Traction Assays for Studies of Cell Mechanotransduction V. Damljanović 1, B. Lagerholm 1, M. Dembo 2 & K. Jacobson 1 1 Cell & Developmental Biology, University of North Carolina, Chapel Hill, NC; 2 Biomedical Engineering, Boston University, Boston, MA

Cell Mechanotransduction Sense environment Correlate w/ cell state Feedback Signal  Cytoskeleton Apply tractions Motion

Cell Tractions Tractions are determined from the deformation of substrate Adhesion molecules Elastic substrate “Frictional” tractions “Propulsive” tractions Direction of migration Substrate tractions Polyacrylamide gel on 22 x 22 mm coverslip (modified protocol of Yu-li Wang)

Experimental Requirements (must match theoretical assumptions) Gel must be flat, with free edges and bottom fixed on the coverslip Gel thickness must be orders of magnitude greater than average displacement, but small enough for optics (  m optimal) Fluorescent markers must be small (we use 0.2  m) and only at the top (not really the case) Must keep the focus always at the same set of beads (difficult) Must be isolated from all vibrations—translation is tolerable, but not rotation

Conditions That Affect Gel Modulus % acrylamide and BIS Swelling in fluid Media ionic content

Assay Basics Top view Deformed Stress-free Cell applies tractions From Theory of Elasticity calculate Cell Tractions Cell shape (phase) Displacement map Integration contour Bead positions (fluorescence ) (null image) – (deformed image) = (displacement map)

Conjugation of ECM Proteins to PA Gels H 2 N — NH 2 Hydrazine hydrate C NH 2 O PA Polyacrylamide H C OH R2R2 R1R1 Hydroxylysin in collagen NaIO 4 C O R2R2 R1R1 Oxidized collagen C R2R2 R1R1 Activated polyacrylamide NH 2 NH PA C N NH R2R2 R1R1 Collagen-coated gel PA more affordable, easier to use and provides more consistent coating than previously used UV-activatable x-linker Sulfo-SANPAH Hydrazine hydrate (reducing agent)

Correspondence Failures in Correlation- Based Optical Flow Gel top 0.6  m lower 1.4  m lower Good correspondence with null-image No correspondence with null-image Slight shift of focus plane results in loss of relevant displacement field  Must always capture images of the same TOP bead layer

Micro-patterning With ECM Proteins  Excellent results, patterns from 5  m to few 100  m  Instant transfer, despite of stamp slipping due to alignment by hand 10  m PA gel H-h activated 30 sec Cells on 25  m stripes Cells on flat-printed area 25  m PDMS stamp Fluorescently labeled protein

Ongoing Projects and Applications

Working on Pax (-/+) MEF & wild type MEF tractions  control Overexpress zyxin, vinculin or FAK, try to recover motility “Control” C3H Pax (-/-) MEF Traction vectors 6.71 kPa 19.4  m Traction magnitude x 0.1 kPa Traction shear (mag. of traction gradient) x 0.1 kPa/cm Strain energy density (traction * displacement) x J/m 2 Paxillin and Mechanotransduction

2 min apart Leading Edge Ruffles Both Push and Pull Ruffles are free (no FAs) and used for probing  alternately push and pull on the substrate One more proof of two distinct actin networks: - Strip along the leading edge has no FAs, can push and pull to probe - Inner part, behind leading edge has FAs and always pulls

Traction vectors Traction magnitude 1030 kPa 21.7  m x 0.1 Pa Used m-patterned gel to: Geometrically enforce cell polarity & unidirectional migration Simultaneously record tractions and process of changing direction Future work: Perturb leading edge (end of stripe, CALI, photoactivation) Record protein activity ?? Green fluorescent collagen stripes Red fluorescent beads at the gel surface C3H (phase) in the moment of hesitation 1-D Constrained Migration What Controls Cell Direction and Polarity?

Time [min] x 0.1 Pa  m Hypothesis: HGF does not directly disrupt E-cadherin function. It increases integrin-mediated ECM adhesion The force of cells pulling apart breaks the junctions HGF HGF and Cadherin Mechanism of MDCK spreading Collaboration with Martin Schwartz, UVa