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1999). The small adhesions can mature into larger, more organized structures,
which tend to inhibit migration (Burridge and Chrzanowska-Wodnicka, 1996;
Chrzanowska-Wodnicka and Burridge, 1996; Zamir and Geiger, 2001).
Disassembly of adhesions at the rear results in the net translocation of the
cell in the direction of movement.
This overview focuses on two issues that are of recent interest to us. The first
is the mechanisms by which adhesions form and disassemble. To address this,
we have initiated a ratio imaging approach using GFP variants fused to key
adhesion, signalling, and cytoskeletal proteins. The second is a comparison
between the mechanisms that regulate migration in vitro and in vivo. We have
developed in situ migration systems that closely mimic the in vivo environment
to answer this question. A major advantage of our in situ system is that we can
express exogenous GFP-tagged proteins in the slices and observe the molecular
and cellular dynamics of migration by time-lapse microscopy.
Adhesion dynamics in migrating cells
Adhesion formation
Most studies examining adhesion formation and disassembly have utilized
quiescent fibroblasts, which are not highly motile and tend to form large
adhesions. Since these processes are poorly understood in migrating cells, we
are studying adhesion dynamics under conditions that promote prominent
migration, such as plating cells on low concentrations of fibronectin for short
periods of time (less than 1 h). Our approach is to visualize the dynamics of
adhesion molecules fused to GFP, expressed either singly or in pairs, in these
migrating cells (Laukaitis et al., 2001). The molecules include a5 integrin, focal
adhesion kinase (FAK), zyxin, paxillin and a-actinin.
Since the leading edge of a cell is a site where new adhesions form, we
examined the dynamics of adhesion formation in protrusive regions of the cell.
At a 60 s time resolution, FAK, paxillin and zyxin enter the nascent adhesions
at about the same time. Surprisingly, we were unable to detect visibly
organized a-actinin or a5 integrin in these nascent adhesions. However, after
protrusive activity ceased, a-actinin entered adhesions, which was subse-
quently followed by detection of visible concentrations of a5 integrin in these
structures. The adhesions containing visibly organized a-actinin and a5
integrin probably represent the more mature adhesions that are studied by
most others (Burridge and Chrzanowska-Wodnicka, 1996; Chrzanowska-
Wodnicka and Burridge, 1996).
The absence of organized a5 integrin in the initial FAK/paxillin containing
adhesions was surprising because it is generally assumed that integrin ligation
and/or clustering serves to nucleate the formation of adhesions. Although it is
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