LSP1 COMPETES WITH SUPERVILLIN FOR F- ACTIN AND MYOSIN IIA REGULATORS

128 From previous studies, LSP1 is known to have the ability to directly bind myosins, such as myosin 1e ¹¹¹, however this does not seem to be the case with myosin IIA, where the interaction is mediated by F-actin, as we demonstrated by myosin im-munoprecipitation experiments in the presence of Mg²⁺ /ATP, which reduce the amount of coprecipitated F-actin with concomitant reduction of LSP1, and myosin cosedimentation assays with pure proteins. We can therefore reason that deple-tion of LSP1 leads to concomitant reducdeple-tion of myosin IIA, both at podosomes and the cell cortex, by decreasing the amount of F-actin bundles and, as a consequence, the number of myosin IIA molecules recruited.

In conclusion, we describe a new key role for LSP1 in regulating the mechanosens-ing activity of podosomes by ensurmechanosens-ing the correct functionality of podosome lateral actin fibers. In addition, impairment of LSP1 activity is not only affecting dynamics of single podosomes, but also alters the stability of the whole podosome network and, in consequence, the overall migratory capability of macrophages.

3. LSP1 competes with supervillin for F-actin and myosin IIA

129 They also differ in myosin IIA activation, with LSP1 inducing only moderate activi-ty, whereas supervillin directly binds contractile myosin IIA and induces further activation, thus acting as a myosin hyper activator ⁷⁹. The preferential distribution of supervillin at successor podosomes leads to enrichment of active myosin IIA at this subset, which likely contributes to podosome dissolution, as observed previ-ously in our lab ⁷⁹.

Interestingly, LSP1 and supervillin not only share the interaction with the same subset of myosin regulators but can also compete for them. In fact, when both proteins are overexpressed in macrophages, we observe a concomitant redis-tribution of L-MLCK, calmodulin and Ser19-phosphorylated myosin light chain (pMLC, which is a direct indicator of myosin activity) from the leading edge, where LSP1 is mostly enriched, towards the trailing edge, where supervillin takes over.

This competition contributes to generate distinct subcellular zones of different myosin IIA activity, in other words LSP1 and supervillin can induce and sustain a symmetry break in the cell by differentially regulating actomyosin contractility.

Normally resting macrophages are round-shaped and do not move, indicating that all the internal forces are dynamically counterbalanced. A symmetry breaking event represents a quick change of the internal equilibrium occurring at the mac-romolecular level right before the establishment of a specific polarization ¹²³. It is a complex process that involves many factors, such as the cytoskeleton, soluble fac-tors and a wide range of proteins, all connected by intra- and extracellular signals.

Interestingly, cells can polarize even in the absence of external stimuli, implying that the system normally operates close to instability threshold and thus is highly sensitive to minimal fluctuations ¹²⁴. Symmetry breaking is an essential process to generate functional polarization and sustain directed cell migration. In many cell types, it is normally achieved by differential recruitment of the myosin II isoforms A and B ¹²⁵. However, macrophages lack the isoform B, thus an alternative machin-ery, such as specific recruitment of myosin IIA regulators with different activities (i.e. LSP1 versus supervillin), becomes essential.

Recently, an interesting model has been proposed to describe migratory cell polarization and symmetry breaking. This model is based on the assumption that cells normally assemble and maintain two major F-actin networks that have

differ-130 ent organization and dynamics: branched filaments at sites of protrusion and con-tractile actomyosin bundles at cell cortex that has also the intrinsic feature to “se-quester” and confine myosin ¹²⁶.

These two networks locally compete for the same resource, that is G-actin, deter-mining the degree of migratory cell polarity, with branched network pushing the edge outward, by actin nucleation, and circumferential actomyosin bundles pulling the edge inward ¹²⁶. Obviously, in such a model, myosin plays a pivotal role as it needs to be moderately active. In fact, extreme activation would lead to immobile cell with total inhibition of cell protrusion, whereas inactivation would result in formation of multiple edge protrusions and inefficient formation of single axis of polarity, an essential condition for functional migration ¹²⁶.

According to this model of symmetry breaking, the formation of multiple protru-sive sites (i.e. non-functional cell migration) observed in LSP1 knockdown condi-tions can be explained by strong reduction of cortical actomyosin contractility, be-ing LSP1 an efficient actin bundler that normally supports moderate myosin IIA ac-tivity at the cell cortex. Further confirmation for this model come from rescue ex-periments using supervillin, which normally competes with LSP1 for F-actin. In ab-sence of LSP1, supervillin is no longer confined to successor podosomes but can instead extend its range of action by localizing to precursors, and in general to the cell periphery, restoring an intermediate level of myosin activation, thanks to its capability of directly binding myosin IIA and regulators.

In conclusion, we provide detailed observations to describe how two actomyo-sin regulators, LSP1 and supervillin, which are localized to different subcellular compartments and differ in their ability to induce moderate or high myosin activi-ty, respectively, can generate and sustain symmetry breaking in macrophages.

However, one challenging question still remains open: what is driving the differen-tial localization of the two actomyosin machineries that is responsible of symmetry breaking?

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