Topic 10: Cell Mechanics 4/26/07 BE112b

Collagenous Tissue Testing: Summary of Key Points Tissue testing considerations include –Various possible configurationsconfigurations –The method of tissue clampingtissue clamping –Methods to measure stress and strainmeasure –Defining the stress-free statestress-free state –Anelastic and viscoelastic tissue properties, the need to precondition, and the effects of strain rateAnelastic viscoelastic strain rate –The effects of age, injury, immobilization, surgical repair and replacement.ageinjuryimmobilizationsurgical repair replacement

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

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

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

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

Actin 3-D structure is determined by binding proteins

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

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

Myosin moves along actin in ATP dependent manner K. Chein 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.

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

Microtubules move by transport or by treadmilling

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

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.

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.

Attachment to ECM

Techniques for measuring cell mechanics Atomic Force Microscopy Traction Force Microscopy

Atomic Force Microscopy Atomic_force_microscope_block_diagram.JPG

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

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

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

Traction Force Microscopy Wong et al. 1999

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

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

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

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

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.