Biophysics of Molecules
Concepts of cell adhesion and cellular
mechanostransduction – part II
Dr. Carsten Grashoff
MPI of Biochemistry
E-mail: [email protected]
Measuring mechanotransduction
The problem
We need to know which molecules are exposed to mechanical
forces within the cells.
How can we measure mechanical forces in cells ?
2
Traction force microscopy – nano-pillars
from Fu et al., Nat Methods, 2010
-
traction force microscopy is used to determine how much force
is exerted by the cell onto an extracellular substrate
-
nano-pillars of known size and elasticity can be used to
determine traction forces microscopically
3
Traction force microscopy – micro-beads
-
micro-beads embedded in polyacrylamid-gels of known elastic
modulues can be similarly used to measure traction forces with
a light microscope
4
Methods to determine mechanical parameters
experimentally in cells
5
Measuring forces across molecules
- single-molecule atomic
force microscopy is
used to measure
mechanical forces
across distinct proteins
- but: we want to
measure mechanical
forces in cells !
from Fisher et al., 1999, Trends in Biochemical Sciences
6
Mechanical aspects of cell migration
FAs
disassemble
FAs grow
7
Traction forces during cell migration
in protrusions:
- FAs grow
- traction forces high
?
Beningo et al., JCB,
2001
in retracting areas:
- FAs disassemble
- traction forces high
8
The mechanical cell migration paradox
9
Fluorescence resonance energy transfer
10
Principle of a FRET-based tension sensor
-
the linker peptide has to be short (< 5 nm long), elastic and
responsive to pN forces
11
Remember entropic elastomers ?
12
A genetically encoded flagelliform sensor
flagelliform (spider silk protein)
from Becker et al., Nat Mater, 2003
from Grashoff et al., Nature, 2010
13
Single molecule calibration
with TJ Ha, University of Illinois, USA
14
First application: a vinculin tension sensor
- the biosensor cDNA is
transfected (or infected)
and the protein is
expressed by the cell
but we need controls:
Vinculin
a „zero-force“ control
that can not be under
force
- a wildtype vinculin
control that behaves as
the endogenous protein
15
Generating a suitable model system
- knockout cells (cells with no
endogenous vinculin) should
be used
- cells are isolated from
genetically modified mice and
immortalized
- the vinculin gene is deleted in
vitro
- the biosensor is transiently or
stably expressed in knockout
cells
16
Tension sensor evaluation in cells
…displays normal localization
staining
The sensor is stably
expressed…
…and shows normal FA dynamics
Western Blotting
FRAP
17
Measuring vinculin forces within cells
Fluorescence lifetime microcopy
18
Forces across vinculin in migrating cells
from Grashoff et al., Nature, 2010
-
no differences in FRET of the zero-force control
-
low FRET (high force) in protrusions
-
high FRET (low force) in retracting areas
19
Automatic quantification of FRET in FAs
- due to use of
endogenous vinculin
levels and small FRET
changes experimental data
are noisy
- using custom-written
software individual FAs are
automatically segmented
and averaged to boost
statistical power
20
Automatic quantification in dynamic FAs
21
Statistical evaluation of data
high forces across vinculin in
assembling FAs
low forces across vinculin in
disassembling FAs
22
Does vinculin regulate cell migration ?
• traction force high
• traction force high
• FA stability low
• FA stability high
• vinculin forces low
• vinculin forces high
Hypothesis: Vinculin stabilizes FAs under force
23
Testing the hypothesis
increases contractility of cells
myosin II
active RhoA
Vinculin does stabilize FAs under force !
24
Metavinculin is a muscle-specific isoform
altered actin
binding
normal
hypertrophic
normal
hypertrophic
- mice with a metavinculin deletion in the heart muscle develop
hypertrophic hearts (cardiomyopathy) and die of heart failure
25
Metavinculin mutations lead to disorders
Mutations in cardiomyopathy patients:
- (Meta)Vinculin L277M
- Metavinculin A934V
- Metavinculin Δ954
- Metavinculin R975W
metavinculin-specific
mutations in patients suffering
from cardiomyopathy
Next question: Do mutations in metavinculin effect the ability of the
protein to bear (and transduce) mechanical force ?
26
Improving the tension sensor technique
Matthias Rief Lab (TU Munich, Germany)
27
Summary – force measurments
- there are many biophysical techniques to investigate mechanical
properties of cells:
- traction force microscopy (using nao-pillars or micro-beads)
- atomic force microscopy
- single-molecule atomic force microscopy
- micro-rheology, etc.
- a FRET-based sensor can be used to visualize and measure forces
across proteins in cells
- vinculin is exposed to mechanical forces of a few pN
- vinculin is exposed high force in assembling FAs but is not bearing
forces in disassembling FAs
28
Mechanobiology is interdisciplinary
29