Biology Reference
In-Depth Information
Figure 6.2 Description of Rac1 complexes in MDCK cells during cell-cell adhesion. (For
details of protein distributions, live cell imaging and biochemical analysis of protein
complexes, please see Ehrlich et al., 2000, and Hansen et al., 2000.) Greater than 95% of
Rac1 is in a GDP bound and inactive form in the cytosol in a complex with Rho-GDI
(Hansen and Nelson, 2001). We detected three Rac1 complexes in membranes of MDCK
cells, termed A, B and C. Complex A comprises a Rac1:PAK complex (Hansen and
Nelson, 2001). Complex B and C are high molecular weight complexes of unknown
components. Complex B is enriched in single, migratory cells, but is almost absent in cells
undergoing cell-cell adhesion (hence it is not coloured). Complex C is enriched during cell-
cell adhesion, and we suggest that it localizes at cell-cell contacts (as shown for RacGFP,
see Ehrlich et al., 2002 and text). Localization of Rac1 complexes at cell-cell contacts
would be expected to increase the number, frequency and perdurance of lamellipodia,
which would lead to a cascade of events involving increased cell surface contacts between
adhering cells, increased opportunity for cadherin-cadherin interactions and thereby
strengthening of cell-cell adhesion. (A colour reproduction of this figure can be found in
the colour plate section)
preferentially bind GDP and sequester machinery required to activate
endogenous Rac1. Constitutively active Rac1 mutants (G12V and Q61L)
have reduced GTPase activity and remain GTP bound for long periods,
thereby generating a persistent signal through downstream effector
proteins. Additionally, sequestration of GTPase activating proteins by active
mutants slows inactivation of endogenous Rac1,
leaving it in the active
conformation.
We expressed RacT17NGFP in MDCK cells and found cell-cell contacts
expanded very slowly compared with wild-type cells (Ehrlich et al., 2002).
Bursts of localized lamellipodia formation at sites of cell-cell contacts were
markedly reduced in RacT17NGFP cells, and RacT17NGFP cells failed to
restrict membrane protrusions to sites of cell-cell contact. In contrast to cells
