Animal cells can move in the body, for example to heal a wound, by protruding a leading edge forwards, attaching it to the surroundings and then pulling against these new attachments while disassembling the older ones. Mechanical forces regulate the assembly and disassembly of these attachments, known as focal adhesions, and so do signals from outside the cell that are transmitted to the adhesions via specialized proteins. However, it was not clear how the assembly and disassembly of adhesions is coordinated.

CRL5 is a ubiquitin ligase, an enzyme that can mark other proteins for destruction. Cells migrate more quickly if CRL5 is inhibited, and so Teckchandani and Cooper set out to uncover whether CRL5 affects the assembly and disassembly of focal adhesions. The experiments showed that human cells lacking a crucial component of the CRL5 complex, SOCS6, disassemble adhesions faster than normal cells, but only at their leading edge and not at the rear.

Teckchandani and Cooper also found that SOCS6 localizes to the leading edge by binding to a focal adhesion protein called Cas. Shortly after the attachments assemble, the Cas protein becomes tagged with a phosphate group and then acts to promote the adhesion to disassemble. Further experiments indicated that Cas was marked by the CRL5 complex and possibly destroyed while in or very close to the leading edge adhesions, slowing their disassembly.

Together, these findings suggest that by binding Cas, SOCS6 regulates the turnover of adhesions, specifically by inhibiting disassembly and allowing adhesions to grow at the leading edge. Since SOCS6 is not present in adhesions outside of the leading edge, this may help explain how the older adhesions are disassembled. Future studies could next focus on the exact sequence of events that occur in focal adhesions after the CRL5 complex binds to Cas as the cell migrates.

During development, wound healing and cancer invasion, migrating cells need to move between other cells and through the dense extracellular matrix (ECM). Cells can attach to and pull on the ECM by using integrins ‒ transmembrane receptors that link ECM outside the cell to focal adhesions (FAs) and the actin cytoskeleton inside the cell (Alexander et al., 2008; Hynes, 2002; Pelham and Wang, 1997; Petrie et al., 2012; Puklin-Faucher and Sheetz, 2009). FAs are dynamic assemblies containing many proteins held together by dense networks of protein-protein interactions (Kanchanawong et al., 2010; Zaidel-Bar et al., 2007a). Nascent FAs (often called focal complexes) initiate when talin and other proteins associate with β integrin tails to stabilize an active integrin conformation and stimulate binding to the ECM (Calderwood et al., 1999; Tadokoro et al., 2003). Talin then binds actin and vinculin and actin flow exerts forces that create additional binding sites for vinculin, which in turn recruits more FA proteins and more actin (del Rio et al., 2009; Jiang et al., 2003). In this way, the force generated by actin flow, resisted by the ECM, creates a positive feedback loop to stabilize and grow the adhesion (Case and Waterman, 2015). In concert, force from the FA acts on actin filaments to induce the formation of contractile stress fibers and actin arcs (Burridge and Wittchen, 2013; Livne and Geiger, 2016; Roca-Cusachs et al., 2013). The contraction of stress fibers and actin arcs provides motive power to advance the cell body. As the cell body moves forwards over an FA, the force vector is redirected and the FA remodels or disassembles, allowing the FA proteins to recycle through the cytosol for reuse at the leading edge (Wehrle-Haller, 2012). Inhibition of Rho kinase or myosin relaxes actomyosin tension and induces rapid FA disassembly (Chrzanowska-Wodnicka and Burridge, 1996; Volberg et al., 1994). These findings support a mechanical model in which increased force drives FA assembly and reduced force stimulates disassembly.

FA dynamics are also regulated by signaling through protein kinases and small GTPases. ECM binding stimulates integrin-associated focal adhesion kinase (FAK) and Src-family kinases (SFKs). These tyrosine kinases phosphorylate several FA proteins, helping to build the FA by creating phosphotyrosine (pY) sites that bind to the SH2 domains of additional FA proteins, as well as by activating RhoA (Parsons et al., 2010; Zaidel-Bar et al., 2003, 2007b). Thus, FA size increases when endogenous SFKs are activated, consistent with a positive role for SFKs during FA assembly (Thomas et al., 1995). However, other evidence suggests that SFKs stimulate FA disassembly. For example, FA size increases and FA turnover is inhibited in fibroblasts with mutations in Src, FAK, or a variety of Src/FAK substrates (Ilic et al., 1995; Ren et al., 2000; Volberg et al., 2001; Webb et al., 2004). Furthermore, FAs grow when kinase-inactive Src is over-expressed (Fincham and Frame, 1998), and activated mutant Src weakens integrin-cytoskeletal interactions and accelerates FA disassembly (Felsenfeld et al., 1999; Fincham and Frame, 1998; Fincham et al., 1995; Galbraith et al., 2002). Therefore, SFKs may regulate both FA assembly and disassembly, perhaps by phosphorylating different substrates.

Cas (p130Cas, BCAR1) is an FA protein that is phosphorylated by Src when integrins engage the ECM (Fonseca et al., 2004; Nojima et al., 1995; Petch et al., 1995; Vuori and Ruoslahti, 1995). Cell motility and invasion are stimulated when Cas is over-expressed and inhibited when Cas is deleted (Brabek et al., 2005; Cary et al., 1998; Honda et al., 1999, 1998; Huang et al., 2002; Patwardhan et al., 2006; Sanders and Basson, 2005). At the molecular level, phosphorylation of Cas by SFKs induces binding to adaptor proteins Crk/CrkL and activation of the Rac1 and Rap1 GTPases (Hasegawa et al., 1996; Sakai et al., 1994; Tanaka et al., 1994; Vuori et al., 1996). Forced relocalization of CrkL to FAs stimulates migration via DOCK1 and Rac1 (Li et al., 2003). A SFK/Cas pathway stimulates protrusive activity, inhibits stress fibers, and stimulates FA disassembly (Webb et al., 2004). Therefore, Cas presents a paradoxical case where phosphorylation is induced by integrin engagement but stimulates FA disassembly and migration.

Interestingly, Cas is mechanosensitive. Cytoskeletal tension induces Cas tyrosine phosphorylation (Janostiak et al., 2011; Tamada et al., 2004). Mechanical extension of purified Cas promotes its phosphorylation by SFKs in vitro (Sawada et al., 2006). Thus integrin signaling and cytoskeletal tension may increase the level of phosphotyrosine Cas (pYCas) and stimulate FA disassembly whilst simultaneously unfolding talin to promote FA assembly. This raises the question of how FA assembly and disassembly are balanced in such a way that assembly predominates near the leading edge and disassembly occurs under the cell body. Here we provide evidence that CRL5^SOCS6, a cullin-RING ubiquitin E3 ligase composed of cullin-5 (Cul5), Rbx2, elonginBC and suppressor of cytokine signaling 6 (SOCS6), inhibits pYCas-dependent FA turnover at the front of migrating epithelial cells. We find that SOCS6 is recruited to pYCas in FAs at the leading edge, and that localization of SOCS6 to leading edge FAs slows their disassembly, dependent on cullin and proteasome activity and on binding of SOCS6 to Cas and CRL5. SOCS6 is not present in adhesions under the cell body, and sustained Cas signaling may facilitate their disassembly. The results suggest that CRL5^SOCS6 targets pYCas within adhesions at the front of the cell, thereby allowing FAs to grow and provide anchorage for stress fibers.

We previously reported that Cul5 inhibits the epidermal growth factor (EGF)-independent migration of MCF10A epithelial cells (Teckchandani et al., 2014). Cul5-depleted cells migrate considerably faster than control cells and have an exaggerated leading lamellipodium bearing tiny FAs, few stress fibers and dynamic membrane ruffles. The increased migration requires Src, suggesting that CRL5 may negatively regulate pY proteins. CRL5 targets pY proteins through the SH2 domains of SOCS substrate receptors. Knock-down experiments suggested that different SOCS proteins regulate different aspects of migration, with SOCS6 targeting pYCas and inhibiting ruffling. We proposed that other SOCS proteins may target additional pY proteins to CRL5 for ubiquitination, and each different target may contribute to a distinct aspect of cell migration (Teckchandani et al., 2014).

Cul5 inhibits adhesion dynamics at the leading edge

To investigate FA dynamics, we wounded monolayers of MCF10A cells that express low levels of EYFP-vinculin and allowed the cells to migrate in the absence of EGF. TIRF microscopy was used to detect EYFP puncta. We previously noted that migrating MCF10A cells contained large FAs and prominent stress fibers along the leading edge, which were lost following Cul5 knockdown (Teckchandani et al., 2014). TIRF microscopy of migrating EYFP-vinculin-expressing cells also revealed large FAs at the cell front, but also detected many much smaller FAs all across the ventral surface (Figure 1a, left). Large FAs were absent from migrating cells that had been treated with Cul5 siRNA (Figure 1a, right, see Figure 1—figure supplement 1a for knockdown efficiency). To quantify FA size, we divided the cell into front and back with a line ~ 6 µm from the leading edge (Figure 1—figure supplement 1b), and quantified the size distribution of front FAs of control and Cul5-deficient cells. While the number of large FAs at the front of control cells was not sufficient to affect the mean or median FA size, approximately 3.6% of FAs at the front of control cells were larger than 200 pixels (5 µm²), but <0.5% of FAs at the front of Cul5-deficient cells were this large (Figure 1b).

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To investigate whether different-sized FAs resulted from different dynamics, we recorded time lapse movies. FAs at the front of Cul5-deficient cells were notably more dynamic than those at the front of control cells (Video 1 and 2). To illustrate this difference, FAs were color-coded based on their presence in different frames, starting with blue at time zero and advancing through teal, green, orange, red and crimson by 100 min (Figure 1c). Many FAs at the front of control cells were white, indicating that they were present during the entire movie, while most FAs at the front of Cul5-deficient cells were singly colored, indicating they were short-lived. Blue FAs, present at the start, marked the original leading edge, and red FAs, born at the end, marked the ending leading edge position. The contrast between stable FAs in control cells and transient FAs in Cul5-deficient cells was also apparent from examination of individual frames from the movies (Figure 1d).

Video 1

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Video 2

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To quantify FA dynamics, movies were analyzed using FA analysis software (FAAS) (Berginski et al., 2011). This software does not distinguish FAs from the smaller focal complexes, so we refer to all EYFP structures larger than 0.05 µm² (two pixels) as FAs. We extracted first-order rate constants for the assembly and disassembly phases of each FA (Figure 1—figure supplement 1c–e). The rate constants for assembly and disassembly were generally lower for front FAs of control cells than front FAs of Cul5-deficient cells (Figure 1e). However, as expected, individual FAs were very heterogeneous. To control for technical or biological variation between experiments, we determined the median assembly and disassembly rate constants of many FAs in each of four to six experiments and then averaged these parameters across independent experiments. We found that FAs at the front and back of control cells assembled and disassembled with similar rate constants (0.012–0.013 min⁻¹ for assembly; 0.008–0.009 min^-1 for disassembly), suggesting that the front FAs were not larger because of increased assembly or decreased disassembly (Figure 1f, Table 1). Indeed, there was no correlation between mean FA area and assembly or disassembly rate constants (Figure 1—figure supplement 2), as expected for first-order reactions. Removing Cul5 had no effect on the assembly or disassembly of back FAs (Figure 1f, Table 1). At the front, however, removing Cul5 stimulated both assembly and disassembly approximately two-fold, to ~0.029 min⁻¹ for assembly and ~0.021 min⁻¹ for disassembly (Figure 1f, Table 1). Similar assembly and disassembly rates were measured in cells in which Cul5 was stably knocked down with shRNA targeting a different sequence, so the effect is unlikely to be off-target (see below, Figure 3—figure supplement 1). The increase in disassembly of leading edge FAs was also apparent from histograms of disassembly rate constants: the percentage of FAs with disassembly rates <0.02 min⁻¹ shifted from >60% to <10% when Cul5 was removed (Figure 1g). While we were unable to directly measure the lifetimes of the large FAs at the front of control cells, most persisted for longer than the 3 hr duration of the movies, suggesting lifetimes exceeding 3 hr. In contrast, front FAs of Cul5-deficient cells had an average 24 min half time for assembly, 36 min stable phase, and 33 min half time for disassembly, for a total 93 min average lifetime. The longer persistence of FAs at the front of control cells may contribute to their increased size.

Table 1

Conditions	Region	Assembly	Disassembly	# of movies	# of FAs
		(X 10−2 min−1)	(X 10−2 min−1)
siCon	front	1.3 (±0.2)	0.8 (±0.03)	4	64
siCul5	front	2.9 (±0.1) p=0.0002	2.1 (±0.9) p=0.00004	6	263
siCon	back	1.2 (±0.2)	0.9 (±0.1)	4	667
siCul5	back	1.3 (±0.05)	1.3 (±0.5) p=0.04	6	811
siCon	front	1.1 (±0.1)	0.8 (±0.1)	5	142
siCul5	front	3.0 (±0.4) p=0.008	1.9 (±0.08) p=0.00008	4	444
siCas	front	1.6 (±0.3)	0.7 (±0.1)	6	151
siCul5+siCas	front	1.1 (±0.2)	0.8 (±0.07)	5	137
siCon	back	0.8 (±0.04)	0.7 (±0.09)	5	632
siCul5	back	0.9 (±0.08)	0.8 (±0.08)	4	505
siCas	back	0.8 (±0.05)	0.6 (±0.08)	6	484
siCul5+siCas	back	0.7 (±0.1)	0.5 (±0.05)	5	541
shCon	front	1.0 (±0.1)	1.0 (±0.1)	4	105
shCul5	front	2.0 (±0.1) p=0.0012	2.0 (±0.09) p=0.0015	4	217
shCas	front	0.9 (±0.1)	1.0 (±0.06)	4	126
shCul5+shCas	front	1.0 (±0.2)	0.8 (±0.07)	4	152
shCon	back	0.9 (±0.06)	0.7 (±0.8)	4	701
shCul5	back	0.8 (±0.04)	0.9 (±0.1)	4	497
shCas	back	0.6 (±0.06)	0.9 (±0.1)	4	559
shCul5+shCas	back	0.7 (±0.1)	0.8 (±0.4)	4	608
siCon	front	1.4 (±0.1)	0.9 (±0.06)	5	185
siSOCS6	front	1.7 (±0.2)	1.4 (±0.03) p=0.0004	5	201
siCon	back	0.9 (±0.09)	0.7 (±0.04)	5	620
siSOCS6	back	0.8 (±0.07)	0.8 (±0.01)	5	469
siCon	front	1.4 (±0.2)	1.0 (±0.1)	4	254
siSOCS6 (alternate)	front	1.7 (±0.08)	1.9 (±0.08) p=0.0007	4	179
siCon	back	1.1 (±0.02)	1.0 (±0.06)	4	640
siSOCS6 (alternate)	back	1.2 (±0.05)	1.2 (±0.1)	4	600
Con	front	1.0 (±0.09)	1.0 (±0.06)	4	76
MLN4924	front	2.0 (±0.08) p=0.00016	1.6 (±0.03) p=0.0004	4	111
Con	back	0.6 (±0.07)	0.6 (±0.05)	4	182
MLN4924	back	1.0 (±0.2)	0.7 (±0.04)	4	293

1. Rate constants are reported as mean ± SEM

Cul5 regulates microtubule-dependent FA disassembly

Microtubules (MTs) are required for FA disassembly and are observed repeatedly engaging with FAs during disassembly (Bershadsky et al., 1996; Kaverina et al., 1999, 1998; Stehbens et al., 2012). We tested whether CRL5-regulated FA disassembly requires MTs. We treated cells with nocodazole to disrupt MTs and then removed nocodazole and assayed adhesion disassembly during MT regrowth (Ezratty et al., 2005). We found that nocodazole stabilized FAs in both control and Cul5-deficient cells (Figure 2a). However, FAs disappeared more rapidly from Cul5-deficient cells than control cells upon nocodazole removal (Figure 2a,b). Stress fibers also disappeared more rapidly from Cul5-deficient cells (Figure 2). Accelerated FA disassembly in Cul5-deficient cells was not due to faster MT regrowth after nocodazole removal because the MT cytoskeleton fully recovered in less than 15 min whether or not Cul5 was present (Figure 2—figure supplement 1a). Indeed, MT plus ends, tracked with fluorescent plus-end binding-protein EB1, reached FAs in the ventral membrane within minutes of removing nocodazole (Figure 2—figure supplement 1b). Therefore, Cul5 slows the disassembly of FAs after MTs have regrown, implying that Cul5 controls the speed of disassembly after MT targeting.

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MT targeting is reported to stimulate FA disassembly by clathrin-dependent integrin endocytosis (Ezratty et al., 2009; Ezratty et al., 2005). Accordingly, depleting clathrin heavy chains (CHC) strongly inhibited FA disassembly in control cells (Figure 2—figure supplement 2a). However, CHC depletion only partly inhibited the more rapid FA disassembly in Cul5-deficient cells (Figure 2—figure supplement 2a), suggesting that Cul5 inhibits a distinct mechanism of disassembly that is clathrin-independent. Consistent with this interpretation, clathrin-coated pits were missing from the leading lamellipodium of migrating Cul5-deficient cells (Figure 2—figure supplement 2b), as recently reported for other cell types (Kural et al., 2015). This suggests that Cul5 regulates FA disassembly through mechanisms unrelated to clathrin-mediated endocytosis.

Cas and SOCS6 regulate FA disassembly at the leading edge

Since we previously identified pYCas as a CRL5 substrate (Teckchandani et al., 2014) and since pYCas is required for FA disassembly in migrating fibroblasts (Webb et al., 2004), we tested whether Cul5-stimulated disassembly requires Cas. We found that removing Cas suppressed the increase in FA disassembly at the front of migrating Cul5-deficient MCF10A cells but had no effect on FAs at the back or F control cells (Figure 3a,b). These effects were unlikely to be due to off-target effects of Cas siRNA because similar results were obtained with cells stably knocked down for Cul5 and Cas using shRNA (Figure 3—figure supplement 1). Consistent with a Cul5-Cas pathway, Cas knockdown restored MT-dependent FA disassembly in Cul5-deficient HeLa cells in the nocodazole washout assay, but had no effect on control cells (Figure 3c,d). Therefore, Cas is required for increased FA disassembly when Cul5 is absent.

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Removal of Cas also suppressed the increased FA assembly of Cul5-deficient cells (Figure 3a,b), consistent with the observation that FA and stress fiber assembly is slowed in cas mutant fibroblasts (Antoku et al., 2008; Honda et al., 1998), potentially due to decreased signaling through a Crk/ C3G/Rap1 signaling pathway (Li et al., 2002; Voss et al., 2003).

SOCS6 is the substrate receptor through which CRL5 binds pYCas (Teckchandani et al., 2014). Accordingly, depleting SOCS6 stimulated FA disassembly at the front but not back of migrating MCF10A cells (Figure 4a,b). This was unlikely to be due to an off-target effect, because a different pool of SOCS6 siRNA had the same effect (Figure 4—figure supplement 1). SOCS6 depletion also stimulated FA disassembly in the nocodazole washout assay and Cas knockdown inhibited this effect (Figure 4c–e). Normal rates of FA disassembly were rescued by re-expression of wildtype SOCS6, but not by mutant SOCS6LCQQ that cannot bind elongin B and the rest of the CRL5 complex (Figure 4—figure supplement 2a). Moreover, expression of CasFF, a mutant that does not bind SOCS6 (Teckchandani et al., 2014), stimulated FA disassembly regardless of the presence or absence of Cul5, while wildtype Cas only stimulated FA disassembly when Cul5 was absent and Cas15F, a mutant that cannot signal downstream, did not stimulate FA disassembly even when Cul5 was absent (Figure 4—figure supplement 2b). These results suggest that SOCS6 inhibits FA disassembly dependent on binding to both Cas and CRL5. However, unlike Cul5, SOCS6 did not impact FA assembly rates (Figure 4b), suggesting that, while an increase in pYCas (and other SOCS6 targets) is sufficient to stimulate disassembly, increased pYCas does not accelerate assembly. Other SOCS proteins may target different pY substrates for ubiquitination by CRL5 to regulate FA assembly.

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SOCS6 localizes to FAs by binding to pYCas

CRL5 may regulate FA dynamics by directly interacting with substrates in FAs or by altering the steady-state concentrations of FA proteins in the cytoplasm. We investigated whether CRL5 localizes to FAs. Available antibodies revealed endogenous Cul5 in the nucleus and throughout the cytosol (data not shown). Cul5 was not detectably enriched in FAs. In addition, none of the available SOCS protein antibodies we tested were sufficiently sensitive or specific to detect endogenous SOCS proteins. However, when we transiently transfected HeLa cells with T7 epitope-tagged SOCS6, the tagged protein was enriched in FAs relative to the rest of the ventral membrane (Figure 5a and Figure 5—figure supplement 1). Enrichment was slight relative to the cytosol, suggesting a low affinity of binding and the potential for rapid exchange.

We investigated the basis for SOCS6 association with FAs. Because FAs contain pYCas and because SOCS6 binds pYCas (Petch et al., 1995; Teckchandani et al., 2014), we tested whether pYCas is required for SOCS6 localization. FA localization of SOCS6 was inhibited in cells treated with Cas siRNA or with the SFK inhibitor SU6656 (Figure 5a). Moreover, a point mutation in the phosphotyrosine-binding SH2 domain of SOCS6 inhibited binding to pYCas (Figure 5—figure supplement 2a) and prevented FA localization (Figure 5a). Therefore, pYCas is required for SOCS6 localization to FAs.

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To test whether SOCS6 also localizes to front FAs in migrating MCF10A cells, we stably expressed wildtype mCherry-tagged SOCS6 (mChSOCS6WT) in MCF10A cells. Unfortunately, stable expression of mChSOCS6WT down-regulated Cas and, perhaps as a consequence, mChSOCS6WT was not detected in FAs (Figure 5—figure supplement 2b and data not shown). We attempted to express the mChSOCS6LCQQ mutant, which does not stimulate Cas degradation. However, this mutant was only expressed at low level in MCF10A cells, perhaps due to instability or toxicity of the mutant (Figure 5—figure supplement 2a,b). Therefore, we tested whether treating mChSOCS6WT-MCF10A cells with MLN4924, which inhibits the neddylation process required for CRL activity (Enchev et al., 2015; Petroski et al., 2005; Soucy et al., 2009), might allow recovery of Cas levels and localize mChSOCS6WT to FAs. We found empirically that incubation with 5 µM MLN4924 for 6 hr followed by normal media for 2 hr allowed Cas levels to recover without disturbing FA stability (Figure 5—figure supplement 2c). Under these conditions, mChSOCS6WT co-localized with vinculin in FAs (Figure 5b). Remarkably, mChSOCS6WT was approximately 1.6-fold more abundant in FAs in the front 6 µm than FAs in the rest of the cell. We tested whether leading edge FAs are also enriched for pYCas. The ratio of pYCas to vinculin was approximately 3-fold higher in FAs in the front 6 µm than back (Fonseca et al., 2004) (Figure 5c). These results suggest that pYCas is enriched in FAs at the front of migrating cells and that SOCS6 preferentially associates with these FAs, as expected if SOCS6 binds to pYCas.

SOCS6 needs access to FAs to regulate their turnover

Since both Cas and SOCS6 exchange between the cytosol and adhesions, SOCS6 could regulate Cas levels or activity in either location. Inhibition of SOCS6 or Cul5 expression slows Cas turnover, causing a 2–3-fold increase in the steady-state level of Cas (Teckchandani et al., 2014) (Figure 1—figure supplement 1a). Accordingly, phosphorylated Cas levels in FAs increased approximately 3-fold when Cul5 expression was inhibited, especially in FAs in the front 6 µm of the cell (Figure 5—figure supplement 3). Since Cas exchanges rapidly between the cytosol and FAs (Janostiak et al., 2011; Machiyama et al., 2014), the increase in pYCas in FAs could be secondary to the increased Cas in the cytosol. Moreover, the chronic increase in pYCas in adhesions could accelerate adhesion turnover regardless of whether SOCS6 targets Cas in the cytosol or in FAs. We therefore tested whether SOCS6 could regulate FA turnover if it was sequestered away from adhesion sites. To this end, we constructed mChS6mito, a fusion of mCherry, SOCS6 and a sequence that targets the mitochondrial outer membrane (Bear et al., 2000). mChS6mito still bound to Cul5 and to Cas (Figure 6—figure supplement 1a), but localized to mitochondria instead of FAs (Figure 6—figure supplement 1b). In the nocodazole washout assay, mChS6 rescued FA disassembly in SOCS6-depleted cells but mChS6mito did not (Figure 6—figure supplement 1c). These results suggested that SOCS6 needs to access FAs in order to inhibit their disassembly.

We used two approaches to test whether CRL5^SOCS6 represses Cas during FA disassembly. First, we used an optogenetic approach. We used the light-regulated association between the CIBN and CRY2 proteins (Kennedy et al., 2010) to sequester SOCS6 away from adhesion sites during the disassembly process. We introduced two plasmids into HeLa cells from which endogenous SOCS6 had been removed with siRNA. One plasmid encoded CRY2 fused to mCherry-tagged SOCS6 (CRY2mChS6), and the second encoded CIBN fused to GFP and the mitochondrial targeting sequence (CIBNGFPmito) (Figure 6a). In the dark, CRY2mChS6 co-localized with FAK in FAs (Figure 6b left). In the light, however, CRY2mChS6 co-localized with CIBNGFPmito at the mitochondria and was not detected in FAs (Figure 6b right). We then tested whether CRY2mChS6 could rescue FA disassembly using the nocodazole washout assay. Transfected cells were kept in the dark during growth and nocodazole treatment, so that CRY2mChS6 could diffuse and maintain normal levels of Cas. We then subjected the cells to light or dark conditions and removed nocodazole. The results showed that light inhibits FA disassembly (Figure 6c). This implies that SOCS6 must have access to FAs during the disassembly process, and that the steady state level of Cas in the cytoplasm only plays a minor role.

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As an independent approach to assess the timing of CRL5^SOCS6 action, we again used the neddylation inhibitor, MLN4924, which inhibits CRL5 and other cullin-RING E3s (Soucy et al., 2009). MT-dependent FA disassembly was stimulated when MLN4924 was added an hour before and during 30 min of nocodazole removal (Figure 6d). A similar result was obtained with epoxomicin, a proteasome inhibitor (Meng et al., 1999) (Figure 6d). The results suggest that one or more CRLs and the proteasome need to be active during the disassembly process. Moreover, two hours of treatment with MLN4924 also increased FA assembly and disassembly rates at front but not back of migrating MCF10A cells (Figure 7a,b). This short exposure to MLN4924 increased pYCas levels in front but not back FAs and had no effect on total Cas levels (Figure 7c). Even though the turnover of many cell proteins is altered by MLN4924 and epoxomicin, the results are consistent with the hypothesis that the activities of Cullins and the proteasome are required during the process of FA disassembly. Combined with the optogenetics results, we suggest that SOCS6 regulates FA turnover locally by direct interaction with pYCas in or near FAs, and not by binding to or altering the steady state concentration of Cas or other proteins in the cytosol.

Figure 7

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The results suggest a model in which FA disassembly is slowed at the cell front by pYCas-dependent binding of SOCS6. SOCS6 recruits CRL5 and ubiquitinates pYCas, which is then removed through proteasomal degradation (Figure 7d). If SOCS6 or Cul5 is absent, if SOCS6 is held away from FAs, or if cullin or proteasomal inhibitors are present, then pYCas stimulates FA disassembly. A different, clathrin-dependent process prevails under the cell body. SOCS6 traffic thus controls the balance between FA assembly and disassembly in different parts of the cell.

The coordination between FA assembly and disassembly is critical for cell migration. We find that Cul5, SOCS substrate receptors, and the proteasome regulate FA dynamics in epithelial cells. While Cul5 regulates both assembly and disassembly, one of the CRL5 substrate receptors, SOCS6, is required for disassembly but not assembly, implying that other SOCS proteins are involved in FA assembly. SOCS6 inhibits FA disassembly by binding to the FA protein Cas and to the rest of the CRL5 complex. Our results suggest that CRL5^SOCS6, in conjunction with the proteasome, locally inhibits the SFK-Cas pathway in leading edge FAs to regulate their stability (Figure 7d). We hypothesize that Cas ubiquitination and degradation occurs close to or in the FA, although we have not shown this directly. Without CRL5^SOCS6 or proteasome activity, FAs become unstable, stress fibers are lost and the lamellipodium becomes unstable. Therefore, feedback control through CRL5^SOCS6 organizes FA and actin dynamics at the wound edge and ensures a normal leading lamellipodium. The environment may further regulate SOCS6, CRL5 or proteasome levels or activity, providing additional inputs to modulate the mode and speed of migration. This mechanism may be important in some cancers, where decreased SOCS6 expression has been associated with aggressive disease and increased metastasis (Fang et al., 2015; Letellier et al., 2014; Li et al., 2015; Wu et al., 2013; Zhu et al., 2013).

In migrating fibroblasts, FA turnover depends on Src and various Src substrates, including FAK, paxillin and Cas (Fincham et al., 1998; Webb et al., 2004). However, this mechanism has not been reported in other cell types. We found that the SFK-Cas pathway is also required for the rapid FA turnover at the leading edge of epithelial cells in which CRL5^SOCS6-dependent ubiquitination has been inhibited. This suggests that removing restraints imposed by CRL5^SOCS6 from migrating epithelial cells unveils a latent mechanism more similar to that in migrating fibroblasts, in which Src and Cas, and potentially also FAK and paxillin, stimulate FA turnover.

We and others have observed that Cas phosphorylation is greatest in the large FAs at the front of normal cells and around the edge of spreading cells (Fonseca et al., 2004; Sawada et al., 2006). Moreover, SOCS6 is specifically recruited to the leading edge adhesions by binding to pYCas, and our optogenetics and inhibitor experiments imply that CRL5^SOCS6 and the proteasome act at the FAs to counteract pYCas and to slow disassembly. Towards the rear, Cas is less highly phosphorylated, there is less SOCS6, and disassembly is slow regardless of SOCS6 or Cul5 activity. This suggests that the regulation of SOCS6 localization is critical for selective stabilization of FAs at the leading edge as cells migrate forwards.

Inhibition of SOCS6-CRL5 did not induce FA disassembly in cells that lack MTs. This suggests that MTs trigger disassembly by a pathway that is inhibited by SFKs and Cas. MT-induced FA disassembly requires the MT motor kinesin-1, presumably to mediate anterograde traffic along MTs towards the FA (Krylyshkina et al., 2002). Indeed, MTs deliver a protein complex including IQSEC1, an activator of the Arf6 small GTPase, to FAs (Yue et al., 2014). Arf6, together with clathrin, dynein, and clathrin adaptors such as Dab2 and Numb, stimulates integrin β1 endocytosis and MT-dependent adhesion turnover in keratinocytes and HeLa cells (Chao et al., 2009; Ezratty et al., 2009; Ezratty et al., 2005; Nishimura et al., 2007; Yue et al., 2014). This suggests that MTs deliver endocytic machinery and that integrin endocytosis stimulates FA disassembly. MTs are also involved in weakening the integrin-ECM connection by delivering matrix metalloproteases to FAs (Stehbens et al., 2014). However, our evidence suggests that MT-dependent integrin endocytosis is not involved in the SFK-Cas FA disassembly pathway. Clathrin-coated pits are absent from the ventral surface of the leading edge of motile glioblastoma cells (Kural et al., 2015) and were absent from the long leading lamellipodium of Cul5-deficient MCF10A cells. Moreover, clathrin knockdown inhibited the slow FA disassembly in normal HeLa cells but not the rapid FA disassembly in Cul5-deficient HeLa cells. Therefore, the MT-dependent signal for FA disassembly in Cul5-deficient cells is unlikely to involve clathrin-dependent endocytosis. The exact nature of the MT trigger for Cas-CRL5^SOCS6 regulated disassembly remains to be determined.

Other studies have implicated ubiquitination in cell migration and FA dynamics. RING protein XRNF185, which destabilizes paxillin via the proteasome, stimulates mesodermal cell migration during convergent extension in Xenopus embryos (Iioka et al., 2007). It is not clear whether paxillin ubiquitination occurs locally in this system. On the other hand, paxillin ubiquitination is also stimulated by ubiquitin ligase RNF5, but RNF5 does not induce paxillin degradation and it slows, rather than stimulates, cell migration (Didier et al., 2003). Other authors reported that TRIM5 stimulates paxillin turnover and increases FA disassembly. This is the opposite of the effect of CRL5^SOCS6 in inhibiting disassembly (Uchil et al., 2014). Ubiquitination may also be involved in an autophagy (ATG) pathway for FA disassembly (Kenific et al., 2016). Inhibiting autophagy slows FA assembly and disassembly and increases FA lifetime and size. While it is unclear how autophagy stimulates FA assembly, the evidence suggests that an ATG cargo receptor, NBR1, recruits autophagosomes to FAs to stimulate FA disassembly. Interestingly, NBR1 contains a ubiquitin-binding domain, suggesting that ubiquitination may localize NBR1 to FAs during disassembly.

Perhaps our most surprising finding is that SOCS6 needs to be present in FAs, and cullins and the proteasome need to be active, to regulate FA dynamics. This implies that the localized ubiquitination and degradation of pYCas regulates FA disassembly. Localized degradation of talin by the protease calpain also triggers FA disassembly (Bhatt et al., 2002; Franco et al., 2004). However, calpain is an endopeptidase that creates specific cleavage products, and the talin fragments remain in the FA and regulate FA stability (Huang et al., 2009). In contrast, ubiquitin-dependent proteolysis typically requires translocation of the ubiquitinated protein into the proteasome (Ravid et al., 2008). Removal of Cas by this mechanism would be expected to open up sites in the FA where other Cas molecules could bind. It is known that most of the Cas in a FA exchanges with Cas from the cytosol with a very rapid (~20 s) rate constant (Janostiak et al., 2011; Machiyama et al., 2014). It seems remarkable that ubiquitination and degradation of pYCas would affect the kinetics of FA disassembly if new Cas molecules could replace degraded molecules with such speed. New approaches will be required to measure the exact sequence of events – Cas binding, phosphorylation, SOCS6 binding, ubiquitination and proteolysis – in FAs during cell migration.

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Author details

1. Anjali Teckchandani

   Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States

   Contribution

   AT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Jonathan A Cooper

   Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States

   Contribution

   JAC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   jcooper@fhcrc.org

   Competing interests

   JAC: Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-8626-7827

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