Cell Mechanosensitivity to Extremely Low-Magnitude Signals Is Enabled by a LINCed Nucleus.

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.

Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity during in vitro Expansion.

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated ß-galactosidase activity (p < 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p < 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging.

Sun-mediated mechanical LINC between nucleus and cytoskeleton regulates ßcatenin nuclear access.

ßcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of ßcatenin in the cytoplasm has been well studied but ßcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of ßcatenin destruction complex element GSK3ß (glycogen synthase kinase 3ß) to increase nuclear ßcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3ß due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, ßcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of ßcatenin, resulting in decreased nuclear ßcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in ßcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of ßcatenin.