ISSN:0192-253X    

DOI:10.1002/dvg.1020110502

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe cellular slime moldDictyostelium discoideumis becoming the premier system for the explication of the biochemical and cellular events that occur during motile processes. Proteins associated with the actin cytoskeleton, in particular, appear to play key roles in cellular responses to many external stimuli. This review summarizes our present understanding of the actin‐associated proteins inDictyostelium, including their in vitro activities and their structural and/or functional analogues in mammalian cell

ISSN:0192-253X    

DOI:10.1002/dvg.1020110503

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

年代:1990

数据来源: WILEY

摘要:

AbstractIn this work we evaluate the cartical expansion model for amoeboid chemo‐taxis with regard to new information about molecular events in the cytoskeleton following chemo‐tactic stimulation ofDictyosteliumamoebae. A rapid upshift in the concentration of chemoattrac‐tant can be used to synchronize the motile behavior of a large population of cells. This synchrony presents an opportunity to study the biochemical basis of morphological changes such as pseudopod extension that are required for amoeboid chemotaxis. Changes in the composition and activity of the cytoskeleton following stimulation can be measured with precision and correlated with important morphological changes. Such studies demonstrate that activation of actin nucleation is one of the first and most crucial events in the actin cytoskeleton following stimulation. This activation is followed by incorporation of specific actin cross‐linking proteins into the cytoskeleton, which are implicated in the extension of pseudopods and filopods. These results, as well as those from studies with mutants deficient in myosin, indicate that cortical expansion, driven by focal actin polymerization, cross‐linking and gel osmotic swelling, is an important force for pseudopod extension.It is concluded that whereas three forces, frontal sliding, tail contraction, and cortical expansion may cooperate to produce amoeboid movement, the cortical expansion model offers the simplest explanation of how focal stimulation with a chemoat‐tractant causes polarized pseudopo

ISSN:0192-253X    

DOI:10.1002/dvg.1020110504

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

年代:1990

数据来源: WILEY

摘要:

AbstractDictyosteliumamoebae were analyzed before and after rapid addition of 10−6M cAMP for cellular motility, dynamic shape changes, and intracellular particle movement. Before cAMP addition, amoebae moved in a persistent anterior fashion and were elongate with F‐actin localized predominantly in the anterior pseudopod. Intracellular particles moved rapidly and anteriorly. Within seconds after 10−6M cAMP addition, cells stopped translocating, pseudopod formation ceased, intra‐cellular particle movement was depressed, and F‐actin was lost from the pseudopod and concomitantly relocalized in the cell cortex After 10 seconds, expansion zones reappeared but were small and no longer anteriorly localized. Vesicle movement partially rebounded but was no longer anteriorly directed. The myosin II null mutant HS2215 exhibited both depressed cellular translocation and vesicle movement. The addition of cAMP to HS2215 cells did not result in any detectable change in the random, depressed movement of particles. The results with HS2215 suggest that myosin II is essential for (1) rapid cellular translocation, (2) cellular polarity, (3) rapid particle movement, (4) anteriorly directed particle movement, and (5) the cAMP response. Electron micrographs suggest that at least half of the particles examined in this study contain in turn smaller membrane bound vesicles or multilameilar membrane bodies. The possible role of these vesicles is

ISSN:0192-253X    

DOI:10.1002/dvg.1020110505

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

年代:1990

数据来源: WILEY

摘要:

AbstractPonticulin is a 17,000‐dalton transmembrane glycoprotein that is involved in the binding and nucleation of actin filaments byDictyostelium discoideumplasma membranes. The major actin‐binding protein isolated from these membranes by F‐actin affinity chromatography, ponticulin also binds F‐actin on blot overlays. The actin‐binding activity of ponticulin in vitro is identical to that observed for purified plasma membranes: it resists extraction with 0.1 N NaOH, is sensitive to high salt concentrations, and is destroyed by heat, proteolysis, and thiol reduction and alkylation. A cytoplasmic domain of ponticulin mediates binding to actin because univalent antibody fragments directed against the cytoplasmic surface of this protein inhibit 96% of the actin‐membrane binding in sedimenlation assays. Antibody specific for ponticulin emoves both ponticu‐lin and the ability to reconstitute actin nucleation activity from detergent extracts of solubilized plasma membranes. Levels of plasma membrane ponticulin increase 2‐ to 3‐fold during aggregation streaming, when cells adhere to each other and are highly motile. Although present throughout the plasma membrane, ponticulin is preferentially localized to some actin‐rich membrane structures, including sites of cell‐cell adhesion and arched regions of the plasma membrane reminiscent of the early stages of pseudopod formation. Ponticulin also is present but not obviously enriched at phagocytic cups of log‐phase amebae. These results indicate that ponticulin may function in vivo to attach and nucleate actin filaments at the cytoplas‐mic surface of the plasma membrane. A 17,000‐dalton analogue of ponticulin has been identified in human polymorphonuclear leukocyte plasma membranes by immunoblotting and immunofluo‐rescence microscopy. These findings suggest that the structure and function of ponticulin in motile cells has

ISSN:0192-253X    

DOI:10.1002/dvg.1020110506

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

年代:1990

数据来源: WILEY

摘要:

AbstractTheDictyostelium discoideum30,000 dalton actin‐binding protein is an actin cross‐linking protein that organizes formation of parallel bundles of actin filaments in vitro, and is present in filopodia in living cells. This protein binds calcium directly and exhibits a decreased affinity for actin filaments in the presence of micromolar calcium. In this work, the existence of antigenic homologs of the 30,000 dalton protein inPhysarum polycephalum, Schistosoma mansoni, Chara carolina, andDrosophila melanogasteris detected by use of affinity purified antibody and electrophoretic blotting methods. The expression of this protein during development ofDictyosteliumis also analyzed, revealing a progressive 3‐fold decrease in the level of this protein in amoebae between the vegetative and slug stages. A highly ordered structure of bundles of actin and the 30,000 dalton protein formed in vitro is inferred from the presence of transverse striations on the bundles with a minor periodicity at 11.4 nm and a major periodicity at 33.9 nm. Finally, we propose a working model of the interaction of this actin cross‐linking protein with actin filaments to form

ISSN:0192-253X    

DOI:10.1002/dvg.1020110507

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe two subunits of the het‐erodimeric protein cop32/34, an actin‐binding protein, are encoded by separate single‐copy genes. We have established the genomic structure of both genes. A sequence comparison of cap32/34 with capZ from chicken skeletal muscle and two partially known sequences fromSaccharomyces cerevisiaeandXenopus laevisshow that heterodimeric capping proteins belong to a highly conserved group of actin‐binding proteins. This conclusion is supported by the cross‐reaction of polyclonal antibodies against cap32 and cap34 with proteins from lower and higher eukaryotes. In addition, a system is presented that allows the expression of truncated cap34 polypeptides under the control of the cap34

ISSN:0192-253X    

DOI:10.1002/dvg.1020110508

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

年代:1990

数据来源: WILEY

摘要:

AbstractDuring development ofDictyostelium discoideum, cells acquire EDTA‐resistant cell‐cell adhesion at the aggregation stage. The EDTA‐resistant cell binding activity is associated with a cell surface glycoprotein of Mr 80,000 (gp80), which mediates cell‐cell binding via ho‐mophilic interaction. Analysis of the structure of gp80 deduced from cDNA sequence reveals the presence of three internally homologous segments in the NH2‐terminal domain, which also contains regions with homology to the neural cell adhesion molecule. Secondary structure predictions show an abundance of β‐structures and very few α‐helices. This is confirmed by circular dichroism measurements. It is likely that the homologous segments are organized into globular structures, extended from the cell surface by a Pro‐rich stalk domain. The cell binding activity of gp80 resides within the first globular repeat of the NH2‐terminal domain and has been mapped to a 51 amino acid region between Val123 and Leu 173. Synthetic oligopeptides corresponding to sequences within this region have been prepared and assayed for their ability to bind to cell surface gp80. Results lead to identification of the homophilic binding site to an octapeptide sequence within this region. Synthetic peptides containing this octapeptide sequence and univalent antibodies directed against this site block the formation of organized cell streams during aggregation. Although cell aggregates are eventually formed, most fail to undergo further development to give rise to slugs and fruiting bodies, indicating that cell‐cell adhesion involving gp80 is an important step

ISSN:0192-253X    

DOI:10.1002/dvg.1020110509

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

年代:1990

数据来源: WILEY

摘要:

AbstractDNA mediated transformation is a critical technique for uniting genetic, molecular genetic and reverse genetic approaches to a wide range of problems in cell and molecular biology. InDictyostelium, there is now the capability not only to manipulate DNA sequences in vitro and put them back into the cell, but also to alter the sequences of endogenous chromosomal genes through high frequency homologous recombination. This means that the range of gene manipulation techniques that have made yeast such a useful system should now be applicable inDictyostelium.For studying problems such as cellular motility, morphogenesis, and gene regulation, few organisms have the combination of features offered byDictyostelium.

ISSN:0192-253X    

DOI:10.1002/dvg.1020110510

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

年代:1990

数据来源: WILEY

摘要:

AbstractWe have studied the transient expression, inDictyosteliumcells growing on a bacterial food source, of a construct containing the coding region of the firefly luciferase gene inserted downstream of aDictyosteliumactin promoter. The fusion gene is not detectably expressed when DNA is introduced by calcium phosphate precipitation or by electroporation, but it is expressed when introduced using cationic liposomes (lipofectin). Using this latter procedure, we are able to transform cells with a G418 resistance vector and select stable, drug‐resistant transformants at a relatively low, but workable, efficiency. This technique will allow molecular genetics to be applied to the many important nonaxenicDictyosteliumstrain

ISSN:0192-253X    

DOI:10.1002/dvg.1020110511

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

年代:1990

数据来源: WILEY