A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSCs), high-magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low-level signals to regulate behavior. Exposing MSC to extremely low-magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here, we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with “outside-in” signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent “inside–inside” signaling. Stem Cells 2013;33:2063–2076

: The cell cytoskeleton interprets and responds to physical cues from the microenvironment. Applying mechanical force to mesenchymal stem cells induces formation of a stiffer cytoskeleton, which biases against adipogenic differentiation and toward osteoblastogenesis. mTORC2, the mTOR complex defined by its binding partner rictor, is implicated in resting cytoskeletal architecture and is activated by mechanical force. We asked if mTORC2 played a role in mechanical adaptation of the cytoskeleton. We found that during bi-axial strain-induced cytoskeletal restructuring, mTORC2 and Akt colocalize with newly assembled focal adhesions (FA). Disrupting the function of mTORC2, or that of its downstream substrate Akt, prevented mechanically induced F-actin stress fiber development. mTORC2 becomes associated with vinculin during strain, and knockdown of vinculin prevents mTORC2 activation. In contrast, mTORC2 is not recruited to the FA complex during its activation by insulin, nor does insulin alter cytoskeletal structure. Further, when rictor was knocked down, the ability of mesenchymal stem cells (MSC) to enter the osteoblastic lineage was reduced, and when cultured in adipogenic medium, rictor-deficient MSC showed accelerated adipogenesis. This indicated that cytoskeletal remodeling promotes osteogenesis over adipogenesis. In sum, our data show that mTORC2 is involved in stem cell responses to biophysical stimuli, regulating both signaling and cytoskeletal reorganization. As such, mechanical activation of mTORC2 signaling participates in mesenchymal stem cell lineage selection, preventing adipogenesis by preserving β-catenin and stimulating osteogenesis by generating a stiffer cytoskeleton. © 2014 American Society for Bone and Mineral Research.

The nucleus has long been postulated to play a critical physical role during cell polarization and migration, but that role has not been defined or rigorously tested. Here, we enucleated cells to test the physical necessity of the nucleus during cell polarization and directed migration. Using enucleated mammalian cells (cytoplasts), we found that polarity establishment and cell migration in one dimension (1D) and two dimensions (2D) occur without the nucleus. Cytoplasts directionally migrate toward soluble (chemotaxis) and surface-bound (haptotaxis) extracellular cues and migrate collectively in scratch-wound assays. Consistent with previous studies, migration in 3D environments was dependent on the nucleus. In part, this likely reflects the decreased force exerted by cytoplasts on mechanically compliant substrates. This response is mimicked both in cells with nucleocytoskeletal defects and upon inhibition of actomyosin-based contractility. Together, our observations reveal that the nucleus is dispensable for polarization and migration in 1D and 2D but critical for proper cell mechanical responses.