1. Topic 12: Cell Mechanics

2. David Rogers, Vanderbilt University Cells are dynamic, constantly reorganizing their cytoskeleton

3. Cytoskeleton Mel-c melanocyte treated with cytochalasin and stained for F-actin (blue), microtubules (red), and the melanosome marker TRP-1 (green). John Hammer, NIH Actin filaments Microtubules Intermediate filaments

4. Actin resists tensile stretching and generates internal tension Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates

5. Measurement of the mechanical properties of actin Kishino & Yanagito. Nature. 1988 The elastic modulus of alpha-actin fibers is about 0.1Pa The tensile strength is around 100pN

6. Actin 3-D structure is determined by binding proteins http://www.bms.ed.ac.uk/research/others/smaciver/lectures/Cs2.htm

7. Review – Actin Polymerization Images of actin networks reveal a polarized structure with a barbed and a pointed end A.B.Verkhovsky,T.M.Svitkina, and G.G. Borisy J. Cell Sci., 110: 1693-1704, 1997

8. Dynamic Instability of Actin leads to catastrophic polymerization and depolymerization and treadmilling

9. Actin treadmilling in a migrating cell Waterman-Storer lab, Scripps Institute of Oceanography

10. Myosin moves along actin in ATP dependent manner K. Chein 1999. Myosin I – Important in cell motility and intracellular transport (assisted diffusion) Myosin II – Important in muscle contraction (forms thick filaments) Each individual myosin type is polarized and only travels 1 direction. Different myosins can move different directions along the actin filament.

11. Microtubules act as cell struts, resisting tension Can form as single tubes as well as doublets or triplets

12. Microtubules move by transport or by treadmilling

13. Motor proteins transport loads across cells and move filaments relative to each other Schliwa and Woehlke. Nature 422, 759-765 (17 April 2003)

14. Intermediate Filaments Various monomers– commonly keratin and desmin Link cells together. Do not have a transport role. Moved around through connections to MTs. Hair and claws are large complexes of intermediate filaments.

15. Theory of tensegrity proposes that a cell can be considered as a stable structure of struts and ropes Controversial due to failure to consider dynamic properties of actin and tubulin.

16. Attachment to ECM http://www.steve.gb.com/images/science/cell_adhesion_summary.png

17. Techniques for measuring cell mechanics Atomic Force Microscopy Traction Force Microscopy

18. Atomic Force Microscopy http://www.chemistrydaily.com/chemistry/upload/thumb/9/9c/350px- Atomic_force_microscope_block_diagram.JPG

20. http://www.nanoscience.com/education/i/F-z_curve.gif

21. Where n is the Poisson’s Ratio, P max is the maximum force, h max is the maximum indentation depth and a is the radius of contact as computed below: Where R is the radius of the indenter sphere. The point of contact is determined from a curve fit of unloading data and the elastic modulus is computed per the following equation using Hertzian assumptions

22. AFM Can be used to measure the compliance of a cell or the adhesion force of cell attachment molecules

24. Issues with AFM Infinite half space assumption Surface tension Dynamics of cytoskeleton

25. Traction Force Microscopy Wong et al. 1999

26. Traction Force Microscopy Measure Displacements Create matrix relating shear stress to displacements Solve inverse problem (d=AT for T) to find traction stress (often called “traction force”) in units of force/area (pressure) Integrate stresses over an area to compute total force

27. Bousinesq Equations (Green’s Functions) relate shear stress to deformation

28. Dynamic Traction Force Microscopy Image fluorescent beads Regional Cross- correlation Compute most-likely traction field

29. Issues with TFM Infinite Half Space Assumption Poorly Defined Inverse Problem Effect of noise

30. Micropost Array Chen lab, University of Pennsylvania

31. The force on a post can be calculated knowing the post’s radius, length and deflection

32. Like TFM, this method can calculate forces under specific areas of a cell

33. Magnetic Bead Twisting Fredberg lab, Harvard Generates a torque on a magnitized bead in order to evaluate a cell stiffness

34. Kas lab, University of Leipzig Optical traps use light diffraction, as well as an induced magnetic dipole, to produce forces up to 100 pN

35. Optical traps can be used to direct neurite extension Ehrlicher et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073

36. Summary Cell cytoskeleton composed of actin filaments, microtubules and intermediate filaments Actin filaments resist tension, are polarized and can catastrophically extend and collapse Microtubules resist compression, are polarized and show treadmilling behavior Atomic force microscopy uses low-force indentation of the cell membrane to study cell mechanics. Traction force microscopy observes a cell’s ability to deform its surroundings to compute shear stress and, indirectly, cell force.

37. Summary Traction force microscopy observes a cell’s ability to deform its surroundings to compute shear stress and, indirectly, cell force. Micropost array deflection can also be used to observe a cell’s traction force. Optical traps can produce very small forces that can be used to direct neurite extension.