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expressing RacT17NGFP, cells expressing constitutively active RacQ61LGFP
exhibited an increased rate of lamellipodia formation, with numerous ru
es
extending around the cell periphery, consistent with previous work
demonstrating that activated Rac1 leads to an increase in lamellipodia
formation in many cell types (Bishop and Hall, 2000). We found that broad
lamellipodia extended over contacting cells and lateral expansion of cell-cell
contacts occurred more quickly when RacQ16L was expressed.
To examine whether expression of Rac1 mutants affected endogenous Rac1
complexes A, B and C, we prepared membrane extracts from mutant Rac1
expressing cells and separated the proteins by rate-zonal centrifugation, and
endogenous Rac1 and mutant Rac1 distributions were examined by Western
blotting with a Rac1 antibody (Hansen and Nelson, 2001; Hansen et al.,
2002). In cells expressing Rac1T17N , endogenous Rac1 appeared as a single
peak corresponding to complex A but was absent from complexes B and C.
Rac1T17N sedimented as a single peak corresponding to the normal
distribution of endogenous Rac1 complex B. In cells expressing Rac1G12V,
endogenous Rac1 was detected in complexes A, B, and C, and Rac1G12V
co-sedimented with endogenous Rac1 in peaks corresponding to complexes B
and C.
Thus, in MDCK cells expressing mutant Rac1 there was a strong positive
correlation between the presence of active Rac1 in complexes B and C, and the
formation of lamellipodia-driven cell-cell contacts. In cells expressing
Rac1T17N, endogenous Rac1 was excluded from complexes B and C;
cell-cell adhesions formed tentatively and without lamellipodia. In cells
expressing Rac1Q61L or Rac1G12V, complexes B and C contained
constitutively active Rac1 and dynamic lamellipodia-driven adhesive contacts
were formed rapidly (Hansen and Nelson, 2001; Hansen et al., 2002).
Although live cell imaging revealed changes in the kinetics and degree of
cell-cell contact formation in cells expressing Rac1T17N or Rac1G12V, we
used a quantitative adhesion assay to measure directly the size of cell
aggregates formed over time from a suspension of single cells, and the
resistance of these aggregates to shearing force (Ehrlich et al., 2002). Greater
than 95% of control cells formed large clusters (>50 cells) within 3 to 6 h that
were completely resistant to trituration. Rac1 T17N cells formed aggregates
with kinetics similar to that of controls, but the aggregates were far less
resistant to trituration than were control cells. Since Rac1 T17N blocks
incorporation of endogenous Rac1 into complexes B and C, we conclude that
Rac1 these complexes are required for the strengthening and compaction
phase of cell-cell adhesion. Rac1 G12V cells displayed rapid kinetics of
adhesion formation and aggregates became resistant to trituration earlier than
control cells. That Rac1G12V incorporates into Rac1 complexes B and C is
further evidence that Rac1 these complexes C are important
for the
strengthening and compaction phase of cell-cell adhesion.
