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Magnetic Tweezer System Development. Probing mechanical properties across multiple scales. Jason Sherfey Senior BME, Vanderbilt University. Advisor: Dr. Franz Baudenbacher. Purpose of device: To make quantifying cell-cell adhesion quick and easy. (i.e., finding mechanical properties) - PowerPoint PPT Presentation
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Magnetic Tweezer System Development
Jason SherfeySenior BME, Vanderbilt University
Probing mechanical properties across multiple scales
Advisor: Dr. Franz Baudenbacher
• Purpose of device:
To make quantifying cell-cell adhesion quick and easy.
(i.e., finding mechanical properties)
• Specific structures to quantify:
1. cell-cell linkage
2. adhesion protein linker system
3. cytoskeleton
Motivation: - Cell-cell adhesion is essential to establishing and
maintaining cell and tissue morphology and in cellular migration
- Specific issue - alterations in cell morphology and migration are essential to tumor growth and metastasis- Idea. Quantify cell-cell adhesion Better understand
cellular morphology and migration Improve diagnostics and treatments for cancer
Principle Components of the Design Process:1. System development2. Model testing (E-cadherin system in p120 KO vs WT
mdck) for error analysis and concept testing & validation
1. Tight Junctions:
*permeability barriers
2. Adherens Junctions
*Classical cadherins (E-Cadherins)
*coordinate actin cytoskeleton
Four Major Types of Junctions
4. Focal Adhesions
* integrins
*coordinate actin cytoskeleton
3. Desmosomes
*desmosomal cadherins
*coordinate intermediate filaments
Actin
Normal epithelium
1st mutation
Tumor Progression
Mutation,Time, Probability
90% of human cancer is Epithelial (E-Cadherins) in origin = carcinoma*
*
Morphological changes
Adherens junctions
Differentiated
- E-cadherin- E-cadherin
+ E-cadherin+ E-cadherin
Dedifferentiated/EMT
Differentiated(Polarized/Adhesive)
- E-cadherin- E-cadherin
+ E-cadherin+ E-cadherinDedifferentiated/Permanent EMT(Reduced or mutated E-cadherin or catenins)
Normal Tissue
Transient EMT
Morphogenesis Cancer Tissue
Invasion and Metastasis
(dissociation from tumor)
Reduced adhesiveness
(Adapted from Meiners et al, 1998 and Hirohashi, 1998).
What is the best way to objectivelymeasure changes in cell-cell adhesion relevant to metastasis?
Is it sufficient to directly quantifyE-cadherin activity?
Cytoplasm
Monomer
LateralDimer
Ca++
JMD/p120RhoA
Phosphorylation?
CBDRac/Cdc42
VASPMenaVinculin
AdhesiveDimer(weak adhesion)
CadherinClustering(strong adhesion)
ActinCrosslinking(compaction)
Actin
E-cadherin is a Ca2+ -dependent adhesion protein
CadherinClustering
P
P
p120 induces cadherin clustering
1. The amount of E-cadherin is directly relevant toadhesive strength (all things being equal).
2. The amount of E-cadherin does not necessarily reflect adhesive activity. (eg, Rac, rho experiments)
Where does p120 fit in to all of this?
Cadherin Stability vs. Motility/Invasion
p120
Extracellular Space
-catenin
actinfilaments
-catenin
Kaiso
Wnt 11MatrilysinMetastasin
SRF AP-1
E-Cad.Increased E-cad
stability and adhesion
tumor suppressor?
JNK, p38, p38
RhoA
Rac1
Cdc42
Vav2?Lamellipodia
Filopodia
Stress FibersFocal Contacts
Cell cycle, proteases, etc.
Increasedmotility
andinvasion
metastasispromoter?
p120
E-c
adhe
rin
p120 is rate limiting for E-cadherin expression
p120 is essential for cadherin stability
Measure the mechanical properties of the E-cadherin adhesion system in cells with and without p120.
There should be big differences!
Target Systems
1. E-cadherin activity: linker system mechanics(magnetic tweezer)
2. Mechanics of underlying actin and the homophilic E-cadherin binding junction(fluorescent beads, inversed microgrippers?)
Magnetic Bead based Rheometry
Force vs. distance to tip (~0.1A)
0.00E+00
5.00E-10
1.00E-09
1.50E-09
2.00E-09
2.50E-09
0 100 200 300 400 500 600
Distance to tip (μm)
Fo
rce
(N)
vDF 6
Force Calibration
Forces up to 1.5nN
Protein-A
bead
Fc-Ecad
Ca++
Ecad-Fc Beads Bind Specificallyto E-cadherin Expressing Cells
MDA-231
MDA-231+ E-cad
Accomplishments
• Implemented particle tracking software
• Fabricated magnetic tweezer
• Protocol to quantify the elastic properties of E-cadherins using magnetic bead based microrheology– Validation of the linker system
F
T=0 s
T=1.5 s
0
1 2 30
1
2
3
dis
pla
cem
en
t [
m]
Time [s]
Fit to Mechanical Analog
Extract Model parameter
Force1 nN
Force displacement measurements on magnetic beads linked to the cell surface through E-Cadherin
Force-induced Displacement
0 10 20 30 40 50 600
10
20
30
40
50
60
50 100 150 200 250
50
100
150
200
250
0 10 20 30 40 50 600
10
20
30
40
50
60
Implementation Protocol• 1. Before initiating cell pulling, cultured MDCK cells are: 1. Before initiating cell pulling, cultured MDCK cells are:
– - Trypsinized - Trypsinized – - Seeded in a PDMS cell chamber- Seeded in a PDMS cell chamber– - Mixed w/ E-cadherin coated paramagnetic beads- Mixed w/ E-cadherin coated paramagnetic beads– - Mounted on the stage of the magnetic tweezer - Mounted on the stage of the magnetic tweezer
microrheometer. microrheometer.
• 2. After locating a suitable bead-bound cell, an automated 2. After locating a suitable bead-bound cell, an automated LabView/C++ routine acquires 3 seconds of images at 122Hz: 0-1s, LabView/C++ routine acquires 3 seconds of images at 122Hz: 0-1s, steady-state; 1-2s, power supply triggered to initiate the force; 2-3s, steady-state; 1-2s, power supply triggered to initiate the force; 2-3s, cell relaxation. This sequence is repeated several times for each cell relaxation. This sequence is repeated several times for each bead.bead.
• 3. The cell pulling videos are then analyzed using a particle tracking 3. The cell pulling videos are then analyzed using a particle tracking program in Matlab that allows high resolution quantification of bead program in Matlab that allows high resolution quantification of bead displacement for each bead pulled. This data is then fit to a displacement for each bead pulled. This data is then fit to a mechanical model that characterizes E-cadherin mechanics.mechanical model that characterizes E-cadherin mechanics.
Particle Tracking Algorithm
Spatial Bandpass Filter
Find coordinates of peak intensities in the current frame
Average around peaks to obtain particle centroid
Final Frame?
NO
YES
Analyze bead trajectories through all frames
Fit bead (i.e., membrane) displacements to a viscoelastic model.
{k, γ, τ}
(x(t),y(t),r(t),v(t),…)
(Peak intensity = beads)
Pre-Processing
Optimize parameters forparticle identification
Invert (if necessary) &normalize the images
Video images acquired from the CCD camera using LabView 7.1
(Raw video data)
,)/exp(1)(010
1
0 t
tkk
k
k
Ftx
where 10
101 )(
kk
kk
k = = Elastic constant10 kk
= Viscosity0 = Relaxation Time
00.005
0.010.0150.02
0.025
0.030.0350.04
0.0450.05
0 2 4 6 8 10 12 14 16 18 20
Experiment #
Ela
stic
ity (
Pa
m)
0
0.02
0.04
0.06
0.08
0.1
0.12
0 2 4 6 8 10 12 14 16 18 20
Re
laxa
tion
Tim
e (s
)
[1] Local Measurements of Viscoelastic Parameters of Adherent Cell Surfaces by Magnetic Bead MicrorheometryAndreas R. Bausch et. al., Biophysical Journal Volume 75 October 1998 2038–2049
0
0.002
0.004
0.006
0.008
0.01
0 2 4 6 8 10 12 14 16 18 20
Vis
cosi
ty (
Pa
s m
)
skk
kk024.0042.0
)(
10
101
msPa 0020.00044.00
mPakkk 0090.0020.0)( 10
Analysis of viscoeleastic response curves based on three observables [1]
Number of different cells = 7
0 0.5 1 1.5 2 2.5 30
0.5
1
1.5
2
2.5
3
3.5
time (s)
disp
lace
men
t (m
icro
ns)
0 0.5 1 1.5 2 2.5 30
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
time (s)
disp
lace
men
t (m
icro
ns)
0 0.2 0.4 0.6 0.8 1 1.2 1.4-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
time (s)
disp
lace
men
t (m
icro
ns)
k=-0.036244Pa m,n0=0.0025302Pa s m,tau=0.053513s
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10
0.5
1
1.5
2
2.5
time (s)
disp
lace
men
t (m
icro
ns)
F~1nN, k=22.2696, n0=7.1283, tau=0.055134
Wild-type P120 Knockout
Force-Displacement Curves MDCK cells
What next?
How to quantify mechanical properties at different scales?
Measurements across multiple scales combined with finite element models
Larger Forces?
Local Measurements of Viscoelastic Parameters of Adherent Cell Surfaces by Magnetic Bead MicrorheometryAndreas R. Bausch, Biophysical Journal Volume 75 October 1998 2038–2049
Intracellular and membrane heterogeneity?
Imaging Brownian Motion
Mechanical deformation of neutrophils into narrow channels induces pseudopod projection and changes in biomechanical properties, Belinda Yap and Roger D. Kamm, J Appl Physiol 98: 1930–1939, 2005.
Capture heterogeneity!
- Membrane
- Cytoskeleton
Larger forces (>100nN) for increased spatial scales?
Dual Pipette Assay
prF 2
Force measurements in E-cadherin–mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42Yeh-Shiu Chu et. Al., JCB • VOLUME 167 • NUMBER 6 • 2004
Inversed Mircogrippers
Overview of Techniques
• Brownian motion – cytoskeletal and membrane heterogeneity
• Magnetic tweezer – adhesion protein linker system mechanics
• Dual pipette (large forces) – cadherin-cadherin separation force
• Inversed microgrippers (large forces?) – cadherin-cadherin separation force
Single device for multi-scale measurements of cell-cell adhesion
Now that the tracking and analysis software works and bead-based microrheology has been validated, how can a device be designed that characterizes the mechanical properties of the adhesion system of any cell line over multiple spatial and temporal scales?
CadherinActivatedSignals
E-cadherin
Signaling from Cadherins
adhesion molecules,receptors, etc. Cadherin
DependentSignals
Integration and Miniaturization!Fluorescent microbeads or quantum dots?
- imaging brownian motion
- tracking force-induced changes in heterogeneities
On chip electrical components (e.g., CMOS)?
- signal conditioning
- increase signal-to-noise ratio
High density GMR sensor array substrate with multiplexing?
- removes need for expensive microscope & CCD
- directly senses XY-position
Microfabricated electromagnets?
- removes need for expensive micromanipulators
Microfluidics with cell traps?- fixed cell positions (don’t have to search for good cells)
Micropatterning? …etc…