scholarly journals Genetic evidence for functional interactions between actin noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 275-286
Author(s):  
D B Vinh ◽  
M D Welch ◽  
A K Corsi ◽  
K F Wertman ◽  
D G Drubin
Keyword(s):  
Synthetic Lethality ◽  
Binding Protein ◽  
Cytoskeletal Proteins ◽  
Genetic Interactions ◽  
Null Alleles ◽  
Actin Binding ◽  
Gene Products ◽  
Actin Binding Protein ◽  
Functional Interactions ◽  
Allele Specific

Abstract We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation.

scholarly journals Mapping actin surfaces required for functional interactions in vivo.

1994 ◽  
Vol 126 (2) ◽  
pp. 423-432 ◽  
Author(s):  
D A Holtzman ◽  
K F Wertman ◽  
D G Drubin
Keyword(s):  
Binding Protein ◽  
Null Alleles ◽  
Actin Binding ◽  
Null Mutation ◽  
Wild Type ◽  
Actin Binding Protein ◽  
Functional Interactions ◽  
Type Gene ◽  
First Time

An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.

scholarly journals Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

2002 ◽  
Vol 159 (6) ◽  
pp. 993-1004 ◽  
Author(s):  
Christine L. Humphries ◽  
Heath I. Balcer ◽  
Jessica L. D'Agostino ◽  
Barbara Winsor ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Complex Activity ◽  
Actin Binding Protein ◽  
Coiled Coil Domain ◽  
Allele Specific ◽  
And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

EFFECTS OF SOME ORGANIC SOLVENTS ON GP lb AND ACTIN-BINDING PROTEIN IN BLOOD PLATELETS

1987 ◽  
Author(s):  
A M Aakhus ◽  
N O Solum ◽  
I Hagen
Keyword(s):  
Organic Solvents ◽  
Von Willebrand Factor ◽  
Early Stage ◽  
Binding Protein ◽  
Blood Platelets ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Von Willebrand ◽  
Willebrand Factor

Effects on filamentous proteins appear to be a central phenomenon in the neuronal toxic effects of organic solvents. We have therefore compared the effects of some organic solvents (particularly isopropylalcohol, IPA) to the previously observed effects of dibucaine (DBC) on platelet cytoskeletal proteins. Incubation of platelets with 6% IPA at 37° C, like DBC, initiates a degradation of actin-binding protein (ABP) as substrate for a calcium activated protease (CAP), shown by SDS-PAGE. IPA leads to an increase followed by a decrease in bovine von Willebrand factor-induced agglutination. The decrease is accompanied by a release of glycocali-cin from the GP lb α-chain. The process was also studied using CIE of Triton X-100 extracts of platelets against antiserum to glycocalicin. Incubation of platelets with IPA before extraction in the presence of 4.2 mM leupeptin leads to a time-dependent transformation of GP Ib-related immunoprecipitates from that of the slow-migrating peak III complex (probably between ABP and GP lb) to the faster migrating GP Ib-precipitate. Our working hypothesis is that IPA induces an activation of the CAP by mobilizing calcium. This leads to degradation of ABP and liberation of GP lb from the cytoskeleton accompanied by an increased tendency for agglutination. The following decrease is explained by degradation of the glycocalicin part of the GP lb enchain which contains the binding-site for von Willebrand factor. We conclude that IPA has a similar effect on GP lb and ABP as DBC. Preliminary studies with 1% DMSO and 0,005% toluene at 37° C revealed that these organic solvents have some similar effects on platelets as described for IPA. Possibly the described effects are characteristic of certain cells at an early stage in a process ultimately leading to cell lysis.

scholarly journals Identification of membrane proteins mediating the interaction of human platelets

1980 ◽  
Vol 86 (1) ◽  
pp. 77-86 ◽  
Author(s):  
D Phillips ◽  
L Jennings ◽  
H Edwards
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Membrane Surface ◽  
Direct Interaction ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Insoluble Residue ◽  
Membrane Glycoproteins ◽  
Actin Binding Protein ◽  
Activated Platelets

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

MONOCLONAL ANTIBODIES RECOGNISING PROTEINS OF THE OUTER AND INNER SURFACE OF THE PLATELET PLASMA MEMBRANE

1987 ◽  
Author(s):  
J M Wilkinson ◽  
N Hack ◽  
L I Thorsen ◽  
J A Thomas
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Binding Protein ◽  
Cytoskeletal Proteins ◽  
Membrane Preparation ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Major Surface ◽  
Surface Glycoproteins

Platelet membrane preparations can be fractionated into two major subpopulations by free flow electrophoresis and these have been shown to correspond to the plasma membrane and the endoplasmic reticulum of the platelet. The plasma membrane fraction can be shown, by two-dimensional electrophoresis, to contain the major surface glycoproteins together with considerable amounts of actin and actin-associated proteins such as the 250 kDa actin-binding protein (filamin), P235 (talin), myosin, α-actinin and tropomyosin (Hack, N. … Crawford, N., Biochem. J. 222, 235 (1984). These cytoskeletal proteins are associated with the cytoplasmic face of the plasma membrane and probably interact with transmembrane glycoproteins. We have raised monoclonal antibodies to the purified plasma membrane preparation in order to investigate the nature of these glycoprotein-cytoskeletal interactions. In two fusion experiments, out of 804 tested, 104 hybrids secreted antibody to the membrane preparation and of these 24 were selected for further study. Initial assays were by ELISA using either the membrane preparation or whole fixed platelets as the target antigen. The specificity of the antibodies was investigated further by immunoblotting of SDS gels of total platelet proteins prepared under reducing and nonreducing conditions, by immunofluorescence, by immunohisto-chemistry and by crossed immunoelectrophoresis. The majority of the antibodies recognise major surface glycoproteins; of these, four bind to glycoprotein Ib under all conditions examined while another seven recognise the glycoprotein IIb/IIIa complex as detected by crossed immunoelectrophoresis. Three antibodies recognise the actin binding protein and these cross-react with the smooth muscle protein filamin in a number of different species. Further characterisation of these antibodies in both structural and functional terms will be presented.We are grateful to the Smith and Nephew Foundation for financial support for these studies

Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

Traffic ◽  
2001 ◽  
Vol 2 (11) ◽  
pp. 851-858 ◽  
Author(s):  
Elizabeth M. Bennett ◽  
Chih-Ying Chen ◽  
Asa E. Y. Engqvist-Goldstein ◽  
David G. Drubin ◽  
Frances M. Brodsky
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Actin Binding ◽  
Coated Pits ◽  
Actin Binding Protein

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

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