Extracellular Matrix Proteins and Mechanical Tension in Regulating FAK activity in
Mesenchymal Stem Cells
Forrest Goodfellow, Taejin Kim, Yingxiao Wang
Department of Bioengineering, College of Engineering, University of Illinois at Urbana-Champaign
Introduction
Aim
Focal Adhesion Kinase (FAK)
FAK is a 125 kD protein responsible for
tyrosine phosphorylation. FAK is activated in
response to environmental stimuli such as
extracellular matrix proteins, and mechanical
tension. FAK can exits in they cytosol or bound
to the plasma membrane.
a
b
.
Previous experiments in the Wang Laboratory
have observed FAK activation under an array of
environmental conditions. The effects of
substrate rigidity and the composition of the
extracellular matrix were observed with the CytoFAK biosensor. Now, the intent is to investigate
FAK activation with the Lyn-FAK biosensor. This
investigation will allow for the observation of FAK
activity within the cytosol as well as bound to the
plasma membrane.
Preliminary Results
Conclusions
The Ctyo-FAK biosensor allows for the
visualization of cytosolic FAK activation and
numerical analysis with Metaflour imaging
software enables the numerical characterization
of FAK activation with the Cyto-FAK biosensor.
On going experiments will allow for the
comparison of Cyto-FAK and Lyn-FAK
biosensors under identical environmental
conditions.
FAK activation observed with the Lyn-FAK
biosensor demonstrated an increase in FAK
activation on harder substrates relative to soft
substrates, and the FAK activation at 1 hour
differs significantly. An explanation for the
difference may be the kinetics of FAK activation
caused by the different ECM proteins. FAK
activation must be observed with the Lyn-FAK
biosensor before any further insights can be
made. Continued research will allow for an
understand of FAK distribution with in the cell
and answer several questions.
www.spinal-research.org/
Is FAK synthesized in response to the
environment?
c
www.nationaldiagnostics.com/
Method
Does FAK transiently bind to the plasma
membrane?
Culture of Mesenchymal Stem Cells
Do ECM proteins effect the kinetics of FAK
activation?
www.neuroniccollagen.com/
Figure 1: (a) Polyacrylamide gel is used to simulate in virto environments of
different mechanical tensions by varying the amount of cross-linked
molecules (b) EM images of fibronectin and (c) collagen are ECM proteins
responsible for cellular adhesion and play a role in FAK activation.
Fluorescent Resonance Energy Transfer (FRET)
FRET biosensors are genetically engineered
proteins containing two fluorescent protein
sequences without disrupting the functionality of
the protein. The Lyn-FAK biosensor allows for
the visualization of membrane bound FAK and
Cyto-FAK allows for the visualization of cytosolic
FAK. FRET biosensors enable live cell imaging.
Type I Collagen
Fibronectin
1h
1h
hMSC cultured in hMSCGM (Lonza) and
passaged 1 day prior to transfection
1h
0.6 kPa
24 h
40 kPa
1h
24 h
24 h
0.6 kPa
40 kPa
0.6 kPa
40 kPa
Cellular Transfection
Lipofectamine 2000 system utilized to introduce
the Lyn-FAK or Cyto-FAK plasmid
Figure 3: FRET Images captured with Cyto-FAK biosensor. Panels
represent Fibronectin and Type I Collagen coated gel substrate dishes
at 1 and 24 hours after hMSC were plated on the gel. The stiffness of
the substrate is indicated in the lower left corner.
Fibronectin
Cells seeded on 0.6 kPa and 40 kPa dishes
coated with Fibronectin or Type I Collagen coating
1.16
1.12
1.08
1.04
1
1
Soft
Live Cell Imaging
1.08
1.04
1
After 1 hour and 24hours hMSC were imaged and
FRET Ratio analyzed with Metaflour software
Soft
1
Hard
2
1.16
P=0.721078
Acknowledgments
1.12
1.08
1.04
1.16
FRET ratio (ECFP/Ypet)
Type I Collagen
FRET ratio (ECFP/Ypet)
1.16
1.12
1.2
1
2
Hard
P=0.958231
Wang Y, Shyy JY, Chien S. Annu Rev Biomed
Eng. 2008;10:1.
1.24
P=0.002264
1.2
Seong J, Ouyang M, Kim T, Sun J, Wen PC, Lu
S et al. Detection of focal adhesion kinase
activation at membrane microdomains by
fluorescence resonance energy transfer. Nat
Commun 2011; 2: 406.
24h after seeding
FRET ratio (ECFP/Ypet)
Passage to Gel Substrate Dishes
FRET ratio (ECFP/Ypet)
1.24
Figure 2: (a) indicates the design of the FAK biosensor and the
phosphorylated tyrosine residue in red (b) Activated FAK will phosphorlate
the biosensor’s substrate peptide and the proximity of ECPF and Ypet
effects a change in FRET fluoresces.
Citations
24 h
1h after seeding
Jihye seong, et al.
40 kPa
0.6 kPa
1
Soft
2
Hard
P=0.301495
Beckman Institute for Advance Science and
Technology
1.12
1.08
1.04
1
EBICS Research Experience for
Undergraduates
Soft
1
Hard
2
Figure 4: FRET ratios were calculated with Metaflour software. The FAK
activation observed with the Cyto-FAK FRET ratio.