Studies of cellular mechano-signaling have often utilized static models that do not fully replicate the dynamics of living tissues. Here, we examine the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 h, 4% CTS at 1 Hz), cell characteristics mimic responses to increased substrate stiffness. As the strain regime is intensified (frequency increased to 5 Hz), we characterize rapid establishment of a broad, structured and reversible protein-level response, even as transcription is apparently downregulated. Protein abundance is quantified coincident with changes to protein conformation and post-translational modification (PTM). Furthermore, we characterize changes to the linker of nucleoskeleton and cytoskeleton (LINC) complex that bridges the nuclear envelope, and specifically to levels and PTMs of Sad1/UNC-84 (SUN) domain-containing protein 2 (SUN2). The result of this regulation is to decouple mechano-transmission between the cytoskeleton and the nucleus, thus conferring protection to chromatin.

Word count, excluding figure captions and references (3391/3500)The stiffness of tissue correlates with its ability to resist mechanical damage, with the structure and integrity of the human body defined by stiff tissues such as skin, muscle, cartilage and bone. Tissue mechanical properties are determined by the extracellular matrix (ECM), in particular by the identities and concentrations of its constitutive proteins 1-3 . ECM properties are further modulated by protein cross-linking, post-translational modifications (PTMs) and higher-order organisation. Cells resident within tissues maintain mechanical equilibrium with their environments 4, 5 , and the mechanical properties of cells are also regulated by the identities, concentrations, conformations and PTMs of structural intracellular proteins 1, 3, 6, 7 . The characteristics of adherent cells can be influenced by physical stimulation from the surrounding ECM, with protein content 1 , morphology 1, 8 , motility 9, 10 and differentiation potential 11, 12 amongst behaviours known to be affected by stiffness. Cells in living tissues experience microenvironments of diverse stiffness 5 , but are also subject to deformation during activity. Cells sense and respond to mechanical signals through pathways of mechanotransduction [13][14][15] , but must also maintain integrity and homeostasis within the tissue. A mismatch between mechanical loading and cellular regulation can contribute to pathology, such as in musculoskeletal and connective tissue disorders 16 , with ageing being a significant risk factor 17 .Here, we sought to compare responses to stiffness and mechanical loading in primary human mesenchymal stem cells (hMSCs), a cell type with important physiological and reparative roles, that have led to investigations of their therapeutic potential in tissues such as muscle 18 and heart 19 . We contrast cellular responses to stiffness and strain cycling, and identify a rapid, reversible and structured regulation of the proteome following high-intensity mechanical loading. Furthermore, we identify SUN domain-containing protein 2 (SUN2) as a strain-induced breakpoint in the linker of nucleo-and cytoskeleton (LINC) complex of proteins that acts as a pathway of intracellular mechano-transmission 13, 20 , thus enabling the nucleus to 'decouple' from the cytoskeleton in response to intense strain. Gilbert et al. Nuclear decoupling during mechanical loading Page 4 of 34 RESULTS Cyclic tensile strain (CTS) uncouples the correlation between cellular and nuclear morphologyPrimary hMSCs were cultured on stiffness-controlled polyacrylamide hydrogels or silicone elastomer sheets that could be subjected to CTS (both collagen-I coated). hMSCs were found to spread increasingly on stiffer substrates over a physiological range (2 -50 kPa, cultured for 3 days; Fig. 1a, Supplementary Fig. 1a), as has been reported previously 1,21 . Cells subjected to sinusoidal, equiaxial CTS for 1 hour at 1 or 2 Hz (change in strain = 4%) showed significantly increased spreading immediately after load...

Cell division is a period of danger for cells, as inaccurate segregation of chromosomes can lead to loss of cell viability or aneuploidy. In order to protect against these dangers, cells ultimately initiate mitochondrial apoptosis if they are unable to correctly exit mitosis. A number of important chemotherapeutics exploit this response to delayed mitotic exit, but despite this, the molecular mechanism of the apoptotic timer in mitosis has proved elusive. Some recent studies have now shed light on this, showing how passage through the cell cycle fine-tunes a cell's apoptotic sensitivity such that it can respond appropriately when errors arise.