Cellular response to the stiffness of the environment central role of cytoskeleton

1
Response of cells to nonlinear elastic substrates
Cellular response to the stiffness of the
environment - central role of cytoskeleton fibro
blasts, stem cells, neurons and astrocytes How
does strain-stiffening of biomaterials affect
cellualr mechanosensing? cells in and on
fibrin gels
Qi Wen, Fitzroy Byfield, Ilya
Levental,Jessamine Winer, Penelope Georges
2
Most cells in the body live in a relatively soft
world (E 10 to 100,000 Pa) compared to plastic
or glass (EgtGPa)
Fibroblasts in collagen ECM (T. Nishida et al.
Invest. Ophthalmol. Vis.)
Red cells and platelets in blood clot. John
Weisel- Penn
Can rheology (stiffness) direct cell function and
differentiation? What magnitude of stiffness do
cells probe (whats hard or soft)? Do all cells
sense stiffness? How do cells measure stiffness?
3
Tissue stiffness is tightly controlled in vivo
and changes with disease or injury
lt5 strain 1s/1Hz
glial scar (GFAP)
CNS injury
Levental et al. Soft Matter, 2007
4
Local stiffness differences in a developing mouse
breast tumor
5
Many cell types sense the rigidity of the
extracellular matrix.
bar 20um green GFP-actin
Glass GPa
10kPa
5kPa
1kPa
Substrate Stiffness
NIH 3T3 fibroblasts on Fn-coated PA gels
Pelham and Wang PNAS 1997
Solon et al, Biophys. J. 2007
6
Cell structure and function is determined by
genetics, chemical signals, and physical factors
7
New tolls to study mechanical effects on
cells Microfuidics method to make gels with
stiffness gradients
Adapted from Zaari et al, Advanced Materials
2005
Byfield, Wen et al, Biophys J 2009
8
Many cell types change shape as the substrate
stiffens
But not all cells . (more later)
9
Atomic force microscopy measures local cell and
substrate stiffness
10
Does the stiffness of cells change with substrate
stiffness?
AFM image and force mapping of fibroblast on glass
Ilya Levental, Jerome Solon, Kheya Sengupta
11
Fibroblasts become softer and more uniform on gels
2 µm
1 µm
2 µm
0 µm
1 µm
12
Fibroblast stiffness matches substrate stiffness
glass
13
Not all cells have the same stiffness response
unlike most other cells neurons prefer soft
substrates
150 Pa
Actin structures branch more frequently from MT
cores in neurons on soft gels
2 kPa
11 kPa
Georges et al, Biophys. J. 2006
14
In contrast to neurons, astrocytes cultured from
CNS explants spread poorly on soft matrices but
well on stiff ones
10,000 Pa
150 Pa
15
Primary astrocytes spread and become stellate on
stiff gels.
16
Can stiffness select for favored cell types?
Co-culture of neurons and glial cells on soft (50
Pa) laminin-coated PA gel
Neurons but not glial cells grow on soft gels
17
Astrocytes, but not neurons dominate on hard
surfaces
18
Preference of neurons for soft materials is also
seen when mixed cultures are grown in fibrin gels
of variable stiffness
19
The mechanics (and sometimes the rate) of cell
division is strongly affected by substrate
stiffness (NIH-3T3 fibroblasts)
4000 Pa
300 Pa
20
Tissue culture (immortalized) cells can divide
on soft substrates but many normal, primary cells
cannot
DNA synthesis and cyclin D expression are blocked
by softness, but other signals downstream of
integrins are not
soft
stiff
0
Cyclin D
Entry into S-phase
Hrs after serum
Erik Klein, Rick Assoian, UPenn
Mouse embryo fibroblasts
21
Mesenchymal stem cells stay quiescent for long
periods on soft gels, and .
200 Pa (stiffness of bone marrow)
100
BrDU incorporation
glass
0
sparse confluent glass (GPa)
200 Pa
4 kPa
gels
Winer et al. Tissue Engin. In press
22
are activated by a stiff surface without any
chemical induction
Beningo and Wang Methods Mol. Biol. 370203
and can still differentiate into osteocytes
Winer JP, et al, 2008 Tissue Eng.
23
How does a cell sense stiffness? Multiple
molecular springs in series create the
possibility of variable signals depending on
which spring is the weakest (most deformed)

• What are the mechanical properties of the
  springs?
• (cytoskeleton and ECM)
• 2. What molecules sense the force and transmit
  signals?

24
Both cytoskeletal and ECM networks are strain
stiffening
biopolymers 0.1 to 0.3 polyacrylamide 5
Shear modulus G (Pa )
Storm, 2005
Shear Strain
Non-linear elasticity allows cytoskeletal and ECM
networks to stiffen by internal stress, without
increasing polymer mass or XLs.
25
Strain-stiffening by fibrin means stiffness
depends on how much the cell deforms its substrate
Small strain Same stiffness (neurons)
Large strain Fibrin is stiffer (hMSCs)
26
Cell spread area on linear or stiffening gels
600 Pa
27
Network is required for cell spreading
28
Fibrin Traction Microscopy

• Modifications for fibrin traction microscopy
• Mixed biotinylated with untreated fibrinogen
• Attached streptavidin coated fluorescent beads
• Employed a variety of cytoskeletal inhibitors
  such as cytochalasin D to study tractions.

100 mm
29
Cells apply long range displacements
30
Cells apply larger strains on softer gels
31
AFM confirms that cells locally stiffen the gel
Before Blebbistatin
After Blebbistatin
32
Strain fields are anisotropic and correlate with
cell shape
33
Fibrin gels induce pattern formation
16 kPa Polyacrylaimde 2 mg/ml fibrin
34
Mesenchymal stem cell size and orientation is
affected by neighbors as far as 400 µm away
35
Conclusions

• Substrate mechanosensitive cells spread on soft
  fibrin gels
• Suggests a cell-gel mechanical feedback loop
• Contractile cells can significantly stiffen
  fibrin gels
• The strain field applied by the cell extends
  several cell lengths away
• Cells can use gel mechanics for local
  communication resulting in cell patterning

36
Cells tune their stiffness to match the substrate
when they bind by collagen receptors - A7
melanoma cells
?1 integrin - filamin
?3 integrin - talin
37
(No Transcript)