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concomitant decrease in lamellipodia activity at membranes lacking Rac1
indicates a tight spatial regulation of Rac1 localization and activity. That
Rac1 localization and activity are tightly regulated was also evident in another
observation that we made, namely that the relative frequency, number and
perdurance of lamellipodia gradually increased at sites of cell-cell adhesion
and concomitantly diminished elsewhere around the cell membrane (Ehrlich
et al., 2002). This change in localization of Rac1 and lamellipodia coincided
with a change from a migratory to a non-migratory, cell-cell adherent
phenotype.
Cell-cell contact induces changes in Rac1 complexes
Changes in RacGFP localization as cells transition from a migratory to a non-
migratory, adherent state could reflect changes in molecular associations of
endogenous Rac1 at the plasma membrane. To investigate this, isolated
membranes from a heterogeneous population of migratory and adherent
MDCK cells were extracted in buffer containing octyl-glucoside to solubilize
membrane-bound Rac1; note that <5% of endogenous Rac1 is membrane-
bound, but this population is tightly associated with membranes and can be
removed only by extraction with detergents (Hansen and Nelson, 2001;
Hansen et al., 2002; see Figure 6.2). Solubilized proteins were separated by
rate-zonal centrifugation in sucrose gradients. The Rac1 profile determined by
Western blotting revealed three separate peaks of Rac1 distribution: a low
molecular weight Rac1:PAK effector complex (Hansen and Nelson, 2001),
and two higher molecular weight complexes of *11S (complex B); and *16S
(complex C) (Hansen and Nelson, 2001; Hansen et al., 2002; see Figure 6.2).
Significantly, we found that Rac1 complex B is enriched in non-contacting,
migratory MDCK cells and complex C is absent, while in contacted MDCK
cells complex C is enriched and complex B is absent (the Rac1:RAK complex
is present in both). This switch in molecular associations of endogenous Rac1
from complex B to complex C following cell-cell adhesion correlates with
the redistribution of RacGFP at non-contacting surfaces to lamellipodia at
cell-cell junctions and the concomitant change in lamellipodia activity
(Hansen et al., 2002; see Figure 6.2).
Effects of Rac1 mutant expression on endogenous Rac1
complexes and cell behaviour
Rac1 mutants are thought to exert biological activity by remaining in a
specific nucleotide binding state (Bishop and Hall, 2000; Farnsworth and
Feig, 1991; Stacey et al., 1991). Dominant negative forms of Rac1 (T17N)
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