The goal of the proposed project is to gain a better understanding at the molecular level of actin-membrane interactions by studying the association of vinculin, an actin binding protein, with membranes of muscle cells. We propose to approach the problem in the following way: 1) Vinculin-containing membranes from either smooth or cardiac muscle tissue will be prepared by homogenization followed by differential and density gradient centrifugation steps. 2) Vinculin will be extracted from these membranes using a number of different conditions known to solubilize peripheral membrane proteins. The extraction of vinculin will be compared with that of actin to determine what proportion of actin-membrane contacts is vinculin-dependent. 3) The binding of radioactively labelled vinculin to vinculin-depleted membranes will be studied and analyzed by Scatchard plot analyses. Specific methods will be used to deal with the question of vinculin-membrane vs. vinculin-actin association of the membranes. 4) Following binding studies to membranes, we will identify and isolate the vinculin receptor. Various extraction methods will be used to remove the vinculin receptor from the membrane. Extraction may be followed by measurement of radoactively labelled vinculin binding to extracted membranes. The identification of the receptor will be accomplished by use of technniques such as gel overlay with radioactively labelled vinculin and affinity chromatography on vinculin and rabbit anti-vinculin resins. Isolation of the receptor will allow characterization of its molecular size, shape and amino acid content. 5) Antibodies to the vinculin receptor will be raised and used to localize the receptor in nonmuscle cells by immunofluorescence techniques. Through these studies we hope to determine how the cellular contractile apparatus is attached to the cell membrane with which it must interact to transmit force. These studies may therefore lead to a better understanding of diseased states and impaired function of muscle. Information gained in the study of muscle will also help to answer questions regarding altered structural and motile functions in nonmuscle cells.
| Wilkins, J A; Lin, S (1986) A re-examination of the interaction of vinculin with actin. J Cell Biol 102:1085-92 |
| Wilkins, J A; Risinger, M A; Lin, S (1986) Studies on proteins that co-purify with smooth muscle vinculin: identification of immunologically related species in focal adhesions of nonmuscle and Z-lines of muscle cells. J Cell Biol 103:1483-94 |
