Motion matters: secretory granule motion adjacent to the plasma membrane and exocytosis.
Ontology highlight
ABSTRACT:
SUBMITTER: Allersma MW
PROVIDER: S-EPMC1446096 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament-bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2-induced actin bundling is apparently essential for generating active exocytotic sites.
| S-EPMC4555831 | biostudies-literature
Project description:The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A ?1A subunit. A twofold reduction in ?1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced ?1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine ?-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-?1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Coimmunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.
| S-EPMC4167924 | biostudies-literature
Project description:Rho GTPases are key regulators of the actin cytoskeleton in membrane trafficking events. We previously reported that Cdc42 facilitates exocytosis in neuroendocrine cells by stimulating actin assembly at docking sites for secretory granules. These findings raise the question of the mechanism activating Cdc42 in exocytosis. The neuronal guanine nucleotide exchange factor, intersectin-1L, which specifically activates Cdc42 and is at an interface between membrane trafficking and actin dynamics, appears as an ideal candidate to fulfill this function. Using PC12 and chromaffin cells, we now show the presence of intersectin-1 at exocytotic sites. Moreover, through an RNA interference strategy coupled with expression of various constructs encoding the guanine nucleotide exchange domain, we demonstrate that intersectin-1L is an essential component of the exocytotic machinery. Silencing of intersectin-1 prevents secretagogue-induced activation of Cdc42 revealing intersectin-1L as the factor integrating Cdc42 activation to the exocytotic pathway. Our results extend the current role of intersectin-1L in endocytosis to a function in exocytosis and support the idea that intersectin-1L is an adaptor that coordinates exo-endocytotic membrane trafficking in secretory cells.
| S-EPMC1538555 | biostudies-literature
Project description:The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine alpha-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation showed efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr(946), Ser(949)) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100-kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins.
| S-EPMC2904833 | biostudies-literature
Project description:Mast cells degranulate and release the contents of intracellular secretory granules in response to the cross-linking of FcepsilonRI by multivalent antigens. These granules contain a variety of biologically active inflammatory mediators; however, it is not clear whether these granules are homogenous or whether there is heterogeneity within the secretory granule population in mast cells. By using genetically altered mice lacking specific vesicle-associated SNARE membrane fusion proteins, we found that VAMP-8-deficient mast cells exhibited defects in FcepsilonRI-regulated exocytosis, whereas synaptobrevin 2- or VAMP-3-deficient mast cells did not. Surprisingly, the defect in secretion in VAMP-8-deficient mice was limited to the subpopulation of mast cell secretory granules containing serotonin and cathepsin D, whereas regulated exocytosis of secretory granules containing histamine and TNF-alpha was normal. Confocal microscopy confirmed that serotonin and histamine were present in distinct intracellular granules and that most serotonin-containing granules were VAMP-8-positive. Thus, this study demonstrates that mast cells do indeed possess distinct subsets of secretory granules and that these subsets use different SNARE isoforms for exocytosis.
| S-EPMC2268179 | biostudies-literature
Project description:Secretory granules labeled with Vamp-green fluorescent protein (GFP) showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy. In approximately 90% of fusion events, we observed a large increase in fluorescence intensity coupled with a transition from a small punctate appearance to a larger, spreading cloud with free diffusion of the Vamp-GFP into the plasma membrane. Quantitation suggests that these events reflect the progression of an initially fused and spherical granule flattening into the plane of the plasma membrane as the Vamp-GFP simultaneously diffuses through the fusion junction. Approximately 10% of the events showed a transition from puncta to ring-like structures coupled with little or no spreading. The ring-like images correspond quantitatively to granules fusing and retaining concavity (recess of approximately 200 nm). A majority of fusion events involved granules that were present in the evanescent field for at least 12 s. However, approximately 20% of the events involved granules that were present in the evanescent field for no more than 0.3 s, indicating that the interaction of the granule with the plasma membrane that leads to exocytosis can occur within that time. In addition, approximately 10% of the exocytotic sites were much more likely to occur within a granule diameter of a previous event than can be accounted for by chance, suggestive of sequential (piggy-back) exocytosis that has been observed in other cells. Overall granule behavior before and during fusion is strikingly similar to exocytosis previously described in the constitutive secretory pathway.
| S-EPMC519157 | biostudies-literature
Project description:Weibel-Palade bodies (WPB) are specialized secretory organelles of endothelial cells that control vascular hemostasis by regulated, Ca2+-dependent exocytosis of the coagulation-promoting von-Willebrand factor. Some proteins of the WPB docking and fusion machinery have been identified but a role of membrane lipids in regulated WPB exocytosis has so far remained elusive. We show here that the plasma membrane phospholipid composition affects Ca2+-dependent WPB exocytosis and von-Willebrand factor release. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] becomes enriched at WPB-plasma membrane contact sites at the time of fusion, most likely downstream of phospholipase D1-mediated production of phosphatidic acid (PA) that activates phosphatidylinositol 4-phosphate (PI4P) 5-kinase γ. Depletion of plasma membrane PI(4,5)P2 or down-regulation of PI4P 5-kinase γ interferes with histamine-evoked and Ca2+-dependent WPB exocytosis and a mutant PI4P 5-kinase γ incapable of binding PA affects WPB exocytosis in a dominant-negative manner. This indicates that a unique PI(4,5)P2-rich environment in the plasma membrane governs WPB fusion possibly by providing interaction sites for WPB-associated docking factors.
| S-EPMC7442956 | biostudies-literature
Project description:In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified "large-capacitance flickers" that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent "compound cavicapture," most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.
| S-EPMC3711296 | biostudies-other
Project description:Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.
| S-EPMC1170832 | biostudies-other
Project description:Regulated intramembrane proteolysis, a highly conserved process employed by diverse regulatory pathways, can release soluble fragments that directly or indirectly modulate gene expression. In this study we used pharmacological tools to identify peptidylglycine ?-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a ?-secretase substrate. PAM, an essential enzyme, catalyzes the final step in the synthesis of the majority of neuropeptides that control metabolic homeostasis. Mass spectroscopy was most consistent with the presence of multiple closely spaced NH(2) termini, suggesting that cleavage occurred near the middle of the PAM transmembrane domain. The luminal domains of PAM must undergo a series of prohormone convertase or ?-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a ?-secretase-like cleavage. Cleavage by ?-secretase generates a soluble fragment of the cytosolic domain (sf-CD) that is known to localize to the nucleus. Although PAM sf-CD is unstable in AtT-20 corticotroph tumor cells, it is readily detected in primary rat anterior pituitary cells. PAM isoform expression, which is tissue-specific and developmentally regulated, affects the efficiency with which sf-CD is produced. sf-CD levels are also modulated by the phosphorylation status of the cytosolic domain and by the ability of the cytosolic domain to interact with cytosolic proteins. sf-CD is produced by primary rat anterior pituitary cells in response to secretogogue, suggesting that sf-CD acts as a signaling molecule relaying information about secretion from the secretory granule to the nucleus.
| S-EPMC2966079 | biostudies-other