Department of Biological Chemistry and Molecular Pharmacology
Dana Farber Cancer Institute
Mayer Building, Room 849
44 Binney Street
Boston, MA 02115
Tel: (617) 632-5102 Fax: (617) 632-5103
Email: pamela_silver@macmailgw.dfci.harvard.edu
6 postdoctoral fellows, 3 graduate students
We are interested in how proper intracellular organization is maintained throughout cell growth and division. One focus is the assembly, structure and behavior of the cell nucleus. Movement of macromolecules in and out of the nucleus is a highly specific, rapid and regulated process. We have conducted experiments to address the mechanisms that govern assembly of proteins into the nucleus, exit of protein-RNA complexes from the nucleus and proper segregation of nuclear components during the cell cycle. Other studies concern the action of molecular chaperones in assembly of protein complexes.
The specificity of protein import is generated in part by signals on nuclear-destined proteins, interactions with the nuclear pore complex and regulatory factors. Much less is known about targeting of RNA for export although our results suggest that the two processes can be co-regulated. The key to the mechanism of bi-directional nuclear transport and its regulation is an understanding of how macromolecules are recognized for transport and what mediates their movement through the pore. To accomplish this, we have taken several complementary approaches employing the yeast Saccharomyces cerevisiae as a model organism. Factors important for movement of proteins and RNAs in and out of the nucleus have been identified by isolation and characterization of mutants blocked in both processes. These include a novel RNA binding protein, a GTP-binding protein, its exchange factor and corresponding GAP. We have also reconstituted nuclear transport in a cell-free system and shown that cytosolic proteins and proteins of the pore complex are important for distinct steps in protein import.
We have also recently developed a novel system to observe in living cells the segregation of proteins during cell division. By expressing a fusion of a yeast centrosomal protein to the naturally green fluorescent protein (GFP), we have observed the behavior of the mitotic spindle by time-lapse fluorescence microscopy of living cells. Observation of fluorescent-tagged spindle-pole bodies required us to combine GFP technology with ultra-sensitive microscopic video-imaging techniques. We anticipate that this combination of technologies will become a standard for study of temporal changes in living cells.
Kahana, J., Schnapp, B. and Silver, P. 1995. Kinetics of spindle pole body separation in budding yeast. Proc. Natl. Acad. Sci. 92:9707-9711.
Corbett, A., Koepp, D., Schlenstedt, G., Lee, M., Hopper, A., and Silver, P. 1995. Rna1p, a Ran/TC4 GTP-activating protein, is required for nuclear import. J. Cell Biol. 130:1-10.
Koepp, D., and Silver, P.A. 1996. A GTPase that controls nuclear trafficking -- running in the right direction or walking RANdomly? Cell 87:1-4.