MIIA and MIIB exert differential effects on polarity and tail retraction
Previous observations have shown the differential cellular localization of MII isoforms. In general, MIIA is present in regions distal to MIIB, and MII is largely absent from the lamellipodium of epithelial cells (Kolega, 1998
; Gupton and Waterman-Storer, 2006
). We have confirmed these observations using migrating CHO.K1 cells and reveal novel details (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1
), as follows: (a) the two isoforms often decorate the same actin filaments in a stippled manner, suggesting that some functions might result from additive activities; (b) MIIA and MIIB likely mediate distinct functions because the two isoforms also occupy distinct areas, and therefore do not readily form cofilaments; and (c) MII resides well away from nascent adhesions; therefore, any effect on adhesion dynamics would result from an indirect rather than a local effect.
To determine whether the spatial segregation of MIIA and MIIB results in different roles during cell migration, we generated knockdown vectors that inhibit MIIA and MIIB expression with high specificity (). For both isoforms, down-regulation was comparable and maximal 96 h after transfection, where it averaged 75–95% by immunoblotting, depending on transfection efficiency (). Immunofluorescence revealed >95% knockdown in individual cells ().
Figure 1. Knockdown of MIIA or MIIB differentially alters cell polarity. (A) CHO.K1 cells were transfected with pSUPER-GFP vector or pSUPER-GFP-RNAi against MIIA or MIIB, and blotted for MIIA or MIIB. GIT1 is a loading control. (B) Representative images of MIIA- (more ...)
When plated using migration-promoting conditions (see Materials and methods), MIIA-deficient cells exhibited broader lamellipodia than control cells and did not retract their trailing edge ( and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1
). This resulted in cells with extended tails (, arrowheads). This phenotype is reminiscent of the effect of Rho-kinase inhibitors in macrophages (Worthylake et al., 2001
) and overexpression of a paxillin mutant with the LD4 domain deleted (West et al., 2001
In contrast, MIIB-deficient cells were round and occupied a large area without distinguishable leading and trailing edges (; and Videos 2 and 4, available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1
), e.g., front–back polarity. Depletion of MIIA or MIIB in Rat2 fibroblasts yielded similar results (Fig. S2). This phenotype differs somewhat from that reported for MEFs from MIIB−/−
knockout mice (Lo et al., 2004
). Although both showed inhibited migration, the MIIB−/−
MEFs also showed long, unstable protrusions. This could arise either from incomplete ablation of MIIB by the RNAi knockdown or an uncharacterized adaptation. Off-target effects of the RNAi seem unlikely because our phenotypes were rescued by ectopic expression of RNAi-insensitive MIIA or MIIB, respectively ( and not depicted).
Figure 2. MIIA and IIB regulate protrusion and differentially control adhesion turnover. (A; top) Kymographs from control (pSUPER), MIIA-depleted (pSUP-IIA), and MIIB-depleted (pSUP-IIB) cells. (bottom) Overlay of periodicity and slope from the kymographs. (B) (more ...)
In addition to the round morphology, the MIIB-deficient cells also showed a defect in nuclear, centrosomal, and Golgi anchoring. More than 95% of the nuclei in the knockdown cells rotated clockwise (~2 h/cycle; and Video 4). The centrosome accompanied this rotation, and the Golgi apparatus was distributed around the nucleus rather than polarized, as observed in nontransfected, migrating cells ( and not depicted), suggesting a more general role of MIIB in cell polarization. Although the origin of this nuclear rotation is not known, it suggests that MIIB is part of a balanced mechanism of nuclear anchoring.
MII depletion increases cell protrusion and inhibits maturation of nascent adhesions at the leading edge
The increased area of MIIB-deficient cells and the broader lamellipodia in MIIA-deficient cells pointed to alterations in protrusion. By kymography, MIIA- and MIIB-deficient cells exhibited 2–3-fold increased rates of protrusion (). In addition, the protrusion was continuous, resulting in kymograms that showed a near linear, uninterrupted slope (, bottom). In contrast, wild-type cells often showed an interrupted, stepwise pattern, as previously reported (Giannone et al., 2004
). Interestingly, during these periodic interruptions in protrusion, the adhesions stabilized and grew as MII began to localize in the previously protrusive region (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1
), suggesting a causal link. Finally, when the protrusions in MIIA- and MIIB-deficient cells stopped advancing, they did not retract efficiently (Videos 6 and 8), suggesting that both MII isoforms regulate retraction of the lamellipodium. Thus, both MII isoforms control the speed, stepwise pattern of extension, and retraction of protrusions.
To determine whether the abnormal protrusion is accompanied by alterations in adhesion dynamics, we knocked down MIIA or MIIB in paxillin-GFP– ( and Videos 6–8, available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1
) or vinculin-GFP–expressing cells (not depicted). Control cells showed numerous well-defined adhesions in protrusions, as well as some small adhesions near the leading edge (, top; and Video 7) that assembled and turned over as described previously (Webb et al., 2004
). In contrast, MIIA- and MIIB-deficient cells showed few discrete adhesions in the protrusions; instead, there was a nearly continuous band of adhesions very close to the leading edge (, and Videos 7 and 8). The small individual adhesions that comprise this band were readily apparent at higher magnification (, , and Video 9). These adhesions disassembled and reformed rapidly (t1/2
< 15 s) as the leading edge progressed (, C and D; and Videos 7–9). More importantly, they did not evolve into larger adhesions when lamellipodial growth halted. The defects were rescued when RNAi-insensitive mCherry (mChe)-MIIA or -MIIB were expressed in MIIA- or MIIB-deficient cells, respectively ().
Figure 4. Adhesive signaling near the leading edge of MII-depleted cells. MIIA- or MIIB-depleted or control cells were plated on fibronectin and then fixed and stained for phosphotyrosine (A), phosphoTyr397-FAK (B), and phosphoTyr31-paxillin (C). Bar, 10 μm. (more ...)
It is interesting that the phenotypes of MIIA- and MIIB-deficient cells on protrusion and the dynamics of adhesions in protrusions were almost indistinguishable because MIIA and MIIB occupy different regions of the cell, and neither is present in protrusions. This suggests that MII “acts at a distance”; that is, MII activity at the base of the lamellipodium or in central regions is transmitted to the leading edge, presumably through actin filaments, and generates periodic contractions that coincide with cessation of protrusion and retraction, adhesion maturation, and the movement (sliding) of larger adhesions toward the center of the cell. It also suggests that myosin activity regulates the behavior of adhesions at the leading edge, regardless of the isoform. Finally, these observations support the notion that protrusion and adhesion turnover are coupled.
MIIA promotes the growth of adhesions in central regions and disassembly at the trailing edge
Because both isoforms of MII regulate adhesion dynamics at the leading edge, but only MIIA inhibits rear retraction, we investigated the effect of MIIA and MIIB knockdowns on adhesions in other cellular regions. MIIB-deficient cells exhibit central adhesions comparable to those in control cells (unpublished data). In contrast, MIIA-deficient cells showed abnormally small, but static, adhesions in the central region of the cell ( and Video 10, available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1
). At the cell rear, where MIIA inhibits retraction, adhesion disassembly is greatly inhibited, e.g., the adhesions slide slowly and do not disassemble ( and Video 10), thereby showing that adhesion sliding and disassembly are coordinately regulated by MIIA.
MIIA is required for the effects of MIIB on nascent, but not central, adhesions
Because both MIIA and MIIB mediate contraction and actin bundling (Kelley et al., 1996
), we used cross-rescue experiments to determine whether their functions were overlapping in the regulation of adhesion assembly and disassembly. First, mChe-MIIA was expressed in MIIB-depleted cells coexpressing paxillin-GFP. mChe-MIIA localized in regions very similar to those in unperturbed cells (not depicted), and it restored the maturation of nascent adhesions (, left). However, the polarity defects and the appearance of multiple protrusions around the cell periphery remained (not depicted).
We then expressed mChe-IIB in MIIA-deficient cells. mChe-MIIB localized in the central areas of the cell, as it does in unperturbed cells. However, it did not rescue the inhibited maturation of nascent adhesions induced by the MIIA knockdown. Instead, a band of small, dynamic adhesions remained near the leading edge, as in MIIB knockdowns (, right). In contrast, mChe-MIIB rescued the effect on the central adhesions, i.e., they were larger (, top right).
Thus, increased MIIB in central areas (where endogenous MIIB resides) of MIIA knockdown cells rescues the maturation of adhesions in the central regions of the cell, but not the nascent adhesions at the cells periphery. In contrast, increased MIIA in MIIB knockdown cells rescues the maturation of adhesions at the leading edge. This points to a mechanism in which the central MIIB activity requires MIIA for its translation to the periphery, perhaps by organizing the actin so that tension produced in the middle of the cell propagates into protrusions. This suggestion is supported by our observation that overexpressed MIIA in wild- type cells localizes primarily in actin bundles and produces more and larger adhesions (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1
), indicating that MII activity can dial up or down adhesion assembly, depending on its expression level.
Adhesion maturation at the leading edge depends on the ATPase activity of MIIA and MIIB
To separate the bundling from the contractile functions of MII on cell migration, we produced ATPase-inhibited mutants of MIIA and MIIB fused to GFP and mChe. The ATPase activity of N93K-MIIA and R709C-MIIB are inhibited 80 and 75%, respectively, in vitro (Heath et al., 2001
; Ma et al., 2004
). However, both mutants bind and cross-link, but do not move, actin filaments in vitro (Kim et al., 2005
We used FRAP to show that the mutants exhibit increased time in the actin-bound state, as expected from their inhibited ATPase cycling. Both MIIA N93K and MIIB R709C showed decreased rates and fractional recoveries (). The fractional recovery observed for both mutants was the same, suggesting that the two isoforms bind actin similarly. Interestingly, wild-type MIIA exhibited faster and higher fractional recovery than MIIB (). This suggests that MIIA is more active than MIIB, and therefore binds actin strongly in a smaller fraction of time, as shown previously in vitro (Kelley et al., 1996
). It also points to the use of FRAP as a method to determine MII activity in living cells.
Figure 3. Contractility-deficient mutants of MIIA and MIIB exhibit differential rescue of actin bundling, protrusion, and adhesion dynamics in MIIA- and MIIB-deficient cells. Inhibited FRAP of GFP-MIIA N93K (A) and GFP-MIIB R709C (B) in actin bundles. Data are (more ...)
When expressed in MIIB-deficient cells, MIIB R709C did not effectively restore adhesion maturation (). However, MIIB R709C partially restored the front–back polarity and localized at the back of the cell (). Thus, maturation of adhesions at the leading edge requires MIIB activity; but its role in determining front– back polarity suggests a cross-linking contribution.
In contrast, MIIA N93K localized like its wild-type counterpart (unpublished data), rather than the rearward localization of MIIB R709C, and did not rescue the increased protrusiveness observed in MIIA-deficient cells. However, it did restore leading edge retraction and the concomitant growth of adhesions in protrusions pointing to its actin-binding function in these activities ().
MII regulates adhesive signaling
Adhesive signaling through integrin receptors both stimulates and responds to tension through Rho GTPases, thus constituting a feedback loop connecting adhesion and contraction through MII regulation (Chrzanowska-Wodnicka and Burridge, 1996
). The phosphorylation of paxillin on Y31, Y118, and S273 and the phosphorylation of FAK on Y397 are part of this signaling mechanism and serve as markers for the activation of this pathway (Katz et al., 2003
; Zaidel-Bar et al., 2003
; Nayal et al., 2006
The small dynamic adhesions near the leading edge of MIIA- and MIIB-depleted cells were prominently phosphorylated on tyrosine (). They also stained positively for Y397-FAK and Y31-paxillin, defining an almost continuous band of adhesions (). The staining of these phosphomarkers decreased in the stable adhesions that reside in regions removed from the leading edge (). Thus, depletion of myosin function at the lamellipodium enhances an adhesive signaling pathway that regulates adhesion turnover and MII activity, providing a mechanistic link between myosin-generated tension in the control of adhesion maturation at the leading edge.
The complex interplay between myosin-mediated contraction, protrusion, adhesion, and polarization underscores the central role of MII and its integrative properties in cell migration. Although the protrusion rate is determined by factors that regulate actin polymerization (Pollard and Borisy, 2003
), it is also influenced by the rate of retrograde flow, which, in turn, is regulated by MII activity and serves to counterbalance actin polymerization (Lin and Forscher, 1995
; Mitchison and Cramer, 1996
). The retrograde flow rate is also influenced by adhesion, through a clutch-like mechanism, which links actin filaments to the substratum and can inhibit retrograde flow (Mitchison and Kirschner, 1988
; Lin et al., 1994
; Smilenov et al., 1999
). The net protrusion rate is also influenced by cycles of retraction and adhesion maturation at the leading edge. Highly motile cells protrude and move nearly continuously (Bear et al., 2002
; Jurado et al., 2005
), whereas other cells can show cycles of protrusion and retraction (Giannone et al., 2004
MII is also involved in a feedback loop that links adhesion, protrusion, and tension. Adhesion initiates signaling through Rho family GTPases that leads to the formation of adhesions and protrusions and generates tension. Tension also acts on adhesions to promote their maturation and the formation of actin filament bundles (Bershadsky et al., 2006
). Highly motile cells tend to have small, highly dynamic adhesions that turnover rapidly, whereas the adhesions in slower moving cells stabilize and grow in response to increased tension before turning over (Nayal et al., 2006
). Signaling components, such as phosphorylated paxillin and PAK, localize in the small, dynamic adhesions near the leading edge, where they function in a signaling pathway that inhibits adhesion maturation and promote protrusion (Nayal et al., 2006
). Interestingly, in retracting regions, MII mediates the disassembly, rather than the assembly, of adhesions.
Finally, MII polarizes and connects spatially segregated activities. Myosin acts “at a distance” in regulating protrusion and adhesion. It also contributes to the overall polarity of the migrating cell and establishes front and rear. The former is through the role of MII in orienting microtubules, Golgi, and the nucleus, and the latter is through actin bundling at the rear and sides (Xu et al., 2003
Thus, MII functions as a master regulator of cell migration. It can integrate spatially separated processes, and it is a key effector of signaling pathways that regulate each of the major component processes that drive migration.