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. Author manuscript; available in PMC: 2012 Jun 7.
Published in final edited form as: Dev Cell. 2002 Aug;3(2):259–270. doi: 10.1016/s1534-5807(02)00216-2

Spatio-Temporal Regulation of Rac1 Localization and Lamellipodia Dynamics during Epithelial Cell-Cell Adhesion

Jason S Ehrlich 1, Marc DH Hansen 1, W James Nelson 1,1
PMCID: PMC3369831  NIHMSID: NIHMS381354  PMID: 12194856

Summary

Cadherin-dependent epithelial cell-cell adhesion is thought to be regulated by Rho family small GTPases and PI 3-kinase, but the mechanisms involved are poorly understood. Using time-lapse microscopy and quantitative image analysis, we show that cell-cell contact in MDCK epithelial cells coincides with a spatio-temporal reorganization of plasma membrane Rac1 and lamellipodia from noncontacting to contacting surfaces. Within contacts, Rac1 and lamellipodia transiently concentrate at newest sites, but decrease at older, stabilized sites. Significantly, Rac1 mutants alter kinetics of cell-cell adhesion and strengthening, but not the eventual generation of cell-cell contacts. Products of PI 3-kinase activity also accumulate dynamically at contacts, but are not essential for either initiation or development of cell-cell adhesion. These results define a role for Rac1 in regulating the rates of initiation and strengthening of cell-cell adhesion.

Introduction

Rho family small GTPases play essential roles in a diversity of cellular functions, many of which depend on cytoskeletal and membrane dynamics (recently reviewed by Bishop and Hall, 2000). The best-studied members of the Rho family that regulate dynamics of distinct actin structures are RhoA (stress fibers), Rac1 (lamellipodia), and Cdc42 (filopodia) (Allen et al., 1997; Nobes and Hall, 1995; Ridley and Hall, 1992; Ridley et al., 1992). Activation of Rho GTPases, however, impinges on a large number of downstream target pathways, including chemotaxis and cell motility, cell division, endo- and exocytosis, cell-matrix adhesion, and cell-cell adhesion (Bishop and Hall, 2000; Nobes and Hall, 1999). Therefore, it is likely that regulation of distinct cellular events depends on the particular subset of Rho effectors acti vated by localized, contextual signaling.

Changes in actin organization and membrane dynamics occur during establishment of cell-cell adhesion, which in epithelial cells is regulated by the adhesion molecule E-cadherin (Gumbiner, 2000). Time-lapse imaging of live MDCK epithelial cells expressing a GFP fusion to E-cadherin demonstrated that contact between migratory cells is an opportunistic event to which cells must respond rapidly in order to convert transient contact into strong adhesion (Adams and Nelson, 1998). The response to cell-cell contact is intimately associated with structural modifications to the actin cytoskeleton and rapid accumulation of E-cadherin at contact sites (Adams et al., 1998; Krendel and Bonder, 1999; Vasioukhin et al., 2000; Yonemura et al., 1995). Signaling pathways activated in response to transmembrane E-cadherin ligation remain poorly understood, but the observed remodeling of cortical actin in response to cell-cell adhesion suggests that Rho family proteins are important in this process.

Rac1 and other small GTPases are present at cell-cell junctions (Akhtar and Hotchin, 2001; Jou and Nelson, 1998; Nakagawa et al., 2001; also reviewed by Fukata and Kaibuchi, 2001), as are known GTPase binding partners and regulators such as IQGAP and Tiam1 (Hordijk et al., 1997; Kuroda et al., 1998; Sander et al., 1998; Vasioukhin et al., 2000). When microinjected into kera tinocytes, dominant-negative Rac1T17N and constitutively active Rac1Q61L diminished accumulation of E-cadherin at cell-cell contacts (Braga et al., 1997, 2000). In MDCK epithelial cells, in contrast, E-cadherin remains localized to cell-cell contact sites in cells expressing dominant-negative or constitutively active Rac1 (Jou and Nelson, 1998). Other work in MDCK cells has demonstrated an increased accumulation of E-cadherin at contact sites in the presence of activated Rac1 (Hordijk et al., 1997; Takaishi et al., 1997), as well as a decrease in E-cadherin levels with dominant-negative Rac1 (Takaishi et al., 1997). In mouse L fibroblasts, expression of exogenous E-cadherin and dominant-negative Rac1 results in decreased cell-cell association (Fukata et al., 1999). Taken together, these data point to an important role for Rac1 and other small GTPases in regulating cadherin-based cell-cell contacts. While recent data implicate IQGAP as one modulator of Rac1 function during cell-cell adhesion (reviewed by Fukata and Kaibuchi, 2001), the mechanisms, sites of action, and effectors by which Rho family members function during this process nonetheless remain unclear.

Amounts of GTP-bound, actively signaling Rac1 increase following cadherin-mediated cell-cell adhesion, in a PI 3-kinase-dependent manner (Kim et al., 2000b; Nakagawa et al., 2001; Noren et al., 2001; Pece et al., 1999). This increase in activity upon cadherin engagement might be important in initiating or regulating actin reorganization. The regulatory subunit of PI 3-kinase associates with the E-cadherin/catenin complex (Espada et al., 1999; Pece et al., 1999; Woodfield et al., 2001). Consistent with PI 3-kinase lipid products acting as intermediates between cell-cell adhesion and small GTPase activation, the Rac1 exchange factor Tiam1, which promotes E-cadherin-based cell-cell adhesion, is regulated by PI 3-kinase activity (Hordijk et al., 1997; Sander et al., 1998). Complicating these data, however, is the observation that inhibition of PI 3-kinase enzymatic activity blocks increased Rac1 activation, but has no effect on E-cadherin or Rac1 localization to sites of cell-cell contact (Nakagawa et al., 2001).

In this work, we present new data on the role of Rac1 in epithelial cell-cell adhesion, using a combination of live cell microscopy, Rac1-GFP fusion proteins, and functional, quantitative assays for development of cell-cell adhesion. We find that Rac1 accumulates at new cell-cell contacts in a dynamic pattern that does not correlate with E-cadherin distribution per se, but rather with lamellipodia activity at contact sites. We also test the role of PI 3-kinase in development of strong cell-cell adhesion. In complementary work (M.D.H. Hansen et al., submitted), we correlate these results with biochemical data that identify endogenous Rac1-containing protein complexes involved in development of MDCK cell-cell adhesion.

Results

Lamellipodia Drive Cell-Cell Contact Formation between MDCK Epithelial Cells

Both lamellipodia and filopodia have been observed at developing cell-cell contacts in different epithelial cell types (Vasioukhin et al., 2000; reviewed by Vasioukhin and Fuchs, 2001). However, with few exceptions (Jacinto et al., 2000), these structures have been identified by electron or immunofluorescence microscopy of fixed specimens and, hence, it is unclear whether they are involved directly in cell-cell adhesion. To observe dy namics of membrane activity at cell-cell contacts, we imaged live MDCK cells using time-lapse phase contrast microscopy (Figure 1A and the corresponding Supplemental Movie S1 at http://www.developmentalcell.com/cgi/content/full/3/2/259/DC1). Initial contacts between cells occurred through exploratory lamellipodia that touched and sometimes withdrew from opposing cells. Concerted formation of cell-cell contacts coincided with rounds of lamellipodia protrusions that swept over the contacting surfaces (Figure 1A, arrows). We did not de tect interdigitated filopodia between early cell-cell contacts in live MDCK cells, in contrast to observations of static images of primary epidermal keratinocytes (Vasioukhin et al., 2000).

Figure 1. Cell-Cell Contact in MDCK Cells Is Driven by Waves of Lamellipodia and Is Accompanied by Rapid Cortical Actin Reorganization.

Figure 1

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(A) Time-lapse, phase contrast microscopy of cells undergoing cell-cell contact. Arrows indicate several sites of lamellipodia extension at the contact site.

(B) Time-lapse, two-photon microscopy of cell-cell adhesion between cells expressing GFP-actin. The cortical actin ring dissolves into a complex actin meshwork at the cell-cell contact site (arrows). Note that the top cell expresses more GFP-actin than the lower cell, which allows better visualization of actin filament dynamics at contacting membranes. The figure displays selected panels from QuickTime movies available online. Time is indicated in minutes. Scale bars are in microns.

Cortical Actin Is Rapidly Remodeled at Sites of Cell-Cell Contact

To correlate dynamic lamellipodia activity with actin remodeling in living cells, we used time-lapse two-photon microscopy of MDCK cells expressing GFP-actin. Low levels of GFP-actin expression did not interfere with cell movement, cell-cell contact, or formation of polarized cell monolayers (J.S.E. and A. Barth, unpublished observations). As observed in Figure 1B and the corresponding Supplemental Movie S2, a local burst of lamellipodia extensions occurs at sites of initial cell-cell contact, and small actin cables can be observed between the main circumferential actin bundle and the cell-cell contact (Figure 1B, 9.6 min). These actin cables followed the trajectories of lamellipodia extending over the contacting cell and were oriented perpendicular to the cell-cell contact, similar to actin structures identified in fixed specimens (Adams et al., 1998). As the length of contacting membranes increased, the circumferential actin cable behind the contact dissolved into a complex mesh of shorter cables. Over time, cell-cell contacts expanded laterally and circumferential actin cables at contact margins adopted an arc-like configuration as described previously (Krendel and Bonder, 1999), suggesting that they are under tension and involved in lateral contact growth.

These observations of dynamic cortical actin reorganization and lamellipodia extension at cell-cell contacts implicate Rac1 as a regulator of cell-cell adhesion in MDCK cells.

RacGFP Localization Changes during Cell-Cell Adhesion

To directly examine Rac1 localization during cell-cell adhesion, we used a functional Rac1-green fluorescent protein fusion (RacGFP; Subauste et al., 2000) and time-lapse, confocal imaging of live MDCK cells (Figures 2A and 2B). Data presented are from analysis of single cells, and are representative of over 40 individual cell-cell contacts visualized in several dozen experiments.

Figure 2. Dynamics of RacGFP Distribution during Cell-Cell Contact.

Figure 2

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(A) Time-lapse confocal microscopy of RacGFP-expressing MDCK epithelial cells. Double arrows, RacGFP at contacting membranes; arrowheads, lamellipodia; asterisks, site of initial (and hence oldest) contact. The figure displays selected panels from QuickTime movies available online. Time is indicated as hr:

(B) Higher magnification of frames from (A). Scale bars are in microns.

(C) Time intensity position (TIP) analysis of the cell-cell contact from (A). RacGFP signal intensities across the cell-cell contact (demarcated by the red line in [B]) were measured over time and encoded on a pseudocolor scale. X and Y axes represent time and position along the cell-cell contact, respectively. GFP signal intensity at the contacting membrane is normalized against the average GFP intensity of noncontacting membranes, so TIP scans from different cells may be compared. Arrows indicate the cell-cell contact boundaries. Color bar: black, lowest GFP intensity; white, highest intensity.

As individual cells crawled, RacGFP localized throughout the cytoplasm and around the nucleus (Figure 2A), consistent with a large cytosolic pool of Rac1 in these cells (Hansen and Nelson, 2001). Transient accumulations of RacGFP occurred at tips of extending lamellipodia at the free surface edge of cells (Figure 2A, solid arrow in 0:11 image), as predicted from static images of single migratory cells (Ridley et al., 1992). RacGFP accumulation at those sites was specific, since a comparison of fluorescence intensities of RacGFP and the membrane dye FM 4-64 revealed that, in most cases, the increase in RacGFP signal was significantly greater than that of FM 4-64; additionally, the increase was also greater than that of cytoplasmic, soluble GFP (J.S.E., unpublished results).

As migratory cells collided, membrane ruffling activities were detected at the site of cell-cell contact (Figure 2A, arrowheads; see higher magnification images in Figure 2B). Subsequently, waves of lamellipodia washed over the contacting membranes. Appearance of these lamellipodia coincided with a substantial accumulation of RacGFP along newly contacting membranes (Figure 2A, double arrows). The contacting membrane area then expanded laterally by repeated extension of RacGFP-containing lamellipodia from one cell over another. Lamellipodia at contact sites were more persistent (appearing in several consecutive movie frames) than transient lamellipodia at noncontacting regions of cells.

In more mature regions of contacts, lamellipodia formation and RacGFP signal intensity diminished over time (Figure 2A, compare sites marked with asterisks). These sites typically were near the middle of a growing contact, and often marked sites of the initial, and hence oldest, part of contacts. This finding is more readily appreciated by examining RacGFP signal intensity along the width of the cell-cell contact site over time. Figure 2C presents these data as a time intensity position (TIP) analysis (Adams et al., 1998). The RacGFP signal decreases substantially over time at the original site of cell-cell contact (asterisks in Figures 2A and 2C), and is highest at sites of substantial lamellipodia activity along the expanding, and hence newest, sites of cell- cell contact.

Although cell-cell adhesion in RacGFP cells occurred with a temporal pattern similar to that reported for wild-type and E-cadherin/GFP-expressing cells (Adams et al., 1998), we sought to verify that E-cadherin distribution was not disrupted as a result of RacGFP expression. We localized E-cadherin retrospectively following development of cell-cell contacts in live RacGFP cells, and found it accumulated in a pattern indistinguishable from that in nontransfected MDCK cells (J.S.E., unpublished observations).

Lamellipodia Formation on Noncontacting Surfaces Decreases during Cell-Cell Contact

Formation of cell-cell contacts has striking consequences on membrane dynamics at noncontacting surfaces of cells. As noted above, single migratory cells often displayed multiple, transient lamellipodia at random locations around the cell perimeter (Figures 1 and 2A). Following cell-cell contact, formation of large lamellipodia at noncontacting cell surfaces diminished. The frequency of transient RacGFP accumulations at those sites also decreased. Thus, as cells switched from solitary migration to nonmigratory cell-cell adhesion, both lamellipodia formation and RacGFP accumulation oriented to sites of cell-cell contact.

To quantify the observed changes in membrane protrusive activity, we performed a protrusion mapping analysis in which we scored occurrence and location of lamellipodia during contact formation. Figure 3A presents an analysis of the RacGFP cell-cell contact movie shown in Figure 2 (see Supplemental Movies S3 and S4 corresponding to Figures 2A and 2B, respectively). The mapping analysis examined all membrane protrusions, to be inclusive and unbiased. Initially, membrane protru sions occurred randomly over the entire cell perimeter (0–20 min). As the cell-cell contact expanded (demar cated by red lines in Figure 3A), there was not only an overall decrease in the number of protrusions, but an increasingly restricted distribution of remaining protru sive activity to the contact site and an area 10 microns on each side of it (the pericontact region). Thus, in the first 60 min of cell-cell contact formation, 19% of all scored membrane protrusions occurred within the contact zone, and 33% occurred within the pericontact zone (between the yellow lines in Figure 3A). In contrast, be tween 60 and 120 min, 29% of all scored lamellipodia occurred within the contact zone, and 76% within the pericontact region. From 120 to 200 min, membrane activity became further polarized, with 46% of protrusions within the contact zone and 71% within the pericontact region. This reorientation of lamellipodia activity was specific, as we did not detect restriction to a specific site on the cell perimeter in single, noncontacting cells (Figure 3B).

Figure 3. Lamellipodia Formation Dynamics during Cell-Cell Adhesion.

Figure 3

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(A) A map of the distribution of lamellipodia around a contacting cell. Distance around the cell perimeter from an origin was identified in microns; the cell is “unwrapped” along a single line for each movie frame to represent data on a linear plot. The cell-cell contact zone is within the red lines, and the 10 micron perimeter bordering the contact is outlined in yellow. Data are presented for the cell shown in Figure 2. Blue plus green marks, complete data set (all protrusions); green marks alone, protrusions that contained RacGFP.

(B) A map of the distribution of lamellipodia around a noncontacting cell. The panels display three consecutive movie frames; lamelli-podia were identified as transient membrane protrusions seen in frame-by-frame analysis (arrow). The scale bar represents 5 μm.

(C) Ratio of protrusions occurring within the contact or pericontact region versus the non-contacting region, normalized for the respective membrane areas of each region. The relative number of lamellipodia within the contacting region becomes higher during development of cell-cell adhesion.

It is not known whether all protrusions scored in the above analysis were dependent on Rac1. Indeed, it is likely that a subset of lamellipodia is Rac1 independent (Spaargaren and Bos, 1999). Nonetheless, a significant trend was observed. However, if only RacGFP-containing lamellipodia are measured, focusing of membrane activity toward contact sites becomes even more apparent. In the first 60 min of cell-cell contact formation, 23% (7/31) of scored lamellipodia occurred within the contact zone, versus 63% (15/24) from 60 to 200 min (Figure 3A, green data points only).

We also examined the lamellipodia maps by measuring the ratio of protrusions formed in contact or pericontact regions to the number in the noncontact region, normalized for relative membrane perimeters of each. This calculates the enrichment of protrusions at the contacting and pericontacting regions, expressed as a fold difference over the number at the noncontacting region (Figure 3C). During the development of cell-cell adhesion, there was a 2-fold increase in the relative protrusive index in the cell-cell contacting region, when all lamellipodia were examined; when only RacGFP-containing protrusions were scored, a greater than 5-fold enrichment was observed.

Rac1 Mutants Induce Distinct Behavioral Changes in Cells Undergoing Cell-Cell Contact

Rac1 mutants display altered signaling activity by failing to cycle properly between nucleotide binding states (Bishop and Hall, 2000; Farnsworth and Feig, 1991; Stacey et al., 1991). Dominant-negative mutant Rac1 (T17N) is mainly GDP bound, and blocks endogenous Rac1 function by sequestering regulatory molecules required to activate wild-type protein. Constitutively active Rac1 mutants (G12V and Q61L) persist in GTP-bound states and inappropriately activate downstream Rac1 effectors. We examined effects of these mutants on cell behavior during cell-cell adhesion. Mutant Rac1 proteins fused to GFP were transiently expressed in MDCK cells, and cell-cell contacts were imaged by time-lapse confocal microscopy.

We were surprised to find that cells expressing RacT17NGFP displayed significant amounts of membrane ruffling activity at noncontacting membranes (Figure 4A and the corresponding Supplemental Movie S5; data are presented from a single cell, which is representative of over 20 individual cell-cell contacts observed), although this has been previously reported (West et al., 2000). However, membrane extensions in RacT17NGFP cells generally did not protrude as far from the cell body as those in wild-type RacGFP cells. A typical protrusion in RacT17NGFP cells is presented in the high-magnification panels of Figure 4A (compare with Figure 3C).

Figure 4. Morphological Effects of Dominant-Negative Rac1 Expression on Cell-Cell Contact Development.

Figure 4

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(A) Left panels, time-lapse confocal microscopy of MDCK cells transiently expressing Rac1T17NGFP. The figure displays selected panels from QuickTime movies available online. Time is indicated as hr:min and site of contact as arrowheads. The scale bar represents 15 μm. Right panels, RacT17NGFP-expressing cells have smaller membrane protrusions than wild-type RacGFP-expressing cells; an example is shown in three consecutive video frames. The scale bar represents 5 μm.

(B) TIP analysis of the cell-cell contact from (A). GFP signal intensities across the cell-cell contact (demarcated by the red line in [A]) were measured over time and encoded as in Figure 2. This panel is presented with the same pseudocolor intensity scale as Figure 2C, and can be compared directly.

(C) Lamellipodia mapping analysis of the RacT17NGFP cell-cell contact from (A).

(D) Ratio of protrusions occurring within the contact or pericontact region versus the noncontacting region, normalized for membrane areas.

(E) Time-lapse movie of Rac1Q61LGFP-expressing MDCK cells during cell-cell adhesion. Most cells exhibit promiscuous lamellipodia extension and fail to migrate (asterisk). Unusually broad lamellipodia were observed between contacting cells, and contacts extended faster than in wild-type RacGFP cells. The figure displays selected stills from a QuickTime movie available online. Time is indicated as hr:min. The scale bar represents 20 μm.

Consistent with the idea that functional Rac1 activity plays some role in cell-cell contact growth, cell-cell contacts in RacT17NGFP cells expanded very slowly. Bursts of localized lamellipodia formation characteristic of wild-type RacGFP cells were also markedly reduced in RacT17NGFP cells. Normalized TIP scan analysis (Figure 4B) revealed that accumulation of RacT17NGFP signal at contacting membranes was not as intense as that of RacGFP in wild-type cells.

RacT17NGFP cells also failed to restrict membrane protrusions to sites of cell-cell contact as shown by protrusion mapping (Figure 4C). Between 0 to 60 min and 60 to 175 min of cell-cell contact in RacT17NGFP cells, 11% and 12% of membrane protrusions occurred in the contact zone, respectively, compared with a change from 19% to 46% in wild-type RacGFP-expressing cells (Figure 3A). In addition, the total number of membrane protrusions did not decrease over time in RacT17NGFP cell-cell contacts, in contrast to wild-type RacGFP cells; 24 events were scored for the first 60 min of contact, versus 28 events in the final 80 min, whereas in the wild-type cell (Figure 3A), 64 total events were scored for 0–60 min, versus 24 for 120–200 min. The relative protrusive index (Figure 4D) also showed that lamellipodia were not enriched at RacT17NGFP cell-cell contacts; in contrast to wild-type cells (Figure 3C), there was no increase in the fold difference of protrusions at contacting versus noncontacting regions.

Given the changes in cell-cell contact dynamics observed in RacT17NGFP cells, we examined whether dominant-negative Rac1 blocked wild-type Rac1 from accumulating at cell-cell contacts. We transiently trans fected wild-type RacGFP into cells stably expressing myc-tagged Rac1T17N under control of a tetracycline repressor (Jou and Nelson, 1998; Jou et al., 1998), and examined RacGFP localization by time-lapse microscopy. Wild-type RacGFP did not substantially accumulate at contact sites between many Rac1T17N/RacGFP cells (6 of 11 cell-cell contacts examined; Figure 5A, arrow), whereas Rac1T17N, specifically followed using the epitope tag, accumulated at both cell-cell contacts and noncontacting membranes. In some cells (5 of 11 contacts examined), RacGFP accumulated at contacting membranes, but not at a level significantly higher than that at noncontacting membranes of the same cell (Figure 5B, left cell; compare membranes marked by arrows and asterisks). We did not observe specific enrichment of RacGFP at contacting membranes (above the level at noncontacting membranes) in any Rac1T17N/RacGFP cell-cell contacts, in contrast to our observations in wild-type cells.

Figure 5. Expression of Dominant-Negative Rac1 Reduces Accumulation of Wild-Type RacGFP at Cell-Cell Contacts.

Figure 5

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(A) RacGFP (arrow) and myc-tagged RacT17N expressed in the same cells. Wild-type RacGFP fails to accumulate at cell-cell contacts in many such cells (arrow), whereas Rac1T17N is present.

(B) In some cases, wild-type RacGFP is present at contacting membranes but only at a level similar to that at noncontacting membranes of the same cell (left cell, compare membranes marked by arrows and asterisks). Cells with higher expression of RacGFP and lower expression of Rac1T17N (right cell, arrowheads) showed accumulation of GFP at the contact site in addition to Rac1T17N.

In contrast to cells expressing RacT17NGFP, cells expressing constitutively active RacQ61LGFP exhibited an increased rate of lamellipodia formation, with numerous ruffles extending around the cell periphery (Figure 4E and the corresponding Supplemental Movie S6). This is consistent with previous work demonstrating that activated Rac1 leads to an increase in lamellipodia formation in many cell types (Akhtar et al., 2000; Allen et al., 1997). MDCK cells expressing constitutively active Rac1G12V had a phenotype similar to that of cells expressing RacQ61LGFP (J.S.E., unpublished observa tions). RacQ61LGFP cells often failed to migrate, perhaps because promiscuous lamellipodia extension prevented directional movement (see cell marked with an asterisk in Figure 4D). It was difficult to observe de novo cell-cell contacts between single cells, and consequently we did not perform detailed protrusion mapping as described above because the time of initial cell-cell adhesion was not known. Nevertheless, at cell-cell contacts that we could observe, unusually broad lamellipodia were extended over contacting cells and lateral expansion of cell-cell contacts occurred more quickly. The extent of membrane ruffling both at contacting and noncontacting regions eventually decreased in contacting RacQ61LGFP cells, suggesting that mechanisms for contact-mediated inhibition are not completely abrogated.

We also examined E-cadherin distribution in contacting RacT17NGFP and RacQ61LGFP cells. In both cases, E-cadherin accumulation at cell-cell contacts was similar to that in wild-type RacGFP-expressing cells (J.S.E., unpublished observations). We conclude that effects of Rac1 mutant expression on formation of cell-cell contacts were the result of changes in Rac1-dependent actin-based membrane protrusions that drive cell-cell contact growth, and not on E-cadherin distribution.

Blockade of PI 3-Kinase Activity Does Not Prevent Formation of Cell-Cell Contacts

Recent studies of signaling pathways downstream of E-cadherin have focused on a potential role for PI 3-kinase as a signaling intermediate (Nakagawa et al., 2001; Noren et al., 2001; Pece et al., 1999; Woodfield et al., 2001). Activation of PI 3-kinase at sites of cell-cell contact potentially could increase activity of Rac1 because many Rho family GEFs contain pleckstrin homology (PH) domains that bind lipid products of PI 3-kinase activity. This notion is supported by the finding that E-cadherin engagement coincides with an increase in Rac1 activity that is PI 3-kinase dependent (Nakagawa et al., 2001; Pece et al., 1999). Using the PH domain of Akt fused to GFP as a molecular probe for PI 3-kinase activation, enrichment of PI 3-kinase lipid products was observed at static MDCK cell-cell contacts (Watton and Downward, 1999). We wished to extend previous studies by examining the spatio-temporal dynamics of PI 3-kinase activation using time-lapse microscopy of MDCK cells expressing PH (Akt)-GFP (Haugh et al., 2000).

PH-GFP is transiently enriched in lamellipodia on non-cell-cell contacting surfaces and regions of the cell body behind them (Figure 6A, black arrows, and the corresponding Supplemental Movie S7). However, PH-GFP is even more strongly enriched in lamellipodia at cell-cell contacts (red arrows), particularly in the initial, protrusive stage of lamellipodia extension. When lamellipodia withdrew or contacting membranes became stabilized, the intensity of PH-GFP signal decreased. As cell-cell contacts expanded laterally, highest levels of fluorescence intensity occurred at the growing edges, as shown in the TIP scan (Figure 6B). Thus, PH-GFP is localized to only a subset of E-cadherin-containing membranes; its distribution coincides with that of lamellipodia activity (and thus also with Rac1), and not with that of E-cadherin per se.

Figure 6. Spatio-Temporal Dynamics of PH-GFP and Effect of PI 3-Kinase Blockade during Cell-Cell Adhesion.

Figure 6

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(A) Time-lapse confocal movie of cells expressing PH-GFP. PI 3-kinase activity is observed in lamellipodia on non-cell-cell contacting surfaces (black arrows) and more strongly at lamellipodia of cell-cell contacts (red arrows). Following addition of 20 μM LY-294002, PH-GFP intensity sharply decreases at cell-cell contact sites (+LY in panels) but cell-cell contacts are maintained.

(B) TIP scan analysis of the movie from (A). LY-294002 was added at the time indicated by the asterisks.

(C) LY-294002 added at initiation of cell-cell contact does not prevent subsequent contact growth. The figure displays selected stills from QuickTime movies available online. Time is indicated as hr:min. The scale bars represent 15 μm.

Using the specific PI 3-kinase inhibitor LY-294002 (LY; 20 μM), we assessed the role of PI 3-kinase in development of cell-cell contacts. Addition of LY to cells in the mid and late stages of cell contact formation caused an immediate decrease in PH-GFP intensity at the plasma membranes, consistent with previous reports (Marshall et al., 2001; Watton and Downward, 1999). The decrease in PH-GFP intensity occurred within one movie frame (2–5 min; see asterisks in TIP scan, Figure 6B). Despite this steep reduction in PI 3-kinase activity, cell-cell contacts maintained their integrity, and lamellipodia formation and retraction were clearly observable. Membrane protrusions also continued to form around the noncontacting perimeter of the cell, although they were generally smaller after addition of LY.

We also evaluated the role of PI 3-kinase activity in early formation of cell-cell contacts, by adding LY to PH-GFP-expressing cells at the beginning of contact formation (Figure 6C and the corresponding Supplemental Movie S8). Lamellipodia activity at contact sites appeared reduced from control cells, but lamellipodia were still present and contact formation was unim peded. Cell-cell contacts that formed in the early pres ence of LY had morphology indistinguishable from control cells. We conclude that PI 3-kinase activity is enriched at cell-cell contacts, but this increase in activity is not absolutely required for contact development and maturation.

Analysis of Rac1 Functions Using a Functional Cell-Cell Adhesion Assay

Formation, development, and organization of individual cell-cell contacts differ in cells expressing Rac1 mutants. To quantify these effects on populations of cells, we used a hanging drop assay that measures sizes of cell aggregates formed over time from a suspension of single cells, and resistance of these aggregates to a shearing force (Kim et al., 2000a). This assay reveals trends in the rate and strength of adhesion. On average, 200–400 cells were examined at each time point in each experiment.

In control experiments (Figure 7A), all cells in hanging drops were initially present as single cells or clusters of fewer than ten cells. The number of cells in large clusters (>50 cells) increased to 58% after 2 hr, and to >95% after 3–6 hr. Resistance to trituration increased from 0% of cells remaining in clusters of >50 cells following trituration at 2 hr, to >95% of cells at 6 hr after cell-cell adhesion. Large aggregates of cells had a web-like organization, as smaller clusters joined and cell-cell contacts became compacted.

Figure 7. Analysis of Rac1 in a Quantitative, Functional Adhesion Assay.

Figure 7

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Control cells (A), Rac1T17N cells (B), Rac1G12V cells (C), and LY-294002-treated cells (D). Graphs show the percentage of cells in clusters of 0–10 cells (gray), 11–50 cells (dark gray), and >50 cells (light gray) at the time points indicated, before and after trituration. For each time point, 200–400 cells were scored and data are presented as the average of three independent experiments. Photographs are representative fields at 0 and 4 hr, before and after trituration. The scale bar represents 200 μm.

Rac1T17N cells developed resistance to trituration more slowly than control cells. At all time points, the majority of Rac1T17N aggregates dispersed into clusters of fewer than 50 cells upon trituration (Figure 7B). In contrast to the web-like organization of control clusters, Rac1T17N aggregates appeared clumpy, with little appearance of compaction or formation of strong cell-cell interactions, similar to single RacT17NGFP cell-cell contacts observed by imaging (Figure 4A).

Rac1G12V cells displayed rapid kinetics of adhesion formation (Figure 7C), and became resistant to trituration earlier than control cells. At 2 hr, 53% of Rac1G12V cells remained as large aggregates (>50 cells) after trituration, versus 0% for controls. In contrast to Rac1T17N cells, Rac1G12V cells formed aggregates with similar morphology to controls.

These results are consistent with our imaging data showing that cells expressing Rac1T17N formed contacts slowly, whereas those expressing Rac1Q61L ex tend contacts more rapidly than that of control cells.

We also performed the cell aggregation assay when PI 3-kinase activity was blocked with LY. Our analysis demonstrated no significant difference between control and LY-treated cells (Figure 7D). LY-treated cells formed aggregates morphologically indistinguishable from that of controls, and with a similar time course.

Discussion

Rac1 regulates formation of lamellipodia, which are actin-based membrane protrusions involved in cell motility (Ridley et al., 1992). Rac1 signaling has also been shown to play an important but unclear role in development and regulation of epithelial cell-cell adhesion (Braga et al., 1997, 2000; Fukata and Kaibuchi, 2001; Nakagawa et al., 2001). In this work, we examined dynamics of lamellipodia activity, actin reorganization, and Rac1 protein localization during early development of E-cadherin-dependent epithelial cell-cell adhesion in living cells. By examining all of these parameters concurrently, we can explain a role for Rac1 in cell-cell adhesion.

In MDCK cells, cell-cell contact is an opportunistic event that occurs when migratory cells collide. However, when contacted, cells respond rapidly and lamellipodia appear to be the primary physical drivers of cell-cell contact development. We observed that lamellipodia become focused to the cell-cell contact zone and a region immediately surrounding it. This change coincided with an increase in Rac1 accumulation at cell-cell contacts (as determined by RacGFP localization) and a concomitant loss of Rac1 from noncontacting sites. Subsequently, RacGFP localization and lamellipodia activity became further restricted to newest sites in cell-cell contacts, whereas older sites had less RacGFP and membrane activity (Figure 2C). Analysis of individual cell-cell contacts at high spatial and temporal resolution ħows that Rac1 does not simply colocalize with E-cadherin/catenin complexes at cell-cell junctions as had been suggested previously (Nakagawa et al., 2001), but is specifically restricted to initiating areas of contact, whereas E-cadherin gradually accumulates along the entire contact length.

The finding that Rac1 is associated with newest contact sites suggests that its fundamental role in cell-cell adhesion may be in initiation of cell-cell contact rather than maintenance of stable cell-cell junctions. By activating Rac1 and hence the perdurance of lamellipodia extension at new cell-cell contact sites, cells could rapidly increase the surface area between contacting membranes and, consequently, increase the probability and quantity of E-cadherin interactions between adjacent cells.

That Rac1 is important in affecting the rate of contact initiation is consistent with observations that Rac1T17N expression does not present an absolute block to formation of cadherin-dependent adhesion (Jou and Nelson, 1998; Takaishi et al., 1997). We found that Rac1T17N-expressing cells formed cell-cell contacts, but extended them slowly and displayed a reduced amount of lamellipodia activity at contact sites as compared to wild-type cells. Note that Rac1T17N displaced wild-type Rac1 from cell-cell contacts (Figure 5), indicating that even the Rac1/PAK complex that remains in Rac1T17N cells is not contributing to cell-cell adhesion (Hansen and Nelson, 2001). In a population-based cell-cell adhesion assay, adhesion occurred between Rac1T17N-expressing cells, but the rate of strengthening was significantly slower than that of wild-type cells (Figure 7). In this assay, cells are forced into proximity by gravity, and this close and constant apposition of cell membranes may be sufficient to allow homophilic E-cadherin binding to drive cell-cell adhesion by mass action. In a physiological setting in which cells move relative to each other, Rac1 may primarily be required to increase the rate of contact formation and stabilization (strengthening) through focused lamellipodia extension. Even then, cadherin-cadherin interactions appear to be sufficient to stabilize adhesion as we did not detect lamellipodia, Rac1, or PI 3-kinase products (below) at older sites of cell-cell contact.

In addition to localization of Rac1 and lamellipodia to cell-cell contacts, we noted a decrease in lamellipodia at noncontacting surfaces, and a transition from brief Rac1 accumulations at noncontacting sites to more persistent accumulations at contacts. These observations suggest corresponding changes in location and persistence of upstream activators and downstream effectors of Rac1 as cells undergo cell-cell adhesion. Although RacGFP reveals only total protein localization rather than areas where signaling is actively occurring, Rac1 activity is inferred in our studies by the cause-effect correlation between RacGFP localization and lamellipodia activity. Recently, FRET-based probes for exam ining GTPase signaling in living cells have been developed (Chamberlain et al., 2000; Kraynov et al., 2000; Mochizuki et al., 2001). We attempted to analyze Rac1 signaling dynamics in live MDCK cells using the FLAIR approach (Kraynov et al., 2000). However, we have been unable to obtain sufficient biosensor signal in lamellipodia and cell-cell contacts without seriously perturbing cell physiology (J.S.E., C. Chamberlain, and K. Hahn, unpublished observations). In complementary work (M.D.H. Hansen et al., submitted), we show changes in Rac1-containing protein complexes, supporting the idea that localized Rac1 signaling occurs during cell-cell adhesion.

Similar to Rac1 localization itself, the actin reorganization associated with cell-cell adhesion localized to regions immediately behind newest sites of cell-cell contact. How do Rac1 localization, lamellipodia activity, and cortical actin remodeling become restricted to specific areas within cell-cell contacts? One possible mecha nism is through localized activation of signaling molecules upstream of Rac1. Our observations of persistent RacGFP signal and lamellipodia activity at new contact sites suggest spatially restricted increases in levels of active Rac1. The loss of RacGFP signal and membrane activity at older contact sites, despite the presence of E-cadherin, indicates that this spatial regulation of Rac1 activity is extremely precise and independent of E-cadherin engagement per se.

Recent studies have demonstrated an interplay between the E-cadherin/catenin complex and PI 3-kinase, and E-cadherin ligation correlates with increased activation of PI 3-kinase (Kovacs et al., 2002; Nakagawa et al., 2001; Noren et al., 2001; Pece et al., 1999). It remains unclear how important this event is to localizing Rac1 activity and/or actin cytoskeleton remodeling. Our live cell imaging data demonstrate an increase in PI 3-kinase activity (PH-GFP) at cell-cell contacts, consistent with previous static images (Watton and Downward, 1999). However, using time-lapse imaging to examine dynamics of PH-GFP, we found that highest levels of PH-GFP occurred at growing regions of cell-cell contacts and not just at sites of E-cadherin ligation, similar to our findings for Rac1 (Figure 6B). Despite this apparent concordance between the localizations of PI 3-kinase activity, Rac1, and lamellipodia activity, both live cell imaging and a functional adhesion assay show that cell-cell adhesion can occur in the presence of the PI 3-kinase inhibitor LY-294002. These data indicate that E-cadherin ligation may initiate transient PI 3-kinase activation as reported, but continued E-cadherin engagement may not require further activation of PI 3-kinase. Our results are also consistent with data indicating that an initial phase of cadherin-induced Rac1 activation is PI 3-kinase independent (Kovacs et al., 2002); this pool of activated Rac1 may be sufficient to drive cell-cell contact growth.

Another potential and complementary mechanism for localizing Rac1 activity during cell-cell adhesion is sequestration to cell-cell contacts of necessary adaptor and regulatory molecules, such as GEFs and GAPs, and downstream effectors (Fukata and Kaibuchi, 2001; Fukata et al., 1999; Sander et al., 1998; Vasioukhin et al., 2000). It remains an open question, however, as to which of the many identified Rac1 regulatory and effector components function endogenously in epithelial cells during establishment of cell-cell adhesion. In complementary work (M.D.H. Hansen et al., submitted), we present identification of three membrane-associated protein com plexes that contain endogenous Rac1 and show a shift in molecular composition during cadherin-mediated cell-cell adhesion. We speculate that this is a molecular mechanism for focusing Rac1 activity to cell-cell contacts. Together with the results of live cell imaging and functional cell-cell adhesion assays presented here, these biochemical studies yield significant new insight into the role of Rac1 during epithelial cell-cell adhesion.

Experimental Procedures

Cell Culture

MDCK cells were maintained at low density in low-glucose DMEM (GIBCO) supplemented with 10% FCS. MDCK cells expressing Rac1T17N or -G12V under tetracycline repressor control were maintained in the presence of 20 ng/ml doxycycline, and mutant protein expression was induced as described (Jou and Nelson, 1998). For PI 3-kinase inhibition, cells were incubated in medium supplemented with 20 μM LY-294002 (Calbiochem), a concentration previously used for studies in MDCK cells (Watton and Downward, 1999).

Expression of GFP Constructs

We used functional GFP fusions to Rac1 (Subauste et al., 2000) in which GFP is fused to the N terminus of human Rac1 such that C-terminal lipid modification of Rac1 is intact. cDNA vector was transfected into MDCK T23 cells using Lipofectamine 2000 (GIBCO), and stably expressing clones were isolated using FACS. Vectors encoding T17N and Q61L mutants of RacGFP and PH (Akt)-GFP were transiently transfected by calcium phosphate. GFP-expressing cells were isolated 2–3 days after transfection by FACS, and used the same day for microscopy. To examine actin dynamics, we used an MDCK cell line stably expressing a GFP fusion to actin (Clontech), transfected and isolated as above.

Time-Lapse Microscopy

MDCK cells were plated at low density onto collagen-coated coverslips, allowed to recover for 3–5 hr, and viewed for 2–6 hr with a robotic confocal microscope specialized for time-lapse imaging (described in (Adams et al., 1998; Ahmari et al., 2000). Typically, images were collected every 2–5 min at each stage location. For imaging of GFP-actin, cells were viewed using a similar microscope equipped for two-photon excitation (Coherent Mira laser). Retrospective immunostaining for E-cadherin (3G8 antibody) was per formed as described (Adams et al., 1998).

Image Analysis

Images from the microscope were processed into movies using QuickTime (Apple). Other analysis was performed in MetaMorph (Universal Imaging), ImageJ (http://rsb.info.nih.gov/ij), and Microsoft Excel.

Hanging Drop Adhesion Assays

The assay was performed as described (Kim et al., 2000a). In brief, MDCK cells were grown at low density and Rac1 mutant expression was induced as appropriate. Cells were trypsinized, centrifuged, and resuspended as single-cell suspensions at 2.5× 105 cells/ml. Twenty microliter drops of cell suspension were pipetted onto inside surfaces of 35 mm culture dish lids, and dishes were filled with 2 ml of media to prevent evaporation. At each time point, the lid was inverted and drops were spread onto a glass slide. One drop was first triturated ten times through a 20 μl pipet. Three random fields from each drop were photographed, and numbers and sizes of clusters were determined.

Supplementary Material

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Movie 2
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Movie 3
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Movie 4
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Movie 5
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Movie 6
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Movie 7
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Acknowledgments

We thank Stephen J. Smith for assistance with microscopy, Klaus Hahn for RacGFP vectors, and members of the Nelson laboratory for discussion. J.S.E. was supported by the Medical Scientist Training Program (grant 5T32GM07365 from the National Institutes of Gen eral Medical Sciences), and work was supported by NIH grant GM35527 to W.J.N.

Footnotes

Online Supplemental Material

The time-lapse images that are presented as panels of still figures are presented in their entirety online as QuickTime movies at http://www.developmentalcell.com/cgi/content/full/3/2/259/DC1.

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Supplementary Materials

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