Traction Assays for Studies of Cell Mechanotransduction

V. Damljanović1, B. Lagerholm1, M. Dembo2 & K. Jacobson1

1Cell & Developmental Biology, University of North Carolina, Chapel Hill, NC; 2Biomedical 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 moleculesElastic

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 (70-100m optimal)

• Fluorescent markers must be small (we use 0.2m) 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

10

20

30

40

50

60

70

80

90

100

0.06 0.08 0.1 0.12 0.14 0.16

% BIS

Mo

du

lus

, E

[k

Pa

]

4.5 %

5 %

6 %

8 % acrylamide

% acrylamide and BIS

14

16

18

20

22

24

26

28

0.5 2 4 6 8 20

Hours of swelling

Mo

du

lus

, E

[k

Pa

]

Swelling in fluid

0

5

10

15

20

25

1 2 3

Mo

du

lus

, E

[k

Pa

]Troom

37 C

DMEM0.5%

DMEM10% FBS PBS

Media ionic content

Assay Basics

Top

vie

w

DeformedStress-free

Cell appliestractions

From Theory of Elasticity calculateCell Tractions

d g T dp p ( ) ( )m r r r

Cell shape(phase)

Displacement map

Integration contour

Bead positions

(fluorescence)

(null image) – (deformed image) = (displacement map)

Conjugation of ECM Proteins to PA Gels

H2N — NH2

Hydrazinehydrate

C

NH2

O

PA

Polyacrylamide

H C OH

R2

R1

Hydroxylysin incollagen

NaIO4C O

R2

R1

Oxidizedcollagen

C

R2

R1

Activatedpolyacrylamide

NH2

NH

PA

C N

NHR2

R1

Collagen-coatedgel

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 0.6m lower 1.4m lower

Good correspondence with null-image No correspondencewith 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 5m to few 100 m

Instant transfer, despite of stamp slipping due to alignment by hand

10m

PA gelH-h activated

30 sec

Cells on 25m stripesCells on flat-printed area

25m

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

“Con

trol

” C

3HP

ax (

-/-)

ME

F

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 10-7 J/m2

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 fluorescentcollagen stripes

Red fluorescent beads at the gel surface

C3H (phase) in themoment of hesitation

1-D Constrained MigrationWhat Controls Cell Direction and Polarity?

Time [min]

x 0.1 Pa

0 30 45 60 75 90

10.4 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 CadherinMechanism of MDCK spreading

Collaboration with Martin Schwartz, UVa