Translocation Of Secretory Granules Research Articles

Munc18-2 and Syntaxin 3 Control Distinct Essential Steps in Mast Cell Degranulation

Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and mammalian uncoordinated18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. In this study, we found that small interfering RNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE-binding partner syntaxin 3 (STX3) also markedly inhibited degranulation, whereas combined knockdown produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion, demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation, clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.

Identification of a translocation deficiency in cortical granule secretion in preovulatory mouse oocytes.

Preovulatory, germinal vesicle (GV)-stage mouse oocytes are unable to undergo normal cortical granule (CG) secretion. Full secretory competence is observed by metaphase II (MII) of meiosis and involves the development of calcium response mechanisms. To identify the deficient or inhibited step in CG secretion, preovulatory GV-stage oocytes were stimulated and tested for their ability to undergo translocation, docking, and/or fusion. The mean CG distance to the plasma membrane was not reduced in fertilized or sperm fraction-injected, GV-stage oocytes relative to that in control GV-stage oocytes. In addition, analysis of individual CG distances to the plasma membrane indicated no subpopulation of CGs competent to translocate. Further analysis demonstrated that secretory incompetence likely is not due to a lack of proximity of CGs to the egg's primary calcium store, the endoplasmic reticulum. Calcium/calmodulin-dependent protein kinase II (CaMKII), which is reportedly involved in secretory granule translocation and secretion in many cells, including eggs, was investigated. A 60-kDa CaMKII isoform detected by Western blot analysis increased 150% during oocyte maturation. The CaMKII activity assays indicated that MII-stage eggs correspondingly have 110% more maximal activity than GV-stage oocytes. These data demonstrate that the primary secretory deficiency is due to a failure of CG translocation, and that a maturation-associated increase in CaMKII correlates with the acquisition of secretory competence and the ability of the egg to undergo normal activation.

Changes in the granule population of gonadotrophs of hypogonadal (hpg) and normal female mice associated with the priming effect of LH-releasing hormone in vitro.

Changes in the size and position of secretory granules in pituitary gonadotrophs have been studied in relationship to LH release and self-priming induced by LH-releasing hormone (LHRH) in pituitary glands from normal and hypogonadal (hpg) female mice. Hemipituitary glands were preincubated and then incubated for either 1 or 2 h in the absence or presence of LHRH (8.5 nmol/l). The glands were either processed for ultrastructural morphometry or homogenized for the determination of pituitary LH content. Morphometry was carried out on gonadotrophs identified by immunocytochemistry for LH beta using the thin/semi-thin section method. Pituitary LH content and the amount of LH released were determined by radioimmunoassay. The amount of LH released in response to the first and second hours of incubation with LHRH were similar in hpg and normal mice with a clear priming effect (three- to fourfold increase in pituitary responsiveness to LHRH) occurring in both strains. Despite a substantially reduced total number of granules (and amount of LH) in unstimulated hpg gonadotrophs, the number of granules in the outer 500 nm marginal zone of the cells was similar to that in normal mice. This could explain the similar amount of LH released from normal and hpg glands by the first LHRH challenge. The initial exposure to LHRH was also associated with a marked translocation of secretory granules from the central to the outer marginal region of cytoplasm subjacent to the gonadotroph plasmalemma, such that in 'primed' glands 60% of granules were found in this marginal zone compared with 40% (hpg) or 33% (normal) in unstimulated glands. The mean diameter of granules in the marginal zone was significantly less than that of granules in the central zone of the gonadotrophs of unstimulated glands from both normal and hpg animals. Exposure to LHRH for 1 h was associated with an increase in the number of small granules in the marginal zone and a significant decrease in the mean diameter of the gonadotroph granule population as a whole. After the primed release of LH, increased proportions of granules were still located in the marginal zone of gonadotrophs, indicating that granule migration continued during the second hour of exposure to LHRH in which primed release occurred. The primed release was associated with a detectable reduction in both the LH and granule content of gonadotrophs in normal, but not hpg glands.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of transglutaminase in proinsulin conversion and insulin release.

Among the biophysical events associated with insulin release, the translocation of secretory granules and their access to the exocytotic site are thought to be controlled by a microtubular-microfilamentous system. Only limited information is available on the enzymes involved in these mechanical events. This report deals with the possible role of transglutaminase in the secretory process.

Microtubule‐dependent transport of secretory granules during stalk secretion in a peritrich ciliate

AbstractThe role of microtubules in secretory granule translocation was studied during stalk secretion in the peritrich ciliate, Zoothamnium arbuscula. In each cell, the release of stalk‐forming secretory materials is restricted to a specialized region of the cytoplasm, the scopula. Many of the membrane‐bound secretory granules that dominate the scopular cytoplasm appear to be aligned along cortical microtubules that converge on the scopular surface. This arrangement is consistent with the hypothesis that microtubules transport granules relative to the sites of exocytosis. To establish the role of microtubules in stalk secretion, telotrochs were exposed to agents with different disruptive effects on microtubule function. Exocytosis itself is not prevented by these drugs, and granules positioned for secretion prior to treatment are released. Maytansine and isopropyl‐n‐phenyl carbamate (IPC) completely inhibit stalk elongation. In maytansine‐treated cells, microtubules are absent from the scopular cytoplasm, and granules are absent from the scopular surface. Microtubules are present in IPC‐treated cells, but the granules are misdirected to the cytoplasm lateral to the scopula where no secretory sites exist. Even though the rate of stalk secretion is decreased by deuterium oxide (D2O), a control length stalk is eventually produced. In D2O‐treated cells microtubules are present and in their normal orientation. The inhibition of secretion when microtubules are absent (maytansine) or misdirected (IPC) and the retardation of secretion when microtubule turnover is reduced (D2O) supports a mechanism of granule transport based on the directional turnover of microtubule subunits.