Mechanotransduction: Examples

1. Mechanotransduction:Examples Eric Salm BIOE 506 April 15, 2009

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

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)

4. Outline • Goal • Introduction • Methods • Results • Discussion

5. Goal • Identify cytoskeletal proteins that change conformation due to physiological stresses

6. Introduction • Protein domain unfolding has been shown to occur; however, limited to single-molecule measurements

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

8. Introduction (2) • Interested in membrane bound proteins • Spectrin (RBC): Two chains, α and β, 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

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 weren’t dye-labeled were alkylated with iodoacetamide (IAM)

10. Results • Analysed with separation of proteins by SDS-PAGE gel electrophoresis

11. Results (2) • Analyzed using Liquid Chromatography-coupled tandem Mass Spectrometry (LC-MS/MS)

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

13. Fits force-dependent Linderstrom-Lang (fLL) scheme Shear stress, proportional to force, shifts the confirmational equilibrium

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

15. Tensed vs Relaxed (Blebbistantin)

16. Results Nonmuscle Myosin IIA↑ Vimentin→

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

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)

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

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

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

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

23. Rapid Src activation: Integrin -Stress +Stess, + 0.3 s

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…

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

26. Results: Microtubules

27. Results: Actin Deformation

28. Results: Endosomal and Microtubulal Co-deformation

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

30. Questions?