Susan Craig - US grants

The objective of this research is to learn how actin interacts with plasma membrane. Three examples of actin-membrane linkages are being studied. (1) We are determining the molecular composition of the actin to membrane cross filament in intestinal microvilli. a) We propose to isolate the microvillar 110K protein, a candidate for the cross filament, and to determine whether and how it interacts with actin and the microvillar membrane in vitro. b) We will test the proposed roles of the microvillar core proteins (fimbrin, villin, 110K, actin, and calmodulin) in the structure and membrane association of the core bundle of actin filaments by immunoultrastructural localization of these proteins in situ, using 50 angstroms Fab' fragments of monoclonal antibodies complexed to 50 angstroms gold particles. c) We will screen for microvillar proteins which link actin to membrane by a reconstitution binding assay employing 3H-actin, microvillar membranes, and fractionated microvillar proteins. (2) We are analyzing the association of vinculin-like proteins with the plasma membrane. We have discovered two new proteins in smooth muscle, 150K meta-vinculin, and 300K vinculin-like polypeptide (300K-VLP) that are antigenically related to 130K vinculin, but which unlike vinculin, have solubility properties of amphiphilic membrane proteins and could be direct links between actin filaments and the cell membrane. We propose to analyze the molecular basis for the antigenic similarity and solubility difference between these proteins by peptide mapping, charge shift electrophoresis, biosynthetic labelling and pulse-chase experiments, isolation of native metavinculin and 300K-VLP and characterization of their interaction with actin, plasma membrane and liposomes in vitro. (3) We have defined a cell surface actin (CSA) on some murine lymphocytes by immunofluorescence and flow cytometry. a) We propose to determine by two-color immunofluorescence and flow cytometry if CSA is a marker for new murine and human lymphocyte subpopulations within the known subdivisions of T and B-cells. b) We will assay for biochemical differences between CSA and intracellular actin by determining if CSA is a glycoprotein and if it has a hydrophobic domain. c) We plan to test for the possible function of CSA in mediating adherence of lymphocytes to fibronectin and to cells of the lymphoreticuloendothelial system, and for the possible function of CSA as part of a cell surface growth control complex.

DESCRIPTION (provided by applicant): The goal of this research is to understand how vinculin exerts a tumor-suppressor-like effect on cell motility. Vinculin is a prominent component of cell and tissue structures that mediate transmembrane connections between the intracellular cytoskeleton and the extracellular matrix. Current models suggest that vinculin stimulates adhesion and inhibits motility by strengthening these connections through bifunctional interactions between talin-integrin complexes at vinculin's head domain and actin filaments at its tail domain. Because purified vinculin is autoinhibited, regulation of the head/tail interaction (HTI) to expose or hide ligand binding sites is hypothesized to be the mechanism by which vinculin regulates attachment of membrane proteins to cytoskeleton to control adhesion and motility. A goal of this proposal is to test this model in living cells. We developed two Forster resonance energy transfer (FRET) probes that report on activated and actin-binding conformations of vinculin, a series of mutants having a graded reduction in the strength of the intramolecular HTI, and a talin- binding mutant. We propose to apply these tools to address the following specific aims: 1). Use vinculin FRET probes to test the hypothesis that there are redundant mechanisms for combinatorial activation of vinculin. Talin and actin filaments together can activate vinculin;we will test the roles of other vinculin ligands, as well as PIP2 to define the signaling and localization cues for vinculin activation. 2). Use the head/tail interaction mutants and the talin-binding mutant to test the hypothesis that activation of vinculin regulates interactions between integrin, talin, vinculin, and actin that control cell adhesion, motility, and transduction of force across the cell membrane. 3). Use the conformation-sensitive vinculin FRET probes to test the hypothesis that activation of vinculin responds to mechanical forces and contractility in living cells. Collaborations have been set up with Sharon Campbell to facilitate analyses of PIP2 in combinatorial activation of vinculin (part of Aim1), with Andres Garcia to measure adhesive force in cells, and with Susan Gunst to measure tension development in smooth muscle tissue (parts of Aim2). We anticipate that these studies will provide substantial new information relevant to the general question of how proteins build structures to transmit force across a membrane, and specifically to the molecular mechanism by which vinculin suppresses cell migration. PUBLIC HEALTH REVELANCE: Abnormal cell adhesion and migration are characteristic of cancers that kill people. This project aims to find out how cell migration and adhesion are regulated by a protein called vinculin. By learning how vinculin works, we can better understand how to control the abnormal cell behaviors of cancer.

[unreadable] DESCRIPTION (provided by applicant): The Iong-term goal of this research is to understand how vinculin regulates cell adhesion and motility. inculin is essential for embryonic development in mice and for regulation of adhesion and motility on xtracellular matrix substrates. Current evidence supports a model in which vinculin's role in a focal adhesion is to provide a bifunctional interaction with talin-integrin complexes at vinculin's head domain and actin filaments at its tail domain. In purified vinculin the actin and talin binding sites are masked by an intramolecular interaction between the head (Vh) and tail (Vt) domains of vinculin. Regulation of the head/tail interaction to expose or hide the ligand binding sites is hypothesized to be the mechanism by which vinculin regulates the attachment of membrane proteins to cytoskeleton to control adhesion and motility in cells. A goal of the present proposal is to test this model in living cells. For this purpose, two mutants deficient in intramolecular head/tail interaction were developed and a fluorescence resonance energy transfer (FRET) probe that reports on the closed, open, and actin-bound conformations of vinculin have been constructed. When expressed in cells the mutants have a localization and dynamic phenotype consistent with the interpretation that they stabilize a subset of focal adhesion plaque proteins representing an intermediate in a process carried out by wild type vinculin. To elucidate this process and to test the requirement for regulation of the intramolecular head/tail interaction in the function of vinculin, we propose the following plan. [unreadable] Aim 1, Use the loss of function mutants to identify the requirement for regulated head/tail interaction in rescue of adhesion and motility phenotypes in vinculin null cells. [unreadable] Aim 2, test the hypothesis that the phenotype generated by the constitutive talin-binding conformation of vinculin represents a stalled intermediate in endocytosis of the a5bl integrin receptor for FN. [unreadable] Aim 3, use the conformation-sensitive vinculin FRET probe to determine when and where in a cell vinculin undergoes conformational regulation and to test the hypothesis that activation of vinculin responds to changes in mechanical forces and contractility. [unreadable] Aim 4, test the hypothesis that vinculin's effects on cell adhesion and motility are mediated through Rac, RhoA, or Cdc42 pathways. [unreadable] [unreadable]