Traction Assays for Studies of Cell Mechanotransduction V' Damljanovic1, B' Lagerholm1, M' Dembo2

1
Traction Assays for Studies of Cell
MechanotransductionV. Damljanovic1, B.
Lagerholm1, M. Dembo2 K. Jacobson11Cell
Developmental Biology, University of North
Carolina, Chapel Hill, NC 2Biomedical
Engineering, Boston University, Boston, MA
2
Cell Mechanotransduction
3
Cell Tractions
Adhesion molecules
Tractions are determined from the deformation of
substrate
Polyacrylamide gel on 22 x 22 mm coverslip
(modified protocol of Yu-li Wang)
4
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 (70-100mm optimal)
• Fluorescent markers must be small (we use 0.2mm)
  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 vibrationstranslation
  is tolerable, but not rotation

5
Conditions That Affect Gel Modulus
6
Assay Basics
Displacement map
Bead positions (fluorescence)
Integration contour
From Theory of Elasticity calculate Cell Tractions
Cell shape (phase)
(null image) (deformed image)
(displacement map)
7
Conjugation of ECM Proteins to PA Gels
8
Correspondence Failures in Correlation-Based
Optical Flow
0.6mm lower
1.4mm lower
Gel top
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
9
Micro-patterning With ECM Proteins

• Excellent results, patterns from 5mm to few 100
  mm
• Instant transfer, despite of stamp slipping due
  to alignment by hand

30 sec
Fluorescently labeled protein
PA gel H-h activated
Cells on 25mm stripes
Cells on flat-printed area
10
Ongoing Projects and Applications
11
Paxillin and Mechanotransduction

• Working on Pax (-/) MEF wild type MEF
  tractions ? control
• Overexpress zyxin, vinculin or FAK, try to
  recover motility

12
Leading Edge Ruffles Both Push and Pull
Ruffles are free (no FAs) and used for probing
? alternately push and pull on the substrate
2 min apart

• 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

13
1-D Constrained MigrationWhat Controls Cell
Direction and Polarity?
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

14
HGF and CadherinMechanism of MDCK spreading
Collaboration with Martin Schwartz, UVa
Time min