摘要:

AbstractCell surface proteoglycans participate in molecular events that regulate cell adhesion, migration, and proliferation. To investigate the organization of these molecules at the cell surface, the distribution of two well‐known proteoglycan ligands has been studied. These ligands, lipoprotein lipase and basic fibroblast growth factor, showed a characteristic binding pattern consisting of highly organized parallel arrays that crossed the upper surface of human skin fibroblasts. The proteoglycan nature of the binding sites was evident from their susceptibility to heparinases, and from ligand displacement by heparin. Parallel localization of the ligands and actin, and treatment of the cells with cytochalasin, showed that the binding proteoglycans are organized by the actin cytoskeleton. The ligands induced a different behaviour of the binding sites on incubation of the cells at 37°C. Lipoprotein lipase produced a movement of the binding proteoglycans along the actin filaments towards the cell center. In contrast, after binding of basic fibroblast growth factor the binding proteoglycans remained spread over the cell surface and actin depolymerization was induced. Since an increasing number of ligands appear to depend on proteoglycans for their interactions with their high affinity receptors, distribution and movement of proteoglycans at the cell surface that is organized by the actin cytoskeleton could direct and enhance the encounters between the ligands and their specific receptors. © 1995 Wiley‐Liss

ISSN:0886-1544    

DOI:10.1002/cm.970300202

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

年代:1995

数据来源: WILEY

摘要:

AbstractDesmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin‐Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylared on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell‐cell contact, in the presence of protein kinase inhibitor, H‐7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H‐7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid‐treated cultures. These results suggest that via reversible protein phosphorylation involving both protein kinase and protein phosphatases. © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970300203

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

年代:1995

数据来源: WILEY

摘要:

AbstractProtein tyrosine phosphorylation plays an important role in cell growth, mitosis, and tumorigenesis. It has also been implicated in meiotic maturation and fertilization. We have used anti‐phosphotyrosine immunofluorescence and immunoblotting to identify sperm and egg proteins which are phosphorylated on tyrosine residues prior to and during sea urchin fertilization. On immunoblots of sperm proteins, the monoclonal anti‐phosphotyrosine antibody detected three major proteins with molecular weights of 44, 82, and 100 kD, and six minor bands at 46, 48, 70, 76, 95, and 150 kD. These phosphotyrosyl proteins were localized to the sperm acrosomal and centriolar fossae. In contrast, staining was found globally in unfertilized eggs, and the antibody recognized two major egg phosphotyrosyl proteins of molecular weights 42 and 50 kD, and five minor bands at 40, 90, 116, 130, and 150 kD. While immunofluorescent staining remained throughout the fertilized egg cytoplasm, there were dynamic changes in the staining intensity of single bands. The 90 kD immunoreactive band increased in intensity, and the 40 and 42 kD bands disappeared by 15 min after fertilization. Loss of the 40 and 42 kD bands was due to dephosphorylation by okadaic acid‐sensitive phosphatase(s). The 50 kD immunoreactive protein was unchanged up to the 8‐cell stage and was still present in blastulae, indicating its importance throughout fertilization and early development. Alterations in the pattern of phosphotyrosine‐containing proteins during fertilization did not depend on nascent proteins and could not be completely mimicked by increasing intracellular calcium, pH, and protein kinase C activity alone. Since changes in the fertilization pattern of phosphotyrosyl proteins occurred during formation of the sperm aster and mitotic spindle, we analyzed the role of protein tyrosine kinase activity in these processes using the tyrosine kinase specific inhibitor, erbstatin. Both the sperm aster and mitotic spindle were disrupted, indicating an involvement of tyrosine phosphorylation in these processes during interphase and mitosis. We conclude that the changes in phosphotyrosyl proteins play an important role in fertilization and early development of sea urchin eggs. Control of microtubule assembly into the sperm aster and mitotic spindle of the first cell cycle are examples of such roles. © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970300204

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

年代:1995

数据来源: WILEY

摘要:

AbstractGelsolin, a Ca++activated, 90 kd actin binding protein, can regulate actin polymerization in polymorphonuclear leukocytes (PMNs) via severing of filaments to dissolve gels or by capping of filament ends to limit polymerization. In Triton‐lysed PMNs, 30% of gelsolin is bound to the Triton‐soluble F‐actin (TSF) pool and none is bound to the Triton‐insoluble F‐actin (TIF) pool. Calcium‐activated PMNs exhibit concurrent temporal and quantitative TIF growth and TSF and total F‐actin loss. To determine if gelsolin plays a role in regulating TSF pool size, we monitored gelsolin‐actin interactions and TIF, TSF and G‐actin content at 5 second intervals in PMNs activated with the calcium ionophore, ionomycin. Actin pools were measured by NBDphallacidin binding and by gel scans and expressed relative to basal; gelsolin‐actin interactions were measured as change in the amount of EGTA‐resistant gelsolin:actin (G:A) complexes and by immunoblot quantification of gelsolin in actin pools. In basal PMNs, 33% of PMN gelsolin is bound in 1:1 EGTA‐resistant G:A complexes and TSF and TIF retain 30% and 0% of PMN gelsolin, respectively. By 20 seconds after ionomycin addition, TSF decreases, TIF increases and a fraction of gelsolin repartitions from the TSF to the TIF pool. At maximum change (60 seconds), total F‐actin (TIF + TSF) and TSF decrease and TIF increases by 25%; gelsolin is bound to both TSF and TIF (35% of total gelsolin in each pool), and 1:1 EGTA‐resistant G:A complexes increase from 33% to 70%. No changes occur in cells activated by ionomycin in the absence of Ca++. The data show Ca++activated TIF growth and TSF loss are temporally and quantitatively associated with an increase in the percent of gelsolin bound to actin and the translocation of gelsolin from TSF to TIF. This is unique, since no other PMN activator is known to repartition gelsolin into TIF actin. Further, the Ca++activated initial increase in TIF concurrent with a fall in TSF without a change in total F‐actin or G‐actin content suggest that TIF grows initially only by TSF annealing/cross‐linking to TIF. Gelsolin may regulate the

ISSN:0886-1544    

DOI:10.1002/cm.970300205

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

年代:1995

数据来源: WILEY

摘要:

AbstractMoving along a microtubule, kinesin follows a course parallel to the protofilaments; but it is not known whether kinesin binds exclusively on a single protofilament. The presence of zinc during tubulin polymerization induces sheets where neighboring protofilaments are antiparallel. If kinesin could support the motility of these zinc‐sheets, then the binding site for a kinesin molecule would be limited to a single protofilament.Kamimura and Mandelkow [1992:J. Cell Biol.118:865–75] reported that kinesin moves along zinc‐sheets. We found that zinc‐sheets grown under their conditions often had a microtubule‐like structure along one edge. We confirmed the possibility that the motility observed by Kamimura and Mandelkow [1992:J. Cell Biol.118:865–75] is attributed to the microtubule‐like structure rather than the zinc‐sheet.To resolve the question of whether kinesin can recognize an antiparallel protofilament lattice, we investigated the kinesin‐mediated motility of zinc‐macrotubes. At higher free zinc concentrations, zinc‐sheets roll up as macrotubes, free of edges. In the presence of 10 m̈M taxol and 100 nM free Zn2+at pH 6.8, the samples were shown by electron microscopy to contain only macrotubes. Under these buffer conditions, kinesin could bind strongly to axonemal doublets in the presence of AMP‐PNP, and generate motility in the presence of ATP, but kinesin did not bind to nor move the macrotubes. This shows that kinesin cannot bind efficiently to nor move on the anti‐parallel lattice; it is possible (though not necessary) that the groove between two parallel protofilaments is required for kinesin's motili

ISSN:0886-1544    

DOI:10.1002/cm.970300206

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

年代:1995

数据来源: WILEY

摘要:

AbstractIsolated microtubules from cod (Gadus morhua) are apparently more stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2‐methoxy‐5‐(2,3,4‐trimethoxyphenyl)‐2,4,6‐cyclo heptatrien‐1‐one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 ± 1.5) × 105M1at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin‐colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 × 10−5to 1 × 10−3M), MTC induced a large amount of cold‐stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 10−5M colchicine no spirals were formed, while at 10−4M and 10−3M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different

ISSN:0886-1544    

DOI:10.1002/cm.970300207

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

年代:1995

数据来源: WILEY

摘要:

AbstractGel‐filtration is commonly used to remove contaminants from conventional actin prepared by the method of Spudich and Watt. It has been shown that this procedure removes the majority of a factor that reduces the low‐shear viscosity of actin. We have previously reported that this factor is Cap Z, a barbed end capping protein. We now establish that, even after gel‐filtration, enough Cap Z can be present in conventionally prepared actin to affect events occurring at the barbed ends of actin filaments. We also demonstrate that the concentration of Cap Z can be reduced to more than a log below the KDfor binding of Cap Z to actin by either (1) immunoabsorbtion of conventionally prepared actin with anti‐Cap Z antibodies, or (2) an additional cycle of polymerization/depolymerization followed by repeat gel‐filtration. © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970300208

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.970300201

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

年代:1995

数据来源: WILEY