Jean-Paul Revel - US grants

The program is an interdisciplinary study of several membrane proteins utilizing in common an electron microscope facility and a laboratory for microsequencing in which the capability for nanomole and subnanomole level analyses are being developed. Research will focus on the structure and physiology of gap junctions and their constituent proteins, the nature of protein diversity in transplantation antigens and in the T/t complex so as to provide insight into the organization and evolution of the genes that code for the surface molecules. Work on the glycoproteins of the membranes of the Sindbis virus and their precursors and immunocytological and biochemical analyses of filamentous proteins in muscle and non-muscle cells is being pursued as well as analyses characterizing membrane proteins from cloned murine cell lines derived from UV-induced skin tumors bearing both specific and cross-reactive antigens.

We propose a multidisciplinary approach for the study of the functional organization of cardiac gap junctions at the molecular level. The protein has been isolated and a partial amino acid sequence obtained. We have also isolated undegraded heart total poly(A)+ RNA with which to build libraries. Purified heart gap junctions containing the native protein as well as synthetic peptides modeled on the sequence will be used to produce antibodies. With these and synthetic oligonucleotide probes based on the known amino acid sequence, we will search an expression library for clones expressing the junction protein. Further protein sequencing will yield data with which to prepare other oligonucleotides and site-directed antibodies. Clone(s) containing cDNA encoding the full length of the gap junction protein will be sequenced. The deduced amino acid sequence will then serve to build models of the organization of the junction protein in the membranes. A combination of specific proteolytic cleavages and reactivity with site-directed antibodies will be used to test these models. The models will permit us to examine physiologically the role of different regions of the molecule in cell-cell signaling and exchanges. In vitro mutagenesis should allow us to explore the consequences of modifications to the junction protein at specific locations. This work should provide data permitting the rational design of approaches to the treatment of heart diseases in which defects in conduction may be involved.

The gap junction is an ubiquitous structure which permits exchanges of ions and small molecules between neighboring cells. It is involved in many important cellular processes, synchronizing heart beat and contraction of smooth muscle, maintaining homeostasis by metabolic coupling and permitting the exchanges of messages which somehow control growth, development and differentiation. Using two separate anti-gap junction antibodies we have isolated a cDNA clone which hybridizes with synthetic oligonucleotide probes directed against known sequence of the liver gap junction protein. We plan: 1. To prove definitely that we have a gap junction coding clone by sequencing and comparing the sequences to know features of the gap junction protein. 2. To deduce an amino acid sequence based on the nucleotide sequence obtained, and use it to determine possible configuration(s) for the molecule. 3. To synthesize peptides (8-15 amino acids long) which will in turn be used to generate antibodies against potentially important and accessible portions of the molecule. The arrangement of the gap junction molecule and the structure of connexons will be examined by using gold label immunocytochemistry at the electron microscopy level, and also immunochemical analyses on proteolytically and chemically cleaved gap junction protein. 4. To use the cDNA clone and antibodies in tests of the presence of the protein and/or message in different organs, and at different stages of development. The extent of homology between the mRNAs found to encode for gap junctions in different places will be determined. We will also study the distribution of the gap junction genes in different organisms. 5. To experimentally manipulate systems where gap junction expression and function can be altered at will in order to study putative changes in amounts and kinds of message expressed. 6. To carry out in vitro translation and other experiments to test for the existence of possible junction protein precursors and analyze the mode of assembly of junctional proteins into functional structures. Eventually, we will carry out manipulations of the sequence in attempts at altering function by alteration of known portions of the protein.

intercellular connection; cell cell interaction;

Gap junctions are intercellular channels which play a major role in synchronizing cellular activities and are involved in the control of growth and development. Because they synchronize heart and other muscle cells, junction defects may be involved in arrhythmias and problems in parturition. There needs to be a better understanding of how they are built and how the physiological factors which causes them to open and close, exert their action. We have carried out preliminary work and shown that we can study details of gap junction structure by using a relatively new tool, scanning (atomic) force microscopy. We now propose to extend this work, capitalizing on the fact that we have just succeeded to examine gap junctions to reveal very fine detail. Using a combination of techniques which includes the use of immunolabeling we would analyze the topology of connexin molecules. By combining an atomic force microscope with fluorescence microscopy we hope to identify gap junction precursors either on cell surfaces, or if this does not succeed on membranes derived from cell surface. We know such precursors exist because we have obtained evidence that the extracellular domains of connexins are exposed before junctions form. We want to visualize the precursor structures in the AFM and compare them to what should be the corresponding structures revealed by dissection in mature junctions. We also hope to get some understanding of gap junction biogenesis by studying the organization of connexins in intracellular organelles.