摘要:

AbstractA 17 kDa protein, designated as coactosin, has been purified from an actinmyosin complex reconstituted in vitro from a soluble fraction ofDictyostelium discoideumcells. The protein binds to F‐actin in vitro without significantly altering its viscosity. Immunoblots labeled with monoclonal antibodies indicate that part of the protein is associated with the detergent‐insoluble cytoskeleton. cDNA clones comprising the entire coding region of coactosin have been isolated from an expression library. The cDNA‐derived amino‐acid sequence reveals similarities of coactosin to the drebrins identified in neurons and to actin‐binding proteins from other organisms, including yeast ABP1p, and yeast and vertebrate cofilins. © 1993 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970260302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractCentral to the function of myosin is its ability to assemble into thick filaments which interact precisely and specifically with other myofibrillar proteins. We have established a novel experimental system for studying myofibrillogenesis using transient transfections of COS cells, a monkey kidney cell line. We have expressed both full‐length rat α cardiac myosin heavy chain (MHC) and a truncated heavy meromyosin‐like α MHC (sHMM) and shown that immunoreactive MHC proteins of the expected sizes were detected in lysates of transfected cells. Surprisingly, the full‐length MHC formed large spindle‐shaped structures throughout the cytoplasm of transfected cells as determined by immunofluorescence microscopy. The structures were not found in cells expressing the sHMM construct, indicating that their formation required an MHC rod. The spindle‐shaped structures ranged in length from approximately 1 μm to over 20 μm in length and were birefringent suggesting that they are ordered arrays of thick filaments. This was confirmed by electron microscopic analysis of the transfected cells which revealed arrays of filamentous structures approximately 12 nm in diameter at their widest point. In addition, the vast majority of transfected MHC did not associate with the endogenous nonmuscle myosin light chains, demonstrating that myosin thick filaments can form in the absence of stoichiometric amounts of myosin light chains. © 1993 W

ISSN:0886-1544    

DOI:10.1002/cm.970260303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractCytoskeletal elements associated with the smooth septate junctions linking the midgut columnar cells ofManduca sextalarvae (Insecta, Lepidoptera) were characterized. Myosin subfragment 1 decoration and immunostaining for actin demonstrated that the filaments associated with the septate junctions were constituted of actin. Moreover, using a combination of immunochemical and immunolocalization techniques, evidence is presented that α‐actinin, myosin II, and vinculin are localized close to the specialized plasma membranes. The insertion of microfilament bundles into submembranous F‐actin/α‐actinin/vinculin complexes, previously described in vertebrate junctions of adherens type, appears to be a more general organization, including the insect septate junction here examined. © 1993 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970260304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have identified a novel .ca 400 kDa cell‐cycle dependent kinetochore associated protein in human cells, designated CENP‐F, using human autoimmune serum. Immunofluorescence staining using the native serum, affinity purified antibodies, or antibodies raised against a cloned portion of CENP‐F first reveals CENP‐F homogeneously distributed throughout the nucleus of HeLa cells in the G2 stage of the cell cycle. Progression into prophase is accompanied by the localization of CENP‐F to all the kinetochore regions of the karyotype. Kinetochore association is maintained throughout metaphase, but at the onset of anaphase CENP‐F is no longer detected in association with the kinetochore but is found at the spindle mid‐zone. By telophase, it is concentrated into a narrow band on either side of the midbody. Studies of the interaction of CENP‐F with the kinetochore indicate that this protein associates with the kinetochore independent of tubulin and dissociation is dependent on events connected with the onset of anaphase. Nuclease digestion studies and immunoelectron‐microscopy indicate that CENP‐F is localized to the kinetochore plates and specifically to the outer surface of the outer kinetochore plate. The distribution of CENP‐F closely parallels that of another high molecular weight kinetochore associated protein, CENP‐E. Comparative studies indicate that there are antibodies in the CENP‐F reactive autoimmune serum that recognize determinants present in the central helical rod domain of CENP‐E. Immune depletion experiments confirm that CENP‐F exhibits the distribution pattern in cells that was seen with the native autoimmune

ISSN:0886-1544    

DOI:10.1002/cm.970260305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractgCap39 is a newly identified member of the Ca2+‐ and polyphosphoinositidemodulated gelsolin family of actin binding proteins which is different from gelsolin in several important respects: it caps filament ends, it does not sever filaments, it binds reversibly to actin, it is phosphorylated in vivo, and it is also present in the nucleus. gCap39 and gelsolin coexist in a variety of cells. To better understand the roles of gCap39 and gelsolin, we have compared their relative amounts and intracellular distributions. We found that gCap39 is very abundant in macrophages (accounting for 0.6% of total macrophage proteins), and is present in 12‐fold molar excess to gelsolin. Both proteins are highly induced during differentiation of the promyelocytic leukemia cell line into macrophages. gCap39 is less abundant in fibroblasts (0.04% total proteins) and is present in equal molar ratio to gelsolin. The two proteins are colocalized in the cytoplasm, but gCap39 is also found in the nucleus while gelsolin is not. Nuclear gCap39 redistributes throughout the cytoplasm during mitosis and is excluded from regions containing chromosomes. Our results demonstrate that gCap39 is a nuclear and cytoplasmic protein which has unique as well as common functions compared with gelsolin. © 1993 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970260306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe behaviour of the centrosome immediately following cell division in tissue culture cells has been investigated. We find that following karyokinesis, but preceding cytokinesis, sister centrosomes relocate from the spindle poles to a position adjacent to the intercellular bridge. This repositioning is accompanied by the appearance of a microtubule bundle that extends from the poleward region of the cell to the centrosome and increases in length as the centrosome approaches the intercellular bridge. Disruption of this bundle with colcemid interrupts centrosome repositioning. In contrast, centrosome repositioning persists in late mitotic cells grown in the presence of cytochalasin D. However, the position of the microtubule‐centrosome complex within the cell is randomized suggesting that the path, but not the process, of centrosome repositioning is dependent on an intact actin filament network. This study points out, for the first time, that the complex migration of the centrosome preceding mitosis is paralleled by an equally complex set of events following cell division. We suggest that post‐mitotic centrosome repositioning may play a role in ensuring that daughter cells have equal but opposite polarity and may reflect an interrelationship between the establishment of the interphase cytoskeleton and the completion of cytokinesis. © 1993 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970260307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractAt least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsmα) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full‐length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5. PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F‐actin was stronger than the PEThTM3 isoform. However, analysis of actin‐binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N‐terminal fragment of hTM5 fused to the C‐terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N‐terminal fragment of hTM3 fused to the C‐terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F‐actin than their parental tropomyosins. A bacterially made C‐terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin‐binding of these bacterially expressed tropomyosins. However, PETCaD39′s enhancement of binding to F‐actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low Mrisoform PEThTM5 appeared to be able to amplify the actin‐activated HMM ATPase activity by 4.7 fold, while the high Mrisoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low Mrisoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the ce

ISSN:0886-1544    

DOI:10.1002/cm.970260308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractSea urchin coelomocytes naturally flatten on a substratum into a discoid morphology and display striking, centripetally directed cortical flow along the radii of the cell when viewed with time lapse, video enhanced microscopy. The rate of cortical flow averaged 4.5 μm/min in the peripheral most 10 μm of cytoplasm but slows considerably in the perinuclear region. Cytochalasin B causes: (1) the flow to stop, (2) the buildup of an actin filament‐rich peripheral ridge of cytoskeletal material, (3) the centrifugal dissolution of a portion of the actin cytoskeleton, and (4) the contraction of other portions of the cytoskeleton into foci. Cytochalasin D (CD), on the other hand, causes the flowing actin meshwork to become severed from the edge of the cell and allows it to be drawn at least part way in towards the nucleus. A smaller peripheral ridge of actin filament buildup is also seen with CD. Colcemid induces another striking change in the cytoskeleton. The centripetal progression of the actin is not stopped by colcemid, but shortly after leaving the periphery of the cell, the linear elements within the flow become reoriented into arcs. The long axis of the arcs is roughly parallel with the cell's edge. The effects of all three drugs are reversible. The results are discussed in light of other systems and potential mechanisms for cortical flow. © 1993 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970260309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970260310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970260301

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY