Effect of laminar shear stress on cell alignment (Dewey et al. 1981) Photos removed due to copyright considerations. See Dewey, C.F., et al. "The dynamic response of vascular endothelial cells to fluid shear stress." J Biomech Eng. 1981 Aug;103(3):177-85. No flow Laminar shear stress? applied external force mechano- conformational sensitive ion changes in channel structural proteins Signaling cascade altered D gene synthesis or post expressionn translational modifications in protein Image removed due to copyright considerations. Hanahan & Weinberg, Cell [2000] We know quite a lot about the signaling cascade that follows the initial biochemical event, leading to morphological changes, variations in various biochemical signals, changes in gene expression and protein synthesis, but relatively little about how the initial event is transduced from physical force to biochemical reaction. Mechanotransduction: Hair cell stimulation stereocilium tip link tension in tip link increases Image removed due to copyright considerations. SEM of the Image removed due to copyright considerations. stereocilia on the surface of a single hair cell (Hudspeth) Tension in the tip link activates a stretch-activated ion channel, leading to intracellular calcium ion fluctuations. Structure of a Mechano-Sensitive Ion Channel (MscL, large conductance) Chang, et al., Science, 1998 Membrane stresses alter pore geometry and control the exchange of ions Images removed due to copyright considerations. ? Steered Molecular Dynamics? For each atom:? i m i d 2 x i dt 2 = F ij j=1 N ? = 1, …N F ij represents the force applied to the i th atom by the j th atom (all the force interactions), determined as the gradient of the potential energy. Steered molecular dynamics (SMD) is the forced unfolding of a protein to reveal new conformational states. Molecular dynamics simulation of channel regulation by membrane tension (Gullingsgrud, et al., Biophys J, 2001) Images removed due to copyright considerations. See Figures 1 and 9 in Gullingsrud, Justin, Dorina Kosztin, and Klaus Schulten. "Structural Determinants of MscL Gating Studied by Molecular Dynamics Simulations." Biophys J, Vol. 80, No. 5 (May 2001), p. 2074-2081. http://www.biophysj.org/cgi/content/full/80/5/2074 But other evidence suggests that the pore increases to >20 angstroms! Structure of MscS (“small” conductance) mechano-sensitive channel, is? now also known and studies are underway to identify the mechanism of? operation. (Bass et al., Science, 2003)? Image removed due to copyright considerations. Selective binding to? cytoskeletons after stretch? (Sawada & Sheetz, 2002) ?? Triton X-100 insoluble cytoskeletons ?? Incubated with cytoskeletal proteins having a photocleavable biotin tag w/ and w/o 10% stretch Image removed due to copyright considerations. ?? Focal adhesion kinase, paxillin, See Figure 3 in Sawada, Y., and M. P. Sheetz. "Force transduction by Triton cytoskeletons." p130Cas, PKB/Akt all preferentially J Cell Biol. 2002 Feb 18;156(4):609-15. bound http://www.jcb.org/cgi/content/full/156/4/609 Binding of proteins is influenced by? stretch of cytoskeleton? Can it be due to induced conformational changes? Strength of integrin bonds to ECM ligands? Images removed due to copyright considerations. See Figure 1A and 2 in Lehenkari, P., and M. Horton. "Single Integrin Molecule Adhesion Forces in Intact Cells Measured by Atomic Force Microscopy." Biochem Biophys Res Commun. 1999 Jun 16;259(3):645-50 AFM used to measure the strength of integrin bonds to various RGD ligands. Single bond forces were 32-97 pN. Lower forces (~ 10 pN or less?) are likely adequate to produce conformational changes. Lehenkari & Horton, BBRC, 1999 Potential transduction mechanisms involving? changes in molecular conformation? Shear-stress induced conformational changes in transmembrane proteins tethered to the CSK sites “Sensing” proteins or at focal adhesion Forces exerted by ECM or CSK attachments Some Force Estimates? A shear stress of 1 Pa exerts a 1 pN force on a 1 mm 2 area. A globular protein complex ~100 nm in diameter would experience a 1 pN force in a linear shear flow corresponding to a shear stress of 1 Pa. Image removed due to copyright considerations. Significant protein conformational changes occur for forces in the range of ~10 pN. Magnetic traps can exert forces of ~10 pN per integrin bond. Davies et al., Ann. Rev. Physiol., 1997 SMD of fibronectin (Gao, Craig, Vogel, Schulten, JMB, 2002) Images removed due to copyright considerations. Applied force = See Figures 2 and 3 in Gao, Mu, David Craig, Viola Vogel, and Klaus Schulten. 500pN "Identifying unfolding intermediates of FN-III10 by steered molecular dynamics." Journal of Molecular Biology, 323:939-950 (2002). Unfolding is important in the exposure of buried cryptic binding sites. TEM of cytoskeleton, Actin is another candidate for force-induced conformational change. When stressed, cells form stress fibers, mediated by a variety of actin-binding proteins (a-actinin, fascin). The structure is known, and computations can take TEM cytoskeleton photograph, J. Hartwig, Harvard University. Courtesy of J. Hartwig. Used with permission. advantage of filament periodicity. But, a force of 10 pN supported by a single actin filament (E~10 9 Pa) produces a strain of ~2x10 -4 !!) Structure of actin.? Image courtesy of Prof. Willy Wriggers, http://biomachina.org.? Used with permission. ?