News and Publications

Gap Junctions: Biosynthesis, Degradation, Structure, and Function

M.M. Falk, P. Lopez, S. Braconnot, V. Lagrée,* B.N.G. Giepmans,** S.-C. Chen,*** D. Segretain****

* University of Cambridge, Cambridge, England
** The Netherlands Cancer Research Institute, Amsterdam, the Netherlands
*** Northwest Hospital, Seattle, WA
**** Université de Paris, Paris, France

We study intracellular protein trafficking and the mechanisms involved in the maturation of these proteins into functional structures. Protein trafficking and maturation are fundamental cell biological processes that must be accomplished precisely for accurate cell function. Several human diseases are related directly to defects in these processes. The structures of oligomeric membrane proteins, such as the gap junction membrane channel, are of particular interest to us.

Previously, we studied the biosynthesis, assembly, trafficking, and function of connexins, the subunit proteins of gap junction channels. We used cell-free translation­membrane translocation assays, molecular biology, biochemical and immunological approaches, and expression of subunit proteins in cells in culture.

To investigate these processes in living cells, we tagged the connexins with green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and a red fluorescent protein. We used multicolor and time-lapse microscopy and electron microscopy to study the fate of connexins in fixed cells and in living cells. Our results revealed a number of exciting new aspects of gap junction biosynthesis, including connexin membrane integration, oligomerization, selectivity among connexin isotypes, composition of gap junction channel clusters assembled from several connexin isotypes, and the mobility and structural composition of gap junction channel clusters in membranes of living cells.

In recent studies, we characterized a direct interaction of microtubule plus ends with gap junctions assembled from connexin 43 subunits, functionally characterized connexins tagged with the red fluorescent protein, and investigated the dynamic mobility of gap junction channels and channel clusters in the plasma membrane. Currently, we are investigating delivery of the precursors of gap junction channels to the plasma membrane, accrual of newly synthesized channels to gap junctions (Fig. 1), and, on a molecular level, signals that lead to specific assembly characteristics of different connexin isoforms (Fig. 2). These studies include investigating the roles that other cellular components, such as the actin cytoskeleton, microtubules, regulatory binding proteins, cell adhesion molecules, endocytic components, and lipids, might have in these processes.

Future plans include using an integrated molecular biology, biochemistry, and imaging approach to characterize the requirements for the formation, clustering, regulation, and degradation of gap junction channels and to investigate a potential direct transfer of large subcellular components between neighboring cells. We would also like to use our established experimental approaches to study the biology of other cellular structures involved in cell-cell contact, cell adhesion, and cell signaling.

PUBLICATIONS

Falk, M. Genetic tags for labelling live cells: gap junctions and beyond. Trends Cell Biol. 12:399, 2002.

Lauf, U., Giepmans, B.N., Lopez, P., Braconnot, S., Chen, S.C., Falk, M.M. Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells. Proc. Natl. Acad. Sci. U. S. A. 99:10446, 2002.

Lopez, P., Balicki, D., Buehler, L.K., Falk. M.M., Chen, S.-C. Distribution and dynamics of gap junction channels in living cells. Cell Adhes. Commun. 8:237, 2001.