ISSN:0886-1544    

DOI:10.1002/cm.970310402

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

年代:1995

数据来源: WILEY

摘要:

AbstractA Chinese hamster β‐tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HAβ1‐tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HAβ1‐tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HAβ1‐tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HAβ1‐tubulin. The synthesis of both endogenous β‐tubulin and HAβ1‐tubulin was repressed by colchicine. The HAβ1‐tubulin incorporated into microtubules to the same extent as the endogenous β‐tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, trasfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HAβ1‐tubulin reduced the synthesis of endogenous wild‐type β‐tubulin but increased the synthesis of α‐tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of α‐tubulin was found. The results indicate that expression of excess exogenous β‐tubulin perturbs the synthesis of endogenous α‐tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of α‐ a

ISSN:0886-1544    

DOI:10.1002/cm.970310403

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

年代:1995

数据来源: WILEY

摘要:

AbstractIn mouse fibroblasts, at least five TM isoforms are identified and they can be grouped into the high (TM1, TM2, and TM3) and low molecular weight TM isoforms (TM4 and TM5). Suppression of one of the high molecular weight tropomyosin (TM) isoforms in nonmuscle cells is implicated to be one of the causes for disorganization of actin microfilament bundles and subsequent changes in cell motility and cell shape. In this study, we studied the expression of tropomyosin isoforms in macrophages that exhibit high motility and ability to change cell shape. Two‐dimensional gel electrophoresis followed by Western blot analysis using polyclonal anti‐TM antiserum revealed that the high molecular weight TM isoforms were lacking in both resident and activated mouse peritoneal macrophages. Analyses of newly synthesized TM isoforms, Northern blot analyses using isoform‐specific cDNA probes, and immunostaining with monoclonal anti‐TM antibody that recognizes only the high molecular weight TM isoforms also demonstrated that the syntheses of the high molecular weight TM isoforms (TM1, TM2, and TM3) were completely suppressed, whereas the low molecular weight TM isoforms (TM4 and TM5) were expressed in macrophages. These results indicate that macrophages intrinsically lack the high molecular weight TM isoforms. In order to obtain information about cellular localization of the low molecular weight TM isoforms in macrophages, they were immunostained with polyclonal anti‐TM antiserum that recognizes both the high and low molecular weight TM isoforms. The results showed that the low molecular weight TM isoforms were co‐localized with F‐actin in punctate and short fibrous structures. In addition, we performed in situ hybridization analysis to examine localizations of the TM mRNAs in fibroblasts and macrophages. The results showed that TM mRNAs were localized throughout the cytoplasm. © 1995 W

ISSN:0886-1544    

DOI:10.1002/cm.970310404

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

年代:1995

数据来源: WILEY

摘要:

AbstractUsing dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for in the dephosphorylated NF‐H than the phosphorylated form of NF‐H was searched for in the porcine brain extract. Most NF‐H kinase activity in the brain extract pelleted with microtubules. The NF‐H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF‐H and NF‐M became reactive to antibodies recoginizing in vivo phosphorylation sites (SM131, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1protein and by its reactivity to anti‐p34cdc2antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/26 did not display M‐phase promoting activity when assayed with a cell‐free system ofXenopusegg extract. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970310405

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe distribution of microfilaments inDrosophilaegg chambers stained with rhodamine (Rh)‐conjugated phallcidin was studied by laser scanning confocal microscopy and transmission electron microscopy. These techniques revealed new details in the pattern of microfilament localization. We observed in stage 1–3 egg chambers accumulation of filamentous actin in the oocyte cytoplasm between the ring canals connecting the oocyte with adjacent nurse cells. Starting from stages 6–7 short microfilament bundles arranged in basket‐like structures were associated with the side of the ring canals facing the nurse cell cytoplasm. We also observed a dramatic decrease in the actin network associated with the cortex of the oocyte in stage 10. During stage 10B the nurse cell cytoplasm was crossed by radial actin bundles that showed a sarcomeric‐like cross striation after Rh‐phalloidin staining. The ring canals also did not uniformly stain but showed a punctate labeling. The implications of the actin cytoskeleton during oocyte growth are discussed. © 1995 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970310406

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

年代:1995

数据来源: WILEY

摘要:

AbstractThymosin β4(Tβ4) binds to G‐actin in vitro and inhibits actin polymerization. We studied the effects of incresing Tβ4concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane‐associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane‐associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of Tβ4at a concentration (approximately 28 μM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular Tβ4concentrations were increased by three different methods: (1) microinjection of an RSV vector containing a cDNA for Tβ4; (2) transfection with the same vector; and (3) microinjection of purified Tβ4protein. The plasmid coding for Tβ4was microinjected into PtK2 cells together with fluorescently labeled alpha‐actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha‐actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2–3 days showed disassembly of stress fibers as detected by rhodamine‐phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure Tβ4protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, Similar microinjection of pure thymosin β4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow re‐gressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these Tβ4‐injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing pr

ISSN:0886-1544    

DOI:10.1002/cm.970310407

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe previously reported the expression of ZO‐1 in cell types that do not form tight junctions. Here we compare the molecular environments of ZO‐1 in epithelial cells, primary cultures of astrocytes and in the non‐epithelial S180 sarcoma cell line. ZO‐1 co‐localizes with a subset of actin filament in all cell types. In astrocytes, ZO‐1 is found concentrated in discrete bands at points of cell‐cell contact. Indirect immunofluorescent microscopy shows that these bands of ZO‐1 co‐localize with the adherens junction proteins vinculin and α‐actinin, and with the antigen recognized by a pan‐cadherin antibody. In contrast, ZO‐1 in S180 cells, which exhibit limited cell‐cell interactions, is diffusely distributed over the plasma membrane, with concentrations in lamellipodia where actin filaments accumulate. ZO‐1 does not co‐localize with vinculin at focal adhesions in this cell type. Analysis of ZO‐1 immunoprecipitation profiles from different cell types, performed under conditions previously demonstrated to maintain interactions between ZO‐1, ZO‐2 and p130 from the MDCK epithelial cell line, show that the proteins which co‐precipitate with ZO‐1 vary with cell type. Precipitation of polypeptides at 165 kDa, potentially ZO‐2, and 65 kDa occurs in both a mouse kidney tubule epithelial cell line and the non‐epithelial S180 cells. No proteins specifically associate with ZO‐1 immunoprecipitated from astrocytes. Spectrin, α‐actinin, vinculin and cadherin are not detected in immunoblots of ZO‐1 immunoprec

ISSN:0886-1544    

DOI:10.1002/cm.970310408

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

年代:1995

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970310401

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

年代:1995

数据来源: WILEY