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et al., 2002), VASP (Vasioukhin et al., 2000)] might link cadherin/catenin
function with actin organization.
Examining E-cadherin distribution during cell-cell adhesion in
live cells
Many studies have examined the subcellular distribution of cadherins,
catenins and associated actin cytoskeleton proteins in static, fixed cells.
However, dynamic cellular events, such as cell migration and cell-cell
adhesion, are best examined by live cell imaging, as championed by
Abercrombie and his contemporaries many decades previously (Abercrombie
et al., 1970a,b). Development of techniques to examine protein distributions
at high resolution in single, living cells, in collaboration with Dr Stephen
Smith of Stanford University, permitted investigation into the temporal and
spatial regulation of assembly and function of the cadherin/catenin complex
in MDCK cells. We developed time-lapse differential interference contrast
(DIC) imaging to observe the development of cell-cell contacts (McNeill
et al., 1993). This was correlated with either quantitative retrospective
immunocytochemistry (Adams et al., 1996) or fluorescence microscopy of
cells expressing GFP-tagged proteins (Adams et al., 1998; Ehrlich et al.,
2002) in order to monitor dynamics of protein localization during cell-cell
contact formation. Our initial analyses showed that E-cadherin, a-catenin
and b-catenin, but not plakoglobin, co-assemble into Triton X-100 insoluble
(TX-insoluble) structures at cell-cell contacts (Adams et al., 1996). The
assembly of the cadherin/catenin complex occurred with kinetics similar
to that for strengthening of E-cadherin-mediated cell adhesion deter-
mined using a centrifugation force-based adhesion assay (Angres et al., 1996).
TX-insoluble E-cadherin, a-catenin and b-catenin co-localize along cell-cell
contacts in spatially discrete micro-domains, which we term puncta, and the
relative amounts of each protein in each punctum increased proportionally
during cell-cell adhesion (Adams et al., 1996). Subsequently, we imaged live
cells expressing E-cadherinGFP (EcadGFP) and found that EcadGFP rapidly
accumulated at cell-cell contacts and became concentrated in puncta identical
to those we had found by retrospective immunohistochemistry (Adams et al.,
1998; see Figure 6.1A). Using photo-bleaching of EcadGFP, we showed
directly that each punctum arose by coalescence of a highly mobile, diffuse
pool of cell surface E-cadherin, and that E-cadherin rapidly became
immobilized within each punctum (Adams et al., 1998; see Figure 6.1B,C).
These E-cadherin puncta were spatially coincident with membrane attachment
sites for actin filaments branching off from circumferential actin cables that
circumscribed each cell (Adams et al., 1996, 1998; see Figure 6.1D). As the
