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1.
We report the cDNA sequence and localization of a protein first identified by actin filament chromatography of Drosophila embryo extracts as ABP8 (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). The cDNA encodes a 1201-amino acid protein which we name anillin. Anillin migrates at 190 kD on SDS-PAGE. Anillin is expressed throughout Drosophila development and in tissue culture cells. By immunofluorescence, anillin localizes to the nucleus of interphase cells, except in the syncytial embryo where it is always cytoplasmic. During metaphase, it is present in the cytoplasm and cortex, and during anaphase-telophase it becomes highly enriched in the cleavage furrow along with myosin II. In the syncytial embryo, anillin, along with myosin-II, is enriched in cortical areas undergoing cell cycle regulated invagination including metaphase furrows and the cellularization front. In contractile rings, metaphase furrows, and nascent ring canals, anillin remains bound to the invaginated cortex suggesting a stabilizing role. Anillin is not expressed in cells that have left the cell cycle. Anillin isolated from embryo extracts binds directly to actin filaments. The domain responsible for this binding has been mapped to a region of 244 amino acids by expression of protein fragments in bacteria. This domain, which is monomeric in solution, also bundles actin filaments. We speculate that anillin plays a role in organizing and/or stabilizing the cleavage furrow and other cell cycle regulated, contractile domains of the actin cytoskeleton.  相似文献   

2.
Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors (reviewed in [1, 2]). Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA [3-7]. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. Here, we show that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis.  相似文献   

3.
Myosin II assembles into force-generating filaments that drive cytokinesis and the organization of the cell cortex. Regulation of myosin II activity can occur through modulation of filament assembly and by targeting to appropriate cellular sites. Here we show, using salt-dependent solubility and a novel fluorescence resonance energy transfer assay, that assembly of the Drosophila non-muscle myosin II heavy chain, zipper, is mediated by a 90-residue region (1849-1940) of the coiled-coil tail domain. This filament assembly domain, transiently expressed in Drosophila S2 cells, does not localize to the interphase cortex or the cytokinetic cleavage furrow, whereas a 500-residue region (1350-1865) that overlaps the NH(2) terminus of the assembly domain localizes to the interphase cortex but not the cytokinetic cleavage furrow. Targeting to these two sites appears to utilize distinct localization mechanisms as the assembly domain is required for cleavage furrow recruitment of a truncated coiled-coil tail region but not targeting to the interphase cortex. These results delineate the requirements for zipper filament assembly and indicate that the ability to form filaments is necessary for targeting to the cleavage furrow but not to the interphase cortex.  相似文献   

4.
The Nima-related kinase 2 (Nek2) has been implicated in the regulation of centrosome integrity and separation in several species and is a candidate for cell transformation. We now show that reduction of levels of the Drosophila Nek2 by RNAi in cultured cells leads to both dispersal of centrosomal antigens and formation of ectopic bodies of centrosomal antigens. Overexpression of the active DmNek2 kinase resulted in an increase in the number of mitotic cells with fragmented centrosomes. The DmNek2 protein kinase is associated with punctuate bodies within the centrosome consistent with its presence on centrioles. In addition, it is present at lower levels on the midbody during cytokinesis. Midbody association was enhanced following overexpression, whereupon the DmNek2 protein kinase also localised to the cell cortex becoming concentrated in the region of the cleavage furrow in late telophase. Many of such cells showed abnormalities in the organisation of anillin and actin in the cleavage furrow that was associated with formation of ectopic membrane protrusions between each daughter cell. We discuss potential roles for DmNek2 in maintaining centrosome integrity in mitosis, during cytokinesis, and consequently for the fidelity of chromosome segregation.  相似文献   

5.
Anillin is a conserved component of the contractile ring that is essential for cytokinesis, and physically interacts with three conserved cleavage furrow proteins, F-actin, myosin II and septins in biochemical assays. We demonstrate that the Drosophila scraps gene, identified as a gene involved in cellularization, encodes Anillin. We characterize defects in cellularization, pole cell formation and cytokinesis in a series of maternal effect and zygotic anillin alleles. Mutations that result in amino acid changes in the C-terminal PH domain of Anillin cause defects in septin recruitment to the furrow canal and contractile ring. These mutations also strongly perturb cellularization, altering the timing and rate of furrow ingression. They cause dramatic vesiculation of new plasma membranes, and destabilize the stalk of cytoplasm that normally connects gastrulating cells to the yolk mass. A mutation closer to the N terminus blocks separation of pole cells with less effect on cellularization, highlighting mechanistic differences between contractile processes. Cumulatively, our data point to an important role for Anillin in scaffolding cleavage furrow components, directly stabilizing intracellular bridges, and indirectly stabilizing newly deposited plasma membrane during cellularization.  相似文献   

6.
Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.  相似文献   

7.
Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)(Pbl)-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF(Pbl)-dependent control themselves. Microscopy of Drosophila melanogaster S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, we uncovered a separate RhoGEF(Pbl)-dependent pathway that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF(Pbl)-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. We propose that such interactions occur dynamically and transiently to promote furrow stability.  相似文献   

8.
Localization of the actin crosslinking protein, alpha-actinin, to the cleavage furrow has been previously reported. However, its functions during cytokinesis remain poorly understood. We have analyzed the functions of alpha-actinin during cytokinesis by a combination of molecular manipulations and imaging-based techniques. alpha-actinin gradually dissipated from the cleavage furrow as cytokinesis progressed. Overexpression of alpha-actinin caused increased accumulation of actin filaments because of inhibition of actin turnover, leading to cytokinesis failure. Global depletion of alpha-actinin by siRNA caused a decrease in the density of actin filaments throughout the cell cortex, surprisingly inducing accelerated cytokinesis and ectopic furrows. Local ablation of alpha-actinin induced accelerated cytokinesis specifically at the site of irradiation. Neither overexpression nor depletion of alpha-actinin had an apparent effect on myosin II organization. We conclude that cytokinesis in mammalian cells requires tightly regulated remodeling of the cortical actin network mediated by alpha-actinin in coordination with actomyosin-based cortical contractions.  相似文献   

9.
Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.  相似文献   

10.
The inner centromeric protein (INCENP) and other chromosomal passenger proteins are known to localize on the cleavage furrow and to play a role in cytokinesis. However, it is not known how INCENP localizes on the furrow or whether this localization is separable from that at the midbody. Here, we show that the association of Dictyostelium INCENP (DdINCENP) with the cortex of the cleavage furrow involves interactions with the actin cytoskeleton and depends on the presence of the kinesin-6-related protein Kif12. We found that Kif12 is found on the central spindle and the cleavage furrow during cytokinesis. Kif12 is not required for the redistribution of DdINCENP from centromeres to the central spindle. However, in the absence of Kif12, DdINCENP fails to localize on the cleavage furrow. Domain analysis indicates that the N terminus of DdINCENP is necessary and sufficient for furrow localization and that it binds directly to the actin cytoskeleton. Our data suggest that INCENP moves from the central spindle to the furrow of a dividing cell by a Kif12-dependent pathway. Once INCENP reaches the equatorial cortex, it associates with the actin cytoskeleton where it then concentrates toward the end of cytokinesis.  相似文献   

11.
The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.  相似文献   

12.
Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.  相似文献   

13.
Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.  相似文献   

14.
The Drosophila Formin Homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, we generated embryos deficient for Diaphanous and analyzed three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. Our findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, we propose that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle.  相似文献   

15.
Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.  相似文献   

16.
Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.  相似文献   

17.
Cell division is achieved by a plasma membrane furrow that must ingress between the segregating chromosomes during anaphase [1-3]. The force that drives furrow ingression is generated by the actomyosin cytoskeleton, which is linked to the membrane by an as yet undefined molecular mechanism. A key component of the membrane furrow is anillin. Upon targeting to the furrow through its pleckstrin homology (PH) domain, anillin acts as a scaffold linking the actomyosin and septin cytoskeletons to maintain furrow stability (reviewed in [4, 5]). We report that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P(2), which is enriched in the furrow. Reduction of cellular PI(4,5)P(2) or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P(2), interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression that can be restored by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P(2) binding module, a condition where the septin cytoskeleton is not recruited to the plasma membrane. These data demonstrate that the anillin PH domain has two functions: targeting anillin to the furrow by binding to PI(4,5)P(2) to maintain furrow organization and recruiting septins to the furrow.  相似文献   

18.
Localization and possible functions of Drosophila septins.   总被引:8,自引:3,他引:5       下载免费PDF全文
The septins are a family of homologous proteins that were originally identified in Saccharomyces cerevisiae, where they are associated with the "neck filaments" and are involved in cytokinesis and other aspects of the organization of the cell surface. We report here the identification of Sep1, a Drosophila melanogaster septin, based on its homology to the yeast septins. The predicted Sep1 amino acid sequence is 35-42% identical to the known S. cerevisiae septins; 52% identical to Pnut, a second D. melanogaster septin; and 53-73% identical to the known mammalian septins. Sep1-specific antibodies have been used to characterize its expression and localization. The protein is concentrated at the leading edge of the cleavage furrows of dividing cells and cellularizing embryos, suggesting a role in furrow formation. Other aspects of Sep1 localization suggest roles not directly related to cytokinesis. For example, Sep1 exhibits orderly, cell-cycle-coordinated rearrangements within the cortex of syncytial blastoderm embryos and in the cells of post-gastrulation embryos; Sep1 is also concentrated at the leading edge of the epithelium during dorsal closure in the embryo, in the neurons of the embryonic nervous system, and at the baso-lateral surfaces of ovarian follicle cells. The distribution of Sep1 typically overlaps, but is distinct from, that of actin. Both immunolocalization and biochemical experiments show that Sep1 is intimately associated with Pnut, suggesting that the Drosophila septins, like those in yeast, function as part of a complex.  相似文献   

19.
mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.  相似文献   

20.
Septins are filament-forming proteins with a conserved role in cytokinesis. In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell-cell separation, a late stage in cytokinesis. Here, we identified a protein Mid2p on the basis of its sequence similarity to S. pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p. Like septin mutants, mid2delta mutants had delays in cell-cell separation. mid2delta mutants were defective in septin organization but not contractile ring closure or septum formation. In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract. In mid2delta cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure. FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2delta cells. Mid2p colocalized with septins and required septins for its localization. A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function. No genetic interactions were found between mid2 and the related gene mid1. Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.  相似文献   

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