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1.
肌动蛋白是盘基网柄菌(Dictyostelium discoideum)细胞吞噬过程中的关键组分,通过其细胞内的定位和多聚化形式在确定的时间和地点连接特定的分子,使吞噬过程得以完成。profilin是肌动蛋白多聚化的重要调节分子,在磷脂酰肌醇信号转导与细胞骨架相交处起关键作用。许多小分子G蛋白参与细胞骨架调节,CAP蛋白是两者间重要连接分子。所以,吞噬作用是细胞内诸分子协同作用的结果。  相似文献   

2.
It is well established that Rho-GTPases regulate vesicle fusion and fission events at the plasma membrane through their modulatory role on the cortical actin cytoskeleton. In contrast, their effects on intracellular transport processes and actin pools are less clear. It was recently shown that cdc42 associates with the Golgi apparatus in an ARF-dependent manner, similarly to coat proteins involved in vesicle formation and to several actin-binding proteins. We report here that mutants of cdc42 inhibited the exit of basolateral proteins from the trans-Golgi network (TGN), while stimulating the exit of an apical marker, in two different transport assays. This regulation may result from modulation of the actin cytoskeleton, as GTPase-deficient cdc42 depleted a perinuclear actin pool that rapidly exchanges with exogenous fluorescent actin.  相似文献   

3.
Cdc42 is a member of the Rho family of GTPases and plays an important role in the regulation of actin cytoskeletal organization. Activation of Cdc42 and associated signal transduction cascades are dependent upon proper localization of this GTPase. The studies described herein address the hypothesis that Rho GDP-dissociation inhibitor, RhoGDI, plays an essential role in the translocation of Cdc42 to signaling complexes at the plasma membrane and is essential for Cdc42-mediated actin cytoskeletal rearrangements. An activating mutant of Cdc42 that is RhoGDI-binding defective (Cdc42(G12V/R66E)) is characterized and used as a tool to study the functional importance of the Cdc42-RhoGDI interaction. Overexpression of mycCdc42(G12V/R66E) in COS-7 cells results in actin cytoskeletal rearrangements that are indistinguishable from those stimulated by overexpression of mycCdc42(G12V). In addition, the G12V activating mutant of Cdc42 was overexpressed in mesangial cells that are null for RhoGDI expression. MycCdc42(G12V) stimulation of filopodia formation in these cells was indistinguishable from that observed in wild-type mesangial cells. Taken together, the results presented herein indicate that although RhoGDI is a critical regulator of guanine nucleotide binding, cycling of Cdc42 between membranes and the cytosol and cellular transformation, it is not essential for Cdc42-mediated organization of the actin cytoskeleton.  相似文献   

4.
The syndecans, a family of transmembrane heparan sulfate proteoglycans, are ubiquitous molecules whose intracellular function is still unknown. To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycan in fibroblasts, we performed transfection studies in COS-1 and Swiss 3T3 cells. Endogenous syndecan-2 colocalized with F-actin in cortical structures. Overexpression of full-length syndecan-2 induced the formation of long filopodia-like structures. These changes correlated with a rearrangement of the actin cytoskeleton, which strongly colocalized with syndecan-2. Overexpression of syndecan-2 lacking the extracellular domain increased the number of microspikes on the cell surface but failed to induce filopodia. Addition of heparin blocked the effect of full-length syndecan-2, suggesting that heparan sulfate chains in the extracellular domain are necessary to induce filopodia. Coexpression of cdc42Hs negative-dominant N17 blocked syndecan-2-induced filopodia and cdc42Hs positive-dominant V12 had a synergic effect. This indicates that active cdc42Hs is necessary for syndecan-2 induction of filopodia. These results provide a link between syndecan-2, actin cytoskeleton, and cdc42Hs.  相似文献   

5.
Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.  相似文献   

6.
Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.  相似文献   

7.
Activation of G protein-coupled receptors (GPCRs) leads to the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gβγ subunits regulate actin assembly and migration, but the protein(s) linking Gβγ to the actin cytoskeleton remains unknown. Here, we identified a Gβγ effector, ElmoE in Dictyostelium, and demonstrated that it is required for GPCR-mediated chemotaxis. Remarkably, ElmoE associates with Gβγ and Dock-like proteins to activate the small GTPase Rac, in a GPCR-dependent manner, and also associates with Arp2/3 complex and F-actin. Thus, ElmoE serves as a link between chemoattractant GPCRs, G-proteins and the actin cytoskeleton. The pathway, consisting of GPCR, Gβγ, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis.  相似文献   

8.
Sprouty (SPRY) protein negatively modulates fibroblast growth factor and epidermal growth factor actions. We showed that human SPRY2 inhibits cell growth and migration in response to serum and several growth factors. Using rat intestinal epithelial (IEC-6) cells, we investigated the involvement of the Rho family of GTPases, RhoA, Rac1, and cdc42 in SPRY2-mediated inhibition of cell migration and proliferation. The ability of TAT-tagged SPRY2 to inhibit proliferation and migration of IEC-6 cells transfected with constitutively active mutants of RhoA(G14V), Rac1(G12V), and cdc42 (F28L) was determined. Constitutively active RhoA(G14V), Rac1(G12V), or cdc42(F28L) did not protect cells from the anti-proliferative actions of TAT-SPRY2. The ability of TAT-hSPRY2 to inhibit migration was not altered by of RhoA(G14V) and cdc42(F28L). However, Rac1(G12V) obliterated the ability of SPRY2 to inhibit cell autonomous or serum-induced migration. Also, the activation of endogenous Rac1 was attenuated by TAT-SPRY2. Thus, SPRY2 mediates its anti-migratory actions by inhibiting Rac1 activation.  相似文献   

9.
We have established an in vitro assay for assembly of the cortical actin cytoskeleton of budding yeast cells. After permeabilization of yeast by a novel procedure designed to maintain the spatial organization of cellular constituents, exogenously added fluorescently labeled actin monomers assemble into distinct structures in a pattern that is similar to the cortical actin distribution in vivo. Actin assembly in the bud of small-budded cells requires a nucleation activity provided by protein factors that appear to be distinct from the barbed ends of endogenous actin filaments. This nucleation activity is lost in cells that lack either Sla1 or Sla2, proteins previously implicated in cortical actin cytoskeleton function, suggesting a possible role for these proteins in the nucleation reaction. The rate and the extent of actin assembly in the bud are increased in permeabilized delta cap2 cells, providing evidence that capping protein regulates the ability of the barbed ends of actin filaments to grow in yeast cells. Actin incorporation in the bud can be stimulated by treating the permeabilized cells with GTP-gamma S, and, significantly, the stimulatory effect is eliminated by a mutation in CDC42, a gene that encodes a Rho-like GTP-binding protein required for bud formation. Furthermore, the lack of actin nucleation activity in the cdc42 mutant can be complemented in vitro by a constitutively active Cdc42 protein. These results suggest that Cdc42 is closely involved in regulating actin assembly during polarized cell growth.  相似文献   

10.
Biliary ducts are lined with epithelial cells, which consist of at least two types of cholangiocytes, small and large. In contrast to large cholangiocytes, which are involved in secretion, the role of small cholangiocytes has not been elucidated. To address this question, we analyzed the migration-based characteristics of these cells that may help to understand their functions in vivo. Interestingly, dibutyryl cAMP induced marked filopodia formation and cdc42 activation in the normal mouse cholangiocyte (NMC)-small cell line compared with the NMC-large cell line. Analysis of members of the ephrin (Eph)A family of guidance molecules revealed a distinct subcellular distribution of EphA5 and EphA8 members: EphA8 was equally expressed by both cell types and localized subcellularly in peripheral cell membranes, whereas EphA5 was expressed predominantly in NMC-S and localized to filopodia. Moreover, cAMP-inducible filopodia formation in these cells was abrogated using EphA5 short interfering RNA. Finally, we found that the Rho family GTPase cdc42 was activated in a manner dependent on EphA5. Wortmannin, a specific inhibitor of phosphotidylinositol 3-kinase (PI3K), abolished the activation of cdc42 dependent on EphA5, suggesting the involvement of PI3K in the EphA5-cdc42 pathway. Together, our findings suggest a cAMP-EphA5-cdc42-dependent regulation of small cholangiocyte migration, which are anticipated to facilitate the understanding of the nature of cholangiocytes and to explain certain general aspects of cAMP-cdc42 activation signaling with regard to cell morphogenesis.  相似文献   

11.
Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.  相似文献   

12.
Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis. We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-1ts mutation. S. pombe Cdc42p is 85% identical in predicted amino acid sequence to S. cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog. The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools. We have disrupted the cdc42+ gene and shown that it is essential for growth. The cdc42 null phenotype is an arrest as small, round, dense cells. In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S. cerevisiae CDC42. In contrast to the S. cerevisiae cdc42 mutations, the S. pombe cdc42 mutant alleles were not lethal when overexpressed. However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S. pombe Cdc42p is involved in controlling polarized cell growth.  相似文献   

13.
Dynamic cellular rearrangements involving the actin cytoskeleton are required of both Sertoli and germ cells during spermatogenesis. Rho family small G proteins have been implicated in the control of the actin cytoskeleton in numerous cell types. Therefore, RhoA and Rac1 were investigated in Sertoli and germ cells. RhoA and Rac1 have been detected at both the mRNA and protein levels in these cells. In addition, Sertoli cell L-selectin is shown to interact with actin binding proteins, potentially providing a link between L-selectin and Rac1 signaling. Finally, inactivation of Sertoli cell Rho family proteins yields disruption of the actin cytoskeleton.  相似文献   

14.
Phagocytosis of opsonized particles by neutrophils involves highly localized alterations in the actin cytoskeleton that result in the formation of prominent pseudopodia and the phagocytic cup. Immunofluorescence microscopy was employed to monitor the distribution of several proteins that can regulate the cytoskeleton in human neutrophils undergoing phagocytosis of opsonized Candida albicans. The small GTPase Cdc42, its inhibitory subunit Rho-GDI, the adapter protein Nck, gamma-p21-activated protein kinase (gamma-Pak), and cofilin were found to undergo rapid association with the developing phagosomes in these cells. In contrast, these signaling proteins were either diffusely distributed in the cytoplasm or enriched in focal points at the base of the cell when optical sections were obtained from regions of the cell not involved in phagocytosis. These results are consistent with Cdc42 being critically involved in initiating the early events in phagocytosis by its ability to activate Pak and the Wiskott-Aldrich Syndrome protein that triggers the localized polymerization of actin. These data provide insights into the molecular mechanisms that trigger changes in the actin cytoskeleton during phagocytosis.  相似文献   

15.
Polarized delivery and incorporation of proteins and lipids to specific domains of the plasma membrane is fundamental to a wide range of biological processes such as neuronal synaptogenesis and epithelial cell polarization. The exocyst complex is specifically localized to sites of active exocytosis and plays essential roles in secretory vesicle targeting and docking at the plasma membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark for polarized exocytosis. In a search for proteins that regulate the localization of the exocyst in the budding yeast Saccharomyces cerevisiae, we found that certain cdc42 mutants affect the polarized localization of the exocyst proteins. In addition, we found that these mutant cells have a randomized protein secretion pattern on the cell surface. Biochemical experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound form. Genetic studies demonstrated synthetically lethal interactions between cdc42 and several exocyst mutants. These results have revealed a role for Cdc42 in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery and the actin cytoskeleton for polarized secretion.  相似文献   

16.
Calcium-binding protein 3 (CBP3) expression was up-regulated under the control of the actin 15 promoter and down-regulated by RNA interference in Dictyostelium discoideum. The overexpression of CBP3 accelerated cell aggregation and formed small aggregates and fruiting body. CBP3-inhibited cells showed uneven aggregation and increased slug trail lengths toward the directed light, whereas CBP3-overexpressing cells showed the opposite phenomena. Under dark condition, the enhanced slug trail length was also observed in the CBP3-inhibited cells. Yeast two-hybrid screening identified actin 8 as interacting protein with CBP3. The interaction between CBP3 and actin was confirmed by beta-galactosidase assay and surface plasmon resonance. CBP3 was associated with Triton X-100-insoluble cytoskeleton in the presence of Ca(2+) and the interaction of CBP3 with cytoskeleton was increased by the addition of Ca(2+). Using fluorescence microscopy, CBP3 was also shown to associate with the actin cytoskeleton during development. Subcellular fractionation indicated that CBP3 was enriched in cytosolic fraction. Taken together, these results suggest that CBP3 interacts with actin cytoskeleton and has a role during cell aggregation and slug migration of Dictyostelium.  相似文献   

17.
Schulte RJ  Sefton BM 《Biochemistry》2003,42(31):9424-9430
The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible.  相似文献   

18.

Background

The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1.

Methodology/Principal Findings

Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis.

Conclusions/Significance

These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.  相似文献   

19.
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.  相似文献   

20.
The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from the fronts. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization and depolymerization in spreading cells. Short-lived actin patches that co-localize with clathrin suggest that membrane internalization occurs even when the substrate-attached cell surface expands. We conclude that non-monotonic cell spreading is characterized by spatiotemporal patterns formed by motor proteins together with regulatory proteins that either promote or terminate actin polymerization on the scale of seconds.Key words: actin cytoskeleton, Arp 2/3 complex, cell adhesion, cell spreading, Coronin, Dictyostelium, myosin, self-organization, clathrin  相似文献   

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