摘要:

AbstractMammalian eggs and embryos possess a major cytoskeletal network composed of large planar “sheets” distributed throughout the cytoplasm. Cytoskeletal sheets are found neither in mammalian somatic cells nor in eggs or embryos of non‐mammals. In this study, we have investigated the structural composition of the sheets in eggs and embryos of the golden Syrian hamster by (1) analysis of replicas from quick‐frozen, deep‐etched specimens, (2) analysis of thick, resinembedded specimens using an intermediate voltage electron microscope (IVEM), (3) laser diffraction of EM images, (4) differential extraction with detergents, and (5) immunocytochemistry. Our results indicate that each sheet is composed of two closely apposed arrays of 10‐nm filaments. Each filament within an array is held in register with its neighbor by lateral cross‐bridges and the two parallel arrays of filaments are interconnected by periodic cross‐bridges about 20 nm in length. Laser diffraction of negatives from IVEM images indicates that each array is composed of fibers that form a square lattice, and the two arrays are positioned in register by cross‐bridges forming a single sheet. This lattice forms the skeleton of the sheets which is covered with a tightly packed layer of particulate material. By incubation in media containing different ratios of mixed‐micelle detergents, it is possible to remove components sequentially from the sheets and to extract the particulate material. Immunocytochemical localization demonstrates that the sheets bind antibodies to keratin, and to a small extent actin, but do not bind antibodies to vimentin or tubulin. Examination of sheets within embryos at the time of embryonic compaction demonstrates that the sheets begin to fragment and disassemble in regions of blastomeres where desmosomes form, but undergo no structural alterations in interior and basal surfaces of the blastomeres. In regions of blastomere—blastomere contact the sheets fragment and associate with granules resembling keratohyalin granules found in keratinocytes.

ISSN:0886-1544    

DOI:10.1002/cm.970240202

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

年代:1993

数据来源: WILEY

摘要:

AbstractActin‐binding protein (ABP) is a well‐characterized polypeptide capable of crosslinking filamentous actin. To date, this polypeptide has been shown to exist in a number of tissues and cultured cell lines. This report shows that by using a panel of three monoclonal antibodies for immunoblotting and immunofluorescence analysis, that ABP is present in bovine erythrocytes. Moreover, the data obtained suggest that this protein is a component of the erythrocyte membrane skeleton. Additionally, bovine erythrocyte ABP is shown to possess both an apparent molecular weight and an isoelectric point identical to that of bovine smooth muscle filamin, implying that these two polypeptides are identical. © 1993 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970240203

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

年代:1993

数据来源: WILEY

摘要:

AbstractIn this work, we examine whether the “nexin” linkages of the flagellum can extend in length to accommodate interdoublet sliding. Flagellar bends of large angle were induced in bull spermatozoa by hypotonic treatment. It is argued that this produces large interdoublet displacements that are, nevertheless, still within physiological limits. Such flagella were examined by the rapid‐freeze, deep‐etch techique and the nexin linkages identified by their position in relation to the inner dynein arms and by their straplike, bipartite, morphology. They were found to bridge perpendicularly (or occasionally at an angle) between the A‐ and B‐tubules of adjacent doublets. The nexin linkages were no more than ∼20 nm in length, even in regions in which ∼200 nm of sliding could be inferred. Variable registration between adjacent nexin rows gave some further support to the assumption that sliding had indeed taken place. From this, it is concluded that elastic deformation of the links, such as would accommodate interdoublet sliding, does not occur; some form of displacement must occur between nexin and the adjacent B‐tubule. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970240204

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe structural requirements for assembly of tropomyosin into stress fibers were investigated by microinjecting wildtype and four mutant striated chicken muscle α‐tropomyosins expressed inE. colias fusion and nonfusion proteins into cultured rat embryo fibroblasts, followed by localization of tropomyosin using indirect immunofluorescence. The results show that the determinants for stress fiber incorporation in living cells correlate with the in vitro actin affinity of these tropomyosins. Wildtype recombinant protein incorporated into stress fibers both when the amino terminus was unacetylated and when it was blocked with an 80‐residue fusion protein [Hitchcock‐DeGregori, S.E., and Heald, R.W. (1987): J. Biol. Chem. 262:9730–9735]. The pattern of incorporation was indistinguishable from that of tropomyosin isolated from chicken pectoral muscle. The striated α‐tropomyosin incorporated into stress fibers, even though this isoform is not found in nonmuscle cells. Three recombinant mutant tropomyosins in which one‐half, two‐thirds, or one actin binding site was deleted were tested [Hitchcock‐DeGregori, S.E., and Varnell, T.A. (1990): J. Mol. Biol. 214:885–896]. Only the fusion protein with a full actin binding site deleted incorporated into stress fibers. However, the unacetylated, nonfusion proteins with one half and one actin binding site deleted incorporated into stress fibers, consistent with the ability of troponin to promote the actin binding in vitro. A fourth mutant, in which the conserved amino‐terminal nine residues were deleted, did not incorporate into stress fibers, consistent with the complete loss of function of this mutant [Cho, Y.J., Liu, J., and Hitchcock‐DeGregori, S.E. (1990): J. Biol. Chem. 265:538–545

ISSN:0886-1544    

DOI:10.1002/cm.970240205

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

年代:1993

数据来源: WILEY

摘要:

AbstractImmunofluorescence staining, electron microscopy, and (51Cr) cytolytic release assays are used to investigate the effects of taxol and taxol/hyperthermia treatments on the microtubule organization and cytolytic activity of cytotoxic T lymphocytes (CTLs). A 4 h treatment of CTLs with 1 μM taxol results in an extensive reorganization of the microtubule system to form one to a few large microtubule bundles that extend from the centrosome. The Golgi apparatus is not disrupted by this treatment and remains associated with the microtubule organizing centre (MTOC). This microtubule reorganization has no effect on the ability of CTLs to orient their MTOC towards a bound target cell, nor on their cytolytic activity. In control CTLs, not treated with taxol, a mild hyperthermia treatment (42°C, 30 min) results in an aggregation of the pericentriolar material, a loss of MTOC orientation, an inhibition of cytolytic activity, and a disorganization of the microtubule system [Knox et al.: Exp. Cell Res. 194:275–283, 1991]. In contrast, in taxol‐treated CTLs the stabilized microtubule bundles are unaffected by such hyperthermia treatment; however, the other effects of hyperthermia appear identical in control and taxol‐treated CTLs. These results indicate that a dynamic, radially arranged microtubule array is not required for the functional polarization of CTLs and suggest that a component of the pericentriolar material may play a key role in effecting MTOC orientation. © 1993 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970240206

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe addition of platelet‐derived growth factor (PDGF) to serum‐starved fibroblasts induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI‐cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI‐cycle, on cultured human foreskin fibroblasts. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent‐extracted cells was labelled with TRITC‐phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after 15 min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF‐ and neomycin‐induced changes in the actin cytoskeleton were also observed in human embryonic lung fibroblasts, human glial cells, and embryonic mouse fibroblasts, all of which are known to express PDGF‐receptors. In conclusion, the present study indicates that an increased turnover of the PI‐cycle is not essential for the changes in actin organization induced by PDGF. ©

ISSN:0886-1544    

DOI:10.1002/cm.970240207

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.970240201

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

年代:1993

数据来源: WILEY