|
|
|
|
|
|
|
||
Article |
Contact-dependent promotion of cell migration by the OL-protocadherin–Nap1 interaction
Correspondence to Shinji Hirano: s-hirano{at}cdb.riken.jp; or Masatoshi Takeichi: takeichi{at}cdb.riken.jp
OL-protocadherin (OL-pc) is a transmembrane protein belonging to the cadherin superfamily, which has been shown to accumulate at cell–cell contacts via its homophilic interaction, but its molecular roles remain elusive. In this study, we show that OL-pc bound Nck-associated protein 1 (Nap1), a protein that regulates WAVE-mediated actin assembly. In astrocytoma U251 cells not expressing OL-pc, Nap1 was localized only along the lamellipodia. However, exogenous expression of OL-pc in these cells recruited Nap1 as well as WAVE1 to cell–cell contact sites. Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions. OL-pc mutants lacking the Nap1-binding site exhibited no such effect. N-cadherin knockdown mimicked OL-pc expression in enhancing cell movement. These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1–WAVE1 complex to these sites and, in turn, promotes the migration of cells.
© 2008 Nakao et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
 |
Introduction |
|---|
 TopAbstract Introduction ResultsDiscussionMaterials and methodsReferences |
|---|
OL-protocadherin (OL-pc; protocadherin-10) is a member of the cadherin superfamily (Hirano et al., 2003). As seen with the classical cadherins, OL-pc shows the homophilic binding nature, which can induce cell aggregation when expressed in cadherin-deficient cells (Hirano et al., 1999). However, the ability of OL-pc to promote cell aggregation is much weaker compared with that of classical cadherins (Hirano et al., 1999), raising the possibility that OL-pc might have other functions than the simple physical linking of cells. Our recent analysis of OL-pc knockout mice demonstrated that in the OL-pc–deficient brain, striatal neurons could not normally extend their axons (Uemura et al., 2007). In the wild-type brain, striatal axons project the substantial nigra, whereas in the OL-pc mutants, this projection was missing, as the mutant axons were stalled by clumping together during the early periods of extension. The axon elongation defects were also observed when striatal tissues had been isolated and cultured in vitro. OL-pc was localized along axons, being most highly concentrated in their growth cones. These observations suggested that OL-pc played a role in sustaining the normal migration of striatal axons.
To investigate the molecular role of OL-pc, we screened for proteins that interact with this protocadherin and identified Nck-associated protein 1 (Nap1) as an OL-pc partner. Nap1, which is widely conserved among various organisms, organizes a molecular complex comprising itself, Sra-1/PIR121/cytoplasmic interacting FMR1 protein (CYFIP), Abl interactor (Abi), and HSPC300 as well as Scar/WAVE (Ibarra et al., 2005). The activity of Scar/WAVE is regulated by the formation of this complex, and through this machinery, upstream Rac1 signals are relayed to the actin-related protein (Arp) 2/3 complex to induce lamellipodia formation (Stradal and Scita, 2006). Although depletion of Nap1 abrogates lamellipodia formation (Steffen et al., 2004), it allows filopodia formation (Steffen et al., 2006), indicating that Nap1 regulates only restricted portions of the cellular motility machinery. Analysis of the Dictyostelium discoideum homologue of the Nap1 gene (napA) suggested that Nap1 regulates not only Scar/WAVE but also other cellular activities (Ibarra et al., 2006). In vivo, Nap1 is important for various morphogenetic events, such as neural tube closure and migration of endoderm and mesoderm (Rakeman and Anderson, 2006). In the nervous system, Nap1 is required for neurite extension (Yokota et al., 2007), and its Drosophila melanogaster homologue, KETTE, regulates axon pathfinding (Hummel et al., 2000).
The aforementioned functions of Nap1 suggested its potential involvement in OL-pc–dependent cell or axon migration. To test this idea, we constructed a model cell system by using U251 cells, a human astrocytoma line (Vaheri et al., 1976), which exhibited high locomotive nature. We transfected these cells with OL-pc cDNA and compared them with the parent U251 cells to assess the role of the OL-pc–Nap1 complex in cell behavior. In control U251 cells, Nap1 was localized along the lamellipodia of moving cells but not at their cell–cell contact sites that were quiescent in terms of membrane ruffling. However, OL-pc expression in these cells caused a recruitment of not only Nap1 but also WAVE1 to the cell–cell contact sites. The OL-pc–expressing cells exhibited accelerated migration only when they were in contact with others, concomitant with altering the assembly of F-actin and N-cadherin at cell–cell contacts. OL-pc mutants, which lacked the Nap1-binding site (NBS), had no such effects on cell behavior. Furthermore, depletion of Nap1 and WAVE1 abrogated, at least in part, the aforementioned effects of OL-pc on cell motility, and N-cadherin knockdown mimicked the effects of OL-pc expression. Based on these observations, we propose that signals generated by the OL-pc–Nap1 complex at cell–cell contact sites modulate junctional actin organization, perturbing cadherin-based adherens junctions and thereby enhancing cell migration.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
|
NBS, in which the NBS was deleted, and transfected U251 cells with HA-tagged OL-pcNBS or full-length OL-pc (OL-pc). Immunoprecipitates of these constructs showed that Nap1 was copurified only with the full-length construct (Fig. 1 D).
OL-pc recruits NAP1 and WAVE1 to cell–cell contacts
To study the biological role of the OL-pc–Nap1 complex, we transfected U251 cells with cDNAs for OL-pc and OL-pcNBS and isolated their stable transfectant lines. Each line was a mixture of uncloned transfectants, which allowed us to avoid observing clone-specific phenotypes. Despite the description in the GEO DataSets (National Center for Biotechnology Information) that OL-pc mRNA is expressed in the U251 line, we did not detect any endogenous OL-pc protein in it. In low cell density cultures, U251 cells were highly motile, exhibiting a polarized fanlike shape with lamellipodia at the leading edge (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). When these cells had collided, they formed transient contacts, although they soon became separated from one another as a result of the high locomotive activities. In higher densities, they more stably contacted each other. Irrespective of the cell densities, the exogenous OL-pc and OL-pcNBS were always concentrated at cell–cell contact sites (Fig. 2 A).
These molecules were also detectable in the lamellipodial regions, where they were well colocalized with actin fibers (Fig. 2 A, right).
|
In the OL-pcNBS transfectants, Nap1 was also localized along the lamellipodia, overlapping with OL-pcNBS (Fig. 2 A). Because these two molecules did not interact with each other biochemically (Fig. 1), Nap1 was likely distributed to the lamellipodia in an OL-pc–independent way. More importantly, in the OL-pcNBS transfectants, Nap1 was not localized at cell–cell contact sites (Fig. 2, A and B). These observations suggest that the OL-pc accumulation at cell–cell contacts resulted in the recruitment of Nap1 to the same sites, which was otherwise present only on the lamellipodia, at least in the cell line used here.
Then, we examined the distribution of WAVE1. Because antibodies useful for immunostaining of this molecule were not available, we transiently transfected the aforementioned cells with a GFP-conjugated WAVE1. The behavior of WAVE1 was similar to that of Nap1: in control U251 cells, WAVE1 was concentrated mainly on the lamellipodia, whereas it became localized at cell junctions when OL-pc was coexpressed (Fig. 2 C). OL-pcNBS coexpression had no such effects. These findings are in accord with the observation that Nap1 and WAVE behave together, forming a complex (Ibarra et al., 2005). On the other hand, the distribution of Arp2/3 complex, which is known to be downstream of the Nap1–WAVE signals, was unique, as revealed by immunostaining for ARPC2 (Arp2/3 complex 34-kD subunit), a component of the Arp2/3 complex (Robinson et al., 2001). In control U251 cells, ARPC2 was localized along the leading edge of cells, with additional irregular signals at cell–cell contacts. However, OL-pc expression did not particularly change this localization pattern. Furthermore, we never observed specific colocalization of OL-pc and ARPC2 at cell–cell contact regions, and this was also the case for OL-pcNBS (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1), suggesting that the Arp2/3 complex was not interacting with OL-pc.
OL-pc has no effect on the migration of isolated cells but up-regulates cell motility within colonies
Nap1 and its associated components are known to regulate cell motility. Therefore, we examined whether OL-pc expression had any effect on cell migration by comparing the parent cells with their transfectants. Cells were plated at low densities to allow their free migration, and their movement was then recorded by time-lapse microscopy. Analysis of the video images did not show any difference in migration speed between the control and OL-pc–transfected cells (Fig. 3 A).
We also measured the directionality of cell migration, which was defined previously (Pankov et al., 2005), but could not find any difference in this parameter either between these cells (Fig. 3 A). Careful observation of the videos, however, made us aware that these cells were different in their contacting behavior. When the parent U251 cells made contact with each other, their membrane ruffling was suppressed at the contact sites, and these contact sites as well as the overall polarity of cells, including the lamellipodial directions, were transiently maintained until detachment (Fig. 3 B and Video 1). OL-pc–expressing cells also formed transient contacts; however, these cells in contact randomly moved relative to each other, rapidly changing the lamellipodial positions, suggesting that their peripheral motile activity was not properly controlled by cell–cell contacts (Fig. 3 B and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). The association pattern of OL-pcNBS–expressing cells was similar to that of the control cells, indicating that the aforementioned action of OL-pc required its binding to Nap1.
|
NBS–expressing cells exhibited rather shorter migration tracks than the controls (Fig. 4 B).
|
|
NBS did not show any accelerated cell migration (Fig. 5, A–C; NBS; and Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1), and these constructs actually rather suppressed colony spreading (Fig. 5 E), as observed in the experiments shown in Fig. 4, suggesting that the undeleted portions of the cytoplasmic domain in OL-pcNBS might have uncharacterized functions to suppress cell motility. To summarize, OL-pc did not alter the motility of solitary U251 cells but accelerated the translocation of cells that were in contact with each other, thus suggesting that OL-pc acted on cell motility via cell–cell contacts, where this molecule was concentrated.
Requirement of Nap1 and WAVE1 for OL-pc action
To test whether Nap1 and WAVE1, which associated with OL-pc, were involved in the aforementioned activity of OL-pc, we looked at the effects of RNAi-mediated depletion of these proteins. siRNAs designed for Nap1 and WAVE1 significantly down-regulated the expression of the respective molecules, and control siRNA had no such effects. In addition, the depletion of Nap1 was accompanied by a decrease in the WAVE1 level (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1), suggesting that the stability of WAVE1 was dependent on Nap1. Knockdown of Nap1 in control U251 or OL-pc transfectants resulted in the suppression of lamellipodial formation as reported previously (Steffen et al., 2004), and that of WAVE1 expression also suppressed formation of their polarized lamellipodia (Fig. S3).
Next, we compared the behavior of control and OL-pc transfectants from which Nap1 or WAVE1 was depleted in the wound-healing assay. At the wound edges of Nap1-depleted control U251 cells, these cells appeared more slender than their Nap1-positive counterparts (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1), and, despite their defective lamellipodia formation, they migrated actively (Fig. 6 A and Video 6). When their migration was compared with that of Nap1-depleted OL-pc transfectants, the latter migrated slightly faster than the former (Fig. 6 B and Video 7). However, these Nap1-depleted OL-pc transfectants did not display the uncoordinated migration that was unique to the Nap1-positive OL-pc transfectants but did migrate in a pattern similar to control cells (Fig. 6, A and C). Thus, the migration profile became indistinguishable between the cells with and without OL-pc, except for the migration speed, in the absence of Nap1. WAVE1 depletion produced similar results to that of Nap1, although the overall migration speed of U251 cells was greatly reduced (Fig. 6 and Fig. S4). These results suggest that OL-pc required Nap1 and WAVE1 in inducing the uncoordinated cell movement, although it did not require them for simple enhancement of cell migration.
|
NBS transfectants. Double immunostaining for N-cadherin and OL-pcNBS showed that although OL-pcNBS was distributed throughout the cell–cell contact regions, N-cadherin tended to be condensed along the apical-most portion of the junctions (Fig. 7 A), as generally seen in epithelial cells.
In OL-pc transfectants, on the other hand, N-cadherin was detected as streaklike signals spread over the OL-pc–positive areas, oriented perpendicularly to the cell borders (Fig. 7 A). Cadherin-associated catenins, such as 
-catenin, showed a localization pattern similar to that of N-cadherin in each transfectant.
|
NBS transfectants, diffuse F-actin signals were localized parallel to cell–cell boundaries, colocalizing with N-cadherin, whereas in OL-pc transfectants, the junctional F-actin stretched over cell–cell contact zones, colocalizing with the streaklike N-cadherin signals (Fig. 7 B). Importantly, the actin signals associated with N-cadherin were continuous with the stress fiber–like actin fibers, resembling radial actins, which had been previously shown to terminate at early cadherin-mediated adherens junctions in keratinocytes (Vaezi et al., 2002). In contrast, N-cadherin–associated F-actins in control or OL-pcNBS transfectants were isolated from the major cytoplasmic actin fibers. In all of these cells, the total level of N-cadherin was similar (Fig. S2). To investigate the dynamic aspects of N-cadherin and F-actin accumulation at cell–cell contacts, we conducted Ca2+-switch assays by treating cells with 1 mM EGTA to disrupt cadherin-mediated junctions and adding normal medium to the culture. N-cadherin was restored to cell–cell boundaries within 15 min, together with OL-pc or OL-pcNBS in the case of their transfectants. Even at these early cell–cell contacts, N-cadherin and F-actin exhibited the localization patterns characteristic of the control, OL-pc, and OL-pcNBS transfectants (Fig. 7 C), indicating that the OL-pc–dependent behavior of N-cadherin and F-actin was tightly linked.
As an attempt to characterize the aforementioned actin organization unique to OL-pc transfectants, we treated cells with cytochalasin D. This treatment eliminated most of the major actin fibers in the cells but left the junctional cortical actins intact. After this treatment, the distribution of N-cadherin–associated actins became indistinguishable between OL-pc and OL-pcNBS transfectants or control cells (Fig. 7 D). Thus, the cytochalasin D treatment abolished the effects of OL-pc–dependent reorganization of junctional actins, suggesting that OL-pc expression primarily altered the actin polymerization states. We also examined the N-cadherin and actin distributions in Nap1 or WAVE1 siRNA–treated cells. In these cells, the OL-pc–dependent streaklike localization of N-cadherin was diminished, being converted to a pattern similar to that seen in control cells (Fig. 7 E), supporting the idea that Nap1 and WAVE were working together with OL-pc.
N-cadherin depletion mimics OL-pc expression
The altered N-cadherin distribution, as shown in Fig. 7, might have been involved in the migration behavior specific to the OL-pc transfectants. To test this possibility, we looked at the effects of RNAi-mediated depletion of N-cadherin (Fig. S2) on cell migration by using the wound-healing assay. Our results showed that N-cadherin–depleted cells, whether OL-pc was expressed or not, became similar to OL-pc–expressing cells in terms of their migratory behavior (i.e., they showed an accelerated movement; Figs. 8 and S4), indicating that N-cadherin removal was sufficient to induce the phenotypes seen by OL-pc expression.
To check whether cadherin activity was altered by OL-pc expression, we dissociated control and OL-pc transfectants by using the classic trypsin-Ca2+ treatment protocol, by which cadherins on the cell surface are preserved (Takeichi, 1977), and measured their aggregation rate in suspension cultures. However, we did not find any significant difference in their aggregating abilities (unpublished data). These results suggest that the role of OL-pc is not to interfere with cadherin-dependent cell adhesion but to perturb other cadherin-mediated signaling processes.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
We found that OL-pc was coprecipitated with components of the Nap1–WAVE complex (Stradal and Scita, 2006). Our results further suggested that Nap1 was the primary element to interact with OL-pc among the components of the complex. Immunostaining experiments showed that in control U251 cells, Nap1 or WAVE1 was localized exclusively on the lamellipodia, except for other diffuse cytoplasmic signals. However, OL-pc expression recruited these molecules to cell–cell contact sites. This recruitment most likely resulted from the autonomous concentration of OL-pc molecules into cell junctions as a result of their homophilic interaction. On the other hand, OL-pc was also detectable on lamellipodia, whose signals partly colocalized with those of Nap1. However, OL-pcNBS, not having the Nap1-binding region, also showed similar lamellipodial localization, suggesting that the association of OL-pc with lamellipodia did not depend on its interaction with Nap1. Both OL-pc and OL-pcNBS colocalized with actin fibers at the lamellipodia, suggesting the possibility that they may be able to associate with F-actin by using the non-NBS portion of their cytoplasmic domain. Although the OL-pc localized on lamellipodia may have some biological roles, in the present experiments, we focused on the NBS-dependent functions of OL-pc.
The Nap1–WAVE complex is known to enhance lamellipodia formation and, in turn, cell migration. It is therefore expected that when this complex has been recruited to cell junctions, even the junction-forming cell peripheries might have acquired lamellipodium-like activities. Indeed, we found that the motile behavior of cells was altered by OL-pc when they were in contact with one another. In low density cultures of control U251 cells, when they had transiently contacted each other, their membrane ruffling was suppressed at their contact sites, but this suppression did not affect their overall polarity, including the lamellipodial sites. On the other hand, OL-pc–expressing cells rapidly changed their association pattern with their partners, actively altering lamellipodial directions. These observations suggest that although the wild-type U251 cells have the ability to control their lamellipodial activity upon their contacts, this ability was perturbed by OL-pc expression. This notion was supported by the finding that cell movement was accelerated within cell colonies by OL-pc expression. Similar differences in cell behavior were also observed for cells undergoing wound healing: contrasted with the coordinated migration of control cells at wound edges, the behavior of OL-pc–expressing cells was uncooperative. For example, their migration was sporadically accelerated in the presence of neighbors. All of these findings support the idea that the OL-pc–Nap1–WAVE1 complex stimulated cell motility by localizing at cell–cell contact sites.
The clearest morphological differences between the cells with and without OL-pc were seen in terms of actin organization at the N-cadherin–based cell junctions: in the absence of OL-pc or its NBS, N-cadherin and F-actin, which are the key partners required for organization of the adherens junction (Mege et al., 2006), were localized parallel to cell–cell interfaces. In contrast, in the presence of OL-pc, they were rearranged to stretch perpendicularly to the cell boundaries, becoming linked with radial actin fibers. However, this unique OL-pc–dependent reorganization of F-actin and N-cadherin was abolished by treatment with cytochalasin D, whose direct targets are the actin filaments. These findings suggest the possibility that the OL-pc–Nap1–WAVE complex primarily remodels F-actin assembly at cell–cell contact sites and that this change, in turn, alters the adherens junctions, as adherens junction formation is known to depend on F-actin organization (Mege et al., 2006; Abe and Takeichi, 2008).
With regard to the signaling systems downstream of the OL-pc–associated Nap1, it seems likely that Nap1 and WAVE1 act together, as is well established (Blagg and Insall, 2004) and supported by the observation that the RNAi-mediated removal of these two molecules abolished OL-pc–dependent phenotypes in a similar fashion. On the other hand, our results imply that the Arp2/3 complex, the classic target of WAVE, was unlikely to be involved in the OL-pc signaling system, as this complex did not show any colocalization with OL-pc at cell junctions. It is probable that the OL-pc–associated WAVE1 may use some other signaling system rather than Arp2/3; in fact, the presence of Arp2/3-independent pathways for WAVE signaling was indeed suggested in a previous study (Sasaki et al., 2000). Our results also suggest that OL-pc had some functions independent of the Nap1–WAVE complex. For example, when Nap1 or WAVE1 had been depleted, OL-pc–positive cells still migrated faster than OL-pc–negative cells. Furthermore, the cell migration speed in confluent cultures or during wound healing was rather reduced by OL-pcNBS expression. It is very likely that other regions on the OL-pc cytoplasmic domain than the NBSs bear uncharacterized biological functions, which need to be identified by future studies.
How, then, did the OL-pc–Nap1 complex affect cell migratory behavior? As discussed in the previous sections, it might have activated the motile machinery at cell–cell contact sites via F-actin remodeling, which was otherwise quiescent, and this could be a mechanism to explain the OL-pc–dependent cell behavior. Interestingly, the behavior of OL-pc–expressing cells was mimicked by N-cadherin depletion. Thus, it should be recalled that the cadherin-based cell junctions are required for contact inhibition of cell movement (Chen and Obrink, 1991; Bracke et al., 1997; Huttenlocher et al., 1998). Thus, the aforementioned proposed role of OL-pc might have been elicited through an alteration of N-cadherin functions. For example, we can infer the following sequence of OL-pc action: OL-pc first altered F-actin organization, which caused N-cadherin redistribution, and the redistributed N-cadherin molecules lost the ability to induce contact inhibition, although they did not lose the cell adhesion function, as shown in the present results. Cells, which are less contact inhibited, would display accelerated movement within their monolayers, as observed in this study. It is noteworthy that paraxial protocadherin inhibits the activity of classical cadherins (Chen and Gumbiner, 2006), and arcadlin enhances endocytosis of N-cadherin (Yasuda et al., 2007). These protocadherins share clusters of conserved cytoplasmic sequences with OL-pc (Redies et al., 2005; Vanhalst et al., 2005), suggesting the possibility that they may have conserved biological functions that affect the classical cadherin system.
Our previous analysis using OL-pc knockout mice revealed that striatal axons could not normally extend not only in vivo but also in vitro, when the OL-pc gene had been removed (Uemura et al., 2007). When striatal neurons are dissociated into single cells and cultured in vitro, they do normally elongate irrespective of the presence or absence of OL-pc (unpublished data), suggesting that the ability of elongation is indistinguishable between wide-type and mutant axons. Notably, in vivo, OL-pc–deficient striatal axons clumped together at the stalled points, which is in contrast with the dispersed arrangements of growing normal axons, suggesting that some signals to repel the axons from each other are missing in the mutants. Immunostaining experiments revealed that OL-pc was distributed along axons, particularly concentrated in the growth cones (Uemura et al., 2007), and Nap1 was localized in a similar pattern (unpublished data). During the course of axon elongation, these growth cones may laterally touch each other as they migrate, forming a fascicle. In such occasions, the homophilic interactions could occur between the OL-pc–Nap1 complexes localized on the contacting growth cones, and they may generate signals to facilitate their migration (for example, by activating the cell locomotive machinery localized at the cell periphery). In conclusion, our results disclosed a novel regulatory mechanism for cell migration, one that operates specifically at the homotypic cell–cell contact sites.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
NBS-HA, a C-terminal portion of OL-pc comprising aa 1,027–1,041 was inserted into the aforementioned HA-tagged OL-pc mutant. The resultant OL-pc-HA and OL-pcNBS-HA were subcloned into the pCA-Sal-IRES-neomycin vector (Kametani and Takeichi, 2007) to obtain stable expression vectors. The WAVE1 expression vector cloned into pEGFP (Clontech Laboratories, Inc.) was provided by T. Takenawa (Kobe University, Kobe, Japan).
Cell culture and transfection
U251 human astrocytoma cells were cultured on collagen-coated dishes (Iwaki) in a 1:1 mixture of DME and Ham's F12 (Wako) supplemented with 10% FCS (abbreviated as DH10). Cells were transfected with various expression vectors by use of TransIT-LT1 Transfection Reagents (Mirus) or an electroporation system (Amaxa). For obtaining stable transfectants, transfected cells were selected with 500 µg/ml G418 for 2–3 wk and maintained as uncloned populations, in which we confirmed that >90% of the cells in each culture expressed the transgene. Cytochalasin D (EMD) was added to the culture medium at a final concentration of 0.4 µg/ml, and the cells were fixed after 30 min. For calcium-switch assays, cell layers were incubated for 15 min at 37°C in a Hepes-buffered saline containing 1 mM EGTA and 1 mM MgCl2. Then, the saline was replaced with prewarmed DH10, and the cells were maintained in it until examined.
Antibodies
mAbs 4F5 and 5E3 against Nap1 were generated by immunizing 7-wk-old Donryu rats (Japan SLC, Inc.) with a keyhole limpet hemocyanin–conjugated mouse Nap1 peptide, CLRNAYHAVYKQSVTSSA, synthesized by Invitrogen. In brief, rats were injected with the synthetic peptides four times at 2-wk intervals, and then the splenocytes of immunized rats were recovered and fused with P3-X63-Ag-U1 myeloma cells (animals were immunized and killed according to the guidelines of the RIKEN Center for Developmental Biology). 4F5 and 5E3 mAbs thus obtained gave a single major band in Western blotting (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1). We mainly used 4F5 for immunohistochemistry and 5E3 for Western blotting. Rat mAb 5G10 against mouse OL-pc was described previously (Aoki et al., 2003). Rabbit pAb against WAVE1 was a gift from T. Takenawa (Kobe University, Kobe, Japan). The following antibodies were purchased: rabbit pAbs against p125Nap1, PIR121-1/Sra-1, and p34-Arc/ARPC2 (Millipore); rabbit pAb against Abi-1 (MBL International); rabbit pAb against N-cadherin (Takara Bio); rat mAb against GFP (Nacalai Tesque); rat anti-HA mAb 3F10 (Roche); mouse anti-HA mAb 16B12 (Covance); rabbit anti-Myc pAb (Santa Cruz Biotechnology, Inc.); and mouse anti-FLAG mAb M2 and anti–-tubulin mAb DM1A (Sigma-Aldrich). The following were used as secondary antibodies: goat AlexaFluor488, -555, or -647–conjugated anti–mouse IgG or anti–rabbit IgG (Invitrogen); goat Cy3-conjugated anti–rat IgG (Millipore); donkey Cy2-conjugated anti–rat IgG and donkey HRP-conjugated anti–rat IgG (Jackson ImmunoResearch Laboratories); and sheep HRP-conjugated anti–mouse IgG and donkey HRP-conjugated anti–rabbit IgG (GE Healthcare). F-actin was visualized by using AlexaFluor488–conjugated phalloidin (Invitrogen).
RNAi
All siRNAs were purchased (Stealth siRNA; Invitrogen). The target sense sequences that were mainly used are as follows: for human Nap1, 5'-CAACCUUGAUAAGUUGCACACUGCA-3'; for human WAVE1, 5'-AACUGGUACAGUCUCACAUACUGGG-3'; and for human N-cadherin, 5'-AAUUAAGGGAGCUCAAGGACCCAGC-3'. We chose these sequences from three different sequences for each gene as those that most effectively knocked down the respective gene expression. Negative control stealth siRNAs were also obtained from Invitrogen. As a control for NAP1 RNAi, the scrambled siRNA sequence 5'-CAAGUUAUAUGACGUACAUCCCGCA-3' was used. All siRNAs were transfected by use of Lipofectamine RNAiMAX reagent (Invitrogen). The efficiency of gene product suppression was assessed by Western blotting and immunofluorescence staining of cells. In each RNAi-treated culture, >90% of the original protein expression was suppressed by 3–4 d after transfection (Fig. S2).
Live cell imaging and data processing
Time-lapse images were obtained by using a microscope (DeltaVision; Applied Precision) with observation for 4 h at 3-min intervals unless otherwise noted. 2 h before taking videos, the medium was changed to L-15 medium (Invitrogen) supplemented with 10% FCS. For videos of low density cell cultures, 104 cells were placed on collagen-coated glass-based dishes (35 mm in diameter; Iwaki) 1 d before the assay. For videos of wound-healing cells, 106 cells were placed on noncoated glass-based dishes (35 mm in diameter; Iwaki) and cultured to confluence for 1–2 d. Then, we changed the medium to L-15 medium supplemented with 10% FCS. Wound edges were generated by scraping the cells with a plastic tip, and time-lapse recordings were subsequently started. For tracing fluorescently labeled cells in confluent cultures, we incubated the cells with 30 mM 5(-6) carboxyfluorescein diacetate succinimidyl ester in Hepes-buffered saline for 1 h at room temperature. The labeled cells were then collected by trypsinization and plated together with nonlabeled cells at a 1:10 ratio. After 24–36 h of incubation, the labeled cells were traced at 9-min intervals for another 6 h. In all of these experiments, the dishes were placed on the stage of a microscope (IX71; Olympus) equipped with a cooled CCD camera (Series 300 CH350; Photometrics). The stage was maintained at 37°C by using a thermal controller (Sanyo). Digital images were processed by using SoftWoRx (Applied Precision). For long-term observations of wound healing, images were taken with a system (CV100; Olympus) equipped with a CCD camera in a CO2 incubator. Trace drawing and statistical analyses were performed with MetaMorph Offline (MDS Analytical Technologies) and Excel (Microsoft), respectively.
Immunofluorescence staining
Cells plated on collagen-coated coverslips (Iwaki) were fixed with 4% PFA in HBSS for 10 min and permeabilized with 0.25% Triton X-100 in TBS for 8 min or 1% Triton X-100 for 10 min. The latter treatment was used for N-cadherin immunostaining. Specimens were incubated with the blocking buffer (5% skim milk in TBS) for 1 h, with the corresponding primary antibodies at several dilutions for 2 h and with the secondary antibodies for 1 h. Coverslips were mounted in FluorSave (EMD) and imaged through plan-Apochromat 63x1.40 NA objectives by use of a fluorescence microscope (Axioplan 2; Carl Zeiss, Inc.) connected with a CCD camera (AxioCam HRc; Carl Zeiss, Inc.) or a laser-scanning confocal microscope (LSM510; Carl Zeiss, Inc.) mounted on an inverted microscope (Axiovert 200M; Carl Zeiss, Inc.), and images were processed using Photoshop (Adobe). For detergent extraction before fixation, cells were treated with a Triton X-100 buffer (50 mM NaCl, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 0.5% Triton X-100, and 300 mM sucrose; Yamazaki et al., 2007) for 10 min on ice and fixed with 4% PFA.
Immunoprecipitation
COS-7 cells transiently cotransfected with FLAG-tagged OL-pc and HA-tagged Nap1 or Myc-tagged CYFIP-2 were harvested and dissolved in the lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 5% glycerol, 1 mM PMSF, and Complete EDTA-free tablet [Roche]). Lysates were precleared with protein G–Sepharose 4FF beads (GE Healthcare) for 1 h, incubated with antibodies against each tag for 2 h, and incubated with newly prepared protein G–Sepharose 4FF beads for 1 h. The beads were washed three times with the lysis buffer. Precipitates were separated by SDS-PAGE and analyzed by immunoblotting.
Pull-down assay and mass spectrometric analysis
GST fusion proteins containing various deletions of OL-pc cytoplasmic domain (Fig. 1) were expressed in DH5 cells with pGEX-6p-1 vector (GE Healthcare) and were purified. 1-d-old ICR mouse brains were homogenized and lysed in the lysis buffer with 1 mM EDTA and 0.5 mM DTT. Then, the lysate was incubated for 1 h with GST-tagged proteins and glutathione–Sepharose 4B beads (GE Healthcare). The beads were subsequently washed three times in the lysis buffer containing 1 mM EDTA and 0.5 mM DTT. Precipitates were separated by SDS-PAGE and detected by use of 2D Silver Stain (Daiichi), Silver Stain MS kit (Wako), or by Western blot analysis. Each silver-stained precipitate was identified by LC-MS/MS analysis in the Mass Spectrometry Analysis Laboratory in the RIKEN Center for Developmental Biology.
Western blot analysis
Samples were eluted by boiling them in SDS sample buffer (0.25 M Tris-HCl, pH 6.8, 4% SDS, 40% glycerol, and 0.002% bromophenol blue), separated by SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). Membranes were blocked with 5% skim milk in TBS for 1 h and subsequently exposed to primary antibodies for 2 h and then to secondary antibodies for 1 h. The proteins were detected by use of the ECL Plus system (GE Healthcare).
Statistical analysis and data presentation
The box and whisker plots were presented in the following ways (Zisman et al., 2007): the top and bottom ends of the boxes are the 75th and 25th percentiles, respectively. The lines and dashed lines across the box identify the mean and median, respectively. Dots out of the whiskers indicate outliers. The horizontal lines above the boxes represent the maximum datum points or, in the case of the existence of outliers, the outermost datum points that fall within the 75th percentile plus 1.5 times the value of the interquartile range (75th – 25th percentile). The same applies for the lines below the boxes. The data were analyzed by conducting Welch's t test.
Online supplemental material
Fig. S1 shows double immunostaining for OL-pc and ARPC2. Fig. S2 shows the knockdown efficiencies of the siRNAs used. Fig. S3 presents still images of cells treated with Nap1 or WAVE1 siRNA. Fig. S4 shows time-lapse images of cells after depletion of Nap1, WAVE1, or N-cadherin. Fig. S5 shows the specificity of anti-Nap1 mAbs. Videos 1–9 show time-lapse images of control (Video 1) and OL-pc transfectants (Video 2) in low density cultures; control (Video 3), OL-pc (Video 4), and OL-pcNBS (Video 5) transfectants during wound healing; Nap1-depleted control (Video 6) and OL-pc (Video 7) transfectants during wound healing; and N-cadherin–depleted control (Video 8) and OL-pc (Video 9) transfectants during wound healing. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200802069/DC1.
|
Acknowledgments |
|---|
This work was supported by a grant from the program Grants-in-Aid for Specially Promoted Research of the Ministry of Education, Science, Sports and Culture of Japan to M. Takeichi and by grants-in-aid for scientific research from the Japan Society for the Promotion of Science for Junior Scientists to S. Nakao and for foreign researchers to A. Platek.
Submitted: 19 February 2008
Accepted: 27 June 2008
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Abe, K., and M. Takeichi. 2008. EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc. Natl. Acad. Sci. USA. 105:13–19.
Abercrombie, M. 1967. Contact inhibition: the phenomenon and its biological implications. Natl. Cancer Inst. Monogr. 26:249–277.[Medline]
Abercrombie, M., and E.J. Ambrose. 1958. Interference microscope studies of cell contacts in tissue culture. Exp. Cell Res. 15:332–345.[CrossRef][Medline]
Aoki, E., R. Kimura, S.T. Suzuki, and S. Hirano. 2003. Distribution of OL-protocadherin protein in correlation with specific neural compartments and local circuits in the postnatal mouse brain. Neuroscience. 117:593–614.[Medline]
Blagg, S.L., and R.H. Insall. 2004. Control of SCAR activity in Dictyostelium discoideum. Biochem. Soc. Trans. 32:1113–1114.[CrossRef][Medline]
Bracke, M.E., H. Depypere, C. Labit, V. Van Marck, K. Vennekens, S.J. Vermeulen, I. Maelfait, J. Philippe, R. Serreyn, and M.M. Mareel. 1997. Functional downregulation of the E-cadherin/catenin complex leads to loss of contact inhibition of motility and of mitochondrial activity, but not of growth in confluent epithelial cell cultures. Eur. J. Cell Biol. 74:342–349.[Medline]
Chen, W.C., and B. Obrink. 1991. Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells. J. Cell Biol. 114:319–327.
Chen, X., and B.M. Gumbiner. 2006. Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity. J. Cell Biol. 174:301–313.
Fujito, T., W. Ikeda, S. Kakunaga, Y. Minami, M. Kajita, Y. Sakamoto, M. Monden, and Y. Takai. 2005. Inhibition of cell movement and proliferation by cell-cell contact-induced interaction of Necl-5 with nectin-3. J. Cell Biol. 171:165–173.
Hirano, S., Q. Yan, and S.T. Suzuki. 1999. Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems of the developing mouse brain. J. Neurosci. 19:995–1005.
Hirano, S., S.T. Suzuki, and C. Redies. 2003. The cadherin superfamily in neural development: diversity, function and interaction with other molecules. Front. Biosci. 8:d306–d355.[Medline]
Hummel, T., K. Leifker, and C. Klämbt. 2000. The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization. Genes Dev. 14:863–873.
Huttenlocher, A., M. Lakonishok, M. Kinder, S. Wu, T. Truong, K.A. Knudsen, and A.F. Horwitz. 1998. Integrin and cadherin synergy regulates contact inhibition of migration and motile activity. J. Cell Biol. 141:515–526.
Ibarra, N., A. Pollitt, and R.H. Insall. 2005. Regulation of actin assembly by SCAR/WAVE proteins. Biochem. Soc. Trans. 33:1243–1246.[CrossRef][Medline]
Ibarra, N., S.L. Blagg, F. Vazquez, and R.H. Insall. 2006. Nap1 regulates Dictyostelium cell motility and adhesion through SCAR-dependent and -independent pathways. Curr. Biol. 16:717–722.[CrossRef][Medline]
Joslin, E.J., L.K. Opresko, A. Wells, H.S. Wiley, and D.A. Lauffenburger. 2007. EGF-receptor-mediated mammary epithelial cell migration is driven by sustained ERK signaling from autocrine stimulation. J. Cell Sci. 120:3688–3699.
Kametani, Y., and M. Takeichi. 2007. Basal-to-apical cadherin flow at cell junctions. Nat. Cell Biol. 9:92–98.[CrossRef][Medline]
Kazmierczak, P., H. Sakaguchi, J. Tokita, E.M. Wilson-Kubalek, R.A. Milligan, U. Muller, and B. Kachar. 2007. Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells. Nature. 449:87–91.[CrossRef][Medline]
Marston, D.J., S. Dickinson, and C.D. Nobes. 2003. Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion. Nat. Cell Biol. 5:879–888.[CrossRef][Medline]
Mege, R.M., J. Gavard, and M. Lambert. 2006. Regulation of cell-cell junctions by the cytoskeleton. Curr. Opin. Cell Biol. 18:541–548.[CrossRef][Medline]
Nakao, S., M. Uemura, E. Aoki, S.T. Suzuki, M. Takeichi, and S. Hirano. 2005. Distribution of OL-protocadherin in axon fibers in the developing chick nervous system. Brain Res. Mol. Brain Res. 134:294–308.[CrossRef][Medline]
Otani, T., T. Ichii, S. Aono, and M. Takeichi. 2006. Cdc42 GEF Tuba regulates the junctional configuration of simple epithelial cells. J. Cell Biol. 175:135–146.
Pankov, R., Y. Endo, S. Even-Ram, M. Araki, K. Clark, E. Cukierman, K. Matsumoto, and K.M. Yamada. 2005. A Rac switch regulates random versus directionally persistent cell migration. J. Cell Biol. 170:793–802.
Pollard, T.D., and G.G. Borisy. 2003. Cellular motility driven by assembly and disassembly of actin filaments. Cell. 112:453–465.[CrossRef][Medline]
Rakeman, A.S., and K.V. Anderson. 2006. Axis specification and morphogenesis in the mouse embryo require Nap1, a regulator of WAVE-mediated actin branching. Development. 133:3075–3083.
Redies, C., K. Vanhalst, and F. Roy. 2005. delta-Protocadherins: unique structures and functions. Cell. Mol. Life Sci. 62:2840–2852.[CrossRef][Medline]
Ridley, A.J., M.A. Schwartz, K. Burridge, R.A. Firtel, M.H. Ginsberg, G. Borisy, J.T. Parsons, and A.R. Horwitz. 2003. Cell migration: integrating signals from front to back. Science. 302:1704–1709.
Robinson, R.C., K. Turbedsky, D.A. Kaiser, J.B. Marchand, H.N. Higgs, S. Choe, and T.D. Pollard. 2001. Crystal structure of Arp2/3 complex. Science. 294:1679–1684.
Sasaki, N., H. Miki, and T. Takenawa. 2000. Arp2/3 complex-independent actin regulatory function of WAVE. Biochem. Biophys. Res. Commun. 272:386–390.[CrossRef][Medline]
Smith, A., V. Robinson, K. Patel, and D.G. Wilkinson. 1997. The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells. Curr. Biol. 7:561–570.[CrossRef][Medline]
Steffen, A., K. Rottner, J. Ehinger, M. Innocenti, G. Scita, J. Wehland, and T.E. Stradal. 2004. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J. 23:749–759.[CrossRef][Medline]
Steffen, A., J. Faix, G.P. Resch, J. Linkner, J. Wehland, J.V. Small, K. Rottner, and T.E. Stradal. 2006. Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes. Mol. Biol. Cell. 17:2581–2591.
Stradal, T.E., and G. Scita. 2006. Protein complexes regulating Arp2/3-mediated actin assembly. Curr. Opin. Cell Biol. 18:4–10.[CrossRef][Medline]
Takeichi, M. 1977. Functional correlation between cell adhesive properties and some cell surface proteins. J. Cell Biol. 75:464–474.
Takeichi, M. 2007. The cadherin superfamily in neuronal connections and interactions. Nat. Rev. Neurosci. 8:11–20.[CrossRef][Medline]
Takenawa, T., and S. Suetsugu. 2007. The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat. Rev. Mol. Cell Biol. 8:37–48.[CrossRef][Medline]
Uemura, M., S. Nakao, S.T. Suzuki, M. Takeichi, and S. Hirano. 2007. OL-protocadherin is essential for growth of striatal axons and thalamocortical projections. Nat. Neurosci. 10:1151–1159.[CrossRef][Medline]
Vaezi, A., C. Bauer, V. Vasioukhin, and E. Fuchs. 2002. Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium. Dev. Cell. 3:367–381.[CrossRef][Medline]
Vaheri, A., E. Ruoslahti, B. Westermark, and J. Ponten. 1976. A common cell-type specific surface antigen in cultured human glial cells and fibroblasts: loss in malignant cells. J. Exp. Med. 143:64–72.
Vanhalst, K., P. Kools, K. Staes, F. van Roy, and C. Redies. 2005. delta-Protocadherins: a gene family expressed differentially in the mouse brain. Cell. Mol. Life Sci. 62:1247–1259.[CrossRef][Medline]
Watanabe, T., J. Noritake, and K. Kaibuchi. 2005. Regulation of microtubules in cell migration. Trends Cell Biol. 15:76–83.[CrossRef][Medline]
Yamazaki, D., T. Oikawa, and T. Takenawa. 2007. Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion. J. Cell Sci. 120:86–100.
Yasuda, S., H. Tanaka, H. Sugiura, K. Okamura, T. Sakaguchi, U. Tran, T. Takemiya, A. Mizoguchi, Y. Yagita, T. Sakurai, et al. 2007. Activity-induced protocadherin arcadlin regulates dendritic spine number by triggering N-cadherin endocytosis via TAO2beta and p38 MAP kinases. Neuron. 56:456–471.[Medline]
Yokota, Y., C. Ring, R. Cheung, L. Pevny, and E.S. Anton. 2007. Nap1-regulated neuronal cytoskeletal dynamics is essential for the final differentiation of neurons in cerebral cortex. Neuron. 54:429–445.[CrossRef][Medline]
Zisman, S., K. Marom, O. Avraham, L. Rinsky-Halivni, U. Gai, G. Kligun, V. Tzarfaty-Majar, T. Suzuki, and A. Klar. 2007. Proteolysis and membrane capture of F-spondin generates combinatorial guidance cues from a single molecule. J. Cell Biol. 178:1237–1249.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
Related In this Issue article
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
