Cell-to-cell channels link many animal cells and provide for the direct movement of small molecules between adjacent cells. These channels, which are associated with structures identified as gap junctions, are thought to play an important role in intercellular communication. This form of communication may be critical for the control of cell growth and cellular differentiation. Only recently has the experimental testing of these hypotheses become possible with the development of specific probes for cell- to-cell channels. Antibodies developed as a part of this project represent one class of specfic probes with which we will begin to address the function of cell-to-cell channels. The studies proposed here relate to the role of junctional channels between lens fiber cells in the vertebrate eye. Varied evidence indicates that a protein of 28kDa, termed MP28, is a lens junctional protein and perhaps a protein of cell-to-cell channels. This includes our preliminary findig that the intracellular injection of MP28 antibodies leads to a reduced dye transfer between cells.
The specific aims for the next five years include further work on the injection of antibodies which are specific for cytoplasmic domains on MP28 to examine the effects on junctional permeability in developing lens cells at different stages. These studies will be carried out in well-characterized lens cultures, where the development of """"""""lentoids"""""""" provides an excellent model for lens fiber cells. In addition, antibodies specific for external domains on MP28 will be screened for any inhibitory effects on the formation of junctions between cells. In both cases, junctional permeability will be assayed by intracellular dye injection and quantitative analysis of dye transfer between cells. Antibodies, which are found to inhibit junction formation in culture (between two lentoids manipulated into contact), will also be injected into the eyes of developing chicken embryos to assess the impact of these probes on lens development. Finally, we will characterize further the binding of existing MP28-specific antibodies (both monoclonal and polyclonal) in terms of specific sequences on the protein, particular sides of the membrane, and different types of cells. This analysis includes a clear demonstration of the binding of antibodies to external domains on MP28. the binding studies involve electrophoresis, immunoreplica methods, protein sequencing and electron microscopy. These varied investigations serve to test: i) our working hypothesis which indicates that MP28 forms cell-to-cell channels between fiber cells, ii) a model for the structure of MP28 within the lens plasma membrane, iii) the idea that cell-to-cell channels provide for critical interactions during lens development and iv) the idea that, during the differentiation of lens epithelial cells into fiber cells, two different types of junctional proteins are expressed, including MP2.
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