Traction Assays for Studies of Cell Mechanotransduction
Download
1 / 14

Cell Mechanotransduction - PowerPoint PPT Presentation


  • 155 Views
  • Uploaded on

Traction Assays for Studies of Cell Mechanotransduction V. Damljanovi ć 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.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Cell Mechanotransduction' - george


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Slide1 l.jpg

Traction Assays for Studies of Cell MechanotransductionV. Damljanović1, B. Lagerholm1, M. Dembo2 & K. Jacobson11Cell & Developmental Biology, University of North Carolina, Chapel Hill, NC; 2Biomedical Engineering, Boston University, Boston, MA


Slide2 l.jpg

Cell Mechanotransduction

Signal  Cytoskeleton

Apply tractions

Sense environment

Motion

Correlate w/ cell state

Feedback


Slide3 l.jpg

Cell Tractions

Direction of migration

“Propulsive” tractions

“Frictional” tractions

Elastic substrate

Substrate 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)


Slide4 l.jpg

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 vibrations—translation is tolerable, but not rotation


Slide5 l.jpg

Swelling in fluid

% acrylamide and BIS

Media ionic content

Conditions That Affect Gel Modulus


Slide6 l.jpg

Deformed

Cell applies

tractions

Stress-free

Top view

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)


Slide7 l.jpg

R2

R1

R2

R2

more affordable, easier to use and provides

more consistent coating than previously used

UV-activatable x-linker Sulfo-SANPAH

Hydrazine hydrate

(reducing agent)

NH2

NH2

Activated

polyacrylamide

O

C

H2N — NH2

NH

Hydrazine

hydrate

PA

PA

Polyacrylamide

R1

N

C

Hydroxylysin in

collagen

R1

R1

NH

R2

C

C

O

PA

NaIO4

OH

H

C

Oxidized

collagen

Collagen-coated

gel

Conjugation of ECM Proteins to PA Gels


Slide8 l.jpg

Correspondence Failures in Correlation-Based Optical Flow

1.4mm lower

0.6mm 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


Slide9 l.jpg

25mm

10mm

PDMS stamp

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



Slide11 l.jpg

19.4 mm

6.71 kPa

Traction shear

(mag. of traction gradient)

Strain energy density (traction * displacement)

Traction vectors

Traction magnitude

x 0.1 kPa

x 10-7 J/m2

x 0.1 kPa/cm

“Control” C3H

Pax (-/-) MEF

Paxillin and Mechanotransduction

  • Working on Pax (-/+) MEF & wild type MEF tractions  control

  • Overexpress zyxin, vinculin or FAK, try to recover motility


Slide12 l.jpg

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


Slide13 l.jpg

C3H (phase) in the

moment of hesitation

Green fluorescent

collagen stripes

Red fluorescent beads at the gel surface

??

Traction magnitude

Traction vectors

x 0.1 Pa

1030 kPa

21.7 mm

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


Slide14 l.jpg

Hypothesis:

HGF does not directly disrupt E-cadherin function.

It increases integrin-mediated ECM adhesion

The force of cells pulling apart breaks the junctions

10.4 mm

HGF

x 0.1 Pa

0 30 45 60 75 90

HGF and CadherinMechanism of MDCK spreading

Collaboration with Martin Schwartz, UVa

Time [min]


ad