Zengzhen
Liu‡
,
Philippe
Bun§
,
Nicolas
Audugé
,
Maïté
Coppey-Moisan
and
Nicolas
Borghi
*
Institut Jacques Monod, Unité Mixe de Recherche 7592, Centre National de la Recherche Scientifique, Université Paris-Diderot, 304B Bât. Buffon, 15 rue Hélène Brion, Paris 75013, France. E-mail: nicolas.borghi@ijm.fr; Tel: +33 1 57 27 80 41
First published on 4th May 2016
Rigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions (FAs) are complexes that anchor cells to the matrix. Among their components, vinculin undergoes an auto-inhibitory head–tail interaction that regulates the recruitment of, and interactions with its partners in a force-dependent manner. It is unknown, however, whether this mechanism is involved in substrate rigidity sensing. Here, we use a range of quantitative fluorescence microscopies on live human Mesenchymal Stem Cells to address this question. We identify two distinct rigidity-sensing molecular modules in FAs, one of which involves vinculin and talin, is regulated by vinculin head–tail interaction, and targets cell morphology. Vinculin and talin are recruited independently in a rigidity-dependent manner to FAs where they directly interact in a rigidity-independent stoichiometry at a site proximal to talin head. Vinculin head–tail interaction is required on soft substrates to destabilize vinculin and talin in FAs, and to allow hMSCs branching. Another module involves paxillin and FAK, which soft substrates also destabilize, but independently of vinculin head–tail interaction. This multi-modularity may be key to allow a versatile response to complex biomechanical cues.
Insight, innovation, integrationRigidity sensing is a critical determinant of cell fate and behavior but its molecular mechanisms are poorly understood. Focal adhesions are complexes that anchor cells to the matrix. Among their components, vinculin undergoes an auto-inhibitory head–tail interaction regulating interactions with its partners in a force-dependent manner. Whether this mechanism is involved in rigidity sensing is unknown. Using quantitative fluorescence microscopies on live human Mesenchymal Stem Cells, we identify two distinct rigidity-sensing molecular modules in FAs, one of which involves vinculin and talin, is regulated by vinculin head–tail interaction, and targets cell morphology. Another module involves paxillin and FAK, but independently of vinculin head–tail interaction. This multi-modularity may be key to allow a versatile response to complex biomechanical cues. |
Focal adhesions (FAs) are macromolecular complexes that transmit cell traction forces to the cell substrate. FAs were early found to respond to substrate rigidity,4,6 and a number of their components respond to mechanical forces in situ or in vitro.7–9 Notably, vinculin recruitment to FAs increases with cell contractility, external forces7,10–12 and substrate rigidity.4,13 Moreover, a recent study has shown the requirement of vinculin for proper rigidity-dependent differentiation of human Mesenchymal Stem Cells (hMSCs).14
Interestingly, vinculin comprises a head and a tail domain that bind a number of FA and actin-regulating proteins and membrane lipids,15 but these interactions are competed by a head–tail homo-interaction.16,17 These functional, as well as structural studies18–20 support a model by which the release of vinculin head–tail interaction in FAs involves at least two binding partners, and possibly tension-dependent conformational changes. Indeed, vinculin undergoes conformation opening in FAs,21 and is also under mechanical tension.22 Talin, in turn, exhibits a number of vinculin-head binding sites (VBS) that recruit vinculin in vitro upon actomyosin-generated stretch,23 and is stretched in cells.24 Importantly, a recent study demonstrated in cells that talin tension depends on rigidity, vinculin association and actomyosin activity.25
In summary, the general picture is that in which vinculin act as a cell contractility-dependent master regulator of FA protein recruitment through modulation of its head–tail interaction, and is involved in a positive feedback between talin stretching and actomyosin contractility as a function of substrate rigidity. It is unknown, however, whether vinculin head–tail interaction is involved in rigidity sensing, whether talin interacts with variable numbers of vinculin proteins as a function of rigidity, and the overall effect of these events on FA growth.
To address these issues, we investigate the molecular mechanisms by which substrate rigidity affects hMSCs morphology, FA proteins recruitment, mobility and interactions, and their modulation by a vinculin interaction mutant (T12) that bears four point-mutations in the head-binding region of the tail.17 From higher-than-bone (glass, >50 GPa) to brain tissue-like (0.2 kPa) rigidity, we show that vinculin and talin are recruited independently in a rigidity-dependent manner to FAs, where they directly interact in a rigidity-independent stoichiometry in a site proximal to talin head. Impairment of vinculin head–tail interaction is not sufficient alone to allow vinculin–talin interaction but it stabilizes vinculin and talin in FAs so that their amount is no longer rigidity-dependent. In addition, the T12 mutant prevents hMSCs branching of soft substrates. In contrast, paxillin and FAK amount and turnover in FAs remain rigidity-sensitive independently of vinculin head–tail interaction.
To avoid intercellular mechanical interactions through the deformable substrate, we only considered in all experiments individual cells without neighbors in the observation field. Thus, as many observation fields as cells were captured in all experiments (see figure legends). Reported rigidities are those indicated in the original protocol.
Cell shape was extracted with ImageJ using standard background smoothing and contour segmentation procedures within custom-written macros. Cell morphology was quantified by its circularity (100 × 4π × area/perimeter2): the more circular, the less branched. Cell morphodynamics were quantified by the area A of the non-overlapping cell surface S (protrusions and retractions) between two consecutive time points (A(S(t + 1)XORS(t))) normalized to the average cell surface area A(S), similar to previously done.30
FA protein recruitment patches were segmented in ImageJ with an intensity-based mask. Patches were sorted in two bins based on their surface area (below or above 0.2 μm2).
Data were fitted with a single-exponential recovery model F = F0 + Fτ(1 − exp(−t/τ)), where F0 is the fluorescence that recovers before the first time point, Fτ the fluorescence of the mobile fraction characterized by the turnover time τ.
For FCS, auto-correlation functions G(τ) were analysed with a two species 3D diffusion model to recover slow and fast apparent diffusion coefficients:
FCCS analysis was performed after fluorescence lifetime filtering between channels. This approach efficiently corrects for spectral bleed-through and enable concentration- and laser power-independent cross-correlation amplitude measurement.31 Therefore, it allows unambiguous discrimination of true positive results from false positive cross-correlation systematically observed on non-filtered data. Presence of a complex involving the proteins of interest was considered positive when the cross-correlation curve could be well fitted with a 1-species 3D diffusion model yielding a physically sound diffusion coefficient. The positive control was a tandem dimer of GFP and mCherry proteins and the negative control co-expressed GFP and mCherry. This approach typically allows to measure interacting fractions as low as 10% of the total amount of either protein. As FCS, we focused on dimmer regions of interest.
FLIM-FRET analysis was performed by fitting the fluorescence decay of protein-tagged GFP with a single-exponential decay model N = N0exp(−t/τD) to yield an average fluorescence lifetime τD (or τDA) of protein-tagged GFP in the absence (or the presence) of the mCherry-tagged second protein of interest. We controlled that fluorescence lifetime differences between conditions were not due to variable expression ratios of GFP- and mCherry-tagged proteins.
To quantify cell branching as a function of substrate rigidity, we measured cell circularity on rigid and soft substrates (Fig. 1B and C). Cell circularity was lower on soft than on rigid substrates, regardless of whether or not cells exogenously expressed GFP-tagged WT FA proteins (Fig. 1C). In contrast, cells expressing the vinculin T12 mutant, impaired for head–tail interaction, exhibited no circularity decrease on soft substrates (Fig. 1C). Due to transient expression, GFP-tagged proteins levels span over ranges larger within than between rigidity conditions, and may not account for differences in results between conditions. Therefore, rigidity-dependent hMSCs branching requires vinculin head–tail interaction.
To test whether substrate rigidity also affected hMSCs morphodynamics, we quantified cell protrusions and retractions (Fig. S1, ESI†). In cells expressing GFP-tagged WT vinculin, cell protrusions and retractions surface area did not exhibit any significant trend as a function of substrate rigidity. Therefore, we hypothesized they do not depend on vinculin head–tail interaction. Indeed, cells expressing T12 vinculin exhibited rigidity-independent morphodynamics indistinguishable from that of WT vinculin cells (Fig. S1, ESI†).
Thus, substrate rigidity specifically affects hMSCs branching in a manner dependent on vinculin head–tail interaction.
To assess the role of vinculin head–tail interaction in the recruitment of other FA proteins as a function of substrate rigidity, we measured the sizes of FA protein recruitment patches in cells co-expressing the T12 mutant. Patches of T12 and of the other FA protein generally colocalized but their sizes were not necessarily the same (Fig. S2B, ESI†). Indeed, the size distribution of mCherry-tagged WT vinculin, paxillin and FAK patches followed the same rigidity-dependence whether or not the cells co-expressed the GFP-T12 mutant (Fig. 2B–D). Thus, T12 mutants do not affect the recruitment of WT vinculin, paxillin or FAK as a function of substrate rigidity. Of note, that cells recruit both WT and T12 vinculin in FAs on rigid substrates shows that T12 is expressed at levels that do not compete with WT vinculin. In contrast, expression of the mCherry-T12 mutant significantly affected GFP-talin recruitment as a function of rigidity (Fig. S2B, ESI†). In those cells, the size distribution of talin recruitment patches did not significantly decrease as substrate rigidity decreased (Fig. 2E). In other words, soft substrates tend to deplete FAs of WT vinculin, paxillin, and FAK but not of T12 and talin in the presence of T12. Therefore, vinculin head–tail interaction appears to control the rigidity-dependence of talin recruitment to FAs, but not that of paxillin or FAK.
While FRAP is well suited to study protein mobility in the sec and min timescale, it misses faster phenomena. To access the ms timescale, we thus performed FCS in the dimmer regions of recruitment patches (see Methods). GFP-vinculin, talin and paxillin all exhibited mobilities best fitted with two apparent diffusion coefficients each: a high coefficient in the 20 μm2 s−1 range, close to that of a freely diffusing protein, and a low coefficient in the 1 μm2 s−1 range, which magnitude reveals a protein mobility hindered by transient interactions with immobile ligands. Slow apparent diffusion coefficients of all tested proteins increased when substrate rigidity decreased (Fig. S3, ESI†). Therefore, ms-scale and s-scale mobilities of FA proteins are similarly rigidity-dependent.
To assess the role of vinculin head–tail interaction on FA protein mobilities as a function of substrate rigidity, we first monitored by FRAP the turnover of the mCherry-T12 mutant as well as that of GFP-talin in cells co-expressing the mCherry-T12 mutant. The mCherry-T12 mutant turnover was about an order of magnitude slower than that of GFP-WT vinculin and did not speed up with decreased substrate rigidity. In addition, the slow mobile fraction of T12 was much larger than that of WT vinculin, at the expense of the fast mobile fraction, and both mobile fractions were no longer rigidity-dependent (Fig. 3B). Similarly, apparent diffusion coefficients of T12 mutant were lower than that of WT vinculin and insensitive to substrate rigidity (Fig. S3B, ESI†). Therefore, vinculin mobility in general, and its rigidity-dependence in particular depend on vinculin head–tail interaction.
Moreover, although GFP-talin turnover time in mCherry-T12 mutant cells was rigidity-dependent, it was overall larger than in WT cells. Interestingly, the T12 mutant decreased the slow mobile fraction of talin, especially on soft substrates, but to the benefit of immobile fraction rather than the fast mobile fraction (Fig. 3C). At the msec timescale, T12 expression abolished the rigidity-dependence of the slow apparent diffusion coefficient of talin (Fig. S3C, ESI†). Therefore, vinculin head–tail interaction regulates talin mobility dependence on substrate rigidity.
GFP-mCherry tandem | GFP-Talin | GFP-Vinculin | GFP-T12 | |
---|---|---|---|---|
GFP-mCherry tandem | Yes (Fig. S3E, ESI) | |||
mCherry-Vinculin | No (Fig. S3G, ESI) | No | ||
mCherry-T12 | No | No |
Then, we sought to assess whether vinculin and talin directly interacted in FAs. To do so, we performed FRET-FLIM on hMSCs expressing either mCherry-WT or T12 vinculin, and GFP-talin where mCherry and GFP are bound to talin-binding vinculin head and talin head, respectively. As expected from FCCS results, we observed in the cytoplasm similar GFP-talin fluorescence lifetimes whether or not mCherry-tagged vinculin WT or T12 mutant was co-expressed (Fig. 4A). In contrast, GFP-talin fluorescence lifetime in recruitment patches was significantly lower when mCherry-tagged vinculin WT or T12 mutant were expressed (Fig. 4A). Fluorescence lifetime differences were not due to differences in relative expression of donor and acceptor (Fig. S4, ESI†). Therefore, talin and vinculin directly interact in recruitment patches but not in the cytoplasm, and the interaction occurs in proximity of talin head. In addition, GFP-talin fluorescence lifetime appeared independent of substrate rigidity or the expression of the T12 mutant (Fig. 4A). Therefore, the stoichiometry of talin and vinculin bound in proximity of talin head is independent of substrate rigidity or the possibility of head–tail interaction in vinculin.
In a recent study, Yamashita et al. have investigated in MEF cells the impact of substrate rigidity on FA proteins.13 In those cells, recruitment of vinculin was rigidity-dependent. Specifically, the total area, number, and intensity of vinculin recruitment patches insoluble in a cytoskeleton stabilization buffer were all higher on rigid substrates than on soft, and, somewhat surprisingly, higher than that of total (soluble + insoluble) vinculin recruitment patches on rigid substrates. Here, we observed in hMSCs that total vinculin recruitment patches were larger on rigid substrates (Fig. 2). Thus, while our conditions and results are not identical, they both are consistent with an increased recruitment of vinculin in FAs by rigid substrates. In addition, we show that recruitment patches of paxillin, talin and FAK are also rigidity-dependent, as they are larger on rigid substrates, similarly to vinculin (Fig. 2). Therefore, substrate rigidity appears to modulate FA protein recruitment in a fashion that retain their overall stoichiometry.
Previous studies have evidenced a key role for vinculin head–tail interaction in regulating cell adhesion and migration. Expression of the T12 mutant, impaired for head–tail interaction, increased cell spread area in vinculin-null MEF cells,33 and impaired the directional persistence of membrane protrusions in mouse melanoma cells.34 Here, we show that in hMSCs, T12 expression does not affect cell morphodynamics, which is moreover rigidity-independent (Fig. S1, ESI†). In contrast, T12 expression does impair increased cell branching on soft substrates (Fig. 1). Therefore, vinculin head–tail interaction is specifically required for the rigidity-sensitivity of cell branching in hMSCs. Importantly, rigidity sensing is significantly abolished by T12 expression even though cells also express endogenous WT vinculin.
The role of vinculin head–tail interaction in cell morphology is thought to result from its function in the recruitment of vinculin and other FA proteins to the cell–substrate interface. Indeed, expression of the T12 mutant in vinculin-null and NIH 3T3 cells induced more numerous vinculin recruitment patches that also recruited paxillin and talin.17,35 Moreover, T12 recruitment, unlike that of WT vinculin, persisted even after myosin activity disruption in vinculin-null cells,34 while vinculin closed conformation was observed to precede vinculin release from disassembling FAs.21 All together, these results indicate that vinculin head–tail interaction is required for the regulation of contractility-dependent vinculin recruitment to, and release from the cell–substrate interface. Here, we find that the T12 mutant forms larger recruitment patches than that of WT vinculin in hMSCs on soft substrates but not on rigid substrates, so that T12 recruitment patches size does not depend on substrate rigidity (Fig. 2). This result suggests that soft substrates favor vinculin conformation closing and head–tail interaction. Therefore, substrate rigidity appears to recapitulate the effect of intracellular contractility on vinculin recruitment, a result consistent with the correlation between substrate rigidity and cell traction forces in fibroblasts and epithelial cells,36,37 and that vinculin head–tail interaction limits the range of cell traction forces.33 Nevertheless, it is noteworthy that there is no one-to-one relationship between vinculin conformation and its contractility-dependent molecular tension22 or between vinculin molecular tension and cell traction forces.38
Additionally, we show that recruitment patch sizes of WT vinculin, paxillin and FAK but not talin are still rigidity-dependent in hMSCs also expressing the T12 mutant (Fig. 2). In contrast, a previous study has shown that paxillin, FAK, talin and other FA proteins still colocalize with the T12 mutant at the cell–substrate interface in cells impaired for actomyosin contractility.34 All together, these results reveal that paxillin and FAK recruitment appears to require both rigidity- and contractility-dependent vinculin recruitment and, independently of vinculin, a rigid substrate. This result also indicates that cell morphology rigidity response depends on vinculin head–tail interaction (Fig. 1) but not on paxillin and FAK recruitment as a function of rigidity (Fig. 4B). As a consequence, rigidity sensing exhibits an early signaling divergence toward cell morphology through talin and vinculin head–tail interaction on one hand, and toward other targets through FAK and paxillin on the other hand.
Protein amounts in FAs are generally likely to reflect recruitment kinetics: the more recruited the protein, the less mobile. Indeed, vinculin, talin, paxillin and FAK, which all exhibited smaller recruitment patches on softer substrates (Fig. 2), concomitantly underwent increased turnover, and most exhibited increased fast mobile fractions and slow apparent diffusion coefficients, and decreased slow mobile fractions (Fig. 3 and Fig. S3, ESI†). Noteworthy, Dumbauld et al. have observed that vinculin turnover in FAs correlates with the magnitude of cell traction forces,33 further supporting the similar roles of substrate rigidity and cell contractility on vinculin recruitment. Consistently, Yamashita et al. have recently found that in FAs of MEF cells, vinculin exhibits on soft substrates a smaller immobile fraction than on rigid substrates. However, they observed that paxillin rather exhibits on rigid substrates more mobile features than on soft substrates.13 All together, these results suggest a fairly robust conservation of the rigidity-dependence of vinculin recruitment through cell lineages, while other FA proteins such as paxillin may exhibit cell-type specific behaviors. These differences further support a model involving multiple rigidity-sensing modules within FAs.
Previous studies have shown that the T12 mutant expressed in vinculin-null or NIH 3T3 cells on rigid substrates exhibits in FAs a much slower turnover and smaller mobile fraction than WT vinculin, and also slowed down the turnover of talin, but not that of paxillin, in vinculin-null cells.17,35 Here, we consistently find that the T12 mutant has a much slower turnover and decreased fast mobile fraction and apparent diffusion coefficients compared to WT vinculin (Fig. 3 and Fig. S3, ESI†). Together with increased amounts in FAs on soft substrates compared to WT vinculin (Fig. 2), this further confirms the correlation between lower mobility and higher protein amount in FAs. In addition, we find that decreased substrate rigidity does not increase mobility features of the T12 mutant. Consistently, Dumbauld et al. have shown that T12 mutant turnover does not correlate with the magnitude of cell traction forces.33 Finally, we find that talin mobility features are generally lower or less rigidity-dependent in cells expressing T12 (Fig. 3 and Fig. S3, ESI†), consistent with larger amounts in FAs on soft substrates when the T12 mutant is expressed (Fig. 2). All together, these results supports that substrate rigidity, similarly to cell contractility, controls vinculin and talin turnover in FAs through a modulation of vinculin head–tail interaction.
Indeed, vinculin head domain interacts with talin in vitro, but vinculin head–tail binding competes with this interaction.39 Consistently, full length vinculin does not form a complex with talin in coimmunoprecipitates from NIH 3T3 cells.35 The T12 mutant from HEK283 cell lysates, however, is able to form a complex with the talin rod (residues 397–2541).17 Therefore, the release of vinculin head–tail interaction may allow full-length vinculin–talin interaction. Here, our FLIM-FRET experiments bring direct evidence that full length vinculin and talin do interact directly in proximity of talin head in FAs of live hMSCs (Table 1 and Fig. 4A). As recently proposed,40,41 we show by FLIM-FRET and FCCS that talin and vinculin do not interact in the cytoplasm (Table 1 and Fig. 4A). Additionally, we also show that the T12 mutant does not interact with talin in the cytoplasm (Table 1 and Fig. 4A). Thus, the mere release of vinculin head–tail interaction is not sufficient to allow interaction between the full-length proteins in cells, possibly because vinculin conformation change also requires tension. This is also consistent with a talin pre-stretch requirement, in agreement with a previous study42 but in contrast with another.9 The lack of cytoplasmic complex between the T12 mutant and vinculin putative direct-binding partner paxillin (Table S1, ESI†) also supports this model and, together with the consistent lack of cytoplasmic complex between WT vinculin and paxillin (Table S1, ESI†), contradicts recently observed vinculin–paxillin cytoplasmic complexes.40 The reason for this latter discrepancy possibly lies in our filtered FCCS method that efficiently discards false positive correlation systematically observed in unfiltered FCCS experiments. Altogether, our results do not support that multi-molecular FA protein modules are pre-assembled in the cytoplasm.
A recent study has shown that full length talin under actomyosin-generated tension recruits full length vinculin in vitro.23 In addition, the more stretched the talin rod, the higher the number of vinculin heads it binds in vitro.9 Since in fibroblast FAs talin stretching occurs24 and talin tension is rigidity-dependent,25 an attractive model would be that talin increasingly stretches and thereby interacts with an increasing number of vinculin proteins as rigidity increases. Talin displays five VBS in the three alpha-helix bundles most proximal to its head (R1 to R3), the farthest of which (VBS1 in R3) binding vinculin under the weakest stretching force.42 Therefore, additional vinculin–talin interactions upon increased stretching should occur closer to talin head than R3, namely in R1 and R2. Nevertheless, our FRET results support that the stoichiometry of the interaction between vinculin and talin in proximity of its head is rigidity- and T12-independent (Fig. 4A). It is therefore unlikely that the VBS most proximal to talin head in R1 and R2 be increasingly occupied with vinculin upon increased rigidity. Whether only VBS1 or additional VBS in R1 and R2 are constitutively occupied regardless of substrate rigidity is, however, unknown. Additionally, we show that the amounts and mobilities in FAs of T12 and talin in the presence of T12 are rigidity-independent (Fig. 2, 3 and Fig. S3, ESI†). Therefore, our results are more consistent with a model where vinculin and talin recruitment and direct interaction in proximity of talin head in FAs involve a rigidity-dependent release of vinculin head–tail interaction and conformation change, and a rigidity-independent, vinculin-binding-sufficient talin constitutive stretching within our rigidity range. Symmetrically, vinculin head–tail interaction, rather than talin relaxation, may trigger vinculin and talin dissociation and concomitant release from FAs. In other words, while vinculin head and talin interact in a ratchet-like fashion upon tension,42 vinculin tail loosens the ratchet as a function of rigidity. Noteworthy, this model is not inconsistent with talin tension dependence on substrate rigidity. Indeed, talin tension substantially changes below 1 kPa but increases only modestly in comparison at higher rigidities.25 Conversely, most vinculin head–tail interaction-dependent recruitment of vinculin and talin beyond the size of nascent FAs occurs at rigidities higher than about 10 kPa (Fig. 2). Consequently, low rigidities may be sufficient for talin tension to allow maximum occupation of VBS near talin head. Finally, rigidity sensing at FAs may rely on talin stretching at very low rigidities and vinculin head–tail interaction at higher rigidities. Conserved stoichiometry between talin and vinculin bound near talin head may be useful to ensure balanced protein recruitment during FA growth beyond nascent FA size.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ib00307e |
‡ Present address: Institut Curie, Unité Mixte de Recherche 144, Centre national de la recherche scientifique, Université P. & M. Curie, Paris 75005, France. |
§ Present address: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany. |
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