traction assays for studies of cell mechanotransduction v. damljanović 1, b. lagerholm 1, m. dembo...
TRANSCRIPT
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
• 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?