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1.
Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.  相似文献   

2.
Chromaffin cell secretion requires cortical F-actin disassembly and it has been suggested that scinderin, a Ca2+ dependent F-actin severing protein, controls cortical actin dynamics. An antisense oligodeoxynucleotide targeting the scinderin gene was used to decrease the expression of the protein and access its role in secretion. Treatment with 2 microM scinderin antisense oligodeoxynucleotide for 4 days produced a significant decrease in scinderin expression and its mRNA levels. The expression of gelsolin, another F-actin severing protein, was not affected. Scinderin decrease was accompanied by concomitant and parallel decreases in depolarization-evoked cortical F-actin disassembly and exocytosis. Similar treatment with a mismatched oligodeoxynucleotide produced no effects. Scinderin antisense oligodeoxynucleotide treatment was also a very effective inhibitor of exocytosis in digitonin-permeabilized cells stimulated with increasing concentrations of Ca2+. This ruled out scinderin antisense interference with stimulation-induced depolarization or Ca2+ channel activation. Scinderin antisense treatment decreased the maximum (B(max)) secretory response to Ca2+ without modifying the affinity (K(m)) of the cation for the exocytotic machinery. Moreover, the antisense treatment did not affect norepinephrine uptake or the expression of dopamine ss-hydroxylase, suggesting that the number and function of chromaffin vesicles was not modified. In addition, scinderin antisense treatment did not alter the expression of proteins involved in vesicle-plasma membrane fusion, such as synaptophysin, synaptotagmin or syntaxin, indicating a lack of effects on the fusion machinery components. These observations strongly suggest that scinderin is a key player in the events involved in the secretory process.  相似文献   

3.
Scinderin, a novel Ca2+-activated actin filament-severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/- 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(-7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(-6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F-actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F-actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament-severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross-react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti-scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+-dependent actin filament-severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament-severing activity of the two proteins might be under the control of different transduction and modulating influences.  相似文献   

4.
Scienderin is a Ca+-dependent actin filament severing protein present in chromaffin cells, platelets and a variety of secretory cells. It has been suggested that scinderin is involved in chromaffin cell F-actin dynamics and that this actin network controls the delivery of secretory vesicles to plasma membrane exocytotic sites. Moreover, scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated cells. Here we describe the molecular cloning, the nucleotide sequence and the expression of bovine chromaffin cell scinderin cDNA. The fusion protein obtained cross-reacts with native scinderin antibodies and binds phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PIP2) and actin in a Ca+-dependent manner. Antibodies raised against the fusion protein produced the same cellular staining patterns for scinderin as anti-native scinderin. Nucleotide and amino acid sequence analysis indicate that scinderin has six domains each containing three internal sequence motifs, two actin and two PIP2 binding sites and has 63 and 53% homology with gelsolin and villin. These data indicate that scinderin is a novel member of the family of Ca2+-dependent F-actin severing proteins which includes gelsolin and villin.Abbreviations PIP2 phosphatidylinositol 4,5 bisphosphate - PKC protein kinase C - Sc scinderin - PS phosphatidyl serine - F-Sc scinderin fusion protein - PCR polymerase chain reaction  相似文献   

5.
Abstract: Histamine is a known chromaffin cell secretagogue that induces Ca2+-dependent release of catecholamines. However, conflicting evidence exists as to the source of Ca2+ utilized in histamine-evoked secretion. Here we report that histamine-H1 receptor activation induces redistribution of scinderin, a Ca2+-dependent F-actin severing protein, cortical F-actin disassembly, and catecholamine release. Histamine evoked similar patterns of distribution of scinderin and filamentous actin. The rapid responses to histamine occurred in the absence of extracellular Ca2+ and were triggered by release of Ca2+ from intracellular stores. The trigger for the release of Ca2+ was inositol 1,4,5-trisphosphate because U-73122, a phospholipase C inhibitor, but not its inactive isomer (U-73343), inhibited the increases in IP3 and intracellular Ca2+ levels, scinderin redistribution, cortical F-actin disassembly, and catecholamine release in response to histamine. Thapsigargin, an agent known to mobilize intracellular Ca2+, blocked the rise in intracellular Ca2+ concentration, scinderin redistribution, F-actin disassembly, and catecholamine secretion in response to histamine. Calphostin C and chelerythrine, two inhibitors of protein kinase C, blocked all responses to histamine with the exception of the release of Ca2+ from intracellular stores. This suggests that protein kinase C is involved in histamine-induced responses. The results also show that in the absence of F-actin disassembly, rises in intracellular Ca2+ concentration are not by themselves capable of triggering catecholamine release.  相似文献   

6.
Nicotinic stimulation and high K(+)-depolarization of chromaffin cells cause disassembly of cortical filamentous actin networks and redistribution of scinderin, a Ca(2+)-dependent actin filament-severing protein. These events which are Ca(2+)-dependent precede exocytosis. Activation of scinderin by Ca2+ may cause disassembly of actin filaments leaving cortical areas of low cytoplasmic viscosity which are the sites of exocytosis (Vitale, M. L., A. Rodríguez Del Castillo, L. Tchakarov, and J.-M. Trifaró. 1991. J. Cell. Biol. 113:1057-1067). It has been suggested that protein kinase C (PKC) regulates secretion. Therefore, the possibility that PKC activation might modulate scinderin redistribution was investigated. Here we report that PMA, a PKC activator, caused scinderin redistribution, although with a slower onset than that induced by nicotine. PMA effects were independent of either extra or intracellular Ca2+ as indicated by measurements of Ca2+ transients, and they were likely to be mediated through direct activation of PKC because inhibitors of the enzyme completely blocked the response to PMA. Scinderin was not phosphorylated by the kinase and further experiments using the Na+/H+ antiport inhibitors and intracellular pH determinations, demonstrated that PKC-mediated scinderin redistribution was a consequence of an increase in intracellular pH. Moreover, it was shown that scinderin binds to phosphatidylserine and phosphatidylinositol 4,5-biphosphate liposomes in a Ca(2+)-dependent manner, an effect which was modulated by the pH. The results suggest that under resting conditions, cortical scinderin is bound to plasma membrane phospholipids. The results also show that during nicotinic receptor stimulation both a rise in intracellular Ca2+ and pH are observed. The rise in intracellular pH might be the result of the translocation and activation of PKC produced by Ca2+ entry. This also would explain why scinderin redistribution induced by nicotine is partially (26-40%) inhibited by inhibitors of either PKC or the Na+/H+ antiport. In view of these findings, a model which can explain how scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated conditions is proposed.  相似文献   

7.
Neurosecretory cells including chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane. We have proposed that the F-actin network acts as a barrier to the secretory vesicles blocking their access to exocytotic sites at the plasma membrane. Disassembly of cortical F-actin in chromaffin cells in response to stimulation is thought to allow the free movement of secretory vesicles to exocytotic sites. Moreover, experiments by us using morphometric analysis of resting and stimulated chromaffin cells together with membrane capacitance measurements have shown that cortical F-actin controls the traffic of vesicles from the vesicle reserve compartment to the release-ready vesicle compartment. The dynamics of the cortical F-actin is controlled by two pathways: A) stimulation-induced Ca(2+) entry and scinderin activation; and B) protein kinase C (PKC) activation and MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation. When chromaffin cells are stimulated through nicotinic receptors, cortical F-actin disassembly is mainly through the intervention of pathway A, since in the presence of PKC inhibitors, F-actin disassembly in response to cholinergic stimulation is only blocked by 20%. Pathway A involves the activation of scinderin by Ca(2+) with a consequent F-actin severing. Pathway B is fully activated by phorbol esters and in this case PKC blockers inhibit by 100% the disruption of cortical F-actin. This pathway operates through MARCKS. A peptide with amino acid sequence corresponding to the phosphorylation site domain of MARCKS, which also corresponds to its actin binding site, blocks PMA potentiation of Ca(2+)-induced catecholamine release. The results suggest that under physiological conditions (i.e., nicotinic receptor stimulation) pathway A is the principal mechanism for the control of cortical F-actin dynamic changes.  相似文献   

8.
Secretory vesicles are localized in specific compartments within neurosecretory cells. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is controlled and regulated by Ca2+, scinderin and the cortical F-actin network. Cortical F-actin disassembly is produced by the filament severing activity of scinderin. This Ca2+-dependent activity of scinderin together with its Ca2+-independent actin nucleating activity, control cortical F-actin dynamics during the secretory cycle. A good understanding of the interaction of actin with scinderin and of the role of this protein in secretion has been provided by the analysis of the molecular structure of scinderin together with the use of recombinant proteins corresponding to its different domains.  相似文献   

9.
肌切蛋白(scinderin)是一种重要的肌动蛋白结合蛋白,在哺乳动物和脊椎动物中广泛表达.肌切蛋白作为凝溶胶蛋白超家族的成员之一,通过肌动蛋白丝切割、肌动蛋白聚集等方式来控制肌动蛋白的结构.肌切蛋白生物活性具有多样性,除影响肌动蛋白丝重组外,肌切蛋白还参与细胞胞吐作用、调节细胞运动、细胞分化等细胞活动.此外,肌切蛋白在慢性炎症、凝血过程、免疫性疾病和肿瘤发生发展中也发挥了重要作用.本文对肌切蛋白的结构特点、参与调节细胞的功能和机制进行概述.  相似文献   

10.
Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+-binding sites. F-actin severing activity of Sc is Ca2+-dependent, whereas Sc-evoked actin nucleation is Ca2+-independent. Sc domain role in secretion was studied by co-transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)-fusion Sc constructs. Cells over-expressing actin severing Sc1-6 or Sc1-2 (first and second actin binding sites) constructs, increased F-actin disassembly and hGH release upon depolarization. Over-expression of nucleating Sc5-6, Sc5 or ScABP3 (third actin site) constructs decreased F-actin disassembly and hGH release upon stimulation. Over-expression of ScL5-6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F-actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly left arrow over right arrow assembly) and release (facilitation left arrow over right arrow inhibition). The position of the switch (severing left arrow over right arrow nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation-induced Ca2+ entry would increase Sc-evoked cortical F-actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc-evoked actin nucleation.  相似文献   

11.
Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.  相似文献   

12.
As a final stage of cell signal transduction, secretory cells release hormones by exocytosis. Before secretory granules contact with the cell membrane for fusion, an actin-network barrier must dissociate as a prelude. To elucidate dynamical behaviors of secretory granules in actin networks, in vitro assembly and disassembly processes of actin networks were examined by means of dynamic light-scattering spectroscopy. We studied actin polymerization in the presence of chromaffin granules isolated from bovine adrenal medullas and found that the entanglement of actin filaments rapidly formed cages that confined granules in them. We also studied the effect of gelsolin, one of actin-severing proteins, on the network of actin filaments preformed in the presence of chromaffin granules. It turned out that the cages that confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. A semiquantitative analysis of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or the translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in the literature, a model is proposed for biophysical situations before, during, and after an exocytotic event.  相似文献   

13.
We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.  相似文献   

14.
Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.  相似文献   

15.
The acrosomal reaction (AR) is a regulated sperm exocytotic process that involves fusion of the plasma membrane (PM) with the outer acrosomal membrane (OAM). Our group has described F-actin cytoskeletons associated to these membranes. It has been proposed that in regulated exocytosis, a cortical cytoskeleton acts as a barrier that obstructs membrane fusion, and must be disassembled for exocytosis to occur. Actin-severing proteins from the gelsolin family have been considered to break this barrier. The present study attempted to determine if gelsolin has a function in guinea pig sperm capacitation and AR. By indirect immunofluorescence (IIF), gelsolin was detected in the apical and postacrosomal regions of the head and in the flagellum in both capacitated and non-capacitated guinea pig spermatozoa. By Western blotting, gelsolin was detected in isolated PM and OAM of non-capacitated spermatozoa. Gelsolin and actin were detected in a mixture of PM-OAM obtained by sonication, and both proteins were absent in membranes of capacitated spermatozoa. Inhibition of three different pathways of PIP2 hydrolysis during capacitation did not cancel gelsolin loss from membranes. Gelsolin was detected by Western blotting associated to membrane cytoskeletons obtained after phalloidin F-actin stabilization and Triton-X treatment; additionally, by immunoprecipitation, it was shown that gelsolin is associated with actin. By electron microscopy we observed that skeletons disassemble during capacitation, but phalloidin prevents disassembly. A three-dimensional skeleton was observed that apparently joins PM with OAM. Exogenous gelsolin stimulates AR assayed in a permeabilized spermatozoa model. Results suggest that gelsolin disassembles F-actin cytoskeletons during capacitation, promoting AR.  相似文献   

16.
Pathways that control cortical F-actin dynamics during secretion   总被引:2,自引:0,他引:2  
Chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane which acts as a barrier to the chromaffin vesicles access to exocytotic sites. Disassembly of cortical F-actin in response to stimulation allows the movement of vesicles from the reserve pool to the release-ready vesicle pool and, therefore, to exocytotic sites. The dynamics of cortical F-actin is controlled by two mechanisms: a) stimulation-induced Ca2+ entry and scinderin activation and b) protein kinase C (PKC) activation and MARCKS phosphorylation as demonstrated here by experiments with recombinant proteins, antisense olygodeoxynucleotides and vector mediated transient expressions. Under physiological conditions (i.e., cholinergic receptor stimulation followed by Ca2+ entry), mechanism (a) is the most important for the control of cortical F-actin network whereas when Ca2+ is released from intracellular stores (i.e., histamine stimulation) cortical F-actin is regulated mainly by mechanism b.  相似文献   

17.
Secretory vesicle exocytosis is the mechanism of release of neurotransmitters and neuropeptides. Secretory vesicles are localized in at least two morphologically and functionally distinct compartments: the reserve pool and the release-ready pool. Filamentous actin networks play an important role in this compartmentalization and in the trafficking of vesicles between these compartments. The cortical F-actin network constitutes a barrier (negative clamp) to the movement of secretory vesicles to release sites, and it must be locally disassembled to allow translocation of secretory vesicles in preparation for exocytosis. The disassembly of the cortical F-actin network is controlled by scinderin (a Ca(2+)-dependent F-actin severing protein) upon activation by Ca2+ entering the cells during stimulation. There are several factors that regulate scinderin activation (i.e., Ca2+ levels, phosphatidylinositol 4,5-bisphosphate (PIP2), etc.). The results suggest that scinderin and the cortical F-actin network are components of the secretory machinery.  相似文献   

18.
The large majority of chromaffin vesicles are excluded from the plasma membrane by a cortical F-actin network. Treatment of chromaffin cells with phorbol 12-myristate 13-acetate produces disassembly of cortical F-actin, increasing the number of vesicles at release sites (Vitale, M. L., Seward, E. P., and Trifaró, J. M. (1995) Neuron 14, 353-363). Here, we provide evidence for involvement of myristoylated alanine-rich protein kinase C substrate (MARCKS), a protein kinase C substrate, in chromaffin cell secretion. MARCKS binds and cross-links F-actin, the latter is inhibited by protein kinase C-induced MARCKS phosphorylation. MARCKS was found in chromaffin cells by immunoblotting. MARCKS was also detected by immunoprecipitation. In intact or permeabilized cells MARCKS phosphorylation increased upon stimulation with 10(-7) m phorbol 12-myristate 13-acetate. This was accompanied by cortical F-actin disassembly and potentiation of secretion. MARCKS phosphorylation, cortical F-actin disassembly, and potentiation of Ca(2+)-evoked secretion were inhibited by a peptide (MARCKS phosphorylation site domain sequence (MPSD)) with amino acid sequence corresponding to MARCKS phosphorylation site. MPSD was phosphorylated in the process. A similar peptide (alanine-substituted phosphorylated site domain) with four serine residues of MPSD substituted by alanines was ineffective. These results provide the first evidence for MARCKS involvement in chromaffin cell secretion and suggest that regulation of cortical F-actin cross-linking might be involved in this process.  相似文献   

19.
Scinderin是一种依赖Ca2+的肌动蛋白丝(F-actin)切割蛋白,在细胞分泌过程中发挥着重要作用。目前,针对scinderin在人类疾病尤其是肿瘤中的生物学功能研究报道的并不多。该实验通过构建scinderin—shRNA慢病毒载体并感染人胃癌细胞株SGC-7901,于荧光显微镜下观测感染效率,利用RT-qPCR和Western blot实验证实scinderin的沉默效果。运用实时细胞分析4K(RTCA)检测细胞的增殖能力,流式细胞术检测细胞周期变化,Transwell小室检测细胞的迁移能力。结果显示,将构建好的病毒载体成功转入了胃癌细胞SGC-7901。感染scinderin—shRNA病毒载体后,scinderin的mRNA和蛋白质表达水平均受到不同程度的抑制(P〈0.01),细胞的增殖和迁移能力均显著降低(P〈0.05),细胞周期阻滞在G2/M期。该研究表明,胃癌细胞SGC-7901中scinderin低表达能有效抑制细胞的增殖和转移能力,这也为scinderin在胃癌演化过程中的机制研究奠定了实验基础。  相似文献   

20.
Localized disassembly of cortical F-actin has long been considered necessary for facilitation of exocytosis. Exposure of permeabilized mast cells to calcium/ATP induces cortical F-actin disassembly (calmodulin-dependent) and secretion (calmodulin-independent). The delay in the onset of secretion is characteristic for the calcium/ATP response and is abolished by GTP. Here we report that a constitutively active mutant of Rho (V14RhoA) enhanced both secretion and cortical F-actin disassembly. In addition, V14RhoA mimicked GTP by abolishing the delay in secretion. Inhibition of Rho by C3 transferase prevented both secretion ( approximately 80%) and F-actin disassembly (approximately 20%). Thus, both Rho GTPase and calcium/calmodulin contribute to the control of cortical F-actin disassembly. Stabilization of actin filaments by high concentrations of phalloidin or by a calmodulin-inhibitory peptide (based on the calmodulin-binding domain of myosin light chain kinase) did not affect the extent of secretion or the secretion-enhancing effects of V14RhoA. These results further support the existence of divergent, Rho-dependent, pathways regulating actin and exocytosis. Furthermore, compound Y-27632, a specific inhibitor of Rho-associated protein kinase (p160(ROCK)), attenuated the Rho-induced loss of cortical F-actin without affecting secretion. A model is presented in which Rho regulates secretion and cortical F-actin in a manner dependent on and/or synergistic with calcium.  相似文献   

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