

  19. Mitochondrial fission: rings around the organelle

    PubMed Central

    Pon, Liza A.

    2014-01-01

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

  20. Ciliary Extracellular Vesicles: Txt Msg Organelles.

    PubMed

    Wang, Juan; Barr, Maureen M

    2016-04-01

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

  1. How cells know the size of their organelles**

    PubMed Central

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

    2013-01-01

    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 both under 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. PMID:22955827

  2. Localization of proteins and organelles using fluorescence microscopy.

    PubMed

    Farre, Jean-Claude; Shirahama-Noda, Kanae; Zhang, Lan; Booher, Keith; Subramani, Suresh

    2007-01-01

    This chapter describes the different methods used for localization of proteins and organelles in Pichia pastoris. A series of plasmids and a modified immunofluorescence protocol for localization and co-localization of proteins and organelles are described. Also included are protocols for the labeling of different subcellular organelles with vital stains.

  3. 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.

  4. 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.

  5. 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…

  6. 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

  7. 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.

  8. Mitochondrion-derived organelles in protists and fungi.

    PubMed

    van der Giezen, Mark; Tovar, Jorge; Clark, C Graham

    2005-01-01

    The mitochondrion is generally considered to be a defining feature of eukaryotic cells, yet most anaerobic eukaryotes lack this organelle. Many of these were previously thought to derive from eukaryotes that diverged prior to acquisition of the organelle through endosymbiosis. It is now known that all extant eukaryotes are descended from an ancestor that had a mitochondrion and that in anaerobic eukaryotes the organelle has been modified into either hydrogenosomes, which continue to generate energy for the host cell, or mitosomes, which do not. These organelles have each arisen independently several times. Recent evidence suggests a shared derived characteristic that may be responsible for the retention of the organelles in the absence of the better-known mitochondrial functions--iron-sulfur cluster assembly. This review explores the events leading to this new understanding of mitochondrion-derived organelles in amitochondriate eukaryotes, the current state of our knowledge, and future areas for investigation.

  9. The Evolution of Per-cell Organelle Number.

    PubMed

    Cole, Logan W

    2016-01-01

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

  10. 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.

  11. 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.

  12. 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. PMID:26967755

  13. The protein shells of bacterial microcompartment organelles

    PubMed Central

    Yeates, Todd O.; Thompson, Michael C.; Bobik, Thomas A.

    2011-01-01

    SUMMARY Details are emerging on the structure and function of a remarkable class of capsid-like protein assemblies that serve as simple metabolic organelles in many bacteria. These bacterial microcompartments consist of a few thousand shell proteins, which encapsulate two or more sequentially acting enzymes in order to enhance or sequester certain metabolic pathways, particularly those involving toxic or volatile intermediates. Genomic data indicate that bacterial microcompartment shell proteins are present in a wide range of bacterial species, where they encapsulate varied reactions. Crystal structures of numerous shell proteins from distinct types of microcompartments have provided keys for understanding how the shells are assembled and how they conduct molecular transport into and out of microcompartments. The structural data emphasize a high level of mechanistic sophistication in the protein shell, and point the way for further studies on this fascinating but poorly appreciated class of subcellular structures. PMID:21315581

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

    PubMed

    Weisman, Lois S

    2006-04-01

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

  15. 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.

  16. 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.

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

    PubMed

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

    2015-11-01

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

  18. 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

  19. 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

  20. The partitioning of cytoplasmic organelles at cell division.

    PubMed

    Birky, C W

    1983-01-01

    When an organism has only one or two mitochondria or chloroplasts per cell, it is probable that their partitioning is always stringently controlled so that each daughter cell always receives half the organelles in the parent cell. When there are more copies of an organelle, the available data suggest that partitioning is stochastic but far from random, with a strong tendency toward equality. The molecular mechanisms that promote equal partitioning are not known in any case, but the great variety of organelle behavior suggests that many different mechanisms are involved in different organisms. As Wilson (1925) pointed out, the precision of partitioning of cytoplasmic organelles rarely if ever equals that of mitosis, but it is still an expression of selection for mechanisms that will ensure the hereditary continuity of the organelles. How cells compensate for unequal partitioning by controlling organelle replication is known for only one case. But when one considers that Tetrahymena and Paramecium use different methods to compensate for unequal partitioning of macronuclear DNA, it would not be surprising if organisms use a variety of different compensating replication modes for organelles as well. What is surprising is that so little attention has been paid to these problems. Nothing could be simpler than counting organelles in dividing cells, but this has been done on a large scale in only two systems. Quantitative techniques in cell biology have been developed to the point where such studies could be done even on cells that have too many organelles for direct counting. Molecular mechanisms of partitioning have scarcely been touched on. Much more has been done on the role of the cytoskeleton in determining cell shape, and some observations have been made on its role in positioning organelles in interphase cells, but these kinds of studies have not been extended to dividing cells. Some experiments and observations have been made on the role of microtubules and

  1. 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.

  2. 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

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

    PubMed

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

    2016-08-01

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

  4. [Analysis of sperm morphology: yes or no?].

    PubMed

    Lu, Jin-Chun

    2013-04-01

    The analysis of sperm morphology can be used to evaluate sperm fertilizing ability and spontaneous conception status, and especially the overall analysis of the sperm head, neck and tail, along with the patient's living habits, occupation and clinical manifestations, may contribute to the primary diagnosis of the patients potentia generandi. It can also be employed to assess the effects of the treatment of semen samples. Although oocyte fertilization can be achieved by the technologies of intracytoplasmic sperm injection (ICSI), motile sperm organelle morphology examination (MSOME) and intracytoplasmic morphologically selected sperm injection (IMSI) regardless of sperm morphology and / or motility, which may somewhat weaken the clinical application of sperm morphology analysis, the standardized procedure and the practice of quality control for the analysis of sperm morphology can significantly improve the accuracy of its results and largely promote its clinical application. Therefore, it is of positive necessity as well as clinical application value to perform sperm morphology analysis in andrology laboratories, reproductive centers, sperm banks and the department of laboratory medicine.

  5. A recording backward scattering meter and camera system for examination of the distribution and morphology of macroaggregates

    NASA Astrophysics Data System (ADS)

    Johnson, B. D.; Wangersky, P. J.

    1985-09-01

    Amorphous macroaggregates may be important as conditioned microhabitats and in addition may provide the major flux of particulate materials to deeper waters. Because of an unknown spatial variability, adequate treatment of the distribution of such aggregates requires continuous sampling, and in a mode that avoids disruption of fragile flocculent materials. We have developed a recording backward scattering meter and camera system for investigation of the morphology and distribution of amorphous macroaggregates. This system provides a continuous record of the distribution of light scatterers, and also permits identification and absolute size determination of larger scatterers such as macroaggregates. Activation of the camera through feedback allows a relatively high magnification (× 1/4) with attendant narrow depth of field. The result is resolution of subject detail of nearly 50 μm and reduction of problems associated with image overlap and errors in sizing due to position in the near or far zone of focus.

  6. 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.

  7. Membrane elongation factors in organelle maintenance: the case of peroxisome proliferation

    PubMed Central

    Koch, Johannes; Brocard, Cécile

    2011-01-01

    Separation of metabolic pathways in organelles is critical for eukaryotic life. Accordingly, the number, morphology and function of organelles have to be maintained through processes linked with membrane remodeling events. Despite their acknowledged significance and intense study many questions remain about the molecular mechanisms by which organellar membranes proliferate. Here, using the example of peroxisome proliferation, we give an overview of how proteins elongate membranes. Subsequent membrane fission is achieved by dynamin-related proteins shared with mitochondria. We discuss basic criteria that membranes have to fulfill for these fission factors to complete the scission. Because peroxisome elongation is always associated with unequal distribution of matrix and membrane proteins, we propose peroxisomal division to be non-stochastic and asymmetric. We further show that these organelles need not be functional to carry on membrane elongation and present the most recent findings concerning members of the Pex11 protein family as membrane elongation factors. These factors, beside known proteins such as BAR-domain proteins, represent another family of proteins containing an amphipathic α-helix with membrane bending activity. PMID:21984887

  8. 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.

  9. 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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

    Maréchal, Alexandre; Brisson, Normand

    2010-04-01

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

  12. 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…

  13. 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

  14. 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. PMID:26633540

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. Lam6 Regulates the Extent of Contacts between Organelles.

    PubMed

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

    2015-07-01

    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.

  1. 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

  2. Adenine nucleotide transporters in organelles: novel genes and functions.

    PubMed

    Traba, Javier; Satrústegui, Jorgina; del Arco, Araceli

    2011-04-01

    In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.

  3. 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

  4. 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

  5. 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.

  6. 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. PMID:17402976

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

    PubMed Central

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

    2015-01-01

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

  8. Osmotic regulation of Rab-mediated organelle docking.

    PubMed

    Brett, Christopher L; Merz, Alexey J

    2008-07-22

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

  9. 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.

  10. 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.

  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. Single-organelle tracking by two-photon conversion

    NASA Astrophysics Data System (ADS)

    Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Fukui, Kiichi; Shin-Ichi Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Isobe, Keisuke; Itoh, Kazuyoshi

    2007-03-01

    Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

  13. [Eukaryotes devoid of the most important cellular organelles (flagella, Golgi apparatus, mitochondria) and the main task of organellology].

    PubMed

    Seravin, L N

    1992-01-01

    Comparative evidence on the lack of three important organelles (flagella, Golgi-complex, mitochondria) in cells and organisms at the cellular level of organization has been summarized for all the four eukaryotic kingdoms--Protista, Fungi, Plantae and Animalia (Metazoa). It is established that in the course of evolution these organelles may undergo the total reduction. There is no cellular organelle to be regarded as universal, indispensable. There are only three main obligatory cell components--the plasmalemma, nucleus and cytoplasm (with applied cytoskeleton, cytomembranes and ribosomes). The reduction of flagella (cilia) is occurring in different taxa independent of the transition of protists from the flagellate type of locomotion to the amoeboid, gliding of metabolizing ones, and in the number of metazoan cells. The members of Protista and Fungi, which line in microaerobic or anaerobic conditions, nearly inevitably lose their mitochondria. The tendency to lose Golgi-complex is demonstrated in protists with parasitic mode of life, especially in combination with anaerobiosis. There is so far no satisfied morphological criterium that could say with certainty whether the lacking of flagella, Golgi complex or mitochondria in the low eukaryotes may be primary or secondary (as the result of reduction). Data on the composition, structure and RNA nucleotide sequences cannot be either the straight evidence. A comparative analysis of these data shows that the ribosomes of the primary eukaryotes were, presumably, of a prokaryotic type. Their eukaryotization was carried out for a long time during the evolution of the low eukaryotes (Protista and Fungi), probably, independently in different phylogenetic lines. It is unknown at what steps and in what main phylogenetic lines the three above mentioned organelles may have appeared. It is proposed to single out a special division of cytology--organellology (organoidology)--as an individual science whose main purpose may be

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  15. An unusual organelle in Cryptococcus neoformans links luminal pH and capsule biosynthesis.

    PubMed

    Yoneda, Aki; Doering, Tamara L

    2009-09-01

    Cryptococcus neoformans is a basidiomycete that causes deadly infections in the immunocompromised. We previously generated a secretion mutant in this fungus by introducing a mutation in the SAV1 gene, which encodes a homolog of the Sec4/Rab8 subfamily GTPases. Under restrictive conditions there are two notable morphological changes in the sav1 mutant: accumulation of post-Golgi vesicles and the appearance of an unusual organelle, which we term the sav1 body (SB). The SB is an electron-transparent structure 0.2-1microm in diameter, with vesicles or other membranous structures associated with the perimeter. Surprisingly, the SB was heavily labeled with anti-glucuronoxylomannan (GXM) antibodies, suggesting that it contains a secreted capsule component, GXM. A structure similar to the SB, also labeled by anti-GXM antibodies, was induced in wild type cells treated with the vacuolar-ATPase inhibitor, bafilomycin A(1). Bafilomycin A(1) and other agents that increase intraluminal pH also inhibited capsule polysaccharide shedding and capsule growth. These studies highlight an unusual organelle observed in C. neoformans with a potential role in polysaccharide synthesis, and a link between luminal pH and GXM biosynthesis.

  16. 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.

  17. Energy dispersive X-ray analyses of organelles of NaCI-treated maize root cells

    NASA Astrophysics Data System (ADS)

    Stelzer, Ralf

    1984-04-01

    NaCl sensitive plants of Zea mays cv. ADOUR were grown in nutrient solutions with or without NaCl. Frozen, hydrated root-tip tissues were investigated by means of an ETEC scanning electron microscope fitted with a KEVEX energy dispersive X-ray analyser. Morphological details of the gently etched but non-coated surface of the cross fractured specimen were easy to identify and to analyse using an electron beam with a low intensity at 10 kV. X-ray data obtained from cell compartments and organelles as nuclei, nucleoli and mitochondria within individual cells establish typical X-ray spectra. Comparisons of these spectra support the hypothesis that Na + ions are predominantly localized in vacuoles and also to a lesser extent in the cytoplasm, e.g. in small vesicles, but not in other cell organelles. Furthermore the analysed cell compartments show differences in the distribution of Mg, P, S, Cl, K and Ca effected by the addition of NaCl to the growth medium. The X-ray data are discussed in relation to the physiological meaning of a NaCl induced redistribution of elements within individual maize root cells.

  18. 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.

  19. Impact of intracytoplasmic morphologically selected sperm injection on assisted reproduction outcome: a review.

    PubMed

    Nadalini, Marco; Tarozzi, Nicoletta; Distratis, Vincenzo; Scaravelli, Giulia; Borini, Andrea

    2009-01-01

    To date, several publications have focused their attention on a new method for observing spermatozoa called 'motile sperm organelle morphology examination' (MSOME), which enables the evaluation of the fine nuclear morphology of motile spermatozoa in real time at high magnification (>x6000). As a consequence, a new microinjection procedure called intracytoplasmic morphologically selected sperm injection (IMSI) has been developed. The aim of the present work is therefore to evaluate the efficacy of the IMSI technique in the light of the current literature, focusing attention on the potential clinical application of the selection of strictly morphologically normal spermatozoa in patients undergoing conventional intracytoplasmic sperm injection treatments. In addition, a brief analysis of preliminary data regarding the relationship between IMSI and assisted reproduction treatment outcome is presented.

  20. Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution

    PubMed Central

    Yeates, Todd O.; Crowley, Christopher S.; Tanaka, Shiho

    2012-01-01

    Some bacteria contain organelles or microcompartments consisting of a large virion-like protein shell encapsulating sequentially acting enzymes. These organized microcompartments serve to enhance or protect key metabolic pathways inside the cell. The variety of bacterial microcompartments provide diverse metabolic functions, ranging from CO2 fixation to the degradation of small organic molecules. Yet they share an evolutionarily related shell, which is defined by a conserved protein domain that is widely distributed across the bacterial kingdom. Structural studies on a number of these bacterial microcompartment shell proteins are illuminating the architecture of the shell and highlighting its critical role in controlling molecular transport into and out of microcompartments. Current structural, evolutionary, and mechanistic ideas are discussed, along with genomic studies for exploring the function and diversity of this family of bacterial organelles. PMID:20192762

  1. The role of microtubule movement in bidirectional organelle transport.

    PubMed

    Kulic, Igor M; Brown, André E X; Kim, Hwajin; Kural, Comert; Blehm, Benjamin; Selvin, Paul R; Nelson, Philip C; Gelfand, Vladimir I

    2008-07-22

    We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFP-tagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks.

  2. 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.

  3. Analysis of two genomes from the mitochondrion-like organelle of the intestinal parasite Blastocystis: complete sequences, gene content, and genome organization.

    PubMed

    Pérez-Brocal, Vicente; Clark, C Graham

    2008-11-01

    Acquisition of mitochondria by the ancestor of all living eukaryotes represented a crucial milestone in the evolution of the eukaryotic cell. Nevertheless, a number of anaerobic unicellular eukaryotes have secondarily discarded certain mitochondrial features, leading to modified organelles such as hydrogenosomes and mitosomes via degenerative evolution. These mitochondrion-derived organelles have lost many of the typical characteristics of aerobic mitochondria, including certain metabolic pathways, morphological traits, and, in most cases, the organellar genome. So far, the evolutionary pathway leading from aerobic mitochondria to anaerobic degenerate organelles has remained unclear due to the lack of examples representing intermediate stages. The human parasitic stramenopile Blastocystis is a rare example of an anaerobic eukaryote with organelles that have retained some mitochondrial characteristics, including a genome, whereas they lack others, such as cytochromes. Here we report the sequence and comparative analysis of the organellar genome from two different Blastocystis isolates as well as a comparison to other genomes from stramenopile mitochondria. Analysis of the characteristics displayed by the unique Blastocystis organelle genome gives us an insight into the initial evolutionary steps that may have led from mitochondria to hydrogenosomes and mitosomes. PMID:18765437

  4. Zoology meets Botany: establishing intracellular organelles by endosymbiosis.

    PubMed

    Prechtl, J; Maier, U G

    2001-01-01

    One of the most citated characteristics of eukaryotic cells are mitochondria and in the case of phototrophic cells, the plastids. These organelles are of eubacterial origin and contain a remnant genome. Here, we present hypotheses concerning the origin of the first mitochondrium-harboring cell and show the evolution of primary, secondary and tertiary plastids. Furthermore we discuss models explaining why plastids have to maintain their own genome.

  5. 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.

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

    DOE PAGES

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

    2016-02-25

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

  7. Some reflections on intracytoplasmic morphologically selected sperm injection.

    PubMed

    Ebner, Thomas; Shebl, Omar; Oppelt, Peter; Mayer, Richard Bernhard

    2014-07-01

    Although intracytoplasmic sperm injection (ICSI) allows proper fertilization in most cases of male sub fertility, it is one of the most unphysiological techniques in assisted reproductive technologies (ART). Thus, over the last decade, researchers have tried to improve sperm observation with higher-resolution microscopy techniques such as the intracytoplasmic morphologically selected sperm injection (IMSI) technique. In order to identify literatures for this review, the PubMed database was searched from 2000 onwards using the terms IMSI, motile sperm organelle morphology examination (MSOME) and sperm vacuole. Approximately 10 years after the introduction of the MSOME and IMSI procedures, several questions related to the prevalence, origin, location, and clinical consequences of sperm vacuoles have not yet been clarified. It seems that IMSI as a routine application is not state of the art and the only confirmed indications for IMSI are recurrent implantation failure following ICSI and severe male factor.

  8. Some Reflections on Intracytoplasmic Morphologically Selected Sperm Injection

    PubMed Central

    Ebner, Thomas; Shebl, Omar; Oppelt, Peter; Mayer, Richard Bernhard

    2014-01-01

    Although intracytoplasmic sperm injection (ICSI) allows proper fertilization in most cases of male sub fertility, it is one of the most unphysiological techniques in assisted reproductive technologies (ART). Thus, over the last decade, researchers have tried to improve sperm observation with higher-resolution microscopy techniques such as the intracytoplasmic morphologically selected sperm injection (IMSI) technique. In order to identify literatures for this review, the PubMed database was searched from 2000 onwards using the terms IMSI, motile sperm organelle morphology examination (MSOME) and sperm vacuole. Approximately 10 years after the introduction of the MSOME and IMSI procedures, several questions related to the prevalence, origin, location, and clinical consequences of sperm vacuoles have not yet been clarified. It seems that IMSI as a routine application is not state of the art and the only confirmed indications for IMSI are recurrent implantation failure following ICSI and severe male factor. PMID:25083173

  9. 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.

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

    PubMed

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

    2008-04-01

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

  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

    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. PMID:25750213

  13. 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

  14. 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.

  15. Guanine nucleotide exchange factors (GEFs) have a critical but not exclusive role in organelle localization of Rab GTPases.

    PubMed

    Cabrera, Margarita; Ungermann, Christian

    2013-10-01

    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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed 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

  18. 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.

  19. Staying in touch: the molecular era of organelle contact sites.

    PubMed

    Elbaz, Yael; Schuldiner, Maya

    2011-11-01

    Membrane contact sites (MCS) are close appositions between two organelles that facilitate both signaling and the passage of ions and lipids from one cellular compartment to another. Despite the fact that MCS have been observed for over 50 years now, we still know very little about the molecular machinery required to create them or their structure, function and regulation. In this review, we focus on the three best-characterized contact sites to date: the nucleus-vacuole junction and mitochondria-ER and plasma membrane-ER contact sites. In addition, we discuss principles arising from recent research and highlight several unanswered questions in the field.

  20. 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

  1. 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

  2. Reactivation of organelle movements along the cytoskeletal framework of a giant freshwater ameba

    PubMed Central

    1986-01-01

    The peripheral feeding network of the giant freshwater ameba Reticulomyxa can be easily and rapidly lysed to produce an extensive, stable, and completely exposed cytoskeletal framework of colinear microtubules and microfilaments. Most of the organelles that remain attached to this framework resume rapid saltatory movements at rates of up to 20 micron/s if ATP is added. This lysed model system is also capable of other forms of motility, namely an active splaying of microtubule bundles and bulk streaming. Reactivation does not occur with other nucleoside triphosphates, requires Mg ions, is insensitive to even high concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine, is sensitive to vanadate only at concentrations of approximately 100 microM, and is inhibited by N-ethylmaleimide at concentrations greater than 100 microM. The physiology of this reactivation suggests an organelle transport motor distinct from cytoplasmic dynein and possibly the recently described kinesin. This system can serve as a model for elucidating the mechanisms of intracellular transport and, in addition, provides a unique opportunity to examine associations between microtubules and microfilaments. PMID:3733883

  3. 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.

  4. 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.

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

    PubMed

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

    2016-05-24

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

  6. 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

  7. 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

  8. Organelle pathology in metabolic neuromuscular disease: an overview.

    PubMed Central

    Becker, L E

    1990-01-01

    The spectrum of metabolic neuromuscular disorders is wide. Most inherited metabolic diseases are related to enzyme defects within lysosomes but recent advances emphasize abnormalities of mitochondria, peroxisomes and intermediate filaments. In this overview, organelle pathology is described in the context of both the clinical manifestations and the biochemical and/or molecular aspects of the disease. Among the many clinical presentations of mitochondrial disorders three emerge as distinctive entities: mitochondrial encephalopathy with lactic acidosis and stroke-like symptoms, mitochondrial encephalopathy with ragged-red fibers, and Kearns-Sayre syndrome. Peroxisomal disorders are associated with numerous biochemical defects, the most frequent of which are Zellweger's syndrome, neonatal adrenoleukodystrophy, and infantile Refsum's disease. Disorders of cytoskeletal proteins are associated with distinctive pathological accumulation of intermediate filaments but are without confirmed evidence of a biochemical defect. Understanding the role that organelle pathology plays in the pathogenesis of cellular disturbance or demise is essential to the elucidation of the pathogenesis of metabolic disorders. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10. PMID:2407327

  9. Autophagy facilitates organelle clearance during differentiation of human erythroblasts

    PubMed Central

    Betin, Virginie M.S.; Singleton, Belinda K.; Parsons, Stephen F.; Anstee, David J.; Lane, Jon D.

    2013-01-01

    Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4BC74A; ATG4DC144A) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4BC74A-expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts. PMID:23508006

  10. 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

  11. Kinesin-mediated organelle translocation revealed by specific cellular manipulations

    PubMed Central

    1994-01-01

    The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'- dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi- associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles. PMID:7962067

  12. 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

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

    PubMed

    Gordon, Natalie K; Gordon, Richard

    2016-01-01

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

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

    PubMed

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

    2016-05-10

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

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

    PubMed

    Melo, Rossana C N; Weller, Peter F

    2014-11-01

    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.

  16. 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.

  17. 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…

  18. 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.

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

    PubMed

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

    2016-01-01

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

  20. 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.

  1. A structural role for lipids in organelle shaping.

    PubMed

    Wang, Alan S; Kundu, Aupola; Fong, Burr; Fitzgerald, Julie; Larijani, Banafshé; Poccia, Dominic

    2013-08-01

    The importance of proteins in shaping the membranes that define the perimeters of organelles is well documented. By forming cross-links, motors, or scaffolds or by inserting into membranes, proteins can harness energy to deform membranes, particularly when high degrees of curvature are necessitated-as in small membrane vesicles, tubules of the endoplasmic reticulum, the edges of endoplasmic reticulum sheets or Golgi apparatus cisternae, and membrane fusion intermediates (stalks). Here we propose that membrane lipids displaying negative curvature act in concert with membrane proteins to contribute to the alteration and maintenance of bending in biological membranes. We emphasize recent data from studies of sea urchin eggs and embryos and suggest how novel approaches can lead to future directions for investigating the roles of such lipids in vivo. PMID:23995745

  2. Protein-based organelles in bacteria: carboxysomes and related microcompartments.

    PubMed

    Yeates, Todd O; Kerfeld, Cheryl A; Heinhorst, Sabine; Cannon, Gordon C; Shively, Jessup M

    2008-09-01

    Many bacteria contain intracellular microcompartments with outer shells that are composed of thousands of protein subunits and interiors that are filled with functionally related enzymes. These microcompartments serve as organelles by sequestering specific metabolic pathways in bacterial cells. The carboxysome, a prototypical bacterial microcompartment that is found in cyanobacteria and some chemoautotrophs, encapsulates ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase, and thereby enhances carbon fixation by elevating the levels of CO2 in the vicinity of RuBisCO. Evolutionarily related, but functionally distinct, microcompartments are present in diverse bacteria. Although bacterial microcompartments were first observed more than 40 years ago, a detailed understanding of how they function is only now beginning to emerge.

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

    PubMed Central

    2013-01-01

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

  4. Mitochondria as signaling organelles in the vascular endothelium

    PubMed Central

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

    2006-01-01

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

  5. 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.

  6. Bridging the gap: Membrane contact sites in signaling, metabolism, and organelle dynamics

    PubMed Central

    2014-01-01

    Regions of close apposition between two organelles, often referred to as membrane contact sites (MCSs), mostly form between the endoplasmic reticulum and a second organelle, although contacts between mitochondria and other organelles have also begun to be characterized. Although these contact sites have been noted since cells first began to be visualized with electron microscopy, the functions of most of these domains long remained unclear. The last few years have witnessed a dramatic increase in our understanding of MCSs, revealing the critical roles they play in intracellular signaling, metabolism, the trafficking of metabolites, and organelle inheritance, division, and transport. PMID:24958771

  7. 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

  8. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. 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

  11. 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

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

    PubMed

    Drage, Harriet B; Daley, Allison C

    2016-10-01

    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

  13. 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

  14. 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.

  15. Examination of some morphologically unusual cultures of Phytophthora species using a mitochondrial DNA miniprep technique and a standardised sporangium caducity assessment.

    PubMed

    Hall, G S

    Using the mitochondrial DNA miniprep technique, the identity of sixteen morphologically unusual cultures allocated to Phytophthora nicotianae, Phytophthora mexicana or Phytophthora porri was determined by comparison with a library of mtDNA band patterns obtained from reference cultures. Seven cultures were identified as Phytophthora nicotianae (including those assigned to Phytophthora mexicana and Phytophthora porri), six as strains of Phytophthora palmivora with small, ovoid, weakly caducous sporangia, and one as Phytophthora citrophthora. Some cultures of P. nicotianae had a low percentage of caducous sporangia. Percentage sporangium caducity, but not sporangium L:B ratio, is considered a useful taxonomic criterion for separating species morphologically similar to Phytophthora nicotianae. One culture from tobacco in New Zealand had a highly unusual morphology and a unique DNA band pattern, but was not identifiable. One culture from Acacia mearnsii in South Africa had a unique DNA band pattern which was identical to that of an isolate from Annona squamosa from Australia previously identified as Phytophthora palmivora, the precise identity of which is still unclear. The identity of most isolates from diseased durian was found to be Phytophthora palmivora, confirming its role as the main pathogen, but P. nicotianae was also identified from this host.

  16. 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.

  17. Characterization of photodynamic therapy responses elicited in A431 cells containing intracellular organelle-localized photofrin.

    PubMed

    Hsieh, Ya-Ju; Yu, Jau-Song; Lyu, Ping-Chiang

    2010-11-01

    Photodynamic therapy (PDT), a photochemotherapeutic regimen used to treat several diseases, including cancer, exerts its effects mainly through induction of cell death. Using human epidermoid carcinoma A431 cells as a model, we previously showed that distinct cell death types could be triggered by protocols that selectively delivered Photofrin (a clinically approved photosensitizer) to different subcellular sites (Hsieh et al. [2003] J Cell Physiol 194: 363-375]. Here, the responses elicited by PDT in A431 cells containing intracellular organelle-localized Photofrin were further characterized. Two prominent cell phenotypes were observed under these conditions: one characterized by perinuclear vacuole (PV) formation 2-8 h after PDT followed by cell recovery or shrinkage within 48 h, and a second characterized by typical apoptotic features appearing within 4 h after PDT. DCFDA-sensitive reactive oxygen species formed proximal to PVs during the response to PDT, covering areas in which both endoplasmic reticulum (ER) and the Golgi complex were located. Biochemical analyses showed that Photofrin-PDT also induced JNK activation and altered the protein secretion profile. A more detailed examination of PV formation revealed that PVs were derived from the ER. The alteration of ER structure induced by PDT was similar to that triggered by thapsigargin, an ER Ca(2+)-ATPase inhibitor that perturbs Ca(2+) homeostasis, suggesting a role for Ca(2+) in the formation of PVs. Microtubule dynamics did not significantly affect PV formation. This study demonstrates that cells in which intracellular organelles are selectively loaded with Photofrin mount a novel response to ER stress induced by PDT.

  18. 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. PMID:18403202

  19. 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. PMID:21744328

  20. 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.

  1. Polymeric vesicles: from drug carriers to nanoreactors and artificial organelles.

    PubMed

    Tanner, Pascal; Baumann, Patric; Enea, Ramona; Onaca, Ozana; Palivan, Cornelia; Meier, Wolfgang

    2011-10-18

    One strategy in modern medicine is the development of new platforms that combine multifunctional compounds with stable, safe carriers in patient-oriented therapeutic strategies. The simultaneous detection and treatment of pathological events through interactions manipulated at the molecular level offer treatment strategies that can decrease side effects resulting from conventional therapeutic approaches. Several types of nanocarriers have been proposed for biomedical purposes, including inorganic nanoparticles, lipid aggregates, including liposomes, and synthetic polymeric systems, such as vesicles, micelles, or nanotubes. Polymeric vesicles--structures similar to lipid vesicles but created using synthetic block copolymers--represent an excellent candidate for new nanocarriers for medical applications. These structures are more stable than liposomes but retain their low immunogenicity. Significant efforts have been made to improve the size, membrane flexibility, and permeability of polymeric vesicles and to enhance their target specificity. The optimization of these properties will allow researchers to design smart compartments that can co-encapsulate sensitive molecules, such as RNA, enzymes, and proteins, and their membranes allow insertion of membrane proteins rather than simply serving as passive carriers. In this Account, we illustrate the advances that are shifting these molecular systems from simple polymeric carriers to smart-complex protein-polymer assemblies, such as nanoreactors or synthetic organelles. Polymeric vesicles generated by the self-assembly of amphiphilic copolymers (polymersomes) offer the advantage of simultaneous encapsulation of hydrophilic compounds in their aqueous cavities and the insertion of fragile, hydrophobic compounds in their membranes. This strategy has permitted us and others to design and develop new systems such as nanoreactors and artificial organelles in which active compounds are simultaneously protected and allowed to

  2. Plectin isoform 1b mediates mitochondrion-intermediate filament network linkage and controls organelle shape.

    PubMed

    Winter, Lilli; Abrahamsberg, Christina; Wiche, Gerhard

    2008-06-16

    Plectin is a versatile intermediate filament (IF)-bound cytolinker protein with a variety of differentially spliced isoforms accounting for its multiple functions. One particular isoform, plectin 1b (P1b), remains associated with mitochondria after biochemical fractionation of fibroblasts and cells expressing exogenous P1b. Here, we determined that P1b is inserted into the outer mitochondrial membrane with the exon 1b-encoded N-terminal sequence serving as a mitochondrial targeting and anchoring signal. To study P1b-related mitochondrial functions, we generated mice that selectively lack this isoform but express all others. In primary fibroblasts and myoblasts derived from these mice, we observe a substantial elongation of mitochondrial networks, whereas other mitochondrial properties remain largely unaffected. Normal morphology of mitochondria could be restored by isoform-specific overexpression of P1b in P1b-deficient as well as plectin-null cells. We propose a model where P1b both forms a mitochondrial signaling platform and affects organelle shape and network formation by tethering mitochondria to IFs.

  3. 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.

  4. 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.

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

    PubMed

    Lei, Lei; Spradling, Allan C

    2016-04-01

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

  6. ER–PM connections: sites of information transfer and inter-organelle communication

    PubMed Central

    Stefan, Christopher J; Manford, Andrew G; Emr, Scott D

    2014-01-01

    Eukaryotic cells are divided into distinct membrane-bound organelles with unique identities and specialized metabolic functions. Communication between organelles must take place to regulate the size, shape, and composition of individual organelles, as well as to coordinate transport between organelles. The endoplasmic reticulum (ER) forms an expansive membrane network that contacts and participates in crosstalk with several other organelles in the cell, most notably the plasma membrane (PM). ER–PM junctions have well-established functions in the movement of small molecules, such as lipids and ions, between the ER and PM. Recent discoveries have revealed additional exciting roles for ER–PM junctions in the regulation of cell signaling, ER shape and architecture, and PM domain organization. PMID:23522446

  7. 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.

  8. 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

  9. Nanopreparations for organelle-specific delivery in cancer.

    PubMed

    Biswas, Swati; Torchilin, Vladimir P

    2014-02-01

    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

  10. 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.

  11. MLT1 links cytoskeletal asymmetry to organelle placement in Chlamydomonas

    PubMed Central

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

    2015-01-01

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

  12. MLT1 links cytoskeletal asymmetry to organelle placement in chlamydomonas.

    PubMed

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

    2015-03-01

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

  13. Recent advances in yeast organelle and membrane proteomics.

    PubMed

    Premsler, Thomas; Zahedi, René Peiman; Lewandrowski, Urs; Sickmann, Albert

    2009-10-01

    Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far. Consequently, numerous studies have focused on gaining insight into the yeast proteome, with protein MS providing a very efficient technology to cope with this task since it enables both protein identification and differential quantification of cellular material. In this review we present an overview of recent advances in yeast organelle and membrane proteomics focusing on the cell wall, plasma membrane, mitochondria and vacuole.

  14. 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

  15. Subcompartmentalized Nanoreactors as Artificial Organelle with Intracellular Activity.

    PubMed

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

    2016-04-01

    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.

  16. Modularity of a carbon-fixing protein organelle.

    PubMed

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

    2012-01-10

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

  17. 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

  18. Lipid composition of organelles from germinating castor bean endosperm.

    PubMed

    Donaldson, R P; Beevers, H

    1977-02-01

    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 mumol of phosphoglyceride per mg of protein; the mitochondria, 0.65 mumol/mg; and the glyoxysome membranes, 0.55 mumol/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 beta-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.

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

    PubMed Central

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

    2015-01-01

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

  20. 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.

  1. 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

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

    PubMed

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

    2016-03-01

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

  3. Trans-membrane area asymmetry controls the shape of cellular organelles.

    PubMed

    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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. DiOC6 staining reveals organelle structure and dynamics in living yeast cells.

    PubMed

    Koning, A J; Lum, P Y; Williams, J M; Wright, R

    1993-01-01

    When present at low concentrations, the fluorescent lipophilic dye, DiOC6, stains mitochondria in living yeast cells [Pringle et al.: Methods in Cell Biol. 31:357-435, 1989; Weisman et al.: Proc. Natl. Acad. Sci. U.S.A. 87:1076-1080, 1990]. However, we found that the nuclear envelope and endoplasmic reticulum were specifically stained if the dye concentration was increased or if certain respiratory-deficient yeast strains were examined. The quality of nuclear envelope staining with DiOC6 was sufficiently sensitive to reveal alterations in the nuclear envelope known as karmellae. These membranes were previously apparent only by electron microscopy. At the high dye concentrations required to stain the nuclear envelope, wild-type cells could no longer grow on non-fermentable carbon sources. In spite of this effect on mitochondrial function, the presence of high dye concentration did not adversely affect cell viability or general growth characteristics when strains were grown under standard conditions on glucose. Consequently, time-lapse confocal microscopy was used to examine organelle dynamics in living yeast cells stained with DiOC6. These in vivo observations correlated very well with previous electron microscopic studies, including analyses of mitochondria, karmellae, and mitosis. For example, cycles of mitochondrial fusion and division, as well as the changes in nuclear shape and position that occur during mitosis, were readily imaged in time-lapse studies of living DiOC6-stained cells. This technique also revealed new aspects of nuclear disposition and interactions with other organelles. For example, the nucleus and vacuole appeared to form a structurally coupled unit that could undergo coordinated movements. Furthermore, unlike the general view that nuclear movements occur only in association with division, the nucleus/vacuole underwent dramatic migrations around the cell periphery as cells exited from stationary phase. In addition to the large migrations or

  9. Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria.

    PubMed

    Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

    2014-01-01

    Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors.

  10. 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

    2015-03-01

    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

  11. 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.

  12. 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.

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

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-01

    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.

  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. PMID:16227976

  15. The mitochondrion--an organelle commonly involved in programmed cell death in Arabidopsis thaliana.

    PubMed

    Yao, Nan; Eisfelder, Bartholomew J; Marvin, James; Greenberg, Jean T

    2004-11-01

    Plant cells undergoing programmed cell death (PCD) at late stages typically show chromatin condensation and endonucleolytic cleavage prior to obvious membrane or organelle ultrastructural changes. To investigate possible early PCD-associated events, we used microscopic observations and flow cytometry to quantitate mitochondrial membrane potential (DeltaPsim) changes during PCD at the single cell and population levels using Arabidopsis protoplasts. A DeltaPsim loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in DeltaPsim and PCD by cyclosporin A, which inhibits mitochondrial permeability transition pores in animal cells. DeltaPsim loss occurred prior to nuclear morphological changes and was only associated with mitochondrial cytochrome c release (an apoptotic trigger in animals) in response to one of three PCD elicitors. Three different stimuli in wild type implicated DeltaPsim changes in PCD: ceramide, protoporphyrin IX, and the hypersensitive response elicitor AvrRpt2. Additionally, the behavior of the conditional ectopic cell death mutant accelerated cell death2 and ACD2-overproducing plants also implicated DeltaPsim alteration as key for PCD execution. Because ACD2 is largely a chloroplast component in mature plants, the observation that the cell death in acd2 mutants requires changes in mitochondrial functions implicates communication between chloroplasts and mitochondria in mediating PCD activation. We suggest that DeltaPsim loss is a common early marker in plant PCD, similar to what has been documented in animals. However, unlike in animal cells, in plant cells, mitochondrial cytochrome c release is not an obligatory step in PCD control.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  17. Real-time molecular imaging of organelles in living cell by multifocus excitation CARS microscope

    NASA Astrophysics Data System (ADS)

    Minamikawa, Takeo; Araki, Tsutomu; Hashimoto, Mamoru

    2010-02-01

    We demonstrated real-time imaging of organelles in a living HeLa cell using a multi-focus excitation CARS (coherent anti-Stokes Raman scattering) microscope. Chemical selective CARS imaging of lipids and proteins was demonstrated by observing CH2 and CH3 vibrations. Real-time imaging of lipid rich organelles such as the plasma membrane, mitochondria, and lipid rich vesicles was achieved by observing CH2 stretching vibrations of lipids. The image acquisition rate of 5 frames per second was achieved without any staining. We also demonstrated real-time CARS imaging of laser-induced disruption and reaction of organelles in a living HeLa cell. A near-infrared pulsed laser beam tightly focused on an organelle in a living cell produces ablation at the focal point, causing local disruption of the organelle. We visualized the spatial and temporal distributions of a lipid rich organelles in the cytoplasm of a living HeLa cell in laser-induced dissection. We also demonstrated real-time CARS imaging of disruption of a plasma membrane and its repair.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    1999-12-01

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

  20. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2010-04-01

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

  4. Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle.

    PubMed

    Mohan, K V K; Som, I; Atreya, C D

    2002-03-01

    Peroxisomes are unimembrane, respiratory organelles of the cell. Transport of cellular proteins to the peroxisomal matrix requires a type 1 peroxisomal targeting signal (PTS1) which essentially constitutes a tripeptide from the consensus sequence S/T/A/G/C/N-K/R/H-L/I/V/M/A/F/Y. Although PTS-containing proteins have been identified in eukaryotes, prokaryotes, and parasites, viral proteins with such signals have not been identified so far. We report here the first instance of a virus, the rotavirus, which causes infantile diarrhea worldwide, containing a functional C-terminal PTS1 in one of its proteins (VP4). Analysis of 153 rotavirus VP4-deduced amino acid sequences identified five groups of conserved C-terminal PTS1 tripeptide sequences (SKL, CKL, GKL, CRL, and CRI), of which CRL is represented in approximately 62% of the sequences. Infection of cells by a CRL-containing representative rotavirus (SA11 strain) and confocal immunofluorescence analysis revealed colocalization of VP4 with peroxisomal markers and morphological changes of peroxisomes. Further, transient cellular expression of green fluorescent protein (GFP)-fused VP4CRL resulted in transport of VP4 to peroxisomes, whereas the chimera lacking the PTS1 signal, GFP-VP4DeltaCRL, resulted in diffuse cytoplasmic staining, suggesting a CRL-dependent targeting of the protein. The present study therefore demonstrates hitherto unreported organelle involvement, specifically of the peroxisomes, in rotaviral infections as demonstrated by using the SA11 strain of rotavirus and opens a new line of investigation toward understanding viral pathogenesis and disease mechanisms. PMID:11836432

  5. Unusual extrusive organelles in karyorelictid ciliates: an argument for the ancient origin of this group.

    PubMed

    Raikov, I B

    1992-01-01

    The karyorelictid ciliates never possess extrusomes that are typical of most other ciliates, i.e. trichocysts, mucocysts, and toxicysts, but instead present unusual types of extrusive organelles, most existing nowhere else. These organelles are: (1) Nematocysts with a filament making only 2-3 coils in the longitudinal plane, in Remanella multinucleata; (2) 'Orthonematocysts' with a short straight internal filament, in Remanella rugosa and R. brunnea; (3) Tiny bottle-shaped organelles somewhat resembling haptocysts, in Remanella granulosa; (4) Rhabdocysts, arrow-shaped extrusomes somewhat resembling certain trichocysts but undergoing no strong elongation during extrusion, in species of Tracheloraphis and in Kentrophoros latum; (5) Ampullocysts, complex vesicular organelles with hyaline secretion, occurring in Kentrophoros latum; (6) pigmentocysts or extrusible pigment granules, often with some internal structure, in almost all karyorelictids (Trachelocerca, Tracheloraphis, Trachelonema, Loxodes, Remanella, Geleia). This is the only type of cortical organelles the karyorelictids share with other ciliates, namely, the Heterotrichida (Stentor, Blepharisma). This highly aberrant set of extrusomes in karyorelictids argues that they are a very ancient branch of ciliates which separated from the main trunk early in evolution, conserving or developing an unusual set of extrusomes independently from the rest of ciliates. There is also some evidence for the relatedness of the Karyorelictida to Heterotrichida, already supposed from studies of the ciliary fibre systems and sequencing of ribosomal RNAs. PMID:1292663

  6. 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.

  7. 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.

  8. Regulation of Mouse Oocyte Microtubule and Organelle Dynamics by PADI6 and the Cytoplasmic Lattices

    PubMed Central

    Kan, Rui; Yurttas, Piraye; Kim, Boram; Jin, Mei; Wo, Luccie; Lee, Bora; Gosden, Roger; Coonrod, Scott A.

    2010-01-01

    Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement. PMID:21147087

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

    PubMed

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

    2010-07-01

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

  10. MLN64 Is Involved in Actin-mediated Dynamics of Late Endocytic OrganellesD⃞V⃞

    PubMed Central

    Hölttä-Vuori, Maarit; Alpy, Fabien; Tanhuanpää, Kimmo; Jokitalo, Eija; Mutka, Aino-Liisa; Ikonen, Elina

    2005-01-01

    MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles. PMID:15930133

  11. Antennal and cephalic organelles in the social wasp Paravespula germanica (Hymenoptera, Vespinae): form and possible function.

    PubMed

    Agmon, Ifaat; Plotkin, Marian; Ermakov, Natalya Y; Barkay, Zahava; Ishay, Jacob S

    2006-01-01

    This paper deals with hairs and organelles present on the head and antennae of the German wasp, Paravespula germanica, and their possible role in sensing the physical and chemical ambience, as well as in intercommunicating both while in flight outside or in the nest. Via scanning electron microscope photography, we detected on the frons plate of the wasp's head, hairs that were about 300 microm long and comprised the longest hairs on the body of the wasps. Additionally, the two antennae bore along their entire length photoreceptors, placoids, campaniforms, trichoids, and agmons. These organelles are located at high but variable density along the antennal segments. The paper provides the dimensions of each of the mentioned organelles, and discusses the possible functions of the organelles as well as of the hairs on the frons. Photographs taken via atomic force microscope reveal that the epicuticle of the antenna is of two typical shapes; one, bearing both longitudinal stripes as well as transverse bands that are about 1 mum in width, and a second granulated form. Conceivably, the wasp uses the various organelles mentioned to communicate with its mates that are some distance away, somewhat like the use of radar by humans.

  12. 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.

  13. 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.

  14. 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

  15. 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

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

    PubMed

    Dumoux, Maud; Hayward, Richard D

    2016-08-01

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

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

    PubMed

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

    2016-05-19

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

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

    PubMed

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

    2016-03-28

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

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

    PubMed

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

    2014-07-01

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

  20. 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

  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. 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.

  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. 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

  5. Flagellar regeneration in Chlamydomonas: a model system for studying organelle assembly.

    PubMed

    Johnson, K A; Rosenbaum, J L

    1993-05-01

    How do the many different components of an organelle assemble into a functional structure at an appropriate place and time? Flagellar regeneration by the biflagellate green alga Chlamydomonas is one experimental system in which genetics, biochemistry and ultrastructural analysis are being combined to investigate the assembly of a microtubule-containing organelle. Recent advances in the molecular biology of this 'green yeast' have made possible several new approaches to the problem of flagellar assembly; insights from these new approaches are the focus of this review.

  6. Vacuole membrane contact sites and domains: emerging hubs to coordinate organelle function with cellular metabolism.

    PubMed

    Malia, Pedro Carpio; Ungermann, Christian

    2016-04-15

    Eukaryotic cells rely on a set of membrane-enclosed organelles to perform highly efficient reactions in an optimized environment. Trafficking of molecules via vesicular carriers and membrane contact sites (MCS) allow the coordination between these compartments, though the precise mechanisms are still enigmatic. Among the cellular organelles, the lysosome/vacuole stands out as a central hub, where multiple pathways merge. Importantly, the delivered material is degraded and the monomers are recycled for further usage, which explains its wide variety of roles in controlling cellular metabolism. We will highlight recent advances in the field by focusing on the yeast vacuole as a model system to understand lysosomal function in general.

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

    PubMed Central

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

    2009-01-01

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

  8. 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.…

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

    PubMed Central

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

    2015-01-01

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

  10. 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.

  11. Effects of 4-aminopyridine on organelle movement in cultured mouse dorsal root ganglion neurites.

    PubMed

    Hiruma, Hiromi; Kawakami, Tadashi

    2010-03-01

    Aminopyridines, widely used as a K(+) channel blocker, are membrane-permeable weak bases and have the ability to form vacuoles in the cytoplasm. The vacuoles originate from acidic organelles such as lysosomes. Here, we investigated the effects of 4-aminopyridine (4-AP) on organelle movement in neurites of cultured mouse dorsal root ganglion (DRG) neurons by using video-enhanced microscopy. Some experiments were carried out using fluorescent dyes for lysosomes and mitochondria and confocal microscopy. Treatment of DRG neurons with 4 mM 4-AP caused Brownian movement of some lysosomes within 5 min. The Brownian movement gradually became rapid and vacuoles were formed around individual lysosomes 10-20 min after the start of treatment. Axonal transport of organelles was inhibited by 4-AP. Lysosomes showing Brownian movement were not transported in longitudinal direction of the neurite and the transport of mitochondria was interrupted by vacuoles. The 4-AP-induced Brownian movement of lysosomes with vacuole formation and inhibition of axonal transport were prevented by the simultaneous treatment with vacuolar H(+) ATPase inhibitor bafilomycin A1 or in Cl(-)-free SO(4)(2-) medium. These results indicate that changes in organelle movement by 4-AP are related to vacuole formation and the vacuolar H(+) ATPase and Cl(-) are required for the effects of 4-AP.

  12. 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

  13. 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. PMID:18757809

  14. 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

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

    PubMed

    Faso, Carmen; Hehl, Adrian B

    2011-04-01

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

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

    PubMed

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

    2016-01-01

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

  17. 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

  18. 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.

  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. 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

  1. 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

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

    PubMed

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

    2016-05-01

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

  3. 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

  4. 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.

  5. 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.

  6. The yellow mutation in the frog Rana rugosa: pigment organelle deformities in the three types of chromatophore.

    PubMed

    Ichikawa, Y; Ohtani, H; Miura, I

    2001-08-01

    Crossing experiments revealed that a single recessive gene mutation (yellow) gives rise to the yellow phenotype of Rana rugosa in Japan. Ultrastructural observation of dermal chromatophores showed that the pigment organelles; melanosomes, pterinosomes, and reflecting platelets, all had structural deformities. This suggests that the yellow gene acts at the level of a primordial pigment organelle common to the three types of chromatophore.

  7. The microtrabecular lattice and its relationships to other organelles in prokaryotes and eukaryotes--high resolution scanning electron microscopy.

    PubMed

    Epling, G P; Blixt, J A; Mahurin, R W; Rinaldi, M G

    1983-01-01

    High resolution scanning electron microscopy demonstrated the microtrabecular lattice in bacteria, fungi, plant and animal cells. It is attached to ribosomes and plasma membranes generally, but to other organelles and the nuclear envelope in eukaryotes. The eukaryotic organelle surface substructure is described. Differentiation of real structure from artifacts of fixation, critical point drying and sputter-coating, is discussed.

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

    PubMed Central

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

    2012-01-01

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

  9. 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.

  10. 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

  11. Plant vacuole morphology and vacuolar trafficking

    PubMed Central

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

    2014-01-01

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

  12. Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis

    PubMed Central

    Ellis, Kathryn; Bagwell, Jennifer

    2013-01-01

    The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H+-ATPase–dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development. PMID:23460678

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  15. Quantitatively mapping cellular viscosity with detailed organelle information via a designed PET fluorescent probe.

    PubMed

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

    2014-01-01

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

  16. Diversity and origins of anaerobic metabolism in mitochondria and related organelles

    PubMed Central

    Stairs, Courtney W.; Leger, Michelle M.; Roger, Andrew J.

    2015-01-01

    Across the diversity of life, organisms have evolved different strategies to thrive in hypoxic environments, and microbial eukaryotes (protists) are no exception. Protists that experience hypoxia often possess metabolically distinct mitochondria called mitochondrion-related organelles (MROs). While there are some common metabolic features shared between the MROs of distantly related protists, these organelles have evolved independently multiple times across the breadth of eukaryotic diversity. Until recently, much of our knowledge regarding the metabolic potential of different MROs was limited to studies in parasitic lineages. Over the past decade, deep-sequencing studies of free-living anaerobic protists have revealed novel configurations of metabolic pathways that have been co-opted for life in low oxygen environments. Here, we provide recent examples of anaerobic metabolism in the MROs of free-living protists and their parasitic relatives. Additionally, we outline evolutionary scenarios to explain the origins of these anaerobic pathways in eukaryotes. PMID:26323757

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-08-13

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2014-02-06

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

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

    PubMed

    Gilmore, Simon; Peakall, Rod; Robertson, James

    2007-10-25

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

  2. Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.

    PubMed

    Ellis, Kathryn; Bagwell, Jennifer; Bagnat, Michel

    2013-03-01

    The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H(+)-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

  3. Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization.

    PubMed

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

    2014-03-10

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

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

    PubMed

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

    2014-08-13

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

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

    PubMed Central

    Craven, C Jeremy

    2016-01-01

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

  6. Diversity and origins of anaerobic metabolism in mitochondria and related organelles.

    PubMed

    Stairs, Courtney W; Leger, Michelle M; Roger, Andrew J

    2015-09-26

    Across the diversity of life, organisms have evolved different strategies to thrive in hypoxic environments, and microbial eukaryotes (protists) are no exception. Protists that experience hypoxia often possess metabolically distinct mitochondria called mitochondrion-related organelles (MROs). While there are some common metabolic features shared between the MROs of distantly related protists, these organelles have evolved independently multiple times across the breadth of eukaryotic diversity. Until recently, much of our knowledge regarding the metabolic potential of different MROs was limited to studies in parasitic lineages. Over the past decade, deep-sequencing studies of free-living anaerobic protists have revealed novel configurations of metabolic pathways that have been co-opted for life in low oxygen environments. Here, we provide recent examples of anaerobic metabolism in the MROs of free-living protists and their parasitic relatives. Additionally, we outline evolutionary scenarios to explain the origins of these anaerobic pathways in eukaryotes.

  7. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed

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

    2014-01-01

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

  10. 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) organelle autophagy as allophagy.

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-12-15

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  15. Visualization and structural analysis of the bacterial magnetic organelle magnetosome using atomic force microscopy.

    PubMed

    Yamamoto, Daisuke; Taoka, Azuma; Uchihashi, Takayuki; Sasaki, Hideaki; Watanabe, Hiroki; Ando, Toshio; Fukumori, Yoshihiro

    2010-05-18

    The unique ability of magnetotactic bacteria to navigate along a geomagnetic field is accomplished with the help of prokaryotic organelles, magnetosomes. The magnetosomes have well-ordered chain-like structures, comprising membrane-enveloped, nano-sized magnetic crystals, and various types of specifically associated proteins. In this study, we applied atomic force microscopy (AFM) to investigate the spatial configuration of isolated magnetosomes from Magnetospirillum magneticum AMB-1 in near-native buffer conditions. AFM observation revealed organic material with a approximately 7-nm thickness surrounding a magnetite crystal. Small globular proteins, identified as magnetosome-associated protein MamA, were distributed on the mica surface around the magnetosome. Immuno-labeling with AFM showed that MamA is located on the magnetosome surface. In vitro experiments showed that MamA proteins interact with each other and form a high molecular mass complex. These findings suggest that magnetosomes are covered with MamA oligomers in near-native environments. Furthermore, nanodissection revealed that magnetosomes are built with heterogeneous structures that comprise the organic layer. This study provides important clues to the supramolecular architecture of the bacterial organelle, the magnetosome, and insight into the function of the proteins localized in the organelle.

  16. Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways.

    PubMed

    Bobik, Thomas A; Lehman, Brent P; Yeates, Todd O

    2015-10-01

    Prokaryotes use subcellular compartments for a variety of purposes. An intriguing example is a family of complex subcellular organelles known as bacterial microcompartments (MCPs). MCPs are widely distributed among bacteria and impact processes ranging from global carbon fixation to enteric pathogenesis. Overall, MCPs consist of metabolic enzymes encased within a protein shell, and their function is to optimize biochemical pathways by confining toxic or volatile metabolic intermediates. MCPs are fundamentally different from other organelles in having a complex protein shell rather than a lipid-based membrane as an outer barrier. This unusual feature raises basic questions about organelle assembly, protein targeting and metabolite transport. In this review, we discuss the three best-studied MCPs highlighting atomic-level models for shell assembly, targeting sequences that direct enzyme encapsulation, multivalent proteins that organize the lumen enzymes, the principles of metabolite movement across the shell, internal cofactor recycling, a potential system of allosteric regulation of metabolite transport and the mechanism and rationale behind the functional diversification of the proteins that form the shell. We also touch on some potential biotechnology applications of an unusual compartment designed by nature to optimize metabolic processes within a cellular context.

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

    PubMed

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

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

  18. Plasma membrane-associated superstructure: Have we overlooked a new type of organelle in eukaryotic cells?

    PubMed

    Rodríguez-Fernández, José Luis; de Lacoba, Mario García

    2015-09-01

    A variety of intriguing plasma membrane-associated regions, including focal adhesions, adherens junctions, tight junctions, immunological synapses, neuromuscular junctions and the primary cilia, among many others, have been described in eukaryotic cells. Emphasizing their importance, alteration in their molecular structures induces or correlates with different pathologies. These regions display surface proteins connected to intracellular molecules, including cytoskeletal component, which maintain their cytoarchitecture, and signalling proteins, which regulate their organization and functions. Based on the molecular similarities and other common features observed, we suggest that, despite differences in external appearances, all these regions are just the same superstructure that appears in different locations and cells. We hypothesize that this superstructure represents an overlooked new type of organelle that we call plasma membrane-associated superstructure (PMAS). Therefore, we suggest that eukaryotic cells include classical organelles (e.g. mitochondria, Golgi and others) and also PMAS. We speculate that this new type of organelle might be an innovation associated to the emergence of eukaryotes. Finally we discuss the implications of the hypothesis proposed.

  19. Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation

    PubMed Central

    Elferich, Johannes; Williamson, Danielle M.; Krishnamoorthy, Bala; Shinde, Ujwal

    2013-01-01

    Eukaryotic cells maintain strict control over protein secretion, in part by using the pH gradient maintained within their secretory pathway. How eukaryotic proteins evolved from prokaryotic orthologs to exploit the pH gradient for biological functions remains a fundamental question in cell biology. Our laboratory previously demonstrated that protein domains located within precursor proteins, propeptides, encode histidine-driven pH sensors to regulate organelle-specific activation of the eukaryotic proteases furin and proprotein convertase-1/3. Similar findings have been reported in other unrelated protease families. By analyzing >10,000 unique proteases within evolutionarily unrelated families, we show that eukaryotic propeptides are enriched in histidines compared with prokaryotic orthologs. On this basis, we hypothesize that eukaryotic proteins evolved to enrich histidines within their propeptides to exploit the tightly controlled pH gradient of the secretory pathway, thereby regulating activation within specific organelles. Enrichment of histidines in propeptides may therefore be used to predict the presence of pH sensors in other proteases or even protease substrates.—Elferich, J., Williamson, D. M., Krishnamoorthy, B., Shinde, U. Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation. PMID:23585398

  20. Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways

    PubMed Central

    Bobik, Thomas A.; Lehman, Brent P.; Yeates, Todd O.

    2016-01-01

    Summary Prokaryotes use subcellular compartments for a variety of purposes. An intriguing example is a family of complex subcellular organelles known as bacterial microcompartments (MCPs). MCPs are widely distributed among bacteria and impact processes ranging global carbon fixation and enteric pathogenesis. Overall, MCPs consist of metabolic enzymes encased within a protein shell, and their function is to optimize biochemical pathways by confining toxic or volatile metabolic intermediates. MCPs are fundamentally different from other organelles in having a complex protein shell rather than a lipid-based membrane as an outer barrier. This unusual feature raises basic questions about organelle assembly, protein targeting and metabolite transport. In this review, we discuss the three best-studied MCPs highlighting atomic-level models for shell assembly, targeting sequences that direct enzyme encapsulation, multivalent proteins that organize the lumen enzymes, the principles of metabolite movement across the shell, internal cofactor recycling, a potential system of allosteric regulation of metabolite transport and the mechanism and rationale behind the functional diversification of the proteins that form the shell. We also touch on some potential biotechnology applications an unusual compartment designed by nature to optimize metabolic processes within a cellular context. PMID:26148529

  1. Characterization of the reflective materials and organelles in the bright irides of North American blackbirds (Icterinae).

    PubMed

    Hudon, J; Muir, A D

    1996-04-01

    The reflective materials in the iris stroma of bright-irised American blackbirds (Icterinae, Emberizidae) and the red-eyed vireo (vireo olivaceus) (Vireonidae) were characterized using high-performance liquid chromatography (HPLC) and diode-array detection. Two purines, guanine and hypoxanthine, and two pteridines, leucopterin and xanthopterin, were detected in large amounts in all bright irides. The brown iris of the red-winged blackbird (Agelaius phoeniceus) by comparison contained only small amounts of these and additional unidentified compounds. The absolute and relative amounts of light-absorbing compounds in the iris varied somewhat among species of blackbirds with bright irides, and markedly within one species (brewer's blackbird, Euphagus cyanocephalus) between sexes and age classes that very in eye color. Differences in the types, numbers, and sizes of pigment organelles in the irides appeared to underlie the differences in amounts of light-absorbing compounds. Guanine was the most abundant light-absorbing compound in all bright irides, accounting for about 90% of the total absorption at 250 nm. A wide range of concentrations of guanine, from 96 to 9 micrograms per iris, produced bright irides. The primary pigment organelles of pigment cells in bright irides were reflecting platelets, which typically appeared as open spaces on electron micrographs. In the red-eyed vireo there were in addition red pterinosome-like pigment organelles in the pigment cells on the anterior surface of the iris stroma. Guanine was present even in irides with no overt reflecting platelets.

  2. The evolution of eukaryotic cilia and flagella as motile and sensory organelles.

    PubMed

    Mitchell, David R

    2007-01-01

    Eukaryotic cilia and flagella are motile organelles built on a scaffold of doublet microtubules and powered by dynein ATPase motors. Some thirty years ago, two competing views were presented to explain how the complex machinery of these motile organelles had evolved. Overwhelming evidence now refutes the hypothesis that they are the modified remnants of symbiotic spirochaete-like prokaryotes, and supports the hypothesis that they arose from a simpler cytoplasmic microtubule-based intracellular transport system. However, because intermediate stages in flagellar evolution have not been found in living eukaryotes, a clear understanding of their early evolution has been elusive. Recent progress in understanding phylogenetic relationships among present day eukaryotes and in sequence analysis of flagellar proteins have begun to provide a clearer picture of the origins of doublet and triplet microtubules, flagellar dynein motors, and the 9+2 microtubule architecture common to these organelles. We summarize evidence that the last common ancestor of all eukaryotic organisms possessed a 9+2 flagellum that was used for gliding motility along surfaces, beating motility to generate fluid flow, and localized distribution of sensory receptors, and trace possible earlier stages in the evolution of these characteristics.

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

    PubMed Central

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

    2012-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

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

    SciTech Connect

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

    2010-09-07

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

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

    PubMed

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

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

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

    PubMed Central

    Leister, Dario

    2016-01-01

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

  8. Characterization of the reflective materials and organelles in the bright irides of North American blackbirds (Icterinae).

    PubMed

    Hudon, J; Muir, A D

    1996-04-01

    The reflective materials in the iris stroma of bright-irised American blackbirds (Icterinae, Emberizidae) and the red-eyed vireo (vireo olivaceus) (Vireonidae) were characterized using high-performance liquid chromatography (HPLC) and diode-array detection. Two purines, guanine and hypoxanthine, and two pteridines, leucopterin and xanthopterin, were detected in large amounts in all bright irides. The brown iris of the red-winged blackbird (Agelaius phoeniceus) by comparison contained only small amounts of these and additional unidentified compounds. The absolute and relative amounts of light-absorbing compounds in the iris varied somewhat among species of blackbirds with bright irides, and markedly within one species (brewer's blackbird, Euphagus cyanocephalus) between sexes and age classes that very in eye color. Differences in the types, numbers, and sizes of pigment organelles in the irides appeared to underlie the differences in amounts of light-absorbing compounds. Guanine was the most abundant light-absorbing compound in all bright irides, accounting for about 90% of the total absorption at 250 nm. A wide range of concentrations of guanine, from 96 to 9 micrograms per iris, produced bright irides. The primary pigment organelles of pigment cells in bright irides were reflecting platelets, which typically appeared as open spaces on electron micrographs. In the red-eyed vireo there were in addition red pterinosome-like pigment organelles in the pigment cells on the anterior surface of the iris stroma. Guanine was present even in irides with no overt reflecting platelets. PMID:8857673

  9. F-actin cytoskeleton and the fate of organelles in chromaffin cells.

    PubMed

    Villanueva, José; Gimenez-Molina, Yolanda; Viniegra, Salvador; Gutiérrez, Luis M

    2016-06-01

    In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular mechanism and regulating the secretory process. Performing such roles, the F-actin network might be essential to first transport, and later locate the cellular organelles participating in the secretory cycle. Chromaffin granules are transported from the internal cytosolic regions to the cell periphery along microtubular and F-actin structures. Once in the cortical region, they are embedded in the F-actin network where these vesicles experience restrictions in motility. Similarly, mitochondria transport is affected by both microtubule and F-actin inhibitors and suffers increasing motion restrictions when they are located in the cortical region. Therefore, the F-actin cortex is a key factor in defining the existence of two populations of cortical and perinuclear granules and mitochondria which could be distinguished by their different location and mobility. Interestingly, other important organelles for controlling intracellular calcium levels, such as the endoplasmic reticulum network, present clear differences in distribution and much lower mobility than chromaffin vesicles and mitochondria. Nevertheless, both mitochondria and the endoplasmic reticulum appear to distribute in the proximity of secretory sites to fulfill a pivotal role, forming triads with calcium channels ensuring the fine tuning of the secretory response. This review presents the contributions that provide the basis for our current view regarding the influence that F-actin has on the distribution of organelles participating in the release of catecholamines in chromaffin cells, and summarizes this knowledge in simple models. In chromaffin cells, organelles such as granules and mitochondria distribute forming cortical and perinuclear populations whereas others like the ER present homogenous distributions. In the present review we discuss

  10. The lumen-facing domain is important for the biological function and organelle-to-organelle movement of bZIP28 during ER stress in Arabidopsis.

    PubMed

    Sun, Le; Lu, Sun-Jie; Zhang, Shuang-Shuang; Zhou, Shun-Fan; Sun, Ling; Liu, Jian-Xiang

    2013-09-01

    The membrane-associated transcription factor, bZIP28, is relocated from the endoplasmic reticulum (ER) to the Golgi and proteolytically released from the membrane mediated by two proteases, S1P and S2P, in response to ER stress in Arabidopsis. The activated N-terminal domain recruits nuclear factor Y (NF-Y) subunits in the nucleus to regulate ER stress downstream genes. Little is known about the functions of the bZIP28 C-terminal lumen-facing domain. Here, we provide novel insights into how the ER lumen-facing domain affects the biological function and organelle-to-organelle movement of bZIP28 in the ER stress response. First, we demonstrated the functional redundancy of bZIP28 and bZIP60 by generation and analysis of the bZIP28 and bZIP60 double mutant zip28zip60. Subsequent genetic complementation experiments in zip28zip60 background with deletions on bZIP28 lumen-facing domain highlighted the importance of lumen-facing domain for its in vivo function of bZIP28 in the ER stress response. The protein subcellular localization and Western blotting results further revealed that the bZIP28 lumen-facing domain contains ER retention signal which is important for the proteolytic activation of bZIP28. Thus, the bZIP28 lumen-facing C-terminus plays important roles in the ER-to-Golgi movement of bZIP28, which may contribute to the sensing of the ER stress.

  11. Children's Morphological Knowledge: Links to Literacy

    ERIC Educational Resources Information Center

    McCutchen, Deborah; Green, Laura; Abbott, Robert D.

    2008-01-01

    Using a reliable and broad-based measure of morphological awareness, which tapped knowledge of relational, syntactic, and distributional morphology, we examined the development of morphological knowledge among older elementary students and the relationship of their morphological knowledge to a range of literacy measures. We found that…

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

    PubMed Central

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

    2015-01-01

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

  13. Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified, intact egg of Xenopus laevis

    PubMed Central

    1990-01-01

    Using double-barreled, Ca2(+)-sensitive microelectrodes, we have examined the characteristics of the Ca2+ release by inositol 1,4,5- trisphosphate (Ins(1,4,5)P3) in the various layers of Xenopus laevis eggs in which the organelles had been stratified by centrifugation. Centrifugation of living eggs stratifies the organelles yet retains them in the normal cytoplasmic milieu. The local increase in intracellular free Ca2+ in each layer was directly measured under physiological conditions using theta-tubing, double-barreled, Ca2(+)- sensitive microelectrodes in which one barrel was filled with the Ca2+ sensor and the other was filled with Ins(1,4,5)P3 for microinjection. The two tips of these electrodes were very close to each other (3 microns apart) enabling us to measure the kinetics of both the highly localized intracellular Ca2+ release and its subsequent removal in response to Ins(1,4,5)P3 injection. Upon Ins(1,4,5)P3 injection, the ER- enriched layer exhibited the largest release of Ca2+ in a dosage- dependent manner, whereas the other layers, mitochondria, lipid, and yolk, released 10-fold less Ca2+ in a dosage-independent manner. The removal of released Ca2+ took place within approximately 1 min. The sensitivity to Ins(1,4,5)P3 and the time course of intracellular Ca2+ release in the unstratified (unactivated) egg is nearly identical to that observed in the ER layer of the stratified egg. Our data suggest that the ER is the major organelle of the Ins(1,4,5)P3-sensitive Ca2+ store in the egg of Xenopus laevis. PMID:2324195

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-06-01

    Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. Like most apicomplexans, T. gondii possesses several plant-like features, such as the chloroplast-like organelle, the apicoplast. We describe and characterize a novel organelle in T. gondii tachyzoites, which is visible by light microscopy and possesses a broad similarity to the plant vacuole. Electron tomography shows the interaction of this vacuole with other organelles. The presence of a plant-like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, a cathepsin L-like protease (TgCPL), an aquaporin (TgAQP1), as well as Ca(2+)/H(+) and Na(+)/H(+) exchange activities, supports similarity to the plant vacuole. Biochemical characterization of TgVP1 in enriched fractions shows a functional similarity to the respective plant enzyme. The organelle is a Ca(2+) store and appears to have protective effects against salt stress potentially linked to its sodium transport activity. In intracellular parasites, the organelle fragments, with some markers colocalizing with the late endosomal marker, Rab7, suggesting its involvement with the endocytic pathway. Studies on the characterization of this novel organelle will be relevant to the identification of novel targets for chemotherapy against T. gondii and other apicomplexan parasites as well.

  17. Review of cytological studies on cellular and molecular mechanisms of uniparental (maternal or paternal) inheritance of plastid and mitochondrial genomes induced by active digestion of organelle nuclei (nucleoids).

    PubMed

    Kuroiwa, Tsuneyoshi

    2010-03-01

    In most sexual organisms, including isogamous, anisogamous and oogamous organisms, uniparental transmission is a striking and universal characteristic of the transmission of organelle (plastid and mitochondrial) genomes (DNA). Using genetic, biochemical and molecular biological techniques, mechanisms of uniparental (maternal and parental) and biparental transmission of organelle genomes have been studied and reviewed. Although to date there has been no cytological review of the transmission of organelle genomes, cytology offers advantages in terms of direct evidence and can enhance global studies of the transmission of organelle genomes. In this review, I focus on the cytological mechanism of uniparental inheritance by "active digestion of male or female organelle nuclei (nucleoids, DNA)" which is universal among isogamous, anisogamous, and oogamous organisms. The global existence of uniparental transmission since the evolution of sexual eukaryotes may imply that the cell nuclear genome continues to inhibit quantitative evolution of organelles by organelle recombination.

  18. Lucifer Yellow as a live cell fluorescent probe for imaging water transport in subcellular organelles.

    PubMed

    Chaurra, Adriana; Gutzman, Brittany M; Taylor, Emily; Ackroyd, P Christine; Christensen, Kenneth A

    2011-01-01

    While the water permeability of the plasma membranes of mammalian cells has been studied extensively, water transport across membranes of subcellular compartments (e.g., lysosomes, macropinosomes) has been difficult to study. Here we demonstrate a new method for measuring water flux in late endosomes and lysosomes of intact living cells using time-lapse fluorescence microscopy. Cells were loaded by fluid-phase uptake with a mixture of the Lucifer Yellow dextran (LY-dex), a D(2)O sensitive dye, and a D(2)O insensitive control dye, Alexa fluor 546 dextran (AF546-dex). LY-dex responded linearly to changes in D(2)O concentration and the LY-dex D(2)O sensitivity was not affected by changes in pH, physiological salt, and protein concentrations. The co-loaded control dye, AF546-dex, showed no signal changes as a function of D(2)O concentration. To measure membrane water flux, the LY-dex fluorescence in labeled organelles was recorded during rapid superfusion of cells with isotonic buffers prepared in D(2)O. The time constant of water exchange across the lysosomal membrane of intact cells was determined by fitting the data to a single exponential function. From these data, together with the measured area of the organelles, observed water permeability for intracellular CHO-K1 lysosomes was calculated to be 5.3 × 10(-3) ± 0.3 × 10(-3) cm/s. This work demonstrates the feasibility of measuring water flux into subcellular organelles in live cells using LY-dex. PMID:21211149

  19. The internal architecture of leukocyte lipid body organelles captured by three-dimensional electron microscopy tomography.

    PubMed

    Melo, Rossana C N; Paganoti, Guillherme F; Dvorak, Ann M; Weller, Peter F

    2013-01-01

    Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their "cores". After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB "cores" and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and

  20. The endoplasmic reticulum exerts control over organelle streaming during cell expansion.

    PubMed

    Stefano, Giovanni; Renna, Luciana; Brandizzi, Federica

    2014-03-01

    Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

  1. The emerging complexity of the vertebrate cilium: new functional roles for an ancient organelle.

    PubMed

    Davis, Erica E; Brueckner, Martina; Katsanis, Nicholas

    2006-07-01

    Cilia and flagella are found on the surface of a strikingly diverse range of cell types. These intriguing organelles, with their unique and highly adapted protein transport machinery, have been studied extensively in the context of cellular locomotion, sexual reproduction, or fluid propulsion. However, recent studies are beginning to show that in vertebrates particularly, cilia have been recruited to perform additional developmental and homeostatic roles. Here, we review advances in deciphering the functional components of cilia, and we explore emerging trends that implicate ciliary proteins in signal transduction and morphogenetic pathways.

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

  3. QUANTITATIVE MORPHOLOGY

    EPA Science Inventory

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

  4. Morphological Analysis of Rubus Seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

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

    2015-11-01

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

  9. Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation.

    PubMed

    Elferich, Johannes; Williamson, Danielle M; Krishnamoorthy, Bala; Shinde, Ujwal

    2013-08-01

    Eukaryotic cells maintain strict control over protein secretion, in part by using the pH gradient maintained within their secretory pathway. How eukaryotic proteins evolved from prokaryotic orthologs to exploit the pH gradient for biological functions remains a fundamental question in cell biology. Our laboratory previously demonstrated that protein domains located within precursor proteins, propeptides, encode histidine-driven pH sensors to regulate organelle-specific activation of the eukaryotic proteases furin and proprotein convertase-1/3. Similar findings have been reported in other unrelated protease families. By analyzing >10,000 unique proteases within evolutionarily unrelated families, we show that eukaryotic propeptides are enriched in histidines compared with prokaryotic orthologs. On this basis, we hypothesize that eukaryotic proteins evolved to enrich histidines within their propeptides to exploit the tightly controlled pH gradient of the secretory pathway, thereby regulating activation within specific organelles. Enrichment of histidines in propeptides may therefore be used to predict the presence of pH sensors in other proteases or even protease substrates.

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    Kustatscher, Georg; Grabowski, Piotr

    2016-01-01

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

  13. An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region.

    PubMed

    Schweizer, Nina; Pawar, Nisha; Weiss, Matthias; Maiato, Helder

    2015-08-31

    The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous "spindle envelope" confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.

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

    NASA Astrophysics Data System (ADS)

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

    2005-04-01

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

  15. Interrelations between the parasitophorous vacuole of Toxoplasma gondii and host cell organelles.

    PubMed

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

    2005-04-01

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

    Steinberg, G; Schliwa, M

    1995-01-01

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

  18. The human peroxisome in health and disease: the story of an oddity becoming a vital organelle.

    PubMed

    Vamecq, Joseph; Cherkaoui-Malki, Mustapha; Andreoletti, Pierre; Latruffe, Norbert

    2014-03-01

    Since the first report by Rhodin in 1954, our knowledge on mammalian microbodies/peroxisomes has known several periods. An initial two decades period (1954-1973) has contributed to the biochemical individualisation of peroxisomes as a new class of subcellular organelles (de Duve, 1965). The corresponding research period failed to define a clear role of mammalian peroxisomes in vital functions and intermediary metabolism, explaining why feeling that peroxisomes might be in the human cell oddities has prevailed during several decades. The period standing from 1973 to nowadays has progressively removed this cell oddity view of peroxisomes by highlighting vital function and metabolic role of peroxisomes in health and disease along with genetic and metabolic regulation of peroxisomal protein content, organelle envelope formation and protein signal targeting mechanisms. Research on peroxisomes and their response to various drugs and metabolites, dietary and physiological conditions has also played a key role in the discovery of peroxisome proliferator activated receptors (PPARs) belonging to the nuclear hormone receptor superfamily and for which impact in science and medicine goes now by far beyond that of the peroxisomes.

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

    PubMed

    Tychinsky, Vladimir P

    2014-12-01

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

  20. An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region

    PubMed Central

    Schweizer, Nina; Pawar, Nisha; Weiss, Matthias

    2015-01-01

    The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous “spindle envelope” confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function. PMID:26304726

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

    PubMed

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

    2014-01-01

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

  2. Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles.

    PubMed

    van den Biggelaar, Maartje; Meijer, Alexander B; Voorberg, Jan; Mertens, Koen

    2009-03-26

    Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that coexpression of factor VIII (FVIII) with VWF results in costorage of both proteins. However, whether cotrafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of cotargeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly with VWF. PMID:19088379

  3. Class XIII myosins from the green alga Acetabularia: driving force in organelle transport and tip growth?

    PubMed

    Vugrek, Oliver; Sawitzky, Heiko; Menzel, Diedrik

    2003-01-01

    The green alga Acetabularia cliftonii (Dasycladales) contains at least two myosin genes, which already have been assigned class XIII of the myosin superfamily (Cope et al., 1996, Structure 4: 969-987). Here we report a complete analysis of their gene structure and their corresponding transcripts Aclmyo1 and Aclmyo2. Despite promising Northern blot data no evidence for alternative splicing could be found. Dissecting the primary structure at complementary deoxyribonucleic acid (cDNA) level we found a myosin typical organization in head, neck and variable tail region. Most striking is the extremely short tail region of Aclmyo1 with only 18 residues and the maximum number of 7 IQ motifs in Aclmyo2. Probing Acetabularia protein extracts with an antibody raised to a synthetic peptide derived from the amino terminal region in Alcmyo1 showed cross-reactivity to a polypeptide with a molecular mass of approximately 100 kD. This corresponds to the predicted molecular weight of Aclmyo1, which is 106 kD as deduced from the amino acid sequence. Additionally, the same cross-reactive protein is capable of binding F-actin as indicated by a co-sedimentation assay. Confocal laser scanning microscopy with raised antibody revealed co-localization with organelles, the budding region of lateral whorls and the cell apex suggesting involvement of putative Acetabularia myosin in organelle transport and tip growth.

  4. Successful childbirth after intracytoplasmic morphologically selected sperm injection without assisted oocyte activation in a patient with globozoospermia.

    PubMed

    Sermondade, N; Hafhouf, E; Dupont, C; Bechoua, S; Palacios, C; Eustache, F; Poncelet, C; Benzacken, B; Lévy, R; Sifer, C

    2011-11-01

    We here report a successful pregnancy and healthy childbirth obtained in a case of total globozoospermia after intracytoplasmic morphologically selected sperm injection (IMSI) without assisted oocyte activation (AOA). Two semen analyses showed 100% globozoospermia on classic spermocytogram. Motile sperm organelle morphology examination (MSOME) analysis at ×10,000 magnification confirmed the round-headed aspect for 100% of sperm cells, but 1% of the spermatozoa seemed to present a small bud of acrosome. This particular aspect was confirmed by transmission electron microscopy and anti-CD46 staining analysis. Results from sperm DNA fragmentation and fluorescence in situ hybridization analyses were normal. The karyotype was 46XY, and no mutations or deletions in SPATA16 and DPY19L2 genes were detected. Considering these results, a single IMSI cycle was performed, and spermatozoa were selected for the absence of vacuoles and the presence of a small bud of acrosome. A comparable fertilization rate with or without calcium-ionophore AOA was observed. Two fresh top-quality embryos obtained without AOA were transferred at Day 2 after IMSI, leading to pregnancy and birth of a healthy baby boy. This successful outcome suggests that MSOME may be useful in cases of globozoospermia in order to carefully evaluate sperm morphology and to maximize the benefit of ICSI/IMSI.

  5. Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica.

    PubMed

    Mai, Z; Ghosh, S; Frisardi, M; Rosenthal, B; Rogers, R; Samuelson, J

    1999-03-01

    Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC. 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic.

  6. Ultrastructure of the intercalated body, a novel organelle associated with fluid forming cells in the organ of Corti.

    PubMed

    Sobkowicz, H M; Holy, J; Scott, G L

    1990-07-01

    The intercalated body is a newly discovered organelle in the inner and outer spiral sulcus cells of the mouse organ of Corti. The organelle was found in the cochleas of 14-day and older intact mice and in organs in culture of corresponding ages. The organelle consists of a stack of interconnected cisternae of endoplasmic reticulum and of membrane bound rodlets that are intercalated between, and run parallel to, the cisternae. The cisternal membranes are predominantly smooth, but some may display ribosomes. Most rodlets are from 1 to 2 microns long, about 0.1 micron wide, and contain electron dense material. Mitochondria are commonly associated with or incorporated into the organelle. Some electron micrographs suggest that the rodlets may originate from modified mitochondria. It is our impression that the formation of the organelle begins with the apposition of cisternae and mitochondria. Cisternal-associated mitochondria appear to constrict, elongate, and lose their inner membranes. In both the intact animal and in culture, the cells of the inner and outer spiral sulci display microvilli, apical junctional complexes, lateral intercellular spaces containing interdigitating cell processes, and appear to be involved in fluid formation. Moreover, in culture, the cells of inner and outer spiral sulci as well as some cells proliferating in the outgrowth zone participate in fluid formation, producing large fluid pockets. All these cells commonly contain intercalated bodies. It is possible that in the intact animal, as in culture, intercalated bodies may play a role in fluid regulation in the immediate vicinity of the hair cells.

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

    PubMed Central

    Morley, Stewart A.; Nielsen, Brent L.

    2016-01-01

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

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

    PubMed Central

    2013-01-01

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

  9. Amorphous areas in the cytoplasm of Dendrobium tepal cells: production through organelle degradation and destruction through macroautophagy?

    PubMed

    van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

    2013-08-01

    In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy.

  10. Coupling acidic organelles with the ER through Ca²⁺ microdomains at membrane contact sites.

    PubMed

    Penny, Christopher J; Kilpatrick, Bethan S; Eden, Emily R; Patel, Sandip

    2015-10-01

    Acidic organelles such as lysosomes serve as non-canonical Ca(2+) stores. The Ca(2+) mobilising messenger NAADP is thought to trigger local Ca(2+) release from such stores. These events are then amplified by Ca(2+) channels on canonical ER Ca(2+) stores to generate physiologically relevant global Ca(2+) signals. Coupling likely occurs at microdomains formed at membrane contact sites between acidic organelles and the ER. Molecular analyses and computational modelling suggest heterogeneity in the composition of these contacts and predicted Ca(2+) microdomain behaviour. Conversely, acidic organelles might also locally amplify and temper ER-evoked Ca(2+) signals. Ca(2+) microdomains between distinct Ca(2+) stores are thus likely to be integral to the genesis of complex Ca(2+) signals. PMID:25866010

  11. The effect of temperature and wing morphology on quantitative genetic variation in the cricket Gryllus firmus, with an appendix examining the statistical properties of the Jackknife-MANOVA method of matrix comparison.

    PubMed

    Bégin, M; Roff, D A; Debat, V

    2004-11-01

    We investigated the effect of temperature and wing morphology on the quantitative genetic variances and covariances of five size-related traits in the sand cricket, Gryllus firmus. Micropterous and macropterous crickets were reared in the laboratory at 24, 28 and 32 degrees C. Quantitative genetic parameters were estimated using a nested full-sib family design, and (co)variance matrices were compared using the T method, Flury hierarchy and Jackknife-manova method. The results revealed that the mean phenotypic value of each trait varied significantly among temperatures and wing morphs, but temperature reaction norms were not similar across all traits. Micropterous individuals were always smaller than macropterous individuals while expressing more phenotypic variation, a finding discussed in terms of canalization and life-history trade-offs. We observed little variation between the matrices of among-family (co)variation corresponding to each combination of temperature and wing morphology, with only one matrix of six differing in structure from the others. The implications of this result are discussed with respect to the prediction of evolutionary trajectories.

  12. B chromosomes of Aegilops speltoides are enriched in organelle genome-derived sequences.

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    SciTech Connect

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

    1983-12-01

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

  15. Selective molecular transport through the protein shell of a bacterial microcompartment organelle.

    PubMed

    Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; Sawaya, Michael R; Sinha, Sharmistha; Yeates, Todd O; Bobik, Thomas A

    2015-03-10

    Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.

  16. From Organelle to Protein Gel: A 6-Wk Laboratory Project on Flagellar Proteins

    PubMed Central

    Graziano, Mary R.

    2006-01-01

    Research suggests that undergraduate students learn more from lab experiences that involve longer-term projects. We have developed a one-semester laboratory sequence aimed at sophomore-level undergraduates. In designing this curriculum, we focused on several educational objectives: 1) giving students a feel for the scientific research process, 2) introducing them to commonly used lab techniques, and 3) building skills in both data analysis and scientific writing. Over the course of the semester, students carry out two project-based lab experiences and write two substantial lab reports modeled on primary literature. Student assessment data indicate that this lab curriculum achieved these objectives. This article describes the first of these projects, which uses the biflagellate alga Chlamydomonas reinhardtii to introduce students to the study of flagellar motility, protein synthesis, microtubule polymerization, organelle assembly, and protein isolation and characterization. PMID:17012215

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

    PubMed

    Nikitin, V A

    2007-01-01

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

  18. New insights into an old organelle: meeting report on biology of cilia and flagella.

    PubMed

    Sengupta, Piali; Barr, Maureen M

    2014-06-01

    The rising interest of the scientific community in cilia biology was evident from the fact that registration for the third FASEB conference on 'The Biology of Cilia and Flagella' closed out before the early bird deadline. Cilia and flagella are organelles of profound medical importance; defects in their structure or function result in a plethora of human diseases called ciliopathies. 240 clinicians and basic scientists from around the world gathered from 23 June 2013 to 28 June 2013 at Sheraton at the Falls, Niagara Falls, NY to present and discuss their research on this intensely studied subcellular structure. The meeting was organized by Gregory Pazour (University of Massachusetts Medical School), Bradley Yoder (University of Alabama-Birmingham), and Maureen Barr (Rutgers University) and was sponsored by the Federation of American Societies for Experimental Biology (FASEB). Here, we report highlights, points of discussion, and emerging themes from this exciting meeting. PMID:24612344

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

    PubMed Central

    Nüsse, Oliver

    2011-01-01

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

  20. Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles.

    PubMed

    Vaisberg, E A; Grissom, P M; McIntosh, J R

    1996-05-01

    We describe two dynein heavy chain (DHC)-like polypeptides (DHCs 2 and 3) that are distinct from the heavy chain of conventional cytoplasmic dynein (DHC1) but are expressed in a variety of mammalian cells that lack axonemes. DHC2 is a distant member of the "cytoplasmic" branch of the dynein phylogenetic tree, while DHC3 shares more sequence similarity with dynein-like polypeptides that have been thought to be axonemal. Each cytoplasmic dynein is associated with distinct cellular organelles. DHC2 is localized predominantly to the Golgi apparatus. Moreover, the Golgi disperses upon microinjection of antibodies to DHC2, suggesting that this motor is involved in establishing proper Golgi organization. DCH3 is associated with as yet unidentified structures that may represent transport intermediates between two or more cytoplasmic compartments. Apparently, specific cytoplasmic dyneins, like individual members of the kinesin superfamily, play unique roles in the traffic of cytomembranes.

  1. Complete circular DNA in the mitochondria-like organelles of Blastocystis hominis.

    PubMed

    Wawrzyniak, Ivan; Roussel, Michaël; Diogon, Marie; Couloux, Arnaud; Texier, Catherine; Tan, Kevin S W; Vivarès, Christian P; Delbac, Frédéric; Wincker, Patrick; El Alaoui, Hicham

    2008-10-01

    Blastocystis hominis is an anaerobic parasite of the human intestinal tract belonging to the Stramenopile group. Using genome sequencing project data, we describe here the complete sequence of a 29,270-bp circular DNA molecule that presents mitochondrial features (such as oxidative phosphorylation complex I subunits) but lacks complexes III, IV and V. Transmission electron microscopy analyses reveal that this molecule, as well as mitochondrial (NADH:ubiquinone oxidoreductase subunit 7 (NAD7), beta-succinyl-CoA synthetase (beta-SCS)) and hydrogenosomal (pyruvate ferredoxin oxido-reductase (PFOR), iron-hydrogenase) proteins, are located within double-membrane surrounded-compartments known as mitochondria-like organelles (MLOs). As there is no evidence for hydrogen production by this organism, we suggest that MLOs are more likely anaerobic mitochondria. PMID:18694756

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

    PubMed

    Margulis, L

    1975-01-01

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

  3. Microbodies from spirogyra: organelles of a filamentous alga similar to leaf peroxisomes.

    PubMed

    Stabenau, H

    1976-11-01

    Organelles from Spirogyra cells were separated on a linear sucrose gradient. After centrifugation, most of the protein was found in the top fraction. Two minor protein peaks at density (g/cm(3)) 1.17 and 1.21 were due to chloroplast particles and mitochondria, respectively. Although there was an extremely low concentration of protein at density 1.25 g/cm(3), a major part of the activity of glycolate oxidase was found in this region. The enzyme was able to transfer electrons to O(2) and only lost 12% of its activity in the presence of 1 mm cyanide. As documented by electron micrographs, microbodies moved to density 1.25 g/cm(3) during centrifugation. This observation, as well as the fact that high activities of hydroxypyruvate reductase and catalase were also found at the same density, suggest that the microbodies from Spirogyra are similar to those in green leaves of higher plants.

  4. Enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers.

    PubMed

    Parodi, Alessandro; Corbo, Claudia; Cevenini, Armando; Molinaro, Roberto; Palomba, Roberto; Pandolfi, Laura; Agostini, Marco; Salvatore, Francesco; Tasciotti, Ennio

    2015-07-01

    Nanocarriers are designed to specifically accumulate in diseased tissues. In this context, targeting of intracellular compartments was shown to enhance the efficacy of many drugs and to offer new and more effective therapeutic approaches. This is especially true for therapies based on biologicals that must be encapsulated to favor cell internalization, and to avoid intracellular endosomal sequestration and degradation of the payload. In this review, we discuss specific surface modifications designed to achieve cell cytoplasm delivery and to improve targeting of major organelles; we also discuss the therapeutic applications of these approaches. Last, we describe some integrated strategies designed to sequentially overcome the biological barriers that separate the site of administration from the cell cytoplasm, which is the drug's site of action.

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

    NASA Astrophysics Data System (ADS)

    Hoffmann, Julia; Fickentscher, Rolf; Weiss, Matthias

    2015-02-01

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

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

    PubMed

    Wainstein, Ehud; Seger, Rony

    2016-04-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-06-28

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

  9. Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora.

    PubMed

    Nowack, Eva C M; Grossman, Arthur R

    2012-04-01

    Endosymbiotic acquisition of bacteria by a protist, with subsequent evolution of the bacteria into mitochondria and plastids, had a transformative impact on eukaryotic biology. Reconstructing events that created a stable association between endosymbiont and host during the process of organellogenesis--including establishment of regulated protein import into nascent organelles--is difficult because they date back more than 1 billion years. The amoeba Paulinella chromatophora contains nascent photosynthetic organelles of more recent evolutionary origin (∼60 Mya) termed chromatophores (CRs). After the initial endosymbiotic event, the CR genome was reduced to approximately 30% of its presumed original size and more than 30 expressed genes were transferred from the CR to the amoebal nuclear genome. Three transferred genes--psaE, psaK1, and psaK2--encode subunits of photosystem I. Here we report biochemical evidence that PsaE, PsaK1, and PsaK2 are synthesized in the amoeba cytoplasm and traffic into CRs, where they assemble with CR-encoded subunits into photosystem I complexes. Additionally, our data suggest that proteins routed to CRs pass through the Golgi apparatus. Whereas genome reduction and transfer of genes from bacterial to host genome have been reported to occur in other obligate bacterial endosymbioses, this report outlines the import of proteins encoded by such transferred genes into the compartment derived from the bacterial endosymbiont. Our study showcases P. chromatophora as an exceptional model in which to study early events in organellogenesis, and suggests that protein import into bacterial endosymbionts might be a phenomenon much more widespread than currently assumed.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2007-01-01

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

  14. Microscopy with spatial filtering for monitoring subcellular morphology

    NASA Astrophysics Data System (ADS)

    Zheng, Jing-Yi

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

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

    SciTech Connect

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

    2014-12-02

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

  16. Dual targeting of a processing peptidase into both endosymbiotic organelles mediated by a transport signal of unusual architecture.

    PubMed

    Baudisch, Bianca; Klösgen, Ralf Bernd

    2012-03-01

    As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts are encoded in the nucleus and synthesized in the cytosol as precursor proteins carrying N-terminal transport signals for the 're-import' into the respective target organelle. Most of these transport signals are monospecific, although some of them have dual targeting properties, that is, they are recognized both by mitochondria and by chloroplasts as target organelles. We have identified alpha-MPP2, one of the two isoforms of the substrate binding subunit of mitochondrial processing peptidase of Arabidopsis thaliana, as a novel member of this class of nuclear-encoded organelle proteins. As demonstrated by in organello transport experiments with isolated organelles and by in vivo localization studies employing fluorescent chimeric reporter proteins, the N-terminal region of the alpha-MPP2 precursor comprises transport signals for the import into mitochondria as well as into chloroplasts. Both signals are found within the N-terminal 79 residues of the precursor protein, where they occupy partly separated and partly overlapping regions. Deletion mapping combined with in organello and in vivo protein transport studies demonstrate an unusual architecture of this transport signal, suggesting a composition of three functionally separated domains.

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

    ERIC Educational Resources Information Center

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

    2016-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

    Bonifacino, Juan S

    2004-12-01

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

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

    DOE PAGES

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

    2014-12-02

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

  1. High-throughput imaging of heterogeneous cell organelles with an X-ray laser (CXIDB ID 25)

    SciTech Connect

    Hantke, Max, F.

    2014-11-17

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

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

    PubMed Central

    MAYHEW, TERRY M.

    1999-01-01

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

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

    PubMed

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

    2016-02-01

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

  4. Ultrastructure of the intercalated body, a novel organelle associated with fluid forming cells in the organ of Corti.

    PubMed

    Sobkowicz, H M; Holy, J; Scott, G L

    1990-07-01

    The intercalated body is a newly discovered organelle in the inner and outer spiral sulcus cells of the mouse organ of Corti. The organelle was found in the cochleas of 14-day and older intact mice and in organs in culture of corresponding ages. The organelle consists of a stack of interconnected cisternae of endoplasmic reticulum and of membrane bound rodlets that are intercalated between, and run parallel to, the cisternae. The cisternal membranes are predominantly smooth, but some may display ribosomes. Most rodlets are from 1 to 2 microns long, about 0.1 micron wide, and contain electron dense material. Mitochondria are commonly associated with or incorporated into the organelle. Some electron micrographs suggest that the rodlets may originate from modified mitochondria. It is our impression that the formation of the organelle begins with the apposition of cisternae and mitochondria. Cisternal-associated mitochondria appear to constrict, elongate, and lose their inner membranes. In both the intact animal and in culture, the cells of the inner and outer spiral sulci display microvilli, apical junctional complexes, lateral intercellular spaces containing interdigitating cell processes, and appear to be involved in fluid formation. Moreover, in culture, the cells of inner and outer spiral sulci as well as some cells proliferating in the outgrowth zone participate in fluid formation, producing large fluid pockets. All these cells commonly contain intercalated bodies. It is possible that in the intact animal, as in culture, intercalated bodies may play a role in fluid regulation in the immediate vicinity of the hair cells. PMID:2374037

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

    PubMed

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

    2006-03-17

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

  6. Overexpression of a mutant form of EhRabA, a unique Rab GTPase of Entamoeba histolytica, alters endoplasmic reticulum morphology and localization of the Gal/GalNAc adherence lectin.

    PubMed

    Welter, B H; Temesvari, L A

    2009-07-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.

  7. The Flagellum Attachment Zone: 'The Cellular Ruler' of Trypanosome Morphology.

    PubMed

    Sunter, Jack D; Gull, Keith

    2016-04-01

    A defining feature of Trypanosoma brucei cell shape is the lateral attachment of the flagellum to the cell body, mediated by the flagellum attachment zone (FAZ). The FAZ is a complex cytoskeletal structure that connects the flagellum skeleton through two membranes to the cytoskeleton. The FAZ acts as a 'cellular ruler' of morphology by regulating cell length and organelle position and is therefore critical for both cell division and life cycle differentiations. Here we provide an overview of the advances in our understanding of the composition, assembly, and function of the FAZ. PMID:26776656

  8. Shifts in oxidation states of cerium oxide nanoparticles detected inside intact hydrated cells and organelles

    PubMed Central

    Szymanski, Craig J.; Munusamy, Prabhakaran; Mihai, Cosmin; Xie, Yumei; Hu, Dehong; Gilles, Mary K.; Tyliszczak, Tolek; Thevuthasan, Suntharampillai; Baer, Donald R.; Orr, Galya

    2015-01-01

    Cerium oxide nanoparticles (CNPs) have been shown to induce diverse biological effects, ranging from toxic to beneficial. The beneficial effects have been attributed to the potential antioxidant activity of CNPs via certain redox reactions, depending on their oxidation state or Ce3+/Ce4+ ratio. However, this ratio is strongly dependent on the environment and age of the nanoparticles and it is unclear whether and how the complex intracellular environment impacts this ratio and the possible redox reactions of CNPs. To identify any changes in the oxidation state of CNPs in the intracellular environment and better understand their intracellular reactions, we directly quantified the oxidation states of CNPs outside and inside intact hydrated cells and organelles using correlated scanning transmission x-ray and super resolution fluorescence microscopies. By analyzing hundreds of small CNP aggregates, we detected a shift to a higher Ce3+/Ce4+ ratio in CNPs inside versus outside the cells, indicating a net reduction of CNPs in the intracellular environment. We further found a similar ratio in the cytoplasm and in the lysosomes, indicating that the net reduction occurs earlier in the internalization pathway. Together with oxidative stress and toxicity measurements, our observations identify a net reduction of CNPs in the intracellular environment, which is consistent with their involvement in potentially beneficial oxidation reactions, but also point to interactions that can negatively impact the health of cells. PMID:26056725

  9. Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation.

    PubMed

    Ramadass, Mahalakshmi; Catz, Sergio D

    2016-09-01

    Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion.

  10. Increase number of mitochondrion-like organelle in symptomatic Blastocystis subtype 3 due to metronidazole treatment.

    PubMed

    Raman, Kalyani; Kumar, Suresh; Chye, Tan Tian

    2016-01-01

    Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery.

  11. Increase number of mitochondrion-like organelle in symptomatic Blastocystis subtype 3 due to metronidazole treatment.

    PubMed

    Raman, Kalyani; Kumar, Suresh; Chye, Tan Tian

    2016-01-01

    Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery. PMID:26481491

  12. Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae.

    PubMed

    Suresh, Harsha Garadi; da Silveira Dos Santos, Aline Xavier; Kukulski, Wanda; Tyedmers, Jens; Riezman, Howard; Bukau, Bernd; Mogk, Axel

    2015-05-01

    Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum-, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum-mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.

  13. Diminished organelle motion in murine Kupffer cells during the erythrocytic stage of malaria

    PubMed Central

    Bellows, Charles F.; Molina, Ramon M.; Brain, Joseph D.

    2011-01-01

    Parasitized erythrocytes are ingested by murine hepatic macrophages during malaria infection. We non-invasively monitored how this altered the motion of intracellular phagosomes in Kupffer cells using magnetometry. Submicrometric γFe2O3 particles were injected prior to malaria infection. They were cleared from the blood, primarily by Kupffer cells, and retained within their phagosomes. The mice were periodically magnetized. After removing this external magnet, the aligned iron particles created a remnant magnetic field (RMF) which then decayed (relaxation), reflecting the motion of particle-containing phagosomes. After baseline measurements of relaxation, the mice were injected intravenously with Plasmodium chabaudi-parasitized or normal murine red blood cells (RBCs). During the next 15 days, relaxation measurements, parasitaemia and haematocrit values were monitored. At 6 days post injection with 3 × 107 parasitized RBCs, relaxation rates had decreased. At this time, all mice had parasitaemias greater than 58 per cent and haematocrits less than 20 per cent. At day 7, while the parasitaemias were declining, the rate of relaxation continued to decrease. Throughout the experiment, relaxation remained constant in animals injected with normal RBCs. Electron microscopy revealed Kupffer cells filled with damaged and parasitized erythrocytes, and haemoglobin degradation pigment. We conclude that ingestion and metabolism of parasitized erythrocytes by liver macrophages during malaria infection decreases their organelle motion with likely consequences of compromised host defences. PMID:21068031

  14. Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics.

    PubMed

    Piro, Amalia; Serra, Ilia Anna; Spadafora, Antonia; Cardilio, Monica; Bianco, Linda; Perrotta, Gaetano; Santos, Rui; Mazzuca, Silvia

    2015-12-01

    Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses.

  15. The dynamics of the mitochondrial organelle as a potential therapeutic target

    PubMed Central

    Anne Stetler, R; Leak, Rehana K; Gao, Yanqin; Chen, Jun

    2013-01-01

    Mitochondria play a central role in cell fate after stressors such as ischemic brain injury. The convergence of intracellular signaling pathways on mitochondria and their release of critical factors are now recognized as a default conduit to cell death or survival. Besides the individual processes that converge on or emanate from mitochondria, a mitochondrial organellar response to changes in the cellular environment has recently been described. Whereas mitochondria have previously been perceived as a major center for cellular signaling, one can postulate that the organelle's dynamics themselves affect cell survival. This brief perspective review puts forward the concept that disruptions in mitochondrial dynamics—biogenesis, clearance, and fission/fusion events—may underlie neural diseases and thus could be targeted as neuroprotective strategies in the context of ischemic injury. To do so, we present a general overview of the current understanding of mitochondrial dynamics and regulation. We then review emerging studies that correlate mitochondrial biogenesis, mitophagy, and fission/fusion events with neurologic disease and recovery. An overview of the system as it is currently understood is presented, and current assessment strategies and their limitations are discussed. PMID:23093069

  16. Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

    PubMed Central

    Suresh, Harsha Garadi; da Silveira dos Santos, Aline Xavier; Kukulski, Wanda; Tyedmers, Jens; Riezman, Howard; Bukau, Bernd; Mogk, Axel

    2015-01-01

    Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes. PMID:25761633

  17. Adjustment of the osmolality of Percoll for the isopycnic separation of cells and cell organelles.

    PubMed

    Vincent, R; Nadeau, D

    1984-09-01

    The addition of 1 part of 1.5 M NaCl or 2.5 M sucrose (10 X concentrate) to 9 parts of Percoll produces a stock solution that is hypertonic (350-360 mOsm/kg H2O). Because the osmolality is a critical variable in the isopycnic separation of cells and cell organelles, the factors accountable for this hypertonicity were investigated. Percoll, a colloidal suspension of silica particles coated with polyvinylpyrrolidone, can be described as a medium composed of two distinct compartments, an aqueous phase and a solid phase. According to this model, solutes (e.g., NaCl, sucrose) should have access to the aqueous phase and add to the intrinsic osmolality of Percoll, but should be excluded from the solid phase. In order to verify this hypothesis, mathematical equations were derived and tested. It was found that the ratio of the aqueous volume to the total volume of Percoll (R value) was dependent on the ionic strength of the stock solution. With this parameter, the osmolality of Percoll stock solutions could be predicted (+/-2%) and, consequently, one could calculate the proper dilution to be used with saline, culture medium, or sucrose concentrates to obtain a truly isotonic Percoll stock solution (congruent to 290 mOsm/kg H2O). The relative importance of an accurate control of the osmolality in the preparation of density gradients made up of Percoll is also discussed.

  18. Shifts in oxidation states of cerium oxide nanoparticles detected inside intact hydrated cells and organelles

    SciTech Connect

    Szymanski, Craig J.; Munusamy, Prabhakaran; Mihai, Cosmin; Xie, Yumei; Hu, Dehong; Gilles, Marry K.; Tyliszczak, T.; Thevuthasan, Suntharampillai; Baer, Donald R.; Orr, Galya

    2015-09-01

    Cerium oxide nanoparticles (CNPs) have been shown to induce diverse biological effects, ranging from toxic to beneficial. The beneficial effects have been attributed to the potential antioxidant activity of CNPs via certain redox reactions, depending on their oxidation state or Ce3+/Ce4+ ratio. However, this ratio is strongly dependent on the environment and age of the nanoparticles and it is unclear whether and how the complex intracellular environment impacts this ratio and the possible redox reactions of CNPs. To identify any changes in the oxidation state of CNPs in the intracellular environment and better understand their intracellular reactions, we directly quantified the oxidation states of CNPs outside and inside intact hydrated cells and organelles using correlated scanning transmission x-ray and super resolution fluorescence microscopies. By analyzing hundreds of small CNP aggregates, we detected a shift to a higher Ce3+/Ce4+ ratio in CNPs inside versus outside the cells, indicating a net reduction of CNPs in the intracellular environment. We further found a similar ratio in the cytoplasm and in the lysosomes, indicating that the net reduction occurs earlier in the internalization pathway. Together with oxidative stress and toxicity measurements, our observations identify a net reduction of CNPs in the intracellular environment, which is consistent with their involvement in potentially beneficial oxidation reactions, but also point to interactions that can negatively impact the health of cells.

  19. Prevention of atherosclerosis by bioactive palmitoleate through suppression of organelle stress and inflammasome activation.

    PubMed

    Çimen, Ismail; Kocatürk, Begüm; Koyuncu, Seda; Tufanlı, Özlem; Onat, Umut I; Yıldırım, Asli D; Apaydın, Onur; Demirsoy, Şeyma; Aykut, Zaliha G; Nguyen, Uyen T; Watkins, Steven M; Hotamışlıgil, Gökhan S; Erbay, Ebru

    2016-09-28

    De novo lipogenesis (DNL), the conversion of glucose and other substrates to lipids, is often associated with ectopic lipid accumulation, metabolic stress, and insulin resistance, especially in the liver. However, organ-specific DNL can also generate distinct lipids with beneficial metabolic bioactivity, prompting a great interest in their use for the treatment of metabolic diseases. Palmitoleate (PAO), one such bioactive lipid, regulates lipid metabolism in liver and improves glucose utilization in skeletal muscle when it is generated de novo from the obese adipose tissue. We show that PAO treatment evokes an overall lipidomic remodeling of the endoplasmic reticulum (ER) membranes in macrophages and mouse tissues, which is associated with resistance of the ER to hyperlipidemic stress. By preventing ER stress, PAO blocks lipid-induced inflammasome activation in mouse and human macrophages. Chronic PAO supplementation also lowers systemic interleukin-1β (IL-1β) and IL-18 concentrations in vivo in hyperlipidemic mice. Moreover, PAO prevents macrophage ER stress and IL-1β production in atherosclerotic plaques in vivo, resulting in a marked reduction in plaque macrophages and protection against atherosclerosis in mice. These findings demonstrate that oral supplementation with a product of DNL such as PAO can promote membrane remodeling associated with metabolic resilience of intracellular organelles to lipid stress and limit the progression of atherosclerosis. These findings support therapeutic PAO supplementation as a potential preventive approach against complex metabolic and inflammatory diseases such as atherosclerosis, which warrants further studies in humans. PMID:27683551

  20. Why does the light-gradient photovoltage from photosynthetic organelles show a wavelength-dependent polarity?

    PubMed Central

    Paillotin, G; Dobek, A; Breton, J; Leibl, W; Trissl, H W

    1993-01-01

    The light-gradient photovoltage from photosynthetic organisms and organelles is thought to arise from the primary charge separation in the reaction centers. The current explanation of the effect is the stronger excitation of the membrane side of a vesicle facing the light source than the one on the opposite side. Together with the known orientation of reaction centers, this explanation predicts unequivocally the polarity of the photovoltage. However, a polarity opposite to the one expected has often been reported. A dependence of the polarity on the wavelength has been published but no explanation was given (Gräber, P., and H.-W. Trissl. 1981. FEBS Lett. 123:95-99). Here we report on a theoretical treatment of light propagation and interference in pigmented and nonpigmented multilayers. A model calculation is carried out for a pair of membranes, demonstrating the wavelength-dependent light distribution as well as the relative photovoltage and its polarity. When the membranes contain no chromophores or when the absorption coefficient is low, the predicted polarity to that expected from a simple macroscopic absorption behavior. The model is tested by comparing new photovoltage data obtained at 532 nm as well as in the blue and red absorption bands of chlorophyll in chloroplasts. It is concluded that outside the main absorption bands the amplitude and polarity of the photovoltage is determined by the ratio of the refractive indices of the membrane and the medium. PMID:8369444

  1. Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

    PubMed Central

    Pramanik, Smritimoy; Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2014-01-01

    Important biological reactions involving nucleic acids occur near the surface of membranes such as the nuclear membrane (NM) and rough endoplasmic reticulum (ER); however, the interactions between biomembranes and nucleic acids are poorly understood. We report here that transcription was facilitated in solution with liposomes, which mimic a biomembrane surface, relative to the reaction in a homogeneous aqueous solution when the template was able to form a G-quadruplex. The G-quadruplex is known to be an inhibitor of transcription, but the stability of the G-quadruplex was decreased at the liposome surface because of unfavourable enthalpy. The destabilization of the G-quadruplex was greater at the surface of NM- and ER-mimicking liposomes than at the surfaces of liposomes designed to mimic other organelles. Thermodynamic analyses revealed that the G-rich oligonucleotides adopted an extended structure at the liposome surface, whereas in solution the compact G-quadruplex was formed. Our data suggest that changes in structure and stability of nucleic acids regulate biological reactions at membrane surfaces. PMID:25336617

  2. Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

    PubMed Central

    Huang, Guozhong; Ulrich, Paul N.; Storey, Melissa; Johnson, Darryl; Tischer, Julie; Tovar, Javier A.; Moreno, Silvia N. J.; Orlando, Ron; Docampo, Roberto

    2014-01-01

    Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate. PMID:25503798

  3. Lipid Droplets: A Key Cellular Organelle Associated with Cancer Cell Survival under Normoxia and Hypoxia

    PubMed Central

    Koizume, Shiro; Miyagi, Yohei

    2016-01-01

    The Warburg effect describes the phenomenon by which cancer cells obtain energy from glycolysis even under normoxic (O2-sufficient) conditions. Tumor tissues are generally exposed to hypoxia owing to inefficient and aberrant vasculature. Cancer cells have multiple molecular mechanisms to adapt to such stress conditions by reprogramming the cellular metabolism. Hypoxia-inducible factors are major transcription factors induced in cancer cells in response to hypoxia that contribute to the metabolic changes. In addition, cancer cells within hypoxic tumor areas have reduced access to serum components such as nutrients and lipids. However, the effect of such serum factor deprivation on cancer cell biology in the context of tumor hypoxia is not fully understood. Cancer cells are lipid-rich under normoxia and hypoxia, leading to the increased generation of a cellular organelle, the lipid droplet (LD). In recent years, the LD-mediated stress response mechanisms of cancer cells have been revealed. This review focuses on the production and functions of LDs in various types of cancer cells in relation to the associated cellular environment factors including tissue oxygenation status and metabolic mechanisms. This information will contribute to the current understanding of how cancer cells adapt to diverse tumor environments to promote their survival. PMID:27589734

  4. Shifts in oxidation states of cerium oxide nanoparticles detected inside intact hydrated cells and organelles.

    PubMed

    Szymanski, Craig J; Munusamy, Prabhakaran; Mihai, Cosmin; Xie, Yumei; Hu, Dehong; Gilles, Mary K; Tyliszczak, Tolek; Thevuthasan, Suntharampillai; Baer, Donald R; Orr, Galya

    2015-09-01

    Cerium oxide nanoparticles (CNPs) have been shown to induce diverse biological effects, ranging from toxic to beneficial. The beneficial effects have been attributed to the potential antioxidant activity of CNPs via certain redox reactions, depending on their oxidation state or Ce(3+)/Ce(4+) ratio. However, this ratio is strongly dependent on the environment and age of the nanoparticles and it is unclear whether and how the complex intracellular environment impacts this ratio and the possible redox reactions of CNPs. To identify any changes in the oxidation state of CNPs in the intracellular environment and better understand their intracellular reactions, we directly quantified the oxidation states of CNPs outside and inside intact hydrated cells and organelles using correlated scanning transmission x-ray and super resolution fluorescence microscopies. By analyzing hundreds of small CNP aggregates, we detected a shift to a higher Ce(3+)/Ce(4+) ratio in CNPs inside versus outside the cells, indicating a net reduction of CNPs in the intracellular environment. We further found a similar ratio in the cytoplasm and in the lysosomes, indicating that the net reduction occurs earlier in the internalization pathway. Together with oxidative stress and toxicity measurements, our observations identify a net reduction of CNPs in the intracellular environment, which is consistent with their involvement in potentially beneficial oxidation reactions, but also point to interactions that can negatively impact the health of the cells.

  5. Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation.

    PubMed

    Ramadass, Mahalakshmi; Catz, Sergio D

    2016-09-01

    Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion. PMID:27558339

  6. Advances and New Concepts in Alcohol-Induced Organelle Stress, Unfolded Protein Responses and Organ Damage.

    PubMed

    Ji, Cheng

    2015-01-01

    Alcohol is a simple and consumable biomolecule yet its excessive consumption disturbs numerous biological pathways damaging nearly all organs of the human body. One of the essential biological processes affected by the harmful effects of alcohol is proteostasis, which regulates the balance between biogenesis and turnover of proteins within and outside the cell. A significant amount of published evidence indicates that alcohol and its metabolites directly or indirectly interfere with protein homeostasis in the endoplasmic reticulum (ER) causing an accumulation of unfolded or misfolded proteins, which triggers the unfolded protein response (UPR) leading to either restoration of homeostasis or cell death, inflammation and other pathologies under severe and chronic alcohol conditions. The UPR senses the abnormal protein accumulation and activates transcription factors that regulate nuclear transcription of genes related to ER function. Similarly, this kind of protein stress response can occur in other cellular organelles, which is an evolving field of interest. Here, I review recent advances in the alcohol-induced ER stress response as well as discuss new concepts on alcohol-induced mitochondrial, Golgi and lysosomal stress responses and injuries. PMID:26047032

  7. Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites?

    PubMed

    Mercier, Corinne; Adjogble, Koku D Z; Däubener, Walter; Delauw, Marie-France-Cesbron

    2005-07-01

    Together with micronemes and rhoptries, dense granules are specialised secretory organelles of Apicomplexa parasites. Among Apicomplexa, Plasmodium represents a model of parasites propagated by way of an insect vector, whereas Toxoplasma is a model of food borne protozoa forming cysts. Through comparison of both models, this review summarises data accumulated over recent years on alternative strategies chosen by these parasites to develop within a parasitophorous vacuole and explores the role of dense granules in this process. One of the characteristics of the Plasmodium erythrocyte stages is to export numerous parasite proteins into both the host cell cytoplasm and/or plasma membrane via the vacuole used as a step trafficking compartment. Whether this feature can be correlated to few storage granules and a restricted number of dense granule proteins, is not yet clear. By contrast, the Toxoplasma developing vacuole is decorated by abundantly expressed dense granule proteins and is characterised by a network of membranous nanotubes. Although the exact function of most of these proteins remains currently unknown, recent data suggest that some of these dense granule proteins could be involved in building the intravacuolar membranous network. Conserved expression of the Toxoplasma dense granule proteins throughout most of the parasite stages suggests that they could also be key elements of the cyst formation.

  8. Advances and New Concepts in Alcohol-Induced Organelle Stress, Unfolded Protein Responses and Organ Damage

    PubMed Central

    Ji, Cheng

    2015-01-01

    Alcohol is a simple and consumable biomolecule yet its excessive consumption disturbs numerous biological pathways damaging nearly all organs of the human body. One of the essential biological processes affected by the harmful effects of alcohol is proteostasis, which regulates the balance between biogenesis and turnover of proteins within and outside the cell. A significant amount of published evidence indicates that alcohol and its metabolites directly or indirectly interfere with protein homeostasis in the endoplasmic reticulum (ER) causing an accumulation of unfolded or misfolded proteins, which triggers the unfolded protein response (UPR) leading to either restoration of homeostasis or cell death, inflammation and other pathologies under severe and chronic alcohol conditions. The UPR senses the abnormal protein accumulation and activates transcription factors that regulate nuclear transcription of genes related to ER function. Similarly, this kind of protein stress response can occur in other cellular organelles, which is an evolving field of interest. Here, I review recent advances in the alcohol-induced ER stress response as well as discuss new concepts on alcohol-induced mitochondrial, Golgi and lysosomal stress responses and injuries. PMID:26047032

  9. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole.

  10. Exosomes as Intercellular Signaling Organelles Involved in Health and Disease: Basic Science and Clinical Applications

    PubMed Central

    Corrado, Chiara; Raimondo, Stefania; Chiesi, Antonio; Ciccia, Francesco; De Leo, Giacomo; Alessandro, Riccardo

    2013-01-01

    Cell to cell communication is essential for the coordination and proper organization of different cell types in multicellular systems. Cells exchange information through a multitude of mechanisms such as secreted growth factors and chemokines, small molecules (peptides, ions, bioactive lipids and nucleotides), cell-cell contact and the secretion of extracellular matrix components. Over the last few years, however, a considerable amount of experimental evidence has demonstrated the occurrence of a sophisticated method of cell communication based on the release of specialized membranous nano-sized vesicles termed exosomes. Exosome biogenesis involves the endosomal compartment, the multivesicular bodies (MVB), which contain internal vesicles packed with an extraordinary set of molecules including enzymes, cytokines, nucleic acids and different bioactive compounds. In response to stimuli, MVB fuse with the plasma membrane and vesicles are released in the extracellular space where they can interact with neighboring cells and directly induce a signaling pathway or affect the cellular phenotype through the transfer of new receptors or even genetic material. This review will focus on exosomes as intercellular signaling organelles involved in a number of physiological as well as pathological processes and their potential use in clinical diagnostics and therapeutics. PMID:23466882

  11. Structure and Mechanisms of a Protein-Based Organelle in Escherichia coli

    SciTech Connect

    Tanaka, Shiho; Sawaya, Michael R.; Yeates, Todd O.

    2010-08-18

    Many bacterial cells contain proteinaceous microcompartments that act as simple organelles by sequestering specific metabolic processes involving volatile or toxic metabolites. Here we report the three-dimensional (3D) crystal structures, with resolutions between 1.65 and 2.5 angstroms, of the four homologous proteins (EutS, EutL, EutK, and EutM) that are thought to be the major shell constituents of a functionally complex ethanolamine utilization (Eut) microcompartment. The Eut microcompartment is used to sequester the metabolism of ethanolamine in bacteria such as Escherichia coli and Salmonella enterica. The four Eut shell proteins share an overall similar 3D fold, but they have distinguishing structural features that help explain the specific roles they play in the microcompartment. For example, EutL undergoes a conformational change that is probably involved in gating molecular transport through shell protein pores, whereas structural evidence suggests that EutK might bind a nucleic acid component. Together these structures give mechanistic insight into bacterial microcompartments.

  12. Unraveling the Secrets of Bacterial Adhesion Organelles Using Single-Molecule Force Spectroscopy

    NASA Astrophysics Data System (ADS)

    Axner, Ove; Björnham, Oscar; Castelain, Mickaël; Koutris, Efstratios; Schedin, Staffan; Fällman, Erik; Andersson, Magnus

    Many types of bacterium express micrometer-long attachment organelles (so-called pili) whose role is to mediate adhesion to host tissue. Until recently, little was known about their function in the adhesion process. Force-measuring optical tweezers (FMOT) have since then been used to unravel the biomechanical properties of various types of pili, primarily those from uropathogenic E. coli, in particular their force-vs.-elongation response, but lately also some properties of the adhesin are situated at the distal end of the pilus. This knowledge provides an understanding of how piliated bacteria can sustain external shear forces caused by rinsing processes, e.g., urine flow. It has been found that many types of pilus exhibit unique and complex force-vs.-elongation responses. It has been conjectured that their dissimilar properties impose significant differences in their ability to sustain external forces and that different types of pilus therefore have dissimilar predisposition to withstand different types of rinsing conditions. An understanding of these properties is of high importance since it can serve as a basis for finding new means to combat bacterial adhesion, including that caused by antibiotic-resistance bacteria. This work presents a review of the current status of the assessment of biophysical properties of individual pili on single bacteria exposed to strain/stress, primarily by the FMOT technique. It also addresses, for the first time, how the elongation and retraction properties of the rod couple to the adhesive properties of the tip adhesin.

  13. Selective molecular transport through the protein shell of a bacterial microcompartment organelle.

    PubMed

    Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; Sawaya, Michael R; Sinha, Sharmistha; Yeates, Todd O; Bobik, Thomas A

    2015-03-10

    Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins. PMID:25713376

  14. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

    PubMed

    Erbilgin, Onur; Sutter, Markus; Kerfeld, Cheryl A

    2016-03-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  15. Mitochondria from the salt-tolerant yeast Debaryomyces hansenii (halophilic organelles?).

    PubMed

    Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Luévano-Martínez, Luís A; Peña, Antonio; Uribe-Carvajal, Salvador

    2010-02-01

    The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na(+) and 10 mM K(+); these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca(2+) or Mg(2+). However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K(+) and Na(+), and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K(+)/Na(+)-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na(+) or K(+). In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a Delta Psi due to a low AOX activity. In D. hansenii mitochondria K(+) and Na(+) optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.

  16. Advances and New Concepts in Alcohol-Induced Organelle Stress, Unfolded Protein Responses and Organ Damage.

    PubMed

    Ji, Cheng

    2015-06-03

    Alcohol is a simple and consumable biomolecule yet its excessive consumption disturbs numerous biological pathways damaging nearly all organs of the human body. One of the essential biological processes affected by the harmful effects of alcohol is proteostasis, which regulates the balance between biogenesis and turnover of proteins within and outside the cell. A significant amount of published evidence indicates that alcohol and its metabolites directly or indirectly interfere with protein homeostasis in the endoplasmic reticulum (ER) causing an accumulation of unfolded or misfolded proteins, which triggers the unfolded protein response (UPR) leading to either restoration of homeostasis or cell death, inflammation and other pathologies under severe and chronic alcohol conditions. The UPR senses the abnormal protein accumulation and activates transcription factors that regulate nuclear transcription of genes related to ER function. Similarly, this kind of protein stress response can occur in other cellular organelles, which is an evolving field of interest. Here, I review recent advances in the alcohol-induced ER stress response as well as discuss new concepts on alcohol-induced mitochondrial, Golgi and lysosomal stress responses and injuries.

  17. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole. PMID:27311484

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

    NASA Astrophysics Data System (ADS)

    Hantke, Max F.; Hasse, Dirk; Maia, Filipe R. N. C.; Ekeberg, Tomas; John, Katja; Svenda, Martin; Loh, N. Duane; Martin, Andrew V.; Timneanu, Nicusor; Larsson, Daniel S. D.; van der Schot, Gijs; Carlsson, Gunilla H.; Ingelman, Margareta; Andreasson, Jakob; Westphal, Daniel; Liang, Mengning; Stellato, Francesco; Deponte, Daniel P.; Hartmann, Robert; Kimmel, Nils; Kirian, Richard A.; Seibert, M. Marvin; Mühlig, Kerstin; Schorb, Sebastian; Ferguson, Ken; Bostedt, Christoph; Carron, Sebastian; Bozek, John D.; Rolles, Daniel; Rudenko, Artem; Epp, Sascha; Chapman, Henry N.; Barty, Anton; Hajdu, Janos; Andersson, Inger

    2014-12-01

    We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.

  19. Organelle Size Scaling of the Budding Yeast Vacuole Is Tuned by Membrane Trafficking Rates

    PubMed Central

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

    2014-01-01

    Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates. PMID:24806931

  20. Assembly, Properties and Function of Synthetic Phase-Separated RNA/Protein Organelles

    NASA Astrophysics Data System (ADS)

    Taylor, Nicole; Elbaum, Shana; Stone, Howard; Brangwynne, Clifford

    2015-03-01

    Non-membrane bound RNA/protein (RNP) bodies play a key role in cellular RNA processing steps. Many RNA helicases, required for RNA processing, are key components of RNPs. Consistent with this, a purified RNA helicase, Laf-1, exhibits a salt and protein concentration dependent phase separation in vitro, resulting in liquid-like droplets. We use such synthetic RNPs to study the biophysics of RNP assembly, and to elucidate the link between their physical properties and function. To accomplish this, we are developing custom microfluidic devices to measure biophysical properties, nucleation and growth kinetics, and RNA processing function of droplets. We measure droplet viscosity by applying a shear stress to protein droplets that adhere to the channel wall; measurements are consistent with those taken using a particle microrheology approach. We also monitor and control protein droplet nucleation using oil/water emulsions. Our results provide a new platform for addressing how the cell regulates organelle assembly and properties through protein, RNA, and ATP concentration. We anticipate that these findings will offer insight into the contribution of RNPs in key RNA processing functions in the cell.

  1. Lipid Droplets: A Key Cellular Organelle Associated with Cancer Cell Survival under Normoxia and Hypoxia.

    PubMed

    Koizume, Shiro; Miyagi, Yohei

    2016-01-01

    The Warburg effect describes the phenomenon by which cancer cells obtain energy from glycolysis even under normoxic (O₂-sufficient) conditions. Tumor tissues are generally exposed to hypoxia owing to inefficient and aberrant vasculature. Cancer cells have multiple molecular mechanisms to adapt to such stress conditions by reprogramming the cellular metabolism. Hypoxia-inducible factors are major transcription factors induced in cancer cells in response to hypoxia that contribute to the metabolic changes. In addition, cancer cells within hypoxic tumor areas have reduced access to serum components such as nutrients and lipids. However, the effect of such serum factor deprivation on cancer cell biology in the context of tumor hypoxia is not fully understood. Cancer cells are lipid-rich under normoxia and hypoxia, leading to the increased generation of a cellular organelle, the lipid droplet (LD). In recent years, the LD-mediated stress response mechanisms of cancer cells have been revealed. This review focuses on the production and functions of LDs in various types of cancer cells in relation to the associated cellular environment factors including tissue oxygenation status and metabolic mechanisms. This information will contribute to the current understanding of how cancer cells adapt to diverse tumor environments to promote their survival. PMID:27589734

  2. Organelle size scaling of the budding yeast vacuole is tuned by membrane trafficking rates.

    PubMed

    Chan, Yee-Hung Mark; Marshall, Wallace F

    2014-05-01

    Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates.

  3. Doubly uniparental inheritance: two mitochondrial genomes, one precious model for organelle DNA inheritance and evolution.

    PubMed

    Passamonti, Marco; Ghiselli, Fabrizio

    2009-02-01

    Eukaryotes have exploited several mechanisms for organelle uniparental inheritance, so this feature arose and evolved independently many times in their history. Metazoans' mitochondria commonly experience strict maternal inheritance; that is, they are only transmitted by females. However, the most noteworthy exception comes from some bivalve mollusks, in which two mitochondrial lineages (together with their genomes) are inherited: one through females (F) and the other through males (M). M and F genomes show up to 30% sequence divergence. This inheritance mechanism is known as doubly uniparental inheritance (DUI), because both sexes inherit uniparentally their mitochondria. Here, we review what we know about this unusual system, and we propose a model for evolution of DUI that might account for its origin as sex determination mechanism. Moreover, we propose DUI as a choice model to address many aspects that should be of interest to a wide range of biological subfields, such as mitochondrial inheritance, mtDNA evolution and recombination, genomic conflicts, evolution of sex, and developmental biology. Actually, as research proceeds, mitochondria appear to have acquired a central role in many fundamental processes of life, which are not only in their metabolic activity as cellular power plants, such as cell signaling, fertilization, development, differentiation, ageing, apoptosis, and sex determination. A function of mitochondria in the origin and maintenance of sex has been also proposed.

  4. The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle

    PubMed Central

    Kerfeld, Cheryl A.

    2016-01-01

    Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. PMID:26959993

  5. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

  6. In situ morphology of the gut microbiota of wood-eating termites [Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki].

    PubMed Central

    Breznak, J A; Pankratz, H S

    1977-01-01

    Light microscopy and scanning and transmission electron microscopy were used to examine the in situ morphology of the gut microbiota of Reticulitermes flavipes and Caoptotermes formosanus. Laboratory-maintained termites were used and, for R. flavipes, specimens were also prepared immediately after collection from a natural infestation. The latter endeavor enabled a study of different castes and developmental stages of R. flavipes and revealed differences in the microbiota of field versus laboratory specimens. The termite paunch microbiota consisted of an abundance of morphologically diverse bacteria and protozoa. Thirteen bacterial morphotypes in the paunch were described in detail: seven were observed only in R. flavipes, three were observed only in C. formosanus, and three were common to both termite species. The paunch epithelium was densely colonized by bacteria, many of which possessed holdfast elements that secured them tightly to this tissue and to other bacterial cells. Besides bacteria, the protozoan Pyrsonympha vertens adhered to the paunch epithelium of R. flavipes by means of an attachment organelle. Cuplike indentations were present on the paunch epithelial surface and were sites of bacterial aggregation. Ultrastructural features of cups suggested their involvement in ion absorption. In addition to the paunch, the midgut was also colonized by bacteria that were situated between epithelial microvilli. Results suggest that bacteria are an integral part of the gut ecosystem. Images PMID:848959

  7. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain.

    PubMed

    Arnold, J E; Tipler, C; Laszlo, L; Hope, J; Landon, M; Mayer, R J

    1995-08-01

    The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.

  8. Periodic gene expression patterns during the highly synchronized cell nucleus and organelle division cycles in the unicellular red alga Cyanidioschyzon merolae.

    PubMed

    Fujiwara, Takayuki; Misumi, Osami; Tashiro, Kousuke; Yoshida, Yamato; Nishida, Keiji; Yagisawa, Fumi; Imamura, Sousuke; Yoshida, Masaki; Mori, Toshiyuki; Tanaka, Kan; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi

    2009-02-01

    Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon merolae. C. merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.

  9. Mitochondrial and plastid genomes of the colonial green alga Gonium pectorale give insights into the origins of organelle DNA architecture within the volvocales.

    PubMed

    Hamaji, Takashi; Smith, David R; Noguchi, Hideki; Toyoda, Atsushi; Suzuki, Masahiro; Kawai-Toyooka, Hiroko; Fujiyama, Asao; Nishii, Ichiro; Marriage, Tara; Olson, Bradley J S C; Nozaki, Hisayoshi

    2013-01-01

    Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion. PMID:23468928

  10. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  11. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    SciTech Connect

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-04-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of approx. = 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4/sup 0/C or in medium containing 5 ..mu..M colchicine or nocodazole at 37/sup 0/C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37/sup 0/C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures.

  12. Polymeric Nucleic Acid Vehicles Exploit Active Inter-Organelle Trafficking Mechanisms

    PubMed Central

    Fichter, Katye M.; Ingle, Nilesh. P.; McLendon, Patrick M.; Reineke, Theresa M.

    2013-01-01

    Materials that self-assemble with nucleic acids into nanocomplexes (polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect, (which slowly degrades via hydrolysis) and linear PEI, (which does not rapidly hydrolyze) to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h, and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical “proton sponge mechanism”. PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides (ODNs) to the Golgi and endoplasmic reticulum, which may be a route taken for nuclear delivery. However, Glycofect polyplexes demonstrated higher colocalization with the ER than PEI polyplexes and colocalization experiments indicate retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery. PMID:23234474

  13. Modeling Yeast Organelle Membranes and How Lipid Diversity Influences Bilayer Properties.

    PubMed

    Monje-Galvan, Viviana; Klauda, Jeffery B

    2015-11-17

    Membrane lipids are important for the health and proper function of cell membranes. We have improved computational membrane models for specific organelles in yeast Saccharomyces cerevisiae to study the effect of lipid diversity on membrane structure and dynamics. Previous molecular dynamics simulations were performed by Jo et al. [(2009) Biophys J. 97, 50-58] on yeast membrane models having six lipid types with compositions averaged between the endoplasmic reticulum (ER) and the plasma membrane (PM). We incorporated ergosterol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol lipids in our models to better describe the unique composition of the PM, ER, and trans-Golgi network (TGN) bilayers of yeast. Our results describe membrane structure based on order parameters (SCD), electron density profiles (EDPs), and lipid packing. The average surface area per lipid decreased from 63.8 ± 0.4 Å(2) in the ER to 47.1 ± 0.3 Å(2) in the PM, while the compressibility modulus (KA) varied in the opposite direction. The high SCD values for the PM lipids indicated a more ordered bilayer core, while the corresponding lipids in the ER and TGN models had lower parameters by a factor of at least 0.7. The hydrophobic core thickness (2DC) as estimated from EDPs is the thickest for PM, which is in agreement with estimates of hydrophobic regions of transmembrane proteins from the Orientation of Proteins in Membranes database. Our results show the importance of lipid diversity and composition on a bilayer's structural and mechanical properties, which in turn influences interactions with the proteins and membrane-bound molecules.

  14. Ricinosomes: an organelle for developmentally regulated programmed cell death in senescing plant tissues

    NASA Astrophysics Data System (ADS)

    Gietl, C.; Schmid, M.

    2001-02-01

    This review describes aspects of programmed cell death (PCD). Present research maps the enzymes involved and explores the signal transduction pathways involved in their synthesis. A special organelle (the ricinosome) has been discovered in the senescing endosperm of germinating castor beans (Ricinus communis) that develops at the beginning of PCD and delivers large amounts of a papain-type cysteine endopeptidase (CysEP) in the final stages of cellular disintegration. Castor beans store oil and proteins in a living endosperm surrounding the cotyledons. These stores are mobilized during germination and transferred into the cotyledons. PCD is initiated after this transfer is complete. The CysEP is synthesized in the lumen of the endoplasmic reticulum (ER) where it is retained by its C-terminal KDEL peptide as a rather inactive pro-enzyme. Large number of ricinosomes bud from the ER at the same time as the nuclear DNA is characteristically fragmented during PCD. The mitochondria, glyoxysomes and ribosomes are degraded in autophagic vacuoles, while the endopeptidase is activated by removal of the propeptide and the KDEL tail and enters the cytosol. The endosperm dries and detaches from the cotyledons. A homologous KDEL-tailed cysteine endopeptidase has been found in several senescing tissues; it has been localized in ricinosomes of withering day-lily petals and dying seed coats. Three genes for a KDEL-tailed cysteine endopeptidase have been identified in Arabidopsis. One is expressed in senescing ovules, the second in the vascular vessels and the third in maturing siliques. These genes open the way to exploring PCD in plants.

  15. Function of the Caenorhabditis elegans ABC Transporter PGP-2 in the Biogenesis of a Lysosome-related Fat Storage Organelle

    PubMed Central

    Schroeder, Lena K.; Kremer, Susan; Kramer, Maxwell J.; Currie, Erin; Kwan, Elizabeth; Watts, Jennifer L.; Lawrenson, Andrea L.

    2007-01-01

    Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(−) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase–containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(−) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red–stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red–stained fat in pgp-2(−) animals as well as insight into the specialized function of this lysosome-related organelle. PMID:17202409

  16. Development and disintegration of tapetum-specific lipid-accumulating organelles, elaioplasts and tapetosomes, in Arabidopsis thaliana and Brassica napus.

    PubMed

    Suzuki, Toshiya; Tsunekawa, Sonomi; Koizuka, Chie; Yamamoto, Kanta; Imamura, Jun; Nakamura, Kenzo; Ishiguro, Sumie

    2013-06-01

    The pollen coat covering the surface of pollen grains has many important roles for pollination. In Brassicaceae plants, the pollen coat components are synthesized and temporarily accumulated in two tapetum-specific organelles, the elaioplast and the tapetosome. Although many biochemical and electron microscopic analyses have been attempted, the structure and biogenesis of these organelles have not been fully elucidated. To resolve this problem, we performed live imaging of these organelles using two markers, FIB1a-GFP and GRP17-GFP. FIB1a is an Arabidopsis fibrillin, a structural protein of elaioplast plastoglobules. In transgenic Arabidopsis, fluorescence of FIB1a-GFP appeared in young elaioplasts, in which small plastoglobules were developing. However, the fluorescence disappeared in later stages, while enlargement of plastoglobules continued. GRP17 is an Arabidopsis oleopollenin, an oleosin-like protein in tapetosomes. Fluorescence microscopy of GRP17-GFP expressed in Arabidopsis and Brassica napus revealed that tapetosomes do not contain oleopollenin-coated vesicles but have an outer envelope, indicating that the tapetosome structure is distinct from seed oil bodies. Visualization of GRP17-GFP also demonstrated that the tapetal cells become protoplasts and migrate into locules before pollen coat formation, and provided live imaging of the foot formation between pollen grains and stigmatic papilla cells. PMID:23602096

  17. De novo assembly of the complete organelle genome sequences of azuki bean (Vigna angularis) using next-generation sequencers.

    PubMed

    Naito, Ken; Kaga, Akito; Tomooka, Norihiko; Kawase, Makoto

    2013-06-01

    Since chloroplasts and mitochondria are maternally inherited and have unique features in evolution, DNA sequences of those organelle genomes have been broadly used in phylogenetic studies. Thanks to recent progress in next-generation sequencer (NGS) technology, whole-genome sequencing can be easily performed. Here, using NGS data generated by Roche GS Titanium and Illumina Hiseq 2000, we performed a hybrid assembly of organelle genome sequences of Vigna angularis (azuki bean). Both the mitochondrial genome (mtDNA) and the chloroplast genome (cpDNA) of V. angularis have very similar size and gene content to those of V. radiata (mungbean). However, in structure, mtDNA sequences have undergone many recombination events after divergence from the common ancestor of V. angularis and V. radiata, whereas cpDNAs are almost identical between the two. The stability of cpDNAs and the variability of mtDNAs was further confirmed by comparative analysis of Vigna organelles with model plants Lotus japonicus and Arabidopsis thaliana.

  18. Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha

    PubMed Central

    Fokina, Anastasia V.; Chechenova, Maria B.; Karginov, Azamat V.; Ter-Avanesyan, Michael D.; Agaphonov, Michael O.

    2015-01-01

    Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole. PMID:26717478

  19. Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha.

    PubMed

    Fokina, Anastasia V; Chechenova, Maria B; Karginov, Azamat V; Ter-Avanesyan, Michael D; Agaphonov, Michael O

    2015-01-01

    Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole.

  20. Development and disintegration of tapetum-specific lipid-accumulating organelles, elaioplasts and tapetosomes, in Arabidopsis thaliana and Brassica napus.

    PubMed

    Suzuki, Toshiya; Tsunekawa, Sonomi; Koizuka, Chie; Yamamoto, Kanta; Imamura, Jun; Nakamura, Kenzo; Ishiguro, Sumie

    2013-06-01

    The pollen coat covering the surface of pollen grains has many important roles for pollination. In Brassicaceae plants, the pollen coat components are synthesized and temporarily accumulated in two tapetum-specific organelles, the elaioplast and the tapetosome. Although many biochemical and electron microscopic analyses have been attempted, the structure and biogenesis of these organelles have not been fully elucidated. To resolve this problem, we performed live imaging of these organelles using two markers, FIB1a-GFP and GRP17-GFP. FIB1a is an Arabidopsis fibrillin, a structural protein of elaioplast plastoglobules. In transgenic Arabidopsis, fluorescence of FIB1a-GFP appeared in young elaioplasts, in which small plastoglobules were developing. However, the fluorescence disappeared in later stages, while enlargement of plastoglobules continued. GRP17 is an Arabidopsis oleopollenin, an oleosin-like protein in tapetosomes. Fluorescence microscopy of GRP17-GFP expressed in Arabidopsis and Brassica napus revealed that tapetosomes do not contain oleopollenin-coated vesicles but have an outer envelope, indicating that the tapetosome structure is distinct from seed oil bodies. Visualization of GRP17-GFP also demonstrated that the tapetal cells become protoplasts and migrate into locules before pollen coat formation, and provided live imaging of the foot formation between pollen grains and stigmatic papilla cells.

  1. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair[C][W

    PubMed Central

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

    2010-01-01

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway. PMID:20551348

  2. A morphometric analysis of the redistribution of organelles in columella cells of horizontally-oriented roots of Zea mays

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1986-01-01

    In order to determine what structural changes in graviperceptive cells are associated with onset of root gravicurvature, the redistribution of organelles in columella cells of horizontally-oriented, graviresponding roots of Zea mays has been quantified. Root gravicurvature began by 15 min after reorientation, and did not involve significant changes in the (i) volume of individual columella cells or amyloplasts, (ii) relative volume of any cellular organelle, (iii) number of amyloplasts per columella cell, or (iv) surface area of cellular location of endoplasmic reticulum. Sedimentation of amyloplasts began within 1 to 2 min after reorientation, and was characterized by an intensely staining area of cytoplasm adjacent to the sedimenting amyloplasts. By 5 min after reorientation, amyloplasts were located in the lower distal corner of columella cells, and, by 15 min after reorientation, overlaid the entire length of the lower cell wall. No consistent contact between amyloplasts and any cellular structure was detected at any stage of gravicurvature. Centrally-located nuclei initially migrated upward in columella cells of horizontally-oriented roots, after which they moved to the proximal ends of the cells by 15 min after reorientation. No significant pattern of redistribution of vacuoles, mitochondria, dictyosomes, or hyaloplasm was detected that correlated with the onset of gravicurvature. These results indicate that amyloplasts and nuclei are the only organelles whose movements correlate positively with the onset of gravicurvature by primary roots of this cultivar of Zea mays.

  3. Morphological awareness in developmental dyslexia.

    PubMed

    Casalis, Séverine; Colé, Pascale; Sopo, Delphine

    2004-06-01

    This study examines morphological awareness in developmental dyslexia. While the poor phonological awareness of dyslexic children has been related to their difficulty in handling the alphabetical principle, less is known about their morphological awareness, which also plays an important part in reading development. The aim of this study was to analyze in more detail the implications of the phonological impairments of dyslexics in dealing with larger units of language such as morphemes. First, the performance of dyslexic children in a series of morphological tasks was compared with the performance of children matched on reading-level and chronological age. In all the tasks, the dyslexic group performed below the chronological age control group, suggesting that morphological awareness cannot be developed entirely independently of reading experience and/or phonological skills. Comparisons with the reading-age control group indicated that, while the dyslexic children were poorer in the morphemic segmentation tasks, they performed normally for their reading level in the sentence completion tasks. Furthermore, they produced more derived words in the production task. This suggests that phonological impairments prevent the explicit segmentation of affixes while allowing the development of productive morphological knowledge. A second study compared dyslexic subgroups defined by their degree of phonological impairment. Our results suggest that dyslexics develop a certain type of morphological knowledge which they use as a compensatory reading strategy.

  4. Nonlinear electromagnetic responses of active membrane protein complexes in live cells and organelles

    NASA Astrophysics Data System (ADS)

    Nawarathna, Dharmakirthi

    observed, possibly due to the F0 domain of ATP synthase. Finally, harmonics generated by chloroplasts, the plant organelles responsible for photosynthesis, were measured, which are similar in structure and function to mitochondria, depend dramatically on incident light, and vanish in the absence of light. Using spinach chloroplasts, light sensitive peaks were detected in the range of 0--12 kHz, again suggesting that these harmonics are indicative of electron processes in the light harvesting complexes, reaction center, and/or photosynthetic electron transport chain.

  5. Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis1[W

    PubMed Central

    Ogawa, Takahisa; Yoshimura, Kazuya; Miyake, Hiroe; Ishikawa, Kazuya; Ito, Daisuke; Tanabe, Noriaki; Shigeoka, Shigeru

    2008-01-01

    Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its Km value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 μm) than for ADP-glucose (1,235 ± 65 μm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 μm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 μm) and NADPH (36.9 ± 3.5 μm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 μm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes. PMID:18815383

  6. Comparison of Long-Term Storage in Chemical Fixatives on Morphology and Anatomy of Super-Dwarf Wheat

    NASA Technical Reports Server (NTRS)

    Bubenheim, David L.; Campbell, W. F.; Salisbury, F. B.; Hole, P. S.; Gillespie, L.; Levbinskikh, M.; Kliss, Mark H. (Technical Monitor)

    1996-01-01

    Wheat plants (Triticum aestivum L. cv Super-Dwarf) are grown in the microgravity of space and harvested for morphological and anatomical comparison with those exposed to gravity on earth. Such plants are subjected to relatively long periods of storage in chemical fixatives. Examination, evaluation and verification that the integrity of the vascular system is maintained for extended periods of storage in fixatives are required. McDowell and Trump's [4% Formaldehyde -- 1 % Glutaraldehyde (4F: 1 G)] or Variant I [(Russian Fixative): Formalin: Acetic Acid: Alcohol] fixatives, adjusted to pH 7.2, were placed in Aclam(TM), FilmORap(TM), or FilmOSun(TM) plastic bags on April 4, 1994. Wheat seedlings were harvested on days 9, 28, and 68 and preserved in these fixatives. Subsamples of leaves and/or seeds were taken from these stocks after various times in storage, dehydrated, and embedded in Spurr's, LR White's or Unicryl resin. Semithin (1 mm) and thin (50-70 nm) sections were examined by light and transmission electron microscopy. In a few sections, we have observed a slight plasmolysis of the cytostol in leaf tissue fixed with the Variant I, but overall there seem to be no major artifacts in the anatomical structure. The plasmalemma and other organelles appeared normal in the McDowell and Trump fixative. Use of differential chromophores suggests that LR White or Unicryl resins may give greater flexibility for enzyme localizations at both the light and electron microscopical levels.

  7. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    SciTech Connect

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; Grimwood, Jane; Grigoriev, Igor V.; Van Etten, James L.; Keeling, Patrick J.

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.

  8. Morphological Awareness and Children's Writing: Accuracy, Error, and Invention

    PubMed Central

    McCutchen, Deborah; Stull, Sara

    2014-01-01

    This study examined the relationship between children's morphological awareness and their ability to produce accurate morphological derivations in writing. Fifth-grade U.S. students (n = 175) completed two writing tasks that invited or required morphological manipulation of words. We examined both accuracy and error, specifically errors in spelling and errors of the sort we termed morphological inventions, which entailed inappropriate, novel pairings of stems and suffixes. Regressions were used to determine the relationship between morphological awareness, morphological accuracy, and spelling accuracy, as well as between morphological awareness and morphological inventions. Linear regressions revealed that morphological awareness uniquely predicted children's generation of accurate morphological derivations, regardless of whether or not accurate spelling was required. A logistic regression indicated that morphological awareness was also uniquely predictive of morphological invention, with higher morphological awareness increasing the probability of morphological invention. These findings suggest that morphological knowledge may not only assist children with spelling during writing, but may also assist with word production via generative experimentation with morphological rules during sentence generation. Implications are discussed for the development of children's morphological knowledge and relationships with writing. PMID:25663748

  9. Universals in Morphological Representation: Evidence from Italian.

    ERIC Educational Resources Information Center

    Orsolini, Margherita; Marslen-Wilson, William

    1997-01-01

    Examines the dual mechanism hypothesis, which argues that the apparent contrast, in English inflectional morphology, between a rule-based default procedure, generating regular past-tense forms, and listed irregular forms stored in an associative net, reflects universal constraints on the properties of possible morphological systems. The study…

  10. Prosodic Morphology in Four Chinese Dialects.

    ERIC Educational Resources Information Center

    Yip, Moira

    1992-01-01

    Yanggu, Anxiang, and Yuanyang diminutives and Cantonese familiar name formation are examined in the light of recent understanding about the role of prosodic categories in phonology and morphology. The results lend strong support to the growing body of research in prosodic morphology, especially the pioneering work of McCarthy and Prince. (52…

  11. [The morphological and ontogenetic changes in the Protozoa during the transition to a sessile mode of life].

    PubMed

    Dovgal', I V

    2000-01-01

    The morphological adaptations of protozoans to sessile mode life and evolutionary changes in ontogeny are considered. There are main morphotypes of sessile protists: stalked organisms that attached to substrate by the extended base of body (basal disk), and unstalked organisms that are flatted on substrate. The origin of the morphotypes was independent in different taxa and involved nonhomologous structures. Adaptation to the sessile mode of life in the protists was connected with the progressive increase in the body size and intensity of organelle functions by polymerisation, subsequent division of function and change of functions. Evolution of adhesive organelles is characterised by growing intensity of their functions by allometric growth (usually without polymerisation), and in some cases with the subsequent division of functions and change of functions. The evolution manifests itself primarily in the organelles that provide interaction of cell with environment. The organelles that ensuring functioning of cell change due to correlations with the organelles of the first group. These two groups of organelles are similar to A.N. Sewertsoff's ecto- and endosomatic organs in multicellular organisms. The ontogeny of the sessile protists included three stages: formation of the migratory stage, distribution and choice of substrate and metamorphosis of the migratory stage after adhesion. As a rule there are no recapitulations on the first stage. The majority of structures tomotes or zoospores are inherited from the parent cell. Thus the present of some ancestral characteristics at the earlier stages of protistean ontogeny is display of the Baer's law. The main features of ontogeny evolution in sessile protists are the anaboly of the additional stages of life cycle, the displays of archallaxis or deviation during the migratory stage formation, and anaboly at the stage of buds morphogenesis after adhesion. At the last stage, the study of recapitulations is most

  12. Abnormal morphology of myelin and axon pathology in murine models of multiple sclerosis.

    PubMed

    Bando, Yoshio; Nomura, Taichi; Bochimoto, Hiroki; Murakami, Koichi; Tanaka, Tatsuhide; Watanabe, Tsuyoshi; Yoshida, Shigetaka

    2015-02-01

    Demyelination and axonal damage are responsible for neurological deficits in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. However, the pathology of axonal damage in MS is not fully understood. In this study, histological analysis of morphological changes of axonal organelles during demyelination in murine models was investigated by scanning electron microscopy (SEM) using an osmium-maceration method. In cuprizone-induced demyelination, SEM showed typical morphology of demyelination in the corpus callosum of mouse brain. In contrast, SEM displayed variations in ultrastructural abnormalities of myelin structures and axonal organelles in spinal cord white matter of experimental autoimmune encephalomyelitis (EAE) mice, an animal model of MS. Myelin detachment and excessive myelin formation were observed as typical morphological myelin abnormalities in EAE. In addition, well-developed axoplasmic reticulum-like structures and accumulated mitochondria were observed in tortuous degenerating/degenerated axons and the length of mitochondria in axons of EAE spinal cord was shorter compared with naïve spinal cord. Immunohistochemistry also revealed dysfunction of mitochondrial fusion/fission machinery in EAE spinal cord axons. Moreover, the number of Y-shaped mitochondria was significantly increased in axons of the EAE spinal cord. Axonal morphologies in myelin basic protein-deficient shiverer mice were similar to those in EAE. However, shiverer mice had "tortuous" (S-curve shaped mitochondria) and larger mitochondria compared with wild-type and EAE mice. Lastly, analysis of human MS patient autopsied brains also demonstrated abnormal myelin structures in demyelinating lesions. These results indicate that morphological abnormalities of myelin and axonal organelles play important role on the pathogenesis of axonal injury in demyelinating diseases.

  13. Melanoregulin, product of the dsu locus, links the BLOC-pathway and OA1 in organelle biogenesis.

    PubMed

    Rachel, Rivka A; Nagashima, Kunio; O'Sullivan, T Norene; Frost, Laura S; Stefano, Frank P; Marigo, Valeria; Boesze-Battaglia, Kathleen

    2012-01-01

    Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg(dsu) gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg(dsu) locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1(ko/ko) mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1.

  14. Low nucleotide diversity for the expanded organelle and nuclear genomes of Volvox carteri supports the mutational-hazard hypothesis.

    PubMed

    Smith, David Roy; Lee, Robert W

    2010-10-01

    The noncoding-DNA content of organelle and nuclear genomes can vary immensely. Both adaptive and nonadaptive explanations for this variation have been proposed. This study addresses a nonadaptive explanation called the mutational-hazard hypothesis and applies it to the mitochondrial, plastid, and nuclear genomes of the multicellular green alga Volvox carteri. Given the expanded architecture of the V. carteri organelle and nuclear genomes (60-85% noncoding DNA), the mutational-hazard hypothesis would predict them to have less silent-site nucleotide diversity (π(silent)) than their more compact counterparts from other eukaryotes-ultimately reflecting differences in 2N(g)μ (twice the effective number of genes per locus in the population times the mutation rate). The data presented here support this prediction: Analyses of mitochondrial, plastid, and nuclear DNAs from seven V. carteri forma nagariensis geographical isolates reveal low values of π(silent) (0.00038, 0.00065, and 0.00528, respectively), much lower values than those previously observed for the more compact organelle and nuclear DNAs of Chlamydomonas reinhardtii (a close relative of V. carteri). We conclude that the large noncoding-DNA content of the V. carteri genomes is best explained by the mutational-hazard hypothesis and speculate that the shift from unicellular to multicellular life in the ancestor that gave rise to V. carteri contributed to a low V. carteri population size and thus a reduced 2N(g)μ. Complete mitochondrial and plastid genome maps for V. carteri are also presented and compared with those of C. reinhardtii. PMID:20430860

  15. Export of cyst wall material and Golgi organelle neogenesis in Giardia lamblia depend on endoplasmic reticulum exit sites.

    PubMed

    Faso, Carmen; Konrad, Christian; Schraner, Elisabeth M; Hehl, Adrian B

    2013-04-01

    Giardia lamblia parasitism accounts for the majority of cases of parasitic diarrheal disease, making this flagellated eukaryote the most successful intestinal parasite worldwide. This organism has undergone secondary reduction/elimination of entire organelle systems such as mitochondria and Golgi. However, trophozoite to cyst differentiation (encystation) requires neogenesis of Golgi-like secretory organelles named encystation-specific vesicles (ESVs), which traffic, modify and partition cyst wall proteins produced exclusively during encystation. In this work we ask whether neogenesis of Golgi-related ESVs during G. lamblia differentiation, similarly to Golgi biogenesis in more complex eukaryotes, requires the maintenance of distinct COPII-associated endoplasmic reticulum (ER) subdomains in the form of ER exit sites (ERES) and whether ERES are also present in non-differentiating trophozoites. To address this question, we identified conserved COPII components in G. lamblia cells and determined their localization, quantity and dynamics at distinct ERES domains in vegetative and differentiating trophozoites. Analogous to ERES and Golgi biogenesis, these domains were closely associated to early stages of newly generated ESV. Ectopic expression of non-functional Sar1 GTPase variants caused ERES collapse and, consequently, ESV ablation, leading to impaired parasite differentiation. Thus, our data show how ERES domains remain conserved in G. lamblia despite elimination of steady-state Golgi. Furthermore, the fundamental eukaryotic principle of ERES to Golgi/Golgi-like compartment correspondence holds true in differentiating Giardia presenting streamlined machinery for secretory organelle biogenesis and protein trafficking. However, in the Golgi-less trophozoites ERES exist as stable ER subdomains, likely as the sole sorting centres for secretory traffic. PMID:23094658

  16. Reconceptualizing the chlamydial inclusion as a pathogen-specified parasitic organelle: an expanded role for Inc proteins.

    PubMed

    Moore, Elizabeth R; Ouellette, Scot P

    2014-01-01

    Chlamydia is an obligate intracellular pathogen that develops in the host cell in a vacuole termed the chlamydial inclusion. The prevailing concept of the chlamydial inclusion is of a parasitophorous vacuole. Here, the inclusion is the recipient of one-way host-pathogen interactions thus draining nutrients from the cell and negatively impacting it. While Chlamydia orchestrates some aspects of cell function, recent data indicate host cells remain healthy up until, and even after, chlamydial egress. Thus, while Chlamydia relies on the host cell for necessary metabolites, the overall function of the host cell, during chlamydial growth and development, is not grossly disturbed. This is consistent with the obligate intracellular organism's interest to maintain viability of its host. To this end, Chlamydia expresses inclusion membrane proteins, Incs, which serve as molecular markers for the inclusion membrane. Incs also contribute to the physical structure of the inclusion membrane and facilitate host-pathogen interactions across it. Given the function of Incs and the dynamic interactions that occur at the inclusion membrane, we propose that the inclusion behaves similarly to an organelle-albeit one that benefits the pathogen. We present the hypothesis that the chlamydial inclusion acts as a pathogen-specified parasitic organelle. This representation integrates the inclusion within existing subcellular trafficking pathways to divert a subset of host-derived metabolites thus maintaining host cell homeostasis. We review the known interactions of the chlamydial inclusion with the host cell and discuss the role of Inc proteins in the context of this model and how this perspective can impact the study of these proteins. Lessons learnt from the chlamydial pathogen-specified parasitic organelle can be applied to other intracellular pathogens. This will increase our understanding of how intracellular pathogens engage the host cell to establish their unique developmental niches.

  17. When, how and why glycolysis became compartmentalised in the Kinetoplastea. A new look at an ancient organelle.

    PubMed

    Gualdrón-López, Melisa; Brennand, Ana; Hannaert, Véronique; Quiñones, Wilfredo; Cáceres, Ana J; Bringaud, Frédéric; Concepción, Juan Luis; Michels, Paul A M

    2012-01-01

    A characteristic, well-studied feature of the pathogenic protists belonging to the family Trypanosomatidae is the compartmentalisation of the major part of the glycolytic pathway in peroxisome-like organelles, hence designated glycosomes. Such organelles containing glycolytic enzymes appear to be present in all members of the Kinetoplastea studied, and have recently also been detected in a representative of the Diplonemida, but they are absent from the Euglenida. Glycosomes therefore probably originated in a free-living, common ancestor of the Kinetoplastea and Diplonemida. The initial sequestering of glycolytic enzymes inside peroxisomes may have been the result of a minor mistargeting of proteins, as generally observed in eukaryotic cells, followed by preservation and its further expansion due to the selective advantage of this specific form of metabolic compartmentalisation. This selective advantage may have been a largely increased metabolic flexibility, allowing the organisms to adapt more readily and efficiently to different environmental conditions. Further evolution of glycosomes involved, in different taxonomic lineages, the acquisition of additional enzymes and pathways - often participating in core metabolic processes - as well as the loss of others. The acquisitions may have been promoted by the sharing of cofactors and crucial metabolites between different pathways, thus coupling different redox processes and catabolic and anabolic pathways within the organelle. A notable loss from the Trypanosomatidae concerned a major part of the typical peroxisomal H(2)O(2)-linked metabolism. We propose that the compartmentalisation of major parts of the enzyme repertoire involved in energy, carbohydrate and lipid metabolism has contributed to the multiple development of parasitism, and its elaboration to complicated life cycles involving consecutive different hosts, in the protists of the Kinetoplastea clade.

  18. Melanoregulin, product of the dsu locus, links the BLOC-pathway and OA1 in organelle biogenesis.

    PubMed

    Rachel, Rivka A; Nagashima, Kunio; O'Sullivan, T Norene; Frost, Laura S; Stefano, Frank P; Marigo, Valeria; Boesze-Battaglia, Kathleen

    2012-01-01

    Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg(dsu) gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg(dsu) locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1(ko/ko) mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1. PMID:22984402

  19. Evidence for lateral gene transfer (LGT) in the evolution of eubacteria-derived small GTPases in plant organelles

    PubMed Central

    Suwastika, I. Nengah; Denawa, Masatsugu; Yomogihara, Saki; Im, Chak Han; Bang, Woo Young; Ohniwa, Ryosuke L.; Bahk, Jeong Dong; Takeyasu, Kunio; Shiina, Takashi

    2014-01-01

    The genomes of free-living bacteria frequently exchange genes via lateral gene transfer (LGT), which has played a major role in bacterial evolution. LGT also played a significant role in the acquisition of genes from non-cyanobacterial bacteria to the lineage of “primary” algae and land plants. Small GTPases are widely distributed among prokaryotes and eukaryotes. In this study, we inferred the evolutionary history of organelle-targeted small GTPases in plants. Arabidopsis thaliana contains at least one ortholog in seven subfamilies of OBG-HflX-like and TrmE-Era-EngA-YihA-Septin-like GTPase superfamilies (together referred to as Era-like GTPases). Subcellular localization analysis of all Era-like GTPases in Arabidopsis revealed that all 30 eubacteria-related GTPases are localized to chloroplasts and/or mitochondria, whereas archaea-related DRG and NOG1 are localized to the cytoplasm and nucleus, respectively, suggesting that chloroplast- and mitochondrion-localized GTPases are derived from the ancestral cyanobacterium and α-proteobacterium, respectively, through endosymbiotic gene transfer (EGT). However, phylogenetic analyses revealed that plant organelle GTPase evolution is rather complex. Among the eubacterium-related GTPases, only four localized to chloroplasts (including one dual targeting GTPase) and two localized to mitochondria were derived from cyanobacteria and α-proteobacteria, respectively. Three other chloroplast-targeted GTPases were related to α-proteobacterial proteins, rather than to cyanobacterial GTPases. Furthermore, we found that four other GTPases showed neither cyanobacterial nor α-proteobacterial affiliation. Instead, these GTPases were closely related to clades from other eubacteria, such as Bacteroides (Era1, EngB-1, and EngB-2) and green non-sulfur bacteria (HflX). This study thus provides novel evidence that LGT significantly contributed to the evolution of organelle-targeted Era-like GTPases in plants. PMID:25566271

  20. Stone Morphology: Implication for Pathogenesis

    NASA Astrophysics Data System (ADS)

    Daudon, Michel; Jungers, Paul; Bazin, Dominique

    2008-09-01

    Urinary stones of similar crystalline composition as identified by X-ray diffraction or Fourier transform infrared spectroscopy (FT-IR) may exhibit distinct structures, which are reflected in distinctive stone morphology. Among factors involved in stone morphology—some reflecting lithogenic activity, others depending on the crystal shape, or on the propensity of crystalline phases to form large aggregates, finally the environment where the stone is growing—all of these factors influence the inner structure of the stone and its superficial characteristics. We present here examples of the clinical interest of refined morphologic examination of stones, in addition to X-ray diffraction or FT-IR identification of its components. Such combination of methods allows the identification of specific etiologies among calcium oxalate stones, especially a morphological type pathognomonic of primary hyperoxaluria and other types related to distinct conditions of stone formation. Among phosphatic stones—in addition to stone composition, which must be considered not only on the basis of the main component, but also taking into account the minor crystalline phases, which often are clinically relevant—morphological types also contribute to diagnosis of the underlying etiology, especially for stones related to distal tubular acidosis. Finally, common purine stones also exhibit different morphologies related to stone composition and etiology: two main structures for uric acid and two for ammonium urate help to distinguish risk factors and lithogenic conditions involved in the formation of these calculi. Morphologic examination is a simple, rapid and cheap method that points to specific diseases or lithogenic factors. Better awareness of its clinical relevance should lead to wider utilization.

  1. [Neotropical plant morphology].

    PubMed

    Pérez-García, Blanca; Mendoza, Aniceto

    2002-01-01

    An analysis on plant morphology and the sources that are important to the morphologic interpretations is done. An additional analysis is presented on all published papers in this subject by the Revista de Biología Tropical since its foundation, as well as its contribution to the plant morphology development in the neotropics.

  2. Assembly of the Biogenesis of Lysosome-related Organelles Complex-3 (BLOC-3) and Its Interaction with Rab9*

    PubMed Central

    Kloer, Daniel P.; Rojas, Raul; Ivan, Viorica; Moriyama, Kengo; van Vlijmen, Thijs; Murthy, Namita; Ghirlando, Rodolfo; van der Sluijs, Peter; Hurley, James H.; Bonifacino, Juan S.

    2010-01-01

    The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs. PMID:20048159

  3. Phagocytic properties and organelle motility of pulmonary macrophages from smokers and nonsmokers estimated in vitro by magnetometric means.

    PubMed

    Im Hof, V; Klauser, M; Gehr, P

    1990-02-01

    Monolayer cultures of pulmonary macrophages (PM) from 11 smokers (S) and 9 nonsmokers (NS) were incubated with submicrometric ferromagnetic Fe3O4 particles for one hour. After magnetization by an externally applied pulse magnetic field, the aligned Fe3O4 particles, located in the phagosomes and secondary lysosomes of the PM, emanated a remanent magnetic field which decayed with time. The initial field strength, B0, which is proportional to the amount of phagocytosed Fe3O4 particles, was 13.24 nT (SE 0.79 nT) in S and 11.74 nT (SE 1.39 nT) in NS. Ten nT correspond to roughly 4 micrograms Fe3O4. The initial rate of decay of the remanent field (during the first 60 s after magnetization), lambda 0, is proportional to the rate of particle misalignment. Therefore, lambda 0, is hypothesized to be an estimate of organelle motility. It was found to be the same in S and in NS, being 3.14 x 10(-3).s-1 (SE 0.18 x 10(-3).s-1) in S and 3.17 x 10(-3).s-1) (SE 0.22 x 10(-3).s-1) in NS. These results suggest that, in vitro, there is no difference in phagocytic activity and organelle motility in PM from S and NS. PMID:2311741

  4. Coordinate synthesis and protein localization in a bacterial organelle by the action of a penicillin-binding-protein

    PubMed Central

    Hughes, H. Velocity; Lisher, John P.; Hardy, Gail G.; Kysela, David T.; Arnold, Randy J.; Giedroc, David P.; Brun, Yves V.

    2013-01-01

    SUMMARY Organelles with specialized form and function occur in diverse bacteria. Within the Alphaproteobacteria, several species extrude thin cellular appendages known as stalks, which function in nutrient uptake, buoyancy and reproduction. Consistent with their specialization, stalks maintain a unique molecular composition compared to the cell body, but how this is achieved remains to be fully elucidated. Here we dissect the mechanism of localization of StpX, a stalk-specific protein in Caulobacter crescentus. Using a forward genetics approach, we identify a penicillin-bindingprotein PbpC, which is required for the localization of StpX in the stalk. We show that PbpC acts at the stalked cell pole to anchor StpX to rigid components of the outer membrane of the elongating stalk, concurrent with stalk synthesis. Stalklocalized StpX in turn functions in cellular responses to copper and zinc, suggesting that the stalk may contribute to metal homeostasis in Caulobacter. Together, these results identify a novel role for a penicillin-binding-protein in compartmentalizing a bacterial organelle it itself helps create, raising the possibility that cell wallsynthetic enzymes may broadly serve not only to synthesize the diverse shapes of bacteria, but also to functionalize them at the molecular level. PMID:24118129

  5. Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism of Mycoplasma pneumoniae

    PubMed Central

    Kawamoto, Akihiro; Matsuo, Lisa; Kato, Takayuki; Yamamoto, Hiroki

    2016-01-01

    ABSTRACT Mycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding. PMID:27073090

  6. Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

    PubMed Central

    Calvo-Garrido, Javier; Carilla-Latorre, Sergio; Lázaro-Diéguez, Francisco; Egea, Gustavo

    2008-01-01

    Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1− Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1− cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised. PMID:18550798

  7. The multifaceted role of Lon proteolysis in seedling establishment and maintenance of plant organelle function: living from protein destruction.

    PubMed

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

    2012-05-01

    Intracellular selective proteolysis is an important post-translational regulatory mechanism maintaining protein quality control by removing defective, damaged or even deleterious protein aggregates. The ATP-dependent Lon protease is a key component of protein quality control that is highly conserved across the kingdoms of living organisms. Major advancements have been made in bacteria and in non-plant organisms to understand the role of Lon in protection against protein oxidation, ageing and neurodegenerative diseases. This review presents the progress currently made in plants. The Lon gene family in Arabidopsis consists of four members that produce distinct protein isoforms localized in several organelles. Lon1 and Lon4 that potentially originate from a recent gene duplication event are dual-targeted to mitochondria and chloroplasts through distinct mechanisms revealing divergent evolution. Arabidopsis mutant analysis showed that mitochondria and peroxisomes biogenesis or maintenance of function is modulated by Lon1 and Lon2, respectively. Consequently, the lack of Lon selective proteolysis leading to growth retardation and impaired seedling establishment can be attributed to defects in the oil reserve mobilization pathway. The current progress in Arabidopsis research uncovers the role of Lon in the proteome homeostasis of plant organelles and stimulates biotechnology scenarios of plant tolerance against harsh abiotic conditions because of climate instability.

  8. Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events.

    PubMed

    Mihalache, Cristina C; Yousefi, Shida; Conus, Sébastien; Villiger, Peter M; Schneider, E Marion; Simon, Hans-Uwe

    2011-06-01

    The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

  9. Non-coding RNA identification based on topology secondary structure and reading frame in organelle genome level.

    PubMed

    Wu, Cheng-Yan; Li, Qian-Zhong; Feng, Zhen-Xing

    2016-01-01

    Non-coding RNA (ncRNA) genes make transcripts as same as the encoding genes, and ncRNAs directly function as RNAs rather than serve as blueprints for proteins. As the function of ncRNA is closely related to organelle genomes, it is desirable to explore ncRNA function by confirming its provenance. In this paper, the topology secondary structure, motif and the triplets under three reading frames are considered as parameters of ncRNAs. A method of SVM combining the increment of diversity (ID) algorithm is applied to construct the classifier. When the method is applied to the ncRNA dataset less than 80% sequence identity, the overall accuracies reach 95.57%, 96.40% in the five-fold cross-validation and the jackknife test, respectively. Further, for the independent testing dataset, the average prediction success rate of our method achieved 93.24%. The higher predictive success rates indicate that our method is very helpful for distinguishing ncRNAs from various organelle genomes. PMID:26697761

  10. Delivery of drugs to intracellular organelles using drug delivery systems: Analysis of research trends and targeting efficiencies.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2015-12-30

    Targeting of drug delivery systems (DDSs) to specific intracellular organelles (i.e., subcellular targeting) has been investigated in numerous publications, but targeting efficiency of these systems is seldom reported. We searched scientific publications in the subcellular DDS targeting field and analyzed targeting efficiency and major formulation parameters that affect it. We identified 77 scientific publications that matched the search criteria. In the majority of these studies nanoparticle-based DDSs were applied, while liposomes, quantum dots and conjugates were used less frequently. The nucleus was the most common intracellular target, followed by mitochondrion, endoplasmic reticulum and Golgi apparatus. In 65% of the publications, DDSs surface was decorated with specific targeting residues, but the efficiency of this surface decoration was not analyzed in predominant majority of the studies. Moreover, only 23% of the analyzed publications contained quantitative data on DDSs subcellular targeting efficiency, while the majority of publications reported qualitative results only. From the analysis of publications in the subcellular targeting field, it appears that insufficient efforts are devoted to quantitative analysis of the major formulation parameters and of the DDSs' intracellular fate. Based on these findings, we provide recommendations for future studies in the field of organelle-specific drug delivery and targeting.

  11. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    PubMed

    Tjaden, Joachim; Haferkamp, Ilka; Boxma, Brigitte; Tielens, Aloysius G M; Huynen, Martijn; Hackstein, Johannes H P

    2004-03-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.

  12. Magnetic nanoparticles to recover cellular organelles and study the time resolved nanoparticle-cell interactome throughout uptake.

    PubMed

    Bertoli, Filippo; Davies, Gemma-Louise; Monopoli, Marco P; Moloney, Micheal; Gun'ko, Yurii K; Salvati, Anna; Dawson, Kenneth A

    2014-08-27

    Nanoparticles in contact with cells and living organisms generate quite novel interactions at the interface between the nanoparticle surface and the surrounding biological environment. However, a detailed time resolved molecular level description of the evolving interactions as nanoparticles are internalized and trafficked within the cellular environment is still missing and will certainly be required for the emerging arena of nanoparticle-cell interactions to mature. In this paper promising methodologies to map out the time resolved nanoparticle-cell interactome for nanoparticle uptake are discussed. Thus silica coated magnetite nanoparticles are presented to cells and their magnetic properties used to isolate, in a time resolved manner, the organelles containing the nanoparticles. Characterization of the recovered fractions shows that different cell compartments are isolated at different times, in agreement with imaging results on nanoparticle intracellular location. Subsequently the internalized nanoparticles can be further isolated from the recovered organelles, allowing the study of the most tightly nanoparticle-bound biomolecules, analogous to the 'hard corona' that so far has mostly been characterized in extracellular environments. Preliminary data on the recovered nanoparticles suggest that significant portion of the original corona (derived from the serum in which particles are presented to the cells) is preserved as nanoparticles are trafficked through the cells.

  13. Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes

    PubMed Central

    2011-01-01

    Many bacterial species contain intracellular nano- and micro-compartments consisting of self-assembling proteins that form protein-only shells. These structures are built up by combinations of a reduced number of repeated elements, from 60 repeated copies of one unique structural element self-assembled in encapsulins of 24 nm to 10,000-20,000 copies of a few protein species assembled in a organelle of around 100-150 nm in cross-section. However, this apparent simplicity does not correspond to the structural and functional sophistication of some of these organelles. They package, by not yet definitely solved mechanisms, one or more enzymes involved in specific metabolic pathways, confining such reactions and sequestering or increasing the inner concentration of unstable, toxics or volatile intermediate metabolites. From a biotechnological point of view, we can use the self assembling properties of these particles for directing shell assembling and enzyme packaging, mimicking nature to design new applications in biotechnology. Upon appropriate engineering of the building blocks, they could act as a new family of self-assembled, protein-based vehicles in Nanomedicine to encapsulate, target and deliver therapeutic cargoes to specific cell types and/or tissues. This would provide a new, intriguing platform of microbial origin for drug delivery. PMID:22046962

  14. ChloroMitoSSRDB: open source repository of perfect and imperfect repeats in organelle genomes for evolutionary genomics.

    PubMed

    Sablok, Gaurav; Mudunuri, Suresh B; Patnana, Sujan; Popova, Martina; Fares, Mario A; Porta, Nicola La

    2013-04-01

    Microsatellites or simple sequence repeats (SSRs) are repetitive stretches of nucleotides (A, T, G, C) that are distributed either as single base pair stretches or as a combination of two- to six-nucleotides units that are non-randomly distributed within coding and in non-coding regions of the genome. ChloroMitoSSRDB is a complete curated web-oriented relational database of perfect and imperfect repeats in organelle genomes. The present version of the database contains perfect and imperfect SSRs of 2161 organelle genomes (1982 mitochondrial and 179 chloroplast genomes). We detected a total of 5838 chloroplast perfect SSRs, 37 297 chloroplast imperfect SSRs, 5898 mitochondrial perfect SSRs and 50 355 mitochondrial imperfect SSRs across these genomes. The repeats have been further hyperlinked to the annotated gene regions (coding or non-coding) and a link to the corresponding gene record in National Center for Biotechnology Information(www.ncbi.nlm.nih.gov/) to identify and understand the positional relationship of the repetitive tracts. ChloroMitoSSRDB is connected to a user-friendly web interface that provides useful information associated with the location of the repeats (coding and non-coding), size of repeat, motif and length polymorphism, etc. ChloroMitoSSRDB will serve as a repository for developing functional markers for molecular phylogenetics, estimating molecular variation across species. Database URL: ChloroMitoSSRDB can be accessed as an open source repository at www.mcr.org.in/chloromitossrdb.

  15. Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus.

    PubMed

    Hornick, C A; Hamilton, R L; Spaziani, E; Enders, G H; Havel, R J

    1985-05-01

    Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor-mediated endocytosis. MVBs also contained numerous small vesicles, 0.05-0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized t