News and Publications

Biosynthesis of Proteins That Mediate Direct Cell-Cell Communication

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

* The Netherlands Cancer Research Institute, Amsterdam, the Netherlands
** Northwest Hospital, Seattle, WA
*** Université de Paris, Paris, France
**** Universität Stuttgart, Stuttgart, Germany

We study intracellular protein trafficking and the mechanisms involved in the maturation of these proteins into functional structures. Oligomeric membrane proteins are of particular interest. Protein trafficking and maturation are fundamental cell biological processes. Their precise accomplishment is crucial for accurate cell function. Several diseases in humans are linked to defects in intracellular protein trafficking and to mutations in gap junction connexins. Understanding the fundamental processes of protein trafficking, biosynthesis of oligomeric membrane proteins, and direct cell-cell communication may provide the knowledge to understand associated diseases, develop efficient drugs, and increase human health.

Previously, we studied the synthesis, assembly, trafficking, and function of the oligomeric protein structure termed the gap junction membrane channel. Primarily, we used cell-free translation/membrane translocation assays, molecular biology, biochemical and immunologic approaches, and expression of subunit proteins in cells in culture. To study these processes in living cells, we recently tagged connexins, the protein subunits of gap junction channels, with green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and a red fluorescent protein. We are studying the fate of connexins in fixed cells and in living cells by using multicolor and time-lapse light microscopy and electron microscopy. For the light microscopy studies, we are using the highest possible resolution techniques available: deconvolution and confocal microscopy and single-particle imaging.

So far, these studies have 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 clusters of gap junction channels in membranes of living cells (Figs. 1 and 2).
Currently, we are studying the selective assembly of connexin isoforms in cultured cells, the signals that drive connexin selectivity, and the dynamic processes involved in the assembly, regulation, and degradation of gap junctions. We are also investigating the roles that other cellular components, such as the actin cytoskeleton, microtubules, regulatory binding proteins, cell adhesion molecules, endolytic 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 also would 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.M. Biosynthesis and structural composition of gap junction intercellular membrane channels. Eur. J. Cell Biol. 79:564, 2000.

Falk, M.M. Connexin-specific distribution within gap junctions revealed in living cells. J. Cell Sci. 113:4109, 2000.

Falk, M.M. Connexins/connexons: Cell-free expression. Methods Mol. Biol. 154:91, 2001.

Falk, M.M., Lauf, U. High-resolution fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells. Microsc. Res. Tech. 52:251, 2001.

Giepmans, B.N.G., Verlaan, I., Hengeveld, T., Janssen, H., Calafat, J., Falk, M.M., Moolenaar, W.H. Gap junction protein connexin-43 interacts directly with microtubules. Curr. Biol. 11:1364, 2001.

Lauf, U., Lopez, P., Falk. M.M. Expression of fluorescently tagged connexins: A novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498:11, 2001.

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

Shen, P., Giepmans, B.N.G., Lauf, U., Falk, M.M. Assembly, structure and degradation of gap junctions in living cells: GFP in motion II [CD-ROM Suppl.]. Trends Cell Biol. 11:183, 2001.