Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim

Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼2 min. This actin reorganization was triggered by an intracellular Ca²⁺ burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca²⁺-dependent perinuclear actin assembly. However, the endoplasmic reticulum- and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca²⁺- and INF2-dependent manner.Cells can sense and adapt to their physical microenvironment through specific mechanosensing mechanisms. These properties are often mediated by the actin cytoskeleton, which can be modulated by a wide range of forces. Fluid shear stress, for example, induces actin stress fiber assembly and realignment along the direction of flow (1–4), whereas the cyclic stretch of an elastic substrate induces a reorientation of stress fibers under some angle to the direction of stretch (5–8). Applying mechanical force to cells by a microneedle results in focal adhesion growth and activation of formin-type actin nucleators (9, 10). Similarly, local application of force through fibronectin or collagen-coated beads trapped by optical or magnetic tweezers leads to the local reorganization of the actin cytoskeleton. This response is associated with reinforcement of bead attachment (11), recruitment of additional actin-associated proteins (12), and activation of a variety of signaling pathways (13–17). Most studies to date have explored the effects of force on actin structures directly associated with the sites of force application, such as focal adhesions and stress fibers. However, the immediate effect of force on the assembly of actin structures distal from the sites of force application has not been assessed. Such process is despite distal effects having potential implications in the transduction of local forces from the cell periphery to nuclear events (18).In this study, we used a local mechanical force application device and examined the large-scale actin reorganization during and after force application. Remarkably, we identified reversible actin polymerization in the perinuclear region within 1 min after mechanical stimulation. Intracellular Ca²⁺ bursts were found to be essential for the perinuclear actin response. Furthermore, we showed that a potent actin polymerization factor, inverted formin-2 (INF2), was involved in the perinuclear actin remodeling. Specifically, INF2 colocalized with a transient actin structure in the perinuclear region. A reduction in the level of INF2 resulted in the attenuation of this actin remodeling process. This work reveals a previously unidentified mechanotransduction response, whereby external mechanical stimulation induces a rapid transient perinuclear actin polymerization mediated by Ca²⁺ and formin.