ABSTRACT
Structural and functional characterization of membrane proteins includes the determination of their orientation within the membrane (integral proteins), or their exposure at either the cytosolic or extracytoplasmic surface of the membrane (peripheral proteins). We have developed an easily handled immunofluorescence-based method to investigate the exposure of antigenic epitopes at either surface of the membranes in situ. We present conditions for permeabilization of p-formaldehyde-fixed cells which allow the discrimination of epitopes exposed either at the cytosolic face of membranes, within the lumen of vesicles, or at the cell surface. The potential applications of this method include (1) the use of domain-specific antibodies as a tool to study integral membrane proteins with regard to the orientation of their carboxy-terminal and amino-terminal ends or the orientation of the loops of multispanning proteins, and (2) the assignment of the epitope of monoclonal antibodies to the cytosolic or luminal domain of integral membrane proteins with the established structure.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Animals , Antibody Specificity , Antigens, Surface/chemistry , Antigens, Surface/metabolism , Cell Line , Cell Membrane Permeability , Cricetinae , Cytosol/metabolism , Epitopes/chemistry , Epitopes/metabolism , Fixatives , Fluorescent Antibody Technique , Formaldehyde , Humans , Membrane Proteins/immunology , Mice , Polymers , Rabbits , Rats , SwineABSTRACT
This article outlines the present knowledge of the architecture, molecular composition, and dynamics of focal contacts of adhesive animal cells. These structures, developed at the plasma membrane at sites where cells touch their substratum, are essential for cellular attachment in tissue formation during embryogenesis and wound healing. In tissue culture, they are particularly prominent and thus amenable to detailed investigation. Focal contacts consist of a cytoplasmic face, comprising cytoskeletal elements, a transmembrane connecting region, and a extracellular face composed of proteins of the extracellular matrix. The molecular anatomy of the numerous proteins involved, the basis for classifying them as structural or regulatory components, and their in vitro interactions are described. Based on this information, current models on the dynamics of their assembly and of possible regulatory mechanisms involving a variety of signal transduction pathways are discussed.
