Cellular Physiology Section
Lois Greene, PhD, Principal Investigator
Evan Eisenberg, MD, PhD, Principal Investigator
Our laboratory is interested in the formation and
dissolution of both normal and pathological protein complexes in
the cell with an emphasis on the role of molecular chaperones in
these processes. To this end, we are studying the formation and
dissolution of clathrin-coated vesicles in mammalian cells and the
formation and dissolution of prion and Huntingtin aggregates in
both mammalian cells and yeast.
In regard to our first research area, we are using GFP-labeled
clathrin and clathrin adaptor proteins to study the dynamics of
clathrin-coated vesicle formation and uncoating both at the plasma
membrane and at the trans-Golgi network. We are carrying out these
studies in real time in tissue culture cells using fluorescence
recovery after photobleaching (FRAP), fluorescence loss in photobleaching
(FLIP) and total internal reflectance fluorescence microscopy (TIRF).
In regard to the molecular chaperone Hsc70, we have shown that it
mediates the ATP-dependent dissociation of clathrin from clathrin-coated
vesicles and may also be involved in the rebinding of clathrin and
clathrin adaptors to form new clathrin-coated pits. Interestingly,
we have discovered that Hsc70 requires a co-chaperone to carry out
these functions; either the J-domain protein auxilin or GAK is required
for Hsc70 to interact with clathrin-coated vesicles and pits. We
are currently investigating the function of these J-domain proteins
both by using RNAi to inhibit their production in the cell and by
producing auxilin and GAK knock-out mice.
In our second major research area, we are using FRAP, internal
reflectance microscopy and fluorescence correlation spectroscopy
to investigate aggregation of Huntingtin and prion protein both
in mammalian cells and yeast. In yeast using the GFP-labeled prion
protein Sup35p, we have been able for the first time to follow
prion aggregation with confocal microscopy. Using FRAP and fluorescence
correlation spectroscopy, we are also attempting to observe for
the first time both in mammalian cells and yeast the existence
of small diffuse aggregates of Huntingtin polyglutamine fragments
in addition to the large granules that are generally observed
in diseased cells. Finally we are investigating the trafficking
of GFP-labeled membrane bound mammalian prion protein and are
also investigating whether infection of the cells by aggregated
prion protein affects the aggregation of the GFP-labeled prion
protein on the plasma membrane.
Questions, comments and suggestions about this page
may be addressed to Lois Greene
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