Project description:Tissue mechanical homeostasis, the concept that cells sense mechanical properties and alter rates of ECM synthesis, assembly and degradation, is of broad interest in biology and medicine, but there is little direct support or insight into mechanisms. Tissue mechanical properties such as elasticity and stiffness are determined primarily by the extracellular matrix (ECM). We therefore set out to test the mechanical homeostasis hypothesis by developing mutations in the mechanosensitive protein talin1 that alter cells’ sensing of ECM stiffness. We identified the side-to-side interaction between talin1 rod domain helix bundles 1 and 2 (R1 and R2) as a novel mechanosensitive site. Mutations that decrease the affinity of the interaction result in a leftward shift in cellular stiffness sensing curves, enabling cells to spread and exert tension on softer substrates. Opening of the R1-R2 interface promotes binding of the Arp2/3 subunit ArpC5L, which is required for the altered stiffness sensing. Introduction of these talin mutations into mice resulted in softer tissue in the ascending aorta with less fibrillar collagen and rupture under lower pressure ex vivo. Together, these results demonstrate that altering cellular stiffness sensing results in altered ECM deposition, tissue stiffness and strength, thus providing direct support for the mechanical homeostasis hypothesis. These results also identify a novel mechanosensitive interaction in the talin rod domain that contributes to this mechanism.
Project description:The mechanical microenvironment of primary breast tumors plays a substantial role in promoting tumor progression. While the transitory response of cancer cells to pathological stiffness in their native microenvironment has been well described, it is unclear whether mechanical stimuli in the primary tumor influence distant, late-stage metastatic phenotypes in absentia. Here, we show that primary tumor stiffness promotes stable yet non-genetically heritable phenotypes in breast cancer cells. This “mechanical memory” instructs cancer cells to adopt and maintain increased cytoskeletal dynamics, traction force, and 3D invasion in vitro, in addition to promoting osteolytic bone metastasis in vivo. We established a “mechanical conditioning score” comprised of mechanically-regulated genes as a proxy measurement of tumor stiffness response, and we show that it is associated with bone metastasis in patients. Using a discovery approach, we mechanistically traced mechanical memory in part to ERK-mediated mechanotransductive activation of RUNX2, an osteogenic gene bookmarker and bone metastasis driver. This combination of traits allows for the stable transactivation of osteolytic target genes which persists after cancer cells disseminate from their activating microenvironment. Using genetic, epigenetic, and functional approaches, RUNX2-mediated mechanical memory can be stimulated, repressed, selected, or extended. In concert with previous studies detailing how biochemical properties of the primary tumor stroma influence distinct metastatic phenotypes, the impact of local biomechanical properties we present here support a generalized model of cancer progression in which the integrated properties of the primary tumor microenvironment govern cell behavior in the metastatic microenvironment.
Project description:Address the effect of mechanical stimulation by confinement on the status of activation of dendritic cells and the role of cPLA2 in orchestrating this response. We performed gene expression analysis using data obtained from RNA-seq of 3 independent experiment of DCs from WT and KO of cPLA2.
Project description:Cells interact with their mechanical environment and respond in consequence. Mechanical cues can have a wide range of influences on cell behaviour, ranging from guidance of differentiation and cell fate to immune activation. The impact of substrate stiffness on primary macrophages - a key player in innate immunity and inflammation - had not been previously studied. We prepared bone marrow-derived macrophage cultures from adult rat hematopoietic stem cells exposed to M-CSF, and cultured these on polyacrylamide substrates of controlled stiffness (ranging from 50 to 0.1 kPa shear modulus, covering the range found in physiological tissues) for 3 days. The RNA from these cells was then extracted and sequenced.
Project description:Mechanical stress is a measure of internal resistance exhibited by a body or material when external forces, such as compression, tension, bending, etc. are applied. The study of mechanical stress on health and aging is a continuously growing field, as major changes to the extracellular matrix and cell-to-cell adhesions can result in dramatic changes to tissue stiffness during aging and diseased conditions. For example, during normal aging, many tissues including the ovaries, skin, blood vessels, and heart exhibit increased stiffness, which can result in a significant reduction in function of that organ. As such, numerous model systems have recently emerged to study the impact of mechanical and physical stress on cell and tissue health, including cell-culture conditions with matrigels and other surfaces that alter substrate stiffness and ex vivo tissue models that can apply stress directly to organs like muscle or tendons. Here, we sought to develop a novel method in an in vivo, model organism setting to study the impact of mechanical stress on aging, by increasing substrate stiffness in solid agar medium of C. elegans. To our surprise, we found shockingly limited impact of growth of C. elegans on stiffer substrates, including limited effects on cellular health, gene expression, organismal health, stress resilience, and longevity. Overall, our studies reveal that altering substrate stiffness of growth medium for C. elegans have only mild impact on animal health and longevity; however, these impacts were not nominal and open up important considerations for C. elegans biologists in standardizing agar medium choice for experimental assays.
Project description:The Rho family GTPases, Rac and Rho, play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac is dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified Activating Transcription Factor 3 (ATF3) as a major target of stiffness/Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1.
Project description:Mechanical feedback from the tumor microenvironment regulates an array of processes underlying cancer biology. Routine culture and adaptation of cancer cell lines to unnaturally rigid plastic or glass substrates leads to profound changes in their growth, metastatic potential and potentially chemotherapeutic response. Microarray studies were conducted to probe the impact of substratum stiffness on the regulation of genetic pathways in mammary tumor cells, and immortalized cancer cell lines, that modulate sensitivity and resistance towards clinically-approved chemotherapeutics We used microarrays to detail the global programme of gene expression underlying cellular response to substrates of different mechanical stiffness.
Project description:BACKGROUND: Previous genomic studies with human tissues have compared differential gene expression between 2 conditions (ie, normal versus diseased) to identify altered gene expression in a binary manner; however, a potentially more informative approach is to correlate the levels of gene expression with quantitative physiological parameters. METHODS AND RESULTS: In this study, we have used this approach to examine genes whose expression correlates with arterial stiffness in human aortic specimens. Our data identify 2 distinct groups of genes, those associated with cell signaling and those associated with the mechanical regulation of vascular structure (cytoskeletal-cell membrane-extracellular matrix). Although previous studies have concentrated on the contribution of the latter group toward arterial stiffness, our data suggest that changes in expression of signaling molecules play an equally important role. Alterations in the profiles of signaling molecules could be involved in the regulation of cell cytoskeletal organization, cell-matrix interactions, or the contractile state of the cell. CONCLUSIONS: Although the influence of smooth muscle contraction/relaxation on arterial stiffness could be controversial, our provocative data would suggest that further studies on this subject are indicated.<br><br>Note that files GSM6179.txt and GSM6182.txt as imported from GEO are identical.