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1 Mechanotransduction: Examples Eric Salm BIOE 506 April 15, 2009 Slide 2 2 Mechanotransduction: Recap Means by which cells convert a mechanical stimulus to some chemical activity http://www.nature.com/nrm/journal/v10/n1/pdf/nrm2605.pdf Slide 3 3 Article: Local Action Forced Unfolding of Proteins within Cells Authors: Colin P. Johnson, Hsin-Yao Tang, Christine Carag, David w. Speicher, Dennis E. Discher Publishing Journal: Science (2007) Slide 4 4 Outline Goal Introduction Methods Results Discussion Slide 5 5 Goal Identify cytoskeletal proteins that change conformation due to physiological stresses Slide 6 6 Introduction Protein domain unfolding has been shown to occur; however, limited to single-molecule measurements Slide 7 7 Introduction (2) Moving beyond single molecule to live cells Cys-shotgun method used Cysteine is a hydrophobic AA which results in frequent AA-shielding As a protein deforms, previously shielded cysteines can now be labeled Slide 8 8 Introduction (2) Interested in membrane bound proteins Spectrin (RBC): Two chains, and , cross-link F-actin. Found to unfold at low forces. Central role in cell deformability. Myosin IIa (MSC): Responsible for actin-based motility Vimentin (MSC): Member of intermediate filament family of proteins. Intermediate filaments play an important role in cell structure Slide 9 9 Methods: RBC analysis 1a. RBCs reversibly lysed to make hemoglobin- depleted cells 2a. Cys-reactive fluorophore (IAEDANS) added to the cells 3a. Resealed entraping the fluorophore 4a. Ranged of tests from static to stressed 5a. Cells relysed releasing unbound fluorophore 6a. Cells denatured and all Cys that werent dye- labeled were alkylated with iodoacetamide (IAM) Slide 10 10 Results Analysed with separation of proteins by SDS-PAGE gel electrophoresis Slide 11 11 Results (2) Analyzed using Liquid Chromatography-coupled tandem Mass Spectrometry (LC-MS/MS) Slide 12 12 Sequential Two-Dye Labeling 1. Static-accessible sites labeled to near saturation with IAEDANS 2. Shear applied with labeling by a secondary cys-flurophore (BODIPY-IA) Done to magnify the difference between shear and static Cys-labeling Slide 13 13 Fits force-dependent Linderstrom-Lang (fLL) scheme Shear stress, proportional to force, shifts the confirmational equilibrium Slide 14 14 Methods: Mesenchymal Stem Cells 1. Membrane-permeable, Cys-labeling fluorophore monobromobimane used to label both tensed and relaxed cells 2. Cell lysates Cys-quenched 3. SDS-PAGE run 4. LC-MS/MS run Slide 15 15 Tensed vs Relaxed (Blebbistantin) Slide 16 16 Results Nonmuscle Myosin IIA Vimentin Slide 17 17 Conclusions Cys-labeling provided evidence of force- induced changes within the structure of cellular membrane proteins Limited because it does not touch on conformational changes in real-time Slide 18 18 Articles: Deep Cytoplasm Rapid Signal Transduction in Living Cells Is a Unique Feature of Mechanotransduction. Authors:Singsoo Na,, Yingxiao Wang Publishing Journal: Proceedings of the National Academy of Sciences (May 2008) Visualizing the Mechanical Activation of Src Authors: Yingxiao Wang et al. Publishing Journal: Nature (April 2005) Slide 19 19 Goal Pick up where last paper left off Move into real-time, live cell imaging Examine the effects of stress within the cell in terms of stress-induced Src activation Determine pathway for deep cytoplasmic Src activation Slide 20 20 Src Background Tyrosine Kinase First TK to be discovered Linked with Rous sarcoma virus Served to link virus with cancer through gene expression Involved in intracellular signalling Oncogene Slide 21 21 Activating Src-Type Protein Use FRET to measure activation of Src by use of a FRET-based Src reporter Measures CFP vs YFP. Enhanced CFP = active Src Slide 22 22 Methods: Stress-Induced Src Activation 1. Transfect CFP-YFP cytosolic Src reporter into SMCs 2. RGD-coated magnetic bead was then bound to integrin receptors on the cell membrane 3. Local stress induced by turning on a magnetic field 4. Quantify changes in Src activities by using FRET ratios of the Src reporters in the membrane Slide 23 23 Rapid Src activation: Integrin -Stress+Stess, + 0.3 s Slide 24 24 Factors Involved in Rapid Src Activation Integrin activation Deep cytosol stimulation requires stress fibers from focal adhesions Substrate stiffness Prestress in actin is necessary for stress transmission F-actin integrity What else Slide 25 25 Methods: Microtubule, Actin and Endosome Deformation 1. Transfect cells with Src reporter and mCherry-tubulin 2. Induce stress 3. Measured Src activation and deformation of component in question 4. Repeat above with Src reporter and mCherry-actin 5. Repeat above with mCherry-tubulin and GFP-endo Slide 26 26 Results: Microtubules Slide 27 27 Results: Actin Deformation Slide 28 28 Results: Endosomal and Microtubulal Co-deformation Slide 29 29 Conclusion Rapid Src activation too fast to be caused by either diffusion or translocational-based mechanisms. Thus, it is unique to mechanotransduction. Mechanism shown to be: Local stress at focal adhesion F-actin bundles transmit stress to deep cytoplasm Microtubules and endosomes deform Src activation Slide 30 30 Questions?