Journal articles on the topic 'Giant intracellular vesicular structures'
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1
Podszywalow-Bartnicka, Paulina, Agnieszka Strzelecka-Kiliszek, Joanna Bandorowicz-Pikula, and Slawomir Pikula. "Calcium- and proton-dependent relocation of annexin A6 in Jurkat T cells stimulated for interleukin-2 secretion." Acta Biochimica Polonica 54, no. 2 (June 4, 2007): 261–71. http://dx.doi.org/10.18388/abp.2007_3246.
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Annexin A6 (AnxA6) is a Ca(2+)-dependent membrane-binding protein involved in vesicular traffic. The likely participation of AnxA6 in the response of lymphocytes to Ca(2+) signals has not been investigated yet. The present study focuses on intracellular relocation of AnxA6 in human Jurkat T lymphoblasts upon stimulation followed by transient increase of intracellular [Ca(2+)] and exocytosis of interleukin-2 (IL-2). Stimulation of the cells under different experimental conditions (by lowering pH and/or by rising extracellular [Ca(2+)] in the presence of ionomycin) induced time-dependent transients of intracellular [Ca(2+)] and concomitant changes in AnxA6 intracellular localization and in IL-2 secretion, with only minor effects on cell viability and apoptosis. In resting conditions (in the presence of EGTA or with no ionophore) AnxA6 was localized uniformly in the cytosol, whereas it translocated to vesicular structures beneath the plasma membrane within 5 min following stimulation of Jurkat T cells and rise of intracellular [Ca(2+)] at pH 7.4. Lowering the extracellular pH value from 7.4 to 6.0 significantly enhanced this process. AnxA6 changed its location from the cytosol to the secretory granules and early endosomes which seem to represent membranous targets for annexin. In conclusion, AnxA6 is sensitive to variations in intracellular [Ca(2+)] upon stimulation of Jurkat T cells, as manifested by a switch in its intracellular localization from the cytosol to vesicular structures located in close proximity to the plasma membrane, suggestive of participation of AnxA6 in calcium- and proton-dependent secretion of cytokines by lymphocytes.
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Hedman, K., K. L. Goldenthal, A. V. Rutherford, I. Pastan, and M. C. Willingham. "Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry." Journal of Histochemistry & Cytochemistry 35, no. 2 (February 1987): 233–43. http://dx.doi.org/10.1177/35.2.3025294.
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Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.
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Ayala, S. J. "Transport and internal organization of membranes: vesicles, membrane networks and GTP-binding proteins." Journal of Cell Science 107, no. 4 (April 1, 1994): 753–63. http://dx.doi.org/10.1242/jcs.107.4.753.
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Eukaryotic cells contain a variety of membranous organelles such as the Golgi complex and endosomes, which are organized to allow the flow of molecules to specific regions within the cell. Well known examples of this targeted flow include the transport of specific molecules to the apical pole of epithelial cells, to the axon terminals of neurons, and the transcytosis of immunoglobulins. The generally accepted model of transport between the different intracellular compartments maintains that transport is mediated by carrier vesicles, but recent data show the participation of tubulovesicular structures in membrane transport, and the assumed discontinuity of some intracellular compartments has come under considerable scrutiny. It seems that for different intracellular pathways, eukaryotic cells use both the vesicular and the tubular (bolus) means of transport. In this article I will discuss the vesicular and the tubular models of transport as well as a hypothesis for the mechanism of action of small GTPases of the rab family in these movements.
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Ding, Jin-Dong, Raquel Y. Salinas, and Vadim Y. Arshavsky. "Discs of mammalian rod photoreceptors form through the membrane evagination mechanism." Journal of Cell Biology 211, no. 3 (November 2, 2015): 495–502. http://dx.doi.org/10.1083/jcb.201508093.
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Photoreceptor discs are membrane organelles harboring components of the visual signal transduction pathway. The mechanism by which discs form remains enigmatic and is the subject of a major controversy. Classical studies suggest that discs are formed as serial plasma membrane evaginations, whereas a recent alternative postulates that discs, at least in mammalian rods, are formed through intracellular vesicular fusion. We evaluated these models in mouse rods using methods that distinguish between the intracellular vesicular structures and plasma membrane folds independently of their appearance in electron micrographs. The first differentiated membranes exposed to the extracellular space from intracellular membranes; the second interrogated the orientation of protein molecules in new discs. Both approaches revealed that new discs are plasma membrane evaginations. We further demonstrated that vesiculation and plasma membrane enclosure at the site of new disc formation are artifacts of tissue fixation. These data indicate that all vertebrate photoreceptors use the evolutionary conserved membrane evagination mechanism to build their discs.
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Thorens, B., and J. Roth. "Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles." Journal of Cell Science 109, no. 6 (June 1, 1996): 1311–23. http://dx.doi.org/10.1242/jcs.109.6.1311.
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In adipocytes and muscle cells, the GLUT4 glucose transporter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the beta cell line RINm5F and determined its intracellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.
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Wubbolts, R., M. Fernandez-Borja, L. Oomen, D. Verwoerd, H. Janssen, J. Calafat, A. Tulp, S. Dusseljee, and J. Neefjes. "Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface." Journal of Cell Biology 135, no. 3 (November 1, 1996): 611–22. http://dx.doi.org/10.1083/jcb.135.3.611.
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Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta-GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway.
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Toth, Andrea E., Mikkel R. Holst, and Morten S. Nielsen. "Vesicular Transport Machinery in Brain Endothelial Cells: What We Know and What We Do not." Current Pharmaceutical Design 26, no. 13 (May 6, 2020): 1405–16. http://dx.doi.org/10.2174/1381612826666200212113421.
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The vesicular transport machinery regulates numerous essential functions in cells such as cell polarity, signaling pathways, and the transport of receptors and their cargoes. From a pharmaceutical perspective, vesicular transport offers avenues to facilitate the uptake of therapeutic agents into cells and across cellular barriers. In order to improve receptor-mediated transcytosis of biologics across the blood-brain barrier and into the diseased brain, a detailed understanding of intracellular transport mechanisms is essential. The vesicular transport machinery is a highly complex network and involves an array of protein complexes, cytosolic adaptor proteins, and the subcellular structures of the endo-lysosomal system. The endo-lysosomal system includes several types of vesicular entities such as early, late, and recycling endosomes, exosomes, ectosomes, retromer-coated vesicles, lysosomes, trans-endothelial channels, and tubules. While extensive research has been done on the trafficking system in many cell types, little is known about vesicular trafficking in brain endothelial cells. Consequently, assumptions on the transport system in endothelial cells are based on findings in polarised epithelial cells, although recent studies have highlighted differences in the endothelial system. This review highlights aspects of the vesicular trafficking machinery in brain endothelial cells, including recent findings, limitations, and opportunities for further studies.
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Fritzius, Thorsten, Gabriela Burkard, Elvira Haas, Jochen Heinrich, Marc Schweneker, Magnus Bosse, Sven Zimmermann, et al. "A WD-FYVE protein binds to the kinases Akt and PKCζ/λ." Biochemical Journal 399, no. 1 (September 13, 2006): 9–20. http://dx.doi.org/10.1042/bj20060511.
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WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein–protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCζ/λ (protein kinase Cζ/λ) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.
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Webster, P., and DJ Grab. "Intracellular colocalization of variant surface glycoprotein and transferrin-gold in Trypanosoma brucei." Journal of Cell Biology 106, no. 2 (February 1, 1988): 279–88. http://dx.doi.org/10.1083/jcb.106.2.279.
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Endocytosis and intracellular transport has been studied in the bloodstream forms of Trypanosoma brucei by light and electron microscopy, using colloidal gold coupled to bovine transferrin (transferrin-gold). The endocytosed transferrin-gold, visualized by silver intensification for light microscopy, was present in vesicular structures between the cell nucleus and flagellar pocket of the organism. At the ultrastructural level, transferrin-gold was present after a 10-min incubation in the flagellar pocket, coated vesicles, cisternal networks, and lysosomelike structures. Endocytosis and intracellular processing of T. brucei variable surface glycoprotein (VSG) was studied using two preparations of affinity-purified rabbit IgG directed against different parts of the VSG. One preparation of IgG was directed against the cross-reacting determinant (CRD): a complex glycolipid side chain covalently linked to the COOH-terminus of the VSG molecule. The other was directed against determinants on the rest of the VSG molecule. When the two IgG preparations were used on thawed, thin cryosections of trypanosomes that had been incubated in transferrin-gold before fixation, the organelles involved with transferrin-gold endocytosis labeled with both antibodies, as well as many vesicular, tubular, and vacuolar structures that did not contain endocytosed transferrin-gold. Both antibodies also labeled the cell surface. In double-labeling experiments both antibodies were closely associated except that IgG directed against the VSG molecule labeled all the cisternae of the Golgi apparatus, whereas anti-CRD IgG was shown to label only half of the Golgi apparatus. Evidence for sorting of VSG molecules from endocytosed transferrin-gold was found. Double-labeling experiments also showed some tubular profiles which labeled on one side with anti-CRD IgG and on the other side with anti-VSG IgG, suggesting a possible segregation of parts of the VSG molecule.
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Arif, Ehtesham, Pankaj Sharma, Ashish Solanki, Leena Mallik, Yogendra S. Rathore, Waleed O. Twal, Samir K. Nath, et al. "Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1." Molecular and Cellular Biology 36, no. 11 (April 4, 2016): 1639–54. http://dx.doi.org/10.1128/mcb.00020-16.
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The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in thein vitroand in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane.
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Kandror, K. V., L. Coderre, A. V. Pushkin, and P. F. Pilch. "Comparison of glucose-transporter-containing vesicles from rat fat and muscle tissues: evidence for a unique endosomal compartment." Biochemical Journal 307, no. 2 (April 15, 1995): 383–90. http://dx.doi.org/10.1042/bj3070383.
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Insulin-sensitive tissues (fat and muscle) express a specific isoform of glucose-transporter protein, GLUT4, which normally resides in intracellular vesicular structures and is translocated to the cell surface in response to insulin. Here we provide a biochemical comparison of GLUT4-containing structures from fat and muscle cells. We demonstrate that, in spite of totally different protocols for cell homogenization and fractionation used for adipocytes as compared with skeletal-muscle tissue, GLUT4-containing vesicles from both sources have identical buoyant densities, sedimentation coefficients, and a very similar, if not identical, protein composition. The individual proteins first identified in GLUT4-containing vesicles from adipocytes (GTV3/SCAMPs proteins and aminopeptidase gp160) are also present in the analogous vesicles from muscle. Intracellular microsomes from rat adipocytes also contain GLUT1, a ubiquitously expressed glucose-transporter isoform. GLUT1 has not been detected in intracellular vesicular pool(s) from skeletal-muscle cells, probably because of its low abundance there. GLUT1 in adipocytes is excluded from GLUT4-containing vesicles, but is found in membrane structures which are indistinguishable from the former by all methods tested and demonstrate the same type of regulation by insulin. That is, the GLUT1- and GLUT4-containing vesicles have identical densities and sedimentation coefficients in sucrose gradients, and translocate to the cell surface in response to hormonal exposure. Also, we describe a simple procedure for the purification of native glucose-transporter vesicles from rat adipocytes. Overall, our data suggest the existence of a unique endosomal compartment in fat and muscle cells which is functionally and compositionally different from other microsomal vesicles and which is responsible for insulin-sensitive glucose transport in these tissues.
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Amiya, Eisuke, Masafumi Watanabe, Norihiko Takeda, Tetsuya Saito, Taro Shiga, Yumiko Hosoya, Tomoko Nakao, et al. "Angiotensin II Impairs Endothelial Nitric-oxide Synthase Bioavailability under Free Cholesterol-enriched Conditions via Intracellular Free Cholesterol-rich Membrane Microdomains." Journal of Biological Chemistry 288, no. 20 (April 2, 2013): 14497–509. http://dx.doi.org/10.1074/jbc.m112.448522.
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Vascular endothelial function is impaired in hypercholesterolemia partly because of injury by modified LDL. In addition to modified LDL, free cholesterol (FC) is thought to play an important role in the development of endothelial dysfunction, although the precise mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms of endothelial dysfunction induced by an FC-rich environment. Loading cultured human aortic endothelial cells with FC induced the formation of vesicular structures composed of FC-rich membranes. Raft proteins such as phospho-caveolin-1 (Tyr-14) and small GTPase Rac were accumulated toward FC-rich membranes around vesicular structures. In the presence of these vesicles, angiotensin II-induced production of reactive oxygen species (ROS) was considerably enhanced. This ROS shifted endothelial NOS (eNOS) toward vesicle membranes and vesicles with a FC-rich domain trafficked toward perinuclear late endosomes/lysosomes, which resulted in the deterioration of eNOS Ser-1177 phosphorylation and NO production. Angiotensin II-induced ROS decreased the bioavailability of eNOS under the FC-enriched condition.
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Giesübel, Ulrike, Benjamin Dälken, Hayat Mahmud, and Winfried S. Wels. "Cell binding, internalization and cytotoxic activity of human granzyme B expressed in the yeast Pichia pastoris." Biochemical Journal 394, no. 3 (February 24, 2006): 563–73. http://dx.doi.org/10.1042/bj20050687.
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Granzyme B (GrB) is an apoptosis-inducing protease of cytotoxic lymphocytes. We have investigated intracellular and extracellular effects of human GrB using recombinant protein expressed in the yeast Pichia pastoris. GrB was rapidly taken up by HeLa cells, and accumulated in vesicular structures in the cytoplasm. There it remained inactive and could not be liberated by the endosomolytic reagent chloroquine, indicating that the vesicular structures are distinct from late endosomes and lysosomes. Direct cytosolic delivery of GrB with a cationic lipid-based transduction reagent, however, resulted in the induction of apoptotic cell death. After prolonged incubation at or above 125 nM, GrB on its own induced pronounced morphological changes in human tumour cells, leading to partial loss of contact to the culture support. This extracellular effect was dependent on enzymatic activity and could be reversed by removal of the protein, suggesting GrB-dependent cleavage of extracellular matrix components as the underlying mechanism.
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Bänfer, Sebastian, and Ralf Jacob. "Galectins in Intra- and Extracellular Vesicles." Biomolecules 10, no. 9 (August 24, 2020): 1232. http://dx.doi.org/10.3390/biom10091232.
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Carbohydrate-binding galectins are expressed in various tissues of multicellular organisms. They are involved in autophagy, cell migration, immune response, inflammation, intracellular transport, and signaling. In recent years, novel roles of galectin-interaction with membrane components have been characterized, which lead to the formation of vesicles with diverse functions. These vesicles are part of intracellular transport pathways, belong to the cellular degradation machinery, or can be released for cell-to-cell communication. Several characteristics of galectins in the lumen or at the membrane of newly formed vesicular structures are discussed in this review and illustrate the need to fully elucidate their contributions at the molecular and structural level.
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Bandorowicz-Pikuła, J., M. Danieluk, A. Wrzosek, R. Buś, R. Buchet, and S. Pikuła. "Annexin VI: an intracellular target for ATP." Acta Biochimica Polonica 46, no. 3 (September 30, 1999): 801–12. http://dx.doi.org/10.18388/abp.1999_4152.
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Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.
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Béthune, Julien, and Felix T. Wieland. "Assembly of COPI and COPII Vesicular Coat Proteins on Membranes." Annual Review of Biophysics 47, no. 1 (May 20, 2018): 63–83. http://dx.doi.org/10.1146/annurev-biophys-070317-033259.
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In eukaryotes, distinct transport vesicles functionally connect various intracellular compartments. These carriers mediate transport of membranes for the biogenesis and maintenance of organelles, secretion of cargo proteins and peptides, and uptake of cargo into the cell. Transport vesicles have distinct protein coats that assemble on a donor membrane where they can select cargo and curve the membrane to form a bud. A multitude of structural elements of coat proteins have been solved by X-ray crystallography. More recently, the architectures of the COPI and COPII coats were elucidated in context with their membrane by cryo-electron tomography. Here, we describe insights gained from the structures of these two coat lattices and discuss the resulting functional implications.
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Cookson, Mark R. "Cellular functions of LRRK2 implicate vesicular trafficking pathways in Parkinson's disease." Biochemical Society Transactions 44, no. 6 (December 2, 2016): 1603–10. http://dx.doi.org/10.1042/bst20160228.
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Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, associated with Parkinson's disease, have been shown to affect intracellular trafficking pathways in a variety of cells and organisms. An emerging theme is that LRRK2 can bind to multiple membranous structures in cells, and several recent studies have suggested that the Rab family of small GTPases might be important in controlling the recruitment of LRRK2 to specific cellular compartments. Once localized to membranes, LRRK2 then influences downstream events, evidenced by changes in the autophagy–lysosome pathway. Here, I will discuss available evidence that supports or challenges this outline, with a specific emphasis on those aspects of LRRK2 function that have been controversial or remain to be fully clarified.
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Berkova, Z., S. E. Crawford, G. Trugnan, T. Yoshimori, A. P. Morris, and M. K. Estes. "Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms." Journal of Virology 80, no. 12 (June 15, 2006): 6061–71. http://dx.doi.org/10.1128/jvi.02167-05.
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ABSTRACT Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.
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Rivero-Ríos, Pilar, Patricia Gómez-Suaga, Belén Fernández, Jesús Madero-Pérez, Andrew J. Schwab, Allison D. Ebert, and Sabine Hilfiker. "Alterations in late endocytic trafficking related to the pathobiology of LRRK2-linked Parkinson's disease." Biochemical Society Transactions 43, no. 3 (June 1, 2015): 390–95. http://dx.doi.org/10.1042/bst20140301.
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Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene comprise the most common cause of familial Parkinson's disease (PD), and variants increase the risk for sporadic PD. LRRK2 displays kinase and GTPase activity, and altered catalytic activity correlates with neurotoxicity, making LRRK2 a promising therapeutic target. Despite the importance of LRRK2 for disease pathogenesis, its normal cellular function, and the mechanism(s) by which pathogenic mutations cause neurodegeneration remain unclear. LRRK2 seems to regulate a variety of intracellular vesicular trafficking events to and from the late endosome in a manner dependent on various Rab proteins. At least some of those events are further regulated by LRRK2 in a manner dependent on two-pore channels (TPCs). TPCs are ionic channels localized to distinct endosomal structures and can cause localized calcium release from those acidic stores, with downstream effects on vesicular trafficking. Here, we review current knowledge about the link between LRRK2, TPC- and Rab-mediated vesicular trafficking to and from the late endosome, highlighting a possible cross-talk between endolysosomal calcium stores and Rab proteins underlying pathomechanism(s) in LRRK2-related PD.
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Kopanchuk, Sergei, Edijs Vavers, Santa Veiksina, Kadri Ligi, Liga Zvejniece, Maija Dambrova, and Ago Rinken. "Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy." PLOS ONE 17, no. 5 (May 18, 2022): e0268563. http://dx.doi.org/10.1371/journal.pone.0268563.
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Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.
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Li, Xinhua, Ting Wang, Zhifang Zhao, and Steven A. Weinman. "The ClC-3 chloride channel promotes acidification of lysosomes in CHO-K1 and Huh-7 cells." American Journal of Physiology-Cell Physiology 282, no. 6 (June 1, 2002): C1483—C1491. http://dx.doi.org/10.1152/ajpcell.00504.2001.
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ClC-3 is a voltage-gated Cl−channel that is highly conserved and widely expressed, although its function, localization, and properties remain a matter of considerable debate. In this study, we have shown that heterologous expression of ClC-3 in either Chinese hamster ovary (CHO-K1) or human hepatoma (Huh-7) cells results in the formation of large, acidic vesicular structures within cells. Vesicle formation is prevented by bafilomycin, an inhibitor of the vacuolar ATPase, and is not induced by an E224A mutant of ClC-3 with altered channel activity. This demonstrates that vesicle formation requires both proton pumping and Cl−channel activity. Manipulation of the intracellular Cl−concentration demonstrated that the ClC-3-associated vesicles shrink and swell consistent with a highly Cl−-permeable membrane. The ClC-3 vesicles were identified as lysosomes based on their colocalization with the lysosome-associated proteins lamp-1, lamp-2, and cathepsin D and on their failure to colocalize with fluorescently labeled endosomes. We conclude that ClC-3 is an intracellular channel that conducts Cl− when it is present in intracellular vesicles. Its overexpression results in its appearance in enlarged lysosome-like structures where it contributes to acidification by charge neutralization.
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Yahiaoui, B., M. Loyens, A. Taibi, R. Schöneck, J. F. Dubremetz, and M. A. Ouaissi. "Characterization of a Leishmania antigen associated with cytoplasmic vesicles resembling endosomal-like structure." Parasitology 107, no. 5 (December 1993): 497–507. http://dx.doi.org/10.1017/s0031182000068074.
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SUMMARYIn the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named ‘megasomes’. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.
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Wilson, J. M., J. A. Whitney, and M. R. Neutra. "Biogenesis of the apical endosome-lysosome complex during differentiation of absorptive epithelial cells in rat ileum." Journal of Cell Science 100, no. 1 (September 1, 1991): 133–43. http://dx.doi.org/10.1242/jcs.100.1.133.
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Absorptive cells of the neonatal rat ileum have an elaborate apical endocytic complex consisting of tubular and vesicular endosomes, multivesicular bodies (MVB), and a giant lysosomal vacuole. This system develops rapidly over the last 3 days (20–22) of gestation. We followed the assembly of this complex by ultrastructural analysis and immunocytochemistry using antigenic markers for microvilli, endosomal tubules and lysosomal membranes. At 19 days gestation, low levels of lactase appeared on microvilli but specialized apical endosomal tubules and lysosomes were absent. At 20 days, expression of microvillar lactase increased and the endosomal marker entubin appeared, in parallel with the appearance of specialized apical endosomal tubules. The compartments of the apical endosome-lysosome system were assembled sequentially after differentiation of the apical plasma membrane domains; first endosomal tubules and vesicles, followed by MVB, and ending with the assembly of the giant lysosome shortly after birth. During early stages of the assembly process, membrane components of the tubular endosomes and lysosomes appeared in the apical plasma membrane before being restricted to their respective intracellular compartments.
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Henze, Darrell A., David B. T. McMahon, Kristen M. Harris, and German Barrionuevo. "Giant Miniature EPSCs at the Hippocampal Mossy Fiber to CA3 Pyramidal Cell Synapse Are Monoquantal." Journal of Neurophysiology 87, no. 1 (January 1, 2002): 15–29. http://dx.doi.org/10.1152/jn.00394.2001.
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The mechanisms generating giant miniature excitatory postsynaptic currents (mEPSCs) were investigated at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse in vitro. These giant mEPSCs have peak amplitudes as large as 1,700 pA (13.6 nS) with a mean maximal mEPSC amplitude of 366 ± 20 pA (mean ± SD; 5 nS; n = 25 cells). This is compared with maximal mEPSC amplitudes of <100 pA typically observed at other cortical synapses. We tested the hypothesis that giant mEPSCs are due to synchronized release of multiple vesicles across the release sites of single MF boutons by directly inducing vesicular release using secretagogues. If giant mEPSCs result from simultaneous multivesicular release, then secretagogues should increase the frequency of small mEPSCs selectively. We found that hypertonic sucrose and spermine increased the frequency of both small and giant mEPSCs. The peptide toxin secretagogues alpha-latrotoxin and pardaxin failed to increase the frequency of giant mEPSCs, but the possible lack of tissue penetration of the toxins make these results equivocal. Because a multiquantal release mechanism is likely to be mediated by a spontaneous increase in presynaptic calcium concentration, a monoquantal mechanism is further supported by results that giant mEPSCs were not affected by manipulations of extracellular or intracellular calcium concentrations. In addition, reducing the temperature of the bath to 15°C failed to desynchronize the rising phases of giant mEPSCs. Together these data suggest that the giant mEPSCs are generated via a monovesicular mechanism. Three-dimensional analysis through serial electron microscopy of the MF boutons revealed large clear vesicles (50 to 160 nm diam) docked presynaptically at the MF synapse in sufficient numbers to account for the amplitude and frequency of giant mEPSCs recorded electrophysiologically. It is concluded that release of the contents of a single large clear vesicle generates giant mEPSCs at the MF to CA3 pyramidal cell synapse.
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El-Husseini, Alaa E., Sarah E. Craven, Dane M. Chetkovich, Bonnie L. Firestein, Eric Schnell, Chiye Aoki, and David S. Bredt. "Dual Palmitoylation of Psd-95 Mediates Its Vesiculotubular Sorting, Postsynaptic Targeting, and Ion Channel Clustering." Journal of Cell Biology 148, no. 1 (January 10, 2000): 159–72. http://dx.doi.org/10.1083/jcb.148.1.159.
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Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH2 terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH2 terminus of PSD-95 with alternative palmitoylation motifs at either the NH2 or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.
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Luiken, J. J. F. P., D. Miskovic, Y. Arumugam, J. F. C. Glatz, and A. Bonen. "Skeletal Muscle Fatty Acid Transport and Transporters." International Journal of Sport Nutrition and Exercise Metabolism 11, s1 (December 2001): S92—S96. http://dx.doi.org/10.1123/ijsnem.11.s1.s92.
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While it has long been assumed that long chain fatty acids (LCFA) can freely diffuse across the plasma membrane, recent work has shown that LCFA uptake also involves a protein-mediated mechanism. Three putative LCFA transporters have been identified (FABPpm, FATP, and FAT/CD36), and all are expressed in rodent and human muscles. In a new model system (giant vesicles), we have demonstrated that (a) LCFA transport rates are scaled with the oxidative capacity of heart and muscle, (b) only FABPpm and FAT/CD36, but not FATP1, correlate with vesicular LCFA transport, and (c) LCFA transport can be increased by increasing (1) the FAT/CD36 protein of muscle (chronic adaptation) or (2) via the translocation of FAT/CD36 from an intracellular pool to the plasma membrane during muscle contraction (acute adaptation).
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Santos, Elena C. dos, Alessandro Angelini, Dimitri Hürlimann, Wolfgang Meier, and Cornelia G. Palivan. "Giant Polymer Compartments for Confined Reactions." Chemistry 2, no. 2 (May 12, 2020): 470–89. http://dx.doi.org/10.3390/chemistry2020028.
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In nature, various specific reactions only occur in spatially controlled environments. Cell compartment and subcompartments act as the support required to preserve the bio-specificity and functionality of the biological content, by affording absolute segregation. Inspired by this natural perfect behavior, bottom-up approaches are the focus to develop artificial cell-like structures, detrimental for understanding relevant bioprocesses and interactions or to produce tailored solutions in the field of therapeutics and diagnostics. In this review, we discuss the benefits of constructing polymer-based single and multicompartments (capsules and giant unilamellar vesicles (GUVs)), equipped with biomolecules as to mimic cells. In this respect, we outline key examples of how such structures have been designed from scratch, namely starting from the application-oriented selection and synthesis of the amphiphilic block copolymer. We then present the state-of-the-art techniques for assembling the supramolecular structure while permitting the encapsulation of active compounds and the incorporation of specific ion channels (peptides/proteins), essential to support in situ reactions, e.g., to replicate intracellular signaling cascades. Finally, we briefly discuss important features that these compartments offer and how they could be applied to engineer the next generation of microreactors, therapeutic solutions, and cell models.
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Balla, Tamas. "Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation." Physiological Reviews 93, no. 3 (July 2013): 1019–137. http://dx.doi.org/10.1152/physrev.00028.2012.
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Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease.
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Vega-Salas, D. E., P. J. Salas, and E. Rodriguez-Boulan. "Modulation of the expression of an apical plasma membrane protein of Madin-Darby canine kidney epithelial cells: cell-cell interactions control the appearance of a novel intracellular storage compartment." Journal of Cell Biology 104, no. 5 (May 1, 1987): 1249–59. http://dx.doi.org/10.1083/jcb.104.5.1249.
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Experimental conditions that abolish or reduce to a minimum intercellular contacts between Madin-Darby canine kidney epithelial cells result in the appearance of an intracellular storage compartment for apical membrane proteins. Subconfluent culture, incubation in 1-5 microM Ca++, or inclusion of dissociated cells within agarose or collagen gels all caused the intracellular accumulation of a 184-kD apical membrane protein within large (0.5-5 micron) vacuoles, rich in microvilli. Influenza virus hemagglutinin, an apically targeted viral glycoprotein, is concentrated within these structures but the basolateral glycoprotein G of vesicular stomatitis virus and a cellular basolateral 63-kD membrane protein of Madin-Darby canine kidney cells were excluded. This novel epithelial organelle (VAC), which we designate the vacuolar apical compartment, may play an as yet unrecognized role in the biogenesis of the apical plasma membrane during the differentiation of normal epithelia.
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Friedrich, Ralf P., Katharina Tepper, Raik Rönicke, Malle Soom, Martin Westermann, Klaus Reymann, Christoph Kaether, and Marcus Fändrich. "Mechanism of amyloid plaque formation suggests an intracellular basis of Aβ pathogenicity." Proceedings of the National Academy of Sciences 107, no. 5 (January 19, 2010): 1942–47. http://dx.doi.org/10.1073/pnas.0904532106.
Full textAbstract:
The formation of extracellular amyloid plaques is a common patho-biochemical event underlying several debilitating human conditions, including Alzheimer’s disease (AD). Considerable evidence implies that AD damage arises primarily from small oligomeric amyloid forms of Aβ peptide, but the precise mechanism of pathogenicity remains to be established. Using a cell culture system that reproducibly leads to the formation of Alzheimer’s Aβ amyloid plaques, we show here that the formation of a single amyloid plaque represents a template-dependent process that critically involves the presence of endocytosis- or phagocytosis-competent cells. Internalized Aβ peptide becomes sorted to multivesicular bodies where fibrils grow out, thus penetrating the vesicular membrane. Upon plaque formation, cells undergo cell death and intracellular amyloid structures become released into the extracellular space. These data imply a mechanism where the pathogenic activity of Aβ is attributed, at least in part, to intracellular aggregates.
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Kobayashi, T., J. M. Robinson, and H. Seguchi. "Identification of intracellular sites of superoxide production in stimulated neutrophils." Journal of Cell Science 111, no. 1 (January 1, 1998): 81–91. http://dx.doi.org/10.1242/jcs.111.1.81.
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In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.
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Moon, E. K., S. T. Lee, D. I. Chung, and H. H. Kong. "Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi." Eukaryotic Cell 5, no. 1 (January 2006): 125–31. http://dx.doi.org/10.1128/ec.5.1.125-131.2006.
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ABSTRACT Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase.
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Iwayama, Tomoaki, Tomoko Okada, Tsugumi Ueda, Kiwako Tomita, Shuji Matsumoto, Masahide Takedachi, Satoshi Wakisaka, et al. "Osteoblastic lysosome plays a central role in mineralization." Science Advances 5, no. 7 (July 2019): eaax0672. http://dx.doi.org/10.1126/sciadv.aax0672.
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Mineralization is the most fundamental process in vertebrates. It is predominantly mediated by osteoblasts, which secrete mineral precursors, most likely through matrix vesicles (MVs). These vesicular structures are calcium and phosphate rich and contain organic material such as acidic proteins. However, it remains largely unknown how intracellular MVs are transported and secreted. Here, we use scanning electron-assisted dielectric microscopy and super-resolution microscopy for assessing live osteoblasts in mineralizing conditions at a nanolevel resolution. We found that the calcium-containing vesicles were multivesicular bodies containing MVs. They were transported via lysosome and secreted by exocytosis. Thus, we present proof that the lysosome transports amorphous calcium phosphate within mineralizing osteoblasts.
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Hedman, K., I. Pastan, and M. C. Willingham. "The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin." Journal of Histochemistry & Cytochemistry 34, no. 8 (August 1986): 1069–77. http://dx.doi.org/10.1177/34.8.3734417.
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The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.
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Collier, N. C., S. P. Adams, H. Weingarten, and M. J. Schlesinger. "Inhibition of Enveloped RNA Virus Formation by Peptides Corresponding to Glycoprotein Sequences." Antiviral Chemistry and Chemotherapy 3, no. 1 (February 1992): 31–36. http://dx.doi.org/10.1177/095632029200300105.
Full textAbstract:
Peptides, corresponding to amino acid sequences in the cytoplasmic domains of the transmembranal glycoproteins encoded by Sindbis and vesicular stomatitis viruses, inhibited release of virus particles and infectious virus when added to infected cells for a 2h period during a one-cycle growth curve. Each inhibitory peptide was specific for its intended virus. The shortest peptide with antiviral activity for Sindbis virus contained six amino acids, and a related peptide that was acylated at the amino terminus with octanoic acid was ten-fold more potent as an inhibitor. Neither of these peptides affected the synthesis of viral structural proteins, but a third peptide inhibited proteolytic processing of the Sindbis virus E2 glycoprotein. Inhibition of vesicular stomatitis virus particle release was dose-dependent in the range of 50–400 μg ml−1 for a peptide corresponding to the G glycoprotein cytoplasmic domain. We postulate that these peptides competitively inhibit attachment of the glycoprotein to intracellular virus structures during assembly of the virion. Thus, drugs, based on peptides that mimic the protein-protein interactions required for virus assembly, may have therapeutic potential.
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Dantuma, Nico P., Marian A. P. Pijnenburg, Jacques H. B. Diederen, and Dick J. Van der Horst. "Electron Microscopic Visualization of Receptor-mediated Endocytosis of DiI-labeled Lipoproteins by Diaminobenzidine Photoconversion." Journal of Histochemistry & Cytochemistry 46, no. 9 (September 1998): 1085–89. http://dx.doi.org/10.1177/002215549804600913.
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We present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized DiI-labeled lipoproteins without the apparent punctate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of DiI-labeled insect lipoproteins in natural recipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to DiI-excitation light of cells that had been incubated with DiI-labeled lipoproteins yielded a light- and electron-dense DAB reaction product. In addition to the expected staining, an apparent punctate background staining of vesicular structures hindered proper identification of DiI-containing vesicles because these background-stained vesicles were indistinguishable from putative late endosomal and lysosomal structures at the electron microscopic level. This background staining was completely abrogated by inhibition of peroxisomal catalase with aminotriazole. The conversion of DAB by the emitted light of DiI was not affected by aminotriazole. We conclude that specific staining of DiI-labeled intracellular structures can be achieved with the modified DAB photoconversion method reported here.
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Yu, Yuansong, Michail Nomikos, Maria Theodoridou, George Nounesis, F. Anthony Lai, and Karl Swann. "PLCζ causes Ca2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P2." Molecular Biology of the Cell 23, no. 2 (January 15, 2012): 371–80. http://dx.doi.org/10.1091/mbc.e11-08-0687.
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Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca2+ oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate–mediated Ca2+ release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P2 targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P2 with a green fluorescent protein–tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P2 from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca2+ oscillations. Specific depletion of this PI(4,5)P2 pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca2+ oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P2, PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P2 after PLCζ injection. Targeted depletion of vesicular PI(4,5)P2 by expression of ciPLCζ-fused Inp54p inhibited the Ca2+ oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca2+ mobilization by hydrolyzing internal PI(4,5)P2 stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.
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Meresse, S., J. P. Gorvel, and P. Chavrier. "The rab7 GTPase resides on a vesicular compartment connected to lysosomes." Journal of Cell Science 108, no. 11 (November 1, 1995): 3349–58. http://dx.doi.org/10.1242/jcs.108.11.3349.
Full textAbstract:
Rab GTPases belong to the Ras GTPase superfamily and are key regulators of membrane traffic. Among them, rab7 has been localized on late endosomes of NRK cells but its function remains unknown. In order to investigate its role, we generated stable HeLa cell lines that express either wild type or a GTPase-defective mutant of rab7 in an inducible manner. A morphological analysis of the intracellular localization of these proteins was performed by confocal laser microscopy. Here we show that, in HeLa cells, rab7 is present on a vesicular compartment that extends from the perinuclear area to the cell periphery and shows only a partial colocalization with the cation-independent mannose 6-phosphate receptor, a marker for late endosomes. The topology of this compartment is dependent on the microtubule network since nocodazole treatment results in its scattering throughout the cytoplasm. In addition, we observed that, in contrast to the wild-type protein, a rab7 mutant with a reduced GTPase activity is in part associated with lysosomal membranes. This observation was confirmed by subcellular fractionation in a Percoll gradient. Our data implicate rab7 as the first GTPase functioning on terminal endocytic structures in mammalian cells.
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Sorice, M., D. Ferro, R. Misasi, V. Pittoni, A. Longo, A. Circella, T. Garofalo, et al. "Evidence for Anticoagulant Activity and β2-GPI Accumulation in Late Endosomes of Endothelial Cells Induced by Anti-LBPA Antibodies." Thrombosis and Haemostasis 87, no. 04 (July 2002): 735–41. http://dx.doi.org/10.1055/s-0037-1613073.
Full textAbstract:
SummaryThis investigation was undertaken to test whether anti-LBPA antibodies and IgG from patients with APS interfere with intracellular β2GPI distribution in EAhy926 endothelial cells and with the coagulation system. Cell incubation with anti-LBPA MoAb or with patients’ IgG resulted in antibody binding to late endosomes and caused β2GPI redistribution and accumulation within perinuclear vesicular structures reminiscent of late endosomes. This finding suggests that aPl may contribute to the pathogenic mechanisms of APS by modifying the intracellular traffic of proteins, by interactions between aPl and LBPA, β2GPI and/or LBPA- β2GPI complexes. The anticoagulant activity of anti-LBPA MoAb was analyzed in a sensitized activated partial thromboplastin time (aPTT) system and in a dilute Russell’s viper venom time (dRVVT). A significant, concentration-dependent effect of the antibody on both aPTT and dRVVT prolongation was found. These observations suggest that LBPA is an important lipid target for aPl with potential functional implications for the immunopathogenesis of APS.
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Harding, Clare R., Corinna Mattheis, Aurélie Mousnier, Clare V. Oates, Elizabeth L. Hartland, Gad Frankel, and Gunnar N. Schroeder. "LtpD Is a Novel Legionella pneumophila Effector That Binds Phosphatidylinositol 3-Phosphate and Inositol Monophosphatase IMPA1." Infection and Immunity 81, no. 11 (September 3, 2013): 4261–70. http://dx.doi.org/10.1128/iai.01054-13.
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ABSTRACTThe Dot/Icm type IV secretion system (T4SS) ofLegionella pneumophilais crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of theLegionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD471-626bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P]in vitroand colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae ofGalleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-bindingL. pneumophilaeffector that has a role in intracellular bacterial replication.
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Rodepeter, Fiona R., Susanne Wiegand, Hans-Georg Lüers, Gabriel A. Bonaterra, Anson W. Lowe, Michael Bette, Ralf Jacob, and Robert Mandic. "Indication for differential sorting of the rat v-SNARE splice isoforms VAMP-1a and -1b." Biochemistry and Cell Biology 95, no. 4 (August 2017): 500–509. http://dx.doi.org/10.1139/bcb-2016-0184.
Full textAbstract:
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential constituents of the intracellular trafficking machinery. The variable C-terminus in the 2 rat VAMP-1 splice isoforms VAMP-1a and -1b potentially acts as a sorting signal, because similar changes at the C-terminal end of a human VAMP-1 splice isoform resulted in its sorting to mitochondria. To evaluate the differences in the subcellular localization of these two v-SNARE proteins, VAMP-1a and -1b proteins tagged with green fluorescent protein (GFP) and red fluorescent protein (RFP) were expressed in HeLa, COS-7, and MDCK cells and evaluated by conventional confocal as well as total internal reflection fluorescence microscopy. Regions consistent with the endoplasmic reticulum and Golgi apparatus demonstrated a major overlap of both signals. In the periphery, vesicular structures were observed that mainly expressed one of the 2 isoforms. Within our experimental settings, we could not observe sorting of any of the 2 isoforms to mitochondria or peroxisomes, whereas both isoforms were found expressed in a minor subset of singular vesicles, which sporadically appeared to co-localize with the exocyst marker EXOC3/Sec6. Because vesicular structures were seen that expressed only one of the two splice variants, it is possible that VAMP-1a and VAMP-1b are sorted to distinct cellular compartments that require further characterization.
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Freyberg, Zachary, David Sweeney, Anirban Siddhanta, Sylvain Bourgoin, Michael Frohman, and Dennis Shields. "Intracellular Localization of Phospholipase D1 in Mammalian Cells." Molecular Biology of the Cell 12, no. 4 (April 2001): 943–55. http://dx.doi.org/10.1091/mbc.12.4.943.
Full textAbstract:
Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.
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Field, H., B. R. Ali, T. Sherwin, K. Gull, S. L. Croft, and M. C. Field. "TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells." Journal of Cell Science 112, no. 2 (January 15, 1999): 147–56. http://dx.doi.org/10.1242/jcs.112.2.147.
Full textAbstract:
The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.
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Shugrue, C. A., E. R. Kolen, H. Peters, A. Czernik, C. Kaiser, L. Matovcik, A. L. Hubbard, and F. Gorelick. "Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat." Journal of Cell Science 112, no. 24 (December 15, 1999): 4547–56. http://dx.doi.org/10.1242/jcs.112.24.4547.
Full textAbstract:
The regulation of intracellular vesicular trafficking is mediated by specific families of proteins that are involved in vesicular budding, translocation, and fusion with target membranes. We purified a vesicle-associated protein from hepatic microsomes using sequential column chromatography and partially sequenced it. Oliogonucleotides based on these sequences were used to clone the protein from a rat liver cDNA library. The clone encoded a novel protein with a predicted mass of 137 kDa (p137). The protein had an N terminus WD repeat motif with significant homology to Sec31p, a member of the yeast COPII coat that complexes with Sec13p. We found that p137 interacted with mammalian Sec13p using several approaches: co-elution through sequential column chromatography, co-immunoprecipitation from intact cells, and yeast two-hybrid analysis. Morphologically, the p137 protein was localized to small punctate structures in the cytoplasm of multiple cultured cell lines. When Sec13p was transfected into these cells, it demonstrated considerable overlap with p137. This overlap was maintained through several pharmacological manipulations. The p137 compartment also demonstrated partial overlap with ts045-VSVG protein when infected cells were incubated at 15 degrees C. These findings suggest that p137 is the mammalian orthologue of Sec31p.
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Wakabayashi, Yoshiyuki, Jennifer Lippincott-Schwartz, and Irwin M. Arias. "Intracellular Trafficking of Bile Salt Export Pump (ABCB11) in Polarized Hepatic Cells: Constitutive Cycling between the Canalicular Membrane and rab11-positive Endosomes." Molecular Biology of the Cell 15, no. 7 (July 2004): 3485–96. http://dx.doi.org/10.1091/mbc.e03-10-0737.
Full textAbstract:
The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.
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Taoka, Azuma, Junya Kondo, Zachery Oestreicher, and Yoshihiro Fukumori. "Characterization of uncultured giant rod-shaped magnetotactic Gammaproteobacteria from a freshwater pond in Kanazawa, Japan." Microbiology 160, no. 10 (October 1, 2014): 2226–34. http://dx.doi.org/10.1099/mic.0.078717-0.
Full textAbstract:
Magnetotactic bacteria (MTB) are widespread aquatic bacteria, and are a phylogenetically, physiologically and morphologically heterogeneous group, but they all have the ability to orientate and move along the geomagnetic field using intracellular magnetic organelles called magnetosomes. Isolation and cultivation of novel MTB are necessary for a comprehensive understanding of magnetosome formation and function in divergent MTB. In this study, we enriched a giant rod-shaped magnetotactic bacterium (strain GRS-1) from a freshwater pond in Kanazawa, Japan. Cells of strain GRS-1 were unusually large (~13×~8 µm). They swam in a helical trajectory towards the south pole of a bar magnet by means of a polar bundle of flagella. Another striking feature of GRS-1 was the presence of two distinct intracellular biomineralized structures: large electron-dense granules composed of calcium and long chains of magnetosomes that surround the large calcium granules. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that this strain belongs to the Gammaproteobacteria and represents a new genus of MTB.
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Fromme, Lilja, Débora R. Yogui, Mario Henrique Alves, Arnaud L. J. Desbiez, Marion Langeheine, André Quagliatto, Ursula Siebert, and Ralph Brehm. "Morphology of the genital organs of male and female giant anteaters (Myrmecophaga tridactyla)." PeerJ 9 (August 11, 2021): e11945. http://dx.doi.org/10.7717/peerj.11945.
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Background The giant anteater belongs to the supraorder Xenarthra which occupies a systematically isolated position among placental mammals. The species is categorized as Vulnerable by the International Union for Conservation of Nature, and understanding its reproductive characteristics is critical for future conservation efforts. Methods Gross and microscopic anatomy of the genital organs of 23 male and 21 female adult and young roadkill giant anteaters in Brazil were studied. Results Male giant anteaters presented a short conical penis, intraabdominal testes, and prostate, vesicular and bulbourethral glands. A tubular remnant of the partially fused Müllerian ducts extended from the seminal colliculus through the prostate gland, continued cranially in the genital fold, bifurcated, and attached with one elongation each to the left and right epididymal corpus. The structure presented a total length of up to 10 cm and contained a yellowish liquid in its lumen. Histologically, the caudal section of this structure resembled the female vagina, the middle portion corresponded to the uterus, and the extensions showed characteristics of uterine tubes. In adult female giant anteaters, ovoid ovaries with occasional seminiferous cord-like structures were observed. The animals possessed a simple uterus, which was directly continuous with the vaginal canal. The caudal portion of the vagina had two lumina, separated by a longitudinal septum and opening into two apertures into the vaginal vestibule, cranial to the urethral opening. In the urethral and the lateral vestibular wall, glandular structures with characteristics of male prostate and bulbourethral glands, respectively, were found. The vestibule opened through a vertical vulvar cleft to the exterior. A pair of well-differentiated Wolffian ducts with a central lumen originated ventrally at the vaginal opening into the vestibule and passed in a cranial direction through the ventral vaginal and uterine wall. Each duct extended highly coiled along the ipsilateral uterine tube until the lateral pole of the ovaries where it merged with the rete ovarii. Discussion The reproductive morphology of giant anteaters reveals characteristics shared with other Xenarthrans: intraabdominal testes, a simple uterus, and a double caudal vagina. The persistence of well-differentiated genital ducts of the opposite sex in both males and females, however, singles them out among other species. These structures are the results of an aberration during fetal sexual differentiation and possess secretory functions. The possibility of a pathological degeneration of these organs should be considered in reproductive medicine of the species. Conclusion Knowledge of the unique reproductive characteristics of the giant anteater is essential for future reproductive management of the species. Additionally, further research on the peculiarities of the persisting genital duct structures might help to understand sexual differentiation in placental mammals in general.
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Jena, Bhanu P. "Molecular Machinery and Mechanism of Cell Secretion." Experimental Biology and Medicine 230, no. 5 (May 2005): 307–19. http://dx.doi.org/10.1177/153537020523000504.
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Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are Packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called poro-somes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamen-tal cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the Porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The Molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.
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Mazheika, Igor S., Nadezhda V. Psurtseva, and Olga V. Kamzolkina. "Lomasomes and Other Fungal Plasma Membrane Macroinvaginations Have a Tubular and Lamellar Genesis." Journal of Fungi 8, no. 12 (December 19, 2022): 1316. http://dx.doi.org/10.3390/jof8121316.
Full textAbstract:
The plasma membrane of filamentous fungi forms large-sized invaginations, which are either tubes or parietal vesicles. Vesicular macroinvaginations at the ultrastructural level correspond to classical lomasomes. There is an assumption that vesicular macroinvaginations/lomasomes may be involved in macrovesicular endocytosis. The original aim of this study was to test for the presence of macroendocytosis in xylotrophic basidiomycetes using time-lapse and Z-stacks fluorescent microscopic technologies. However, the results were unexpected since most of the membrane structures labeled by the endocytic tracer (FM4-64 analog) are various types of plasma membrane macroinvaginations and not any endomembranes. All of these macroinvaginations have a tubular or lamellar genesis. Moreover, under specific conditions of a microscopic preparation, the diameter of the tubes forming the macroinvaginations increases with the time of the sample observation. In addition, the morphology and successive formation of the macroinvaginations mimic the endocytic pathway; these invaginations can easily be mistaken for endocytic vesicles, endosomes, and vacuole-lysosomes. The paper analyzes the various macroinvagination types, suggests their biological functions, and discusses some features of fungal endocytosis. This study is a next step toward understanding complex fungal physiology and is a presentation of a new intracellular tubular system in wood-decaying fungi.
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Rodrigues, Marcio L., Leonardo Nimrichter, Debora L. Oliveira, Joshua D. Nosanchuk, and Arturo Casadevall. "Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism for the Delivery of Virulence-Associated Macromolecules?" Lipid Insights 2 (January 2008): LPI.S1000. http://dx.doi.org/10.4137/lpi.s1000.
Full textAbstract:
Fungal cells are encaged in rigid, complex cell walls. Until recently, there was remarkably little information regarding the trans-fungal cell wall transfer of intracellular macromolecules to the extracellular space. Recently, several studies have begun to elucidate the mechanisms that fungal cells utilize to secrete a wide variety of macromolecules through the cell wall. The combined use of transmission electron microscopy, serology, biochemistry, proteomics and lipidomics have revealed that the fungal pathogens Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida parapsilosis and Sporothrix schenckii, as well as the model yeast Saccharomyces cerevisiae, each produces extracellular vesicles that carry lipids, proteins, polysaccharides and pigment-like structures of unquestionable biological significance. Compositional analysis of the C. neoformans and H. capsulatum extracellular vesicles suggests that they may function as ‘virulence bags’, with the potential to modulate the host-pathogen interaction in favor of the fungus. The cellular origin of the extracellular vesicles remains unknown, but morphological and biochemical features indicate that they are similar to the well-described mammalian exosomes.