Добірка наукової літератури з теми "Giant intracellular vesicular structures"

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Inflammation is a complex set of immune responses that is critical for survival during injury or infection. It is also crucial for maintenance of tissue homeostasis under myriad conditions caused by cellular stress and tissue malfunction. A successful inflammatory response after the removal of noxious agents culminates into the resolution phase to promote tissue repair and induce adaptive immunity for efficient responses upon second encounter. Based on various immune factors involved and the duration of the processes, inflammation can be broadly classified into acute and chronic inflammation. Acute inflammation generally lasts from a few days to a week and involves activation of non-specific innate immune responses. Acute inflammation is best characterized during microbial infections and often ends with the eradication of infection. However, uncontrolled acute inflammatory responses can be detrimental, a classic example of which is sepsis. The persistence of stimuli or incomplete resolution of inflammation can lead to chronic inflammation, mediated by activation of adaptive immune cells. For example, failure to remove monosodium urate crystals leads to gout, a form of chronic inflammation-induced arthritis. Another example of chronic inflammation is rheumatoid arthritis, an autoimmune disorder affecting the joints in bones. Recent studies have shown the involvement of inflammation in a variety of modern human diseases such as obesity, diabetes and neurodegenerative disorders. Therefore, inflammatory responses are an important area of investigation that affects a wide variety of biological responses. The process of inflammation is driven by a variety of mediators, of which the cytokine network occupies a key regulatory role. Most of the pathological inflammatory conditions are aggravated by pro-inflammatory cytokines like TNFα, IL-1β, IL-6, IL-17 etc. Accordingly, the treatment of rheumatoid arthritis and inflammatory bowel disease patients with neutralizing antibodies or inhibitors to cytokines, e.g. TNFα, IL-1 etc, is beneficial. Although, cytokine signalling is known to be involved in pathogenesis of variety of inflammatory disorders, the roles of these cytokines in regulation of homeostasis and inflammatory responses is still not clear. Other than that, studies on inflammatory mechanisms revealed the critical roles of signalling proteins involved in inflammation, e.g. p38 Mitogen Activated Protein Kinase (p38 MAPK), c-Jun N-Terminal Kinase (JNK), prostaglandins, mediators of nitrosative and oxidative stress etc. In order to obtain a better understanding of the inflammatory responses, this study aims to elucidate the roles of two pro-inflammatory mediators: Interferon-gamma (IFNγ) and Nitric Oxide Synthase 2 (NOS2). In the first part of this work, studies with IFNγ were performed in an in vitro cellular model of macrophage activation. In the second part, the roles of NOS2 in an in vivo setting, i.e. Salmonella Typhimurium (S. Typhimurium) infection-induced model of sepsis, were investigated. I) IFNγ, a type II Interferon, has pleiotropic roles in immunity, cancer biology, autoimmunity etc. IFNγ, a T helper (Th)-1 type effector cytokine, is a key activator of macrophages. It activates the macrophages towards the M1 phenotype, which are characterized by increased expression of pro-inflammatory cytokines like TNFα, IL-1β, IL-12 and production of reactive nitrogen and oxygen intermediates. Concomitantly, IFNγ upregulates Major Histocompatibility Complex (MHC) encoded class I (MHC-I) and class II (MHC-II) molecules for antigen presentation. Also, IFNγ inhibits the differentiation of regulatory T cells and Th-2 type T cells. IFNγ post binding to its receptor induces rapid responses via activation of the canonical Janus Kinase (JAK)-Signal Transducer and Activator 1 (STAT1) pathway. Additionally, IFNγ can also activate various non-canonical pathways independent of STAT1 in a cell- and context-dependent manner. For example, IFNγ mediated activation of p38 MAPK is involved in the regulation of autophagy, phagosome maturation and killing of intra-cellular pathogens. In the present study, the cross talk between IFNγ dependent p38 MAPK activation and its effects on cellular responses of macrophages were investigated. The treatment of RAW 264.7, a mouse macrophage cell line, with the combination of IFNγ and p38 MAPK inhibitors (SB202190 or SB203580) led to the formation of giant intracellular vesicular structures (GIVS). Notably, treatment of RAW 264.7 with IFNγ or p38 MAPK inhibitors alone or the combination of IFNγ with ERK or JNK inhibitors did not lead to the generation of GIVS. The generation of GIVS, upon treatment with IFNγ and SB202190, was also observed in primary mouse peritoneal macrophages but to a lesser extent in other cells, e.g. L929 (a mouse fibroblast cell line) and was negligible in CT26 (a colon carcinoma cell line). Further investigations revealed that the genesis of these GIVS in RAW 264.7 cells was independent of IFNγ-induced nitrite production, autophagy and cell viability. Immunofluorescence microscopic analysis demonstrated that these GIVS were positive for the late endosomal and lysosomal membrane markers such as CD63, Lysosome Associated Membrane Protein (LAMP) 1/ LAMP2 respectively. Importantly, the generation of GIVS was blocked by chloroquine (an inhibitor of endosomal and lysosomal acidification) and Ly294002 (a PI-3K inhibitor). Kinetic studies using live cell imaging showed increased fusion but not fission of LAMP1 positive vesicles, resulting in the formation of GIVS upon treatment of RAW 264.7 cells with IFNγ and SB202190. Interestingly, the cells with large lysosomes showed normal phagocytosis and cell surface expression of MHC I and transferrin receptors (CD71), but lower cell surface expression of MHC II and LAMP1, upon IFNγ stimulation. Surprisingly, IFNγ-induced mammalian Target of Rapamycin (mTOR) activation is inhibited with SB20190 treatment with simultaneous increase in total amounts of transcription factor EB (TFEB), a master regulator of lysosome biogenesis. TFEB was majorly translocated to the nucleus and showed increased transcription of lysosomal biogenesis genes, Naglu1 and Neu1, in RAW 264.7 cells treated with the combination of IFNγ and SB202190. Altogether, the study elucidated the possible roles of inhibition of p38 MAPK during IFNγ mediated regulation of lysosomal biogenesis and/or homeostasis. The implications of formation of enlarged lysosomes are discussed in the context of macrophage functions. II) Sepsis is a syndrome with a considerable global burden in terms of morbidity and mortality. It is characterized as a life-threatening organ damage due to dysregulated host immune responses during infection. Enhanced innate immune responses and suppressed adaptive immunity together contribute to persistent organ damage and recurrent infections, which often contribute to impaired recovery and mortality of sepsis patients. The overwhelming inflammatory responses due to hyper-activated innate immune cells are a hallmark of sepsis. Therefore, molecules that attenuate inflammatory responses are potential therapeutic targets to treat sepsis. Nitric Oxide (NO) is a highly reactive molecule that has multiple functions in the host, ranging from cellular signaling and modulation of gene expression to innate antimicrobial responses. Although, the protective roles of NO in infectious diseases are well documented, its role in pathogenesis of sepsis is controversial. Based on this, the focus of the study was to investigate the role of NOS2 during S. Typhimurium infection-induced peritonitis (inflammation of the peritoneum) leading to sepsis in mice. A S. Typhimurium infection-induced sepsis model in mice was established, which was characterized by cytokine storm, neutrophil recruitment at the site of infection and organ damage leading to death of mice. The roles of NOS2 during S. Typhimurium infection-induced sepsis model in mice were elucidated using a mouse strain harbouring a genetic deletion in Nos2, the enzyme responsible for generation of NO in immune cells. Upon sepsis, Nos2-/- mice had attenuated responses, such as induction of Reactive Oxygen Species (ROS), pro-inflammatory cytokines TNFα, IL-6 and IFNγ as well as reduced neutrophil recruitment into the peritoneal cavity. However, responses such as induction of activation markers on neutrophils, infection-induced glucocorticoids in sera, phagocytic ability of recruited neutrophils and apoptosis of peritoneal cells were largely independent of NOS2-derived NO. Importantly, the dampened initial immune responses in Nos2-/- mice correlated with increased microbial burden in peritoneal lavage, spleen and liver, which contributed to greater liver damage, cardiac dysfunction and reduced survival of Nos2-/- mice upon sepsis. Therefore, NOS2 regulates the initial innate immune responses in this mouse model of S. Typhimurium infection-induced sepsis. To further establish the precise roles of NO, studies with exogenous supplementation of DETA-NO, a NO donor, in Nos2-/- mice were performed. DETA-NO supplementation 3 hours prior to infection significantly restored innate immune responses, including neutrophil recruitment, increase in serum IL-6 and CCL2 amounts etc. Administration of DETA-NO also led to significant reduction in microbial burden in peritoneal lavage, spleen and liver. Consequently, there was reduced organ damage and increased survival of Nos2-/- mice upon sepsis. However, no beneficial effects of DETA-NO were observed in C57BL/6 (wild type) mice. The finding from this study demonstrates that NOS2-derived NO is required to promote beneficial host innate immune responses in this model of peritonitis induced sepsis. The important implications of these findings are discussed in the context of the complex roles of NO during sepsis. Overall, the present study elucidated the uncharacterized roles of small molecule inhibitors of p38 MAPK in functional responses of macrophages to IFNγ and has demonstrated NOS2 to be a critical regulator of host innate immune responses during S. Typhimurium infection-induced sepsis.