麻省理工大学：《生物工程》教学讲义（英文版）lec 24 slid me

Effect of laminar shear stress on cell alignment(Dewey etal.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 aminar shear stress

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 sensitive ior conformational chann structural proteins Signaling altered △ In protein synthesis or post expression translational modifications

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 [20001 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

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 removed due to copyright considerations ension in the tip link activates a SEM of the stretch-activated Image removed due to stereocilia on the copyright considerations ion channel, leading to intracellular surtace of a single hair cell (hudspeth calcium ion fluctuations

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 stereocilia on the copyright considerations. 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 lon 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

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 aton:□ n2-=∑E1N Fi represents the force applied to the ith atom by he h 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

† Steered Molecular Dynamics� For each atom:� m i i d 2 xi dt 2 = Fij j=1 N Â = 1, …N Fij 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!

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, isu now also known and studies are underway to identify the mechanism ofo operation. Bass et al., Science, 2003)o Image removed due to copyright considerations

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 tol cytoskeletons after stretch (Sawada sheetz, 2002) a Triton X-100 insoluble cytoskeletons u Incubated with cytoskeletal proteins having a photocleavable biotin tag w/ and w/o 10% stretch Image removed due to copyright considerations a Focal adhesion kinase, paxillin See Figure 3 in Sawada, Y, and M. P Sheetz p130Cas, PKB/Akt all preferentially Force transduction by Triton cytoskeletons bound J Cell bio.2002Feb18;156(4)609-15 http://wwwjcb.org/cgi/content/full/156/4/609 Binding of proteins is influenced byn stretch of cytoskeleton Can it be due to induced conformational changes?

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 nages removed due to copyright cons 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 pr Lower forces(- 10 pn or less? )are likely adequate to produce conformational changes Lehenkari horton BbRC. 1999

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