Only individual adhesions that could be followed from the point at which they were initially visible all the way through until complete disassembly were quantified for lifetime analysis. Membrane Dynamics Analysis For the membrane dynamics analysis, high magnification confocal movies of cells transfected with tagged FA markers were analyzed using the QuimP11 plugin for Dextrorotation nimorazole phosphate ester ImageJ. minutes, with movies playing back at ten frames per second.(AVI) pone.0091815.s005.avi (2.7M) GUID:?F870182D-1B76-4AA7-8FCA-1E75713A89CD Movie S5: U2OS cells stably expressing vinculin-YFP treated with control siRNA migrating on soft PDMS. Fluorescence images acquired every two minutes, with movies playing back at ten frames per second.(AVI) pone.0091815.s006.avi (6.4M) GUID:?BDF0A91A-D37F-415B-8DD0-7D84698F8615 Movie S6: U2OS cells stably expressing vinculin-YFP treated with control siRNA migrating on hard PDMS. Fluorescence images acquired every Dextrorotation nimorazole phosphate ester two minutes, with movies playing back at ten frames per second.(AVI) pone.0091815.s007.avi (7.3M) GUID:?2AC41BF8-3204-4CE8-9BCA-86832709F3E2 Movie S7: U2OS cells stably expressing vinculin-YFP treated with cdGAP siRNA migrating on soft PDMS. Fluorescence images acquired every two minutes, with movies playing back at ten frames per second.(AVI) pone.0091815.s008.avi (12M) GUID:?60A641D5-7631-46FB-B32E-E10CEA79AF76 Movie S8: U2OS cells stably expressing vinculin-YFP CCNB2 treated with cdGAP siRNA migrating on hard PDMS. Fluorescence images acquired every two minutes, with movies playing back at ten frames per second.(AVI) pone.0091815.s009.avi (4.5M) GUID:?6A0ED417-52CB-4AAD-8F62-929FCC10A85C Movie S9: Phase contrast time-lapse of control siRNA-treated U2OS cells plated in durotaxis chambers. The boundary between soft and hard matrix is usually marked in frame one. Phase contrast images acquired every ten minutes, with movies playing back at ten frames per second.(AVI) pone.0091815.s010.avi (5.3M) GUID:?620B2E48-017D-49F1-8884-7CE97C10984A Movie S10: Phase contrast time-lapse of cdGAP siRNA-treated U2OS cells plated in durotaxis chambers. The boundary between soft and hard matrix is usually marked in frame one. Phase contrast images acquired every ten minutes, with movies playing back at ten frames per second.(AVI) pone.0091815.s011.avi (3.3M) GUID:?6F939F8D-78EB-4E8D-BAE5-B8975B92B591 Abstract Motile cells are capable of sensing the stiffness of the surrounding extracellular matrix through integrin-mediated focal adhesions and migrate towards regions of higher rigidity in a process known as durotaxis. Durotaxis plays an important role in normal development and disease progression, including tumor invasion and metastasis. However, the signaling mechanisms underlying focal Dextrorotation nimorazole phosphate ester adhesion-mediated rigidity sensing and durotaxis are poorly comprehended. Utilizing matrix-coated polydimethylsiloxane gels to manipulate substrate compliance, we show that cdGAP, an adhesion-localized Rac1 and Cdc42 specific GTPase activating protein, is necessary for U2OS osteosarcoma cells to Dextrorotation nimorazole phosphate ester coordinate cell shape changes and migration as a function of extracellular matrix stiffness. CdGAP regulated rigidity-dependent motility by controlling membrane protrusion and adhesion dynamics, as well as by modulating Rac1 activity. CdGAP was also found to be necessary for U2OS cell durotaxis. Taken together, these data identify cdGAP as an important component of an integrin-mediated signaling pathway that senses and responds to mechanical cues in the extracellular matrix in order to coordinate directed cell motility. Introduction Cells derive signals from conversation with the surrounding extracellular matrix (ECM) to regulate crucial functions including cell growth, differentiation and motility [1]. Integrin binding to glycoproteins present in the ECM, such as collagen and fibronectin, stimulates cell motility and promotes the formation of focal adhesions (FAs) in part by signaling to the intracellular Rho family of GTPases, including Rac1, RhoA, and Cdc42 [2]. These molecular switches are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase activating proteins (GAPs) during cell migration to coordinate signaling to the cellular migration machinery, including the regulation of FA dynamics and the remodeling of the actomyosin cytoskeleton through activation of downstream Rho family effectors such as PAK, Arp2/3, and non-muscle myosin II isoforms [3]C[7]. In addition to its chemical composition, recent studies have shown that this mechanical properties of the ECM also influence integrin signaling to promote directed cell migration [8]C[10]. Specifically, cell motility rates are enhanced by increased matrix rigidity and cell migration is usually directed towards more rigid substrates in a process known as durotaxis [8]C[10]. Artificially changing ECM compliance or exerting experimentally derived pressure on integrins can regulate the Rho family GTPases RhoA and Rac1, suggesting that ECM rigidity activates integrin signaling to control the Rho family of GTPases [11]C[14]. However, the function Dextrorotation nimorazole phosphate ester and activity of Rho GTPases during mechanically directed cell migration remains unclear and furthermore, the specific GEFs and GAPs that modulate their spatial and temporal activity to promote durotaxis have not been identified. CdGAP is usually a Rac1.