Detection of vinculin-binding proteins with an 125I-vinculin gel overlay technique

Vinculin is an adhesion plaque component localized on the cytoplasmic side of the cell membrane where stress fibers end. To detect vinculin- binding proteins, we have developed an 125I-vinculin gel overlay method. SDS PAGE was used to separate different protein preparations. After fixing the proteins in the gel with methanol-acetic acid, the SDS was removed with ethanol and the proteins renatured in buffer. The gel was then incubated with 125I-vinculin. After extensive washing to remove nonspecifically associated label, the gel was dried and autoradiographed. Chick embryo fibroblasts, their Rous sarcoma virus transformants, and HeLa cells were found to contain two proteins (Mr 220,000 and 130,000) that bound 125I-vinculin strongly and another (Mr 42,000) that bound it moderately. The 130,000-mol-wt protein was identified as vinculin itself, which suggests that it may self- associate. The 42,000-mol-wt protein was identified as actin with which vinculin is known to interact. The identity of the 220,000-mol-wt protein is not known. It is not cellular fibronectin, myosin, or filamin. When fibroblast proteins were separated into Triton X-100 soluble and insoluble fractions, most of the vinculin and the 220,000- mol-wt protein was found to be in the soluble fraction. Chicken gizzard also contained these vinculin-binding proteins along with three others of Mr 190,000, 170,000, and 100,000.

[1]  J. Siliciano,et al.  Meta-vinculin—a vinculin-related protein with solubility properties of a membrane protein , 1982, Nature.

[2]  T. Kelly,et al.  Proteins involved in the attachment of actin to the plasma membrane. , 1982, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[3]  D. Helfman,et al.  Co-existence of vinculin and a vinculin-like protein of higher molecular weight in smooth muscle. , 1982, The Journal of biological chemistry.

[4]  K. Weber,et al.  Erythroid spectrin, brain fodrin, and intestinal brush border proteins (TW-260/240) are related molecules containing a common calmodulin-binding subunit bound to a variant cell type-specific subunit. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[5]  B. Jockusch,et al.  Structural aspects of vinculin-actin interactions. , 1982, Journal of molecular biology.

[6]  Klaus Weber,et al.  An F-actin- and calmodulin-binding protein from isolated intestinal brush borders has a morphology related to spectrin , 1982, Cell.

[7]  Shin Lin,et al.  High-affinity interaction of vinculin with actin filaments in vitro , 1982, Cell.

[8]  J. Feramisco,et al.  Alpha-actinin and vinculin from nonmuscle cells: calcium-sensitive interactions with actin. , 1982, Cold Spring Harbor symposia on quantitative biology.

[9]  B. Geiger,et al.  Substrate-attached membranes of cultured cells isolation and characterization of ventral cell membranes and the associated cytoskeleton. , 1981, Journal of molecular biology.

[10]  B. Geiger,et al.  Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells , 1981, The Journal of cell biology.

[11]  J. Bryan,et al.  Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels , 1981, The Journal of cell biology.

[12]  J. Levine,et al.  Fodrin: axonally transported polypeptides associated with the internal periphery of many cells , 1981, The Journal of cell biology.

[13]  B. Jockusch,et al.  Interaction of alpha-actinin and vinculin with actin: opposite effects on filament network formation. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[14]  T. Hunter,et al.  Vinculin: A cytoskeletal target of the transforming protein of rous sarcoma virus , 1981, Cell.

[15]  K. Weber,et al.  Calmodulin-binding proteins of the microfilaments present in isolated brush borders and microvilli of intestinal epithelial cells. , 1980, The Journal of biological chemistry.

[16]  S. Singer,et al.  Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[17]  B. Geiger,et al.  Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Feramisco,et al.  Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin , 1980, Cell.

[19]  J. Feramisco,et al.  A rapid purification of alpha-actinin, filamin, and a 130,000-dalton protein from smooth muscle. , 1980, The Journal of biological chemistry.

[20]  B. Geiger A 130K protein from chicken gizzard: Its localization at the termini of microfilament bundles in cultured chicken cells , 1979, Cell.

[21]  E. Engvall,et al.  Binding of soluble form of fibroblast surface protein, fibronectin, to collagen , 1977, International journal of cancer.

[22]  C. S. Izzard,et al.  Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique. , 1976, Journal of cell science.

[23]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[24]  J. Spudich,et al.  Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. , 1976, Journal of supramolecular structure.

[25]  I. Pastan,et al.  The major cell surface glycoprotein of chick embryo fibroblasts is an agglutinin. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Abercrombie,et al.  Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. , 1975, Experimental cell research.

[27]  M. Abercrombie,et al.  The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. , 1971, Experimental cell research.

[28]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[29]  M. Bárány,et al.  Chicken gizzard myosin. , 1966, Archives of biochemistry and biophysics.

[30]  F. Greenwood,et al.  THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. , 1963, The Biochemical journal.

[31]  F. Greenwood,et al.  Preparation of Iodine-131 Labelled Human Growth Hormone of High Specific Activity , 1962, Nature.