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Falk Lab

Department of Cell Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037    -     USA Tel.  ++1 (858) 784-2850 Fax  ++1 (858) 784-9927 mfalk@scripps.edu

Our current research interests are:

•  Intracellular Protein Transport
•  Oligomeric Membrane Proteins
•  Protein-Protein Interactions
•  Gap Junctions
•  Cell-free translation/membrane protein translocation
•  High resolution deconvolution fluorescence microscopy and live-cell imaging

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Gap junctions assembled from GFP-tagged connexins (green) in transfected  human cells. DAPI-stained nuclei in blue, endoplasmic reticulum (calnexin)  stained red.

Biological membranes play a key role for life.  Membranes not only surround cells, but also divide eukaryotic cells into sub-cellular compartments allowing the formation of different environments within a cell.  The membrane lipids, however, form a tight seal and prevent simple exchange of biological molecules.  We are interested in understanding what role membrane proteins might play in trans-membrane transport processes.  Polytopic membrane protein subunits of biological channels are of particular interest.  These subunits are specialized membrane proteins that bear charged amino acid residues within the transmembrane domains to allow formation of hydrophilic pores within the lipid bilayer.  Oligomeric gap junctions are an example of such biological channels.  They cluster in large plaques in the adjoining plasma membranes of neighboring cells and provide direct cell-to-cell communication.  Some untypical features associated with their unique function are extremely interesting.

We have demonstrated that connexins are cotranslationally integrated into the endoplasmic reticulum where they achieve their functional transmembrane topology, and subsequently transported through the Golgi apparatus to reach the plasma membrane.  Investigating gap junction channel subunit assembly in a cell-free translation system, we found that different connexin isotypes interact selectively by only allowing the assembly of homo-oligomeric and certain types of hetero-oligomeric channels.  The results also suggest that “assembly” signals regulating principal connexin subunit recognition, as well as "selectivity" signals regulating specific assembly of hetero-oligomeric channels may exist in the connexin polypeptide sequence.

To study the biosynthesis of gap junctions in live cells we have tagged connexins with green fluorescent protein (GFP) (Fig. 1).  The connexin-GFP fusion proteins were efficiently expressed, oligomerized, and were transported with great efficiency to the plasma membrane, where they assembled into functional gap junction channels and gap junction plaques (Fig. 2).  Deconvolved, high-resolution fluorescence images of gap junction plaques assembled from GFP-tagged a1Cx43 revealed an exiting rich plaque morphology in two as well as three dimensional image reconstruction.

Time-lapse images showed assembly of large gap junction plaques by the fusion of smaller plaques, and that gap junction plaques are not rigid structures, but are extremely dynamic in their appearance and shape.  These dynamic live cell images add a new dimension to the classical static picture of gap junctions and provoke a number of interesting questions on their structure and function.

Current research has three primary aims:  (1) characterize chaperones and other factors that play a role in the biosynthesis of gap junction channels, (2) further characterize the signals that regulate the assembly of gap junction channel subunits, and (3) use the GFP-tagged connexins in combination with high resolution, multi-dimensional fluorescence deconvolution microscopy to investigate the biosynthesis of gap junction channels in live cells.

For more information about our research click on Scientific Report 97/98 / Inside Front cover

Prof. M. Falk, Principal Investigator

Peijuan Shen
 

   1. Synthesis, assembly and structure of gap junction intercellular channels.
     Yeager M, Unger VM, Falk MM
     Curr Opin Struct Biol 1998 Aug 8:4 517-24

   2. Connexin membrane protein biosynthesis is influenced by polypeptide positioning within
     the translocon and signal peptidase access.
     Falk MM, Gilula NB
     J Biol Chem 1998 Apr 3 273:14 7856-64 ; see Abstract

   3. Cell-free synthesis and assembly of connexins into functional gap junction membrane
     channels.
     Falk MM, Buehler LK, Kumar NM, Gilula NB
     EMBO J 1997 May 15 16:10 2703-16

   4. Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo
     Kahn TW, Beachy RN, Falk MM
     Curr Biol 1997 Apr 1 7:4 R207-8

   5. Membrane insertion of gap junction connexins: polytopic channel forming membrane
     proteins.
     Falk MM, Kumar NM, Gilula NB
     J Cell Biol 1994 Oct 127:2 343-55

   6. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.
     Zibert A, Maass G, Strebel K, Falk MM, Beck E
     J Virol 1990 Jun 64:6 2467-73

   7. VPg gene amplification correlates with infective particle formation in foot-and-mouth
     disease virus.
     Falk MM, Sobrino F, Beck E
     J Virol 1992 Apr 66:4 2251-60

   8. Foot-and-mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3.
     Falk MM, Grigera PR, Bergmann IE, Zibert A, Multhaup G, Beck E
     J Virol 1990 Feb 64:2 748-56

Thanks for visiting us. Return whenever time permits for more cool images and live-cell movies!

All rights reserved © 1999 Matthias M. Falk

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