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Gap Junctions: A Model for the Biosynthesis of Complex Plasma Membrane Signaling Structures

* Université de Bordeaux I, Talence, France
** Centre de Recherche du CHUM, Montreal, Canada
*** University of California, San Diego, California
**** Northwest Hospital, Seattle, Washington
***** Université de Paris V, Paris, France
^§ Thomas Jefferson University, Philadelphia, Pennsylvania

Cells within a tissue are autonomous units yet constantly depend on signals from their surrounding. This information exchange is mediated by membrane proteins that assemble into localized and temporally organized multiunit complexes, such as tight junctions, adherens junctions, desmosomes, gap junctions, focal adhesions, hemidesmosomes, and chemical and immunologic synapses.

We are using gap junctions as a model system to investigate how cells regulate the biosynthesis and complex assembly of protein subunits. Previously, we studied the biosynthesis of gap junction proteins, elucidated the compatibility of subunits of the proteins, characterized signals that regulate subunit interaction, determined cellular components involved in these processes, and characterized how newly assembled channels are accrued to gap junctions.

Gap junction channels typically cluster in large numbers in the adjoining membranes of neighboring cells to provide direct cell-cell communication. The results of previous investigations suggested that gap junctions are degraded by the unusual invagination of entire, or large, parts of the double-membrane clusters into 1 of the 2 coupled cells. However, in contrast, our recent observations on the assembly of clusters of gap junction channels indicated that newly synthesized channels are accrued along the outer edge while older channels are simultaneously released from the center. Therefore, we used high-resolution fluorescence and time-lapse microscopy combined with ultrastructural analyses to reinvestigate the degradation of gap junctions.

Our studies indicate that gap junctions are degraded by at least 2 distinct mechanisms: (1) the previously described slow (about 1 hour) invagination and pinching off of entire membrane clusters or of large areas that mature into typical cytoplasmic annular junctions (Fig. 1) and (2) a constant and fast (within seconds) release of small (¾0.5 µm in diameter) vesicular structures that bud from the plane of the clusters (Fig. 2). Ultrastructural analysis indicated that the invaginated junctional membranes include adjacent parts of nonjunctional membrane, suggesting that the adjacent membrane regions may contain the signal for degradation. Early products of the degradation of gap junctions mature into light and then dark endosomes; the endosomes fuse with lysosomes, and thus, channel clusters are completely degraded. Examination of growing cells that express connexin 41, the protein subunit of gap junctions, labeled with either cyan or yellow fluorescent protein in mixed cultures finally indicated that invagination and subsequent formation of annular junctions result in the direct transfer of plasma membrane from one cell into the other.

In the future, we will characterize cellular components that mediate the degradation of gap junctions. Preliminary results indicate that clathrin and actin might be involved, and thus degradation of gap junctions may be similar to the processes involved in classical endocytosis. We also want to investigate whether formation of annular junctions is a vehicle for direct cell-to-cell spread of viral pathogens. Finally, we plan to investigate the biology of other cellular structures involved in cell-cell contact, cell adhesion, and cell signaling.

Publications

Falk, M.M., Segretain, D., DeCrouy, X. Regulation of connexin biosynthesis, assembly, gap junction formation, and removal. Biochim. Biophys. Acta, in press.

Lagrée, V., Brunschwig, K., Lopez, P., Gilula, N.B., Richard, G., Falk, M.M. Specific amino-acid residues in the N-terminus and TM3 implicated in channel function and oligomerization compatibility of connexin43. J. Cell Sci. 116(Pt. 15):3189, 2003.