1. Magnetic Tweezer System Development
Probing mechanical properties across multiple scales
Jason Sherfey
Senior BME, Vanderbilt University
Advisor: Dr. Franz Baudenbacher

2. 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

3. 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 development
2. Model testing (E-cadherin system in p120 KO vs WT mdck) for error analysis and concept testing & validation

4. Four Major Types of Junctions
1. Tight Junctions:
*permeability barriers
Actin
2. Adherens Junctions
*Classical cadherins (E-Cadherins)
*coordinate actin cytoskeleton
4. Focal Adhesions
* integrins
*coordinate actin cytoskeleton
3. Desmosomes
*desmosomal cadherins
*coordinate intermediate filaments

5. * * Tumor Progression Mutation, Time, Probability 2nd mutation
Adherens junctions *
Normal epithelium
1st mutation
Morphological changes
Mutation,
Time,
Probability
2nd mutation *
90% of human cancer is Epithelial (E-Cadherins) in origin = carcinoma

6. Dedifferentiated/EMT
- E-cadherin
Dedifferentiated/EMT
Differentiated
+ E-cadherin

7. - E-cadherin + E-cadherin Differentiated
(Polarized/Adhesive)
- E-cadherin
+ E-cadherin
Dedifferentiated/Permanent EMT
(Reduced or mutated E-cadherin or catenins)
Normal Tissue
Transient EMT
Morphogenesis
Cancer Tissue
Invasion and Metastasis
(dissociation from tumor)
Reduced adhesiveness
Differentiated:
Well organized cell polarity
due to tight cell-cell adhesion)
Morphogenesis
(Adapted from Meiners et al, 1998 and Hirohashi, 1998).

8. What is the best way to objectively
measure changes in cell-cell adhesion
relevant to metastasis?
Differentiated:
Well organized cell polarity
due to tight cell-cell adhesion)
Morphogenesis
Is it sufficient to directly quantify
E-cadherin activity?

9. E-cadherin is a Ca2+ -dependent adhesion protein
Clustering
(strong adhesion)
Adhesive
Dimer
(weak adhesion)
Actin
Crosslinking
(compaction)
Lateral
Dimer
Ca++
Monomer
JMD/p120
CBD
Actin
RhoA
Rac/Cdc42
Phosphorylation?
VASP
Mena
Vinculin
Cytoplasm

10. p120 induces cadherin clustering

11. The amount of E-cadherin is directly relevant to
adhesive strength (all things being equal).
The amount of E-cadherin does not necessarily
reflect adhesive activity. (eg, Rac, rho experiments)
Differentiated:
Well organized cell polarity
due to tight cell-cell adhesion)
Morphogenesis

12. Where does p120 fit in to all of this?
Differentiated:
Well organized cell polarity
due to tight cell-cell adhesion)
Morphogenesis

13. stability and adhesion Cell cycle, proteases, etc.
Cadherin Stability vs. Motility/Invasion
E-Cad.
Extracellular Space
Increased E-cad
stability and adhesion
tumor suppressor?
p120
JNK, p38, p38
RhoA
Rac1
Cdc42
Vav2?
Lamellipodia
Filopodia
Stress Fibers
Focal Contacts
Cell cycle, proteases, etc.
Increased
motility
and
invasion
metastasis
promoter?
-catenin
actin
filaments
a-catenin
Wnt 11
Matrilysin
Metastasin
Kaiso
AP-1
SRF

14. p120 is rate limiting for E-cadherin expression

15. 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!

16. Target Systems E-cadherin activity: linker system mechanics
(magnetic tweezer)
Mechanics of underlying actin and the homophilic
E-cadherin binding junction
(fluorescent beads, inversed microgrippers?)
Differentiated:
Well organized cell polarity
due to tight cell-cell adhesion)
Morphogenesis

17. Magnetic Bead based Rheometry
Force Calibration
Forces up to 1.5nN

18. Ecad-Fc Beads Bind Specifically to E-cadherin Expressing Cells
MDA-231 Ca ++
bead
MDA-231+ E-cad
Protein-A
Fc-Ecad

19. 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

20. Force displacement measurements on magnetic beads linked to the cell surface through E-Cadherin
1 nN 1 2 3
displacement [mm]
Time [s] F
T=0 s
T=1.5 s
Fit to Mechanical Analog
Extract Model parameter

21. Force-induced Displacement

22. Implementation Protocol
1. Before initiating cell pulling, cultured MDCK cells are:
- Trypsinized
- Seeded in a PDMS cell chamber
- Mixed w/ E-cadherin coated paramagnetic beads
- Mounted on the stage of the magnetic tweezer microrheometer.
2. After locating a suitable bead-bound cell, an automated 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, cell relaxation. This sequence is repeated several times for each bead.
3. The cell pulling videos are then analyzed using a particle tracking program in Matlab that allows high resolution quantification of bead displacement for each bead pulled. This data is then fit to a mechanical model that characterizes E-cadherin mechanics.

23. 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 for
particle identification
Invert (if necessary) &
normalize the images
Video images acquired from the CCD camera using LabView 7.1
(Raw video data)
where
k = = Elastic constant
= Viscosity
= Relaxation Time

24. Analysis of viscoeleastic response curves based on three observables [1]
Number of different cells = 7
0.002
0.004
0.006
0.008
0.01 2 4 6 8 10 12 14 16 18 20
Viscosity (Pa s m)
[1] Local Measurements of Viscoelastic Parameters of Adherent Cell Surfaces by Magnetic Bead Microrheometry
Andreas R. Bausch et. al., Biophysical Journal Volume 75 October –2049

25. Force-Displacement Curves MDCK cells
Wild-type
P120 Knockout

26. What next? How to quantify mechanical properties at different scales?

27. Measurements across multiple scales combined with finite element models
Larger Forces?
Local Measurements of Viscoelastic Parameters of Adherent Cell Surfaces by Magnetic Bead Microrheometry
Andreas R. Bausch, Biophysical Journal Volume 75 October –2049

28. Intracellular and membrane heterogeneity?

29. Imaging Brownian Motion
Capture heterogeneity!
Membrane
Cytoskeleton
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.

30. Larger forces (>100nN) for increased spatial scales?

31. Dual Pipette Assay
Force measurements in E-cadherin–mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42
Yeh-Shiu Chu et. Al., JCB • VOLUME 167 • NUMBER 6 • 2004

32. Inversed Mircogrippers

33. 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

34. 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?
Differentiated:
Well organized cell polarity
due to tight cell-cell adhesion)
Morphogenesis

35. Signaling from Cadherins
Activated
Signals
E-cadherin
adhesion molecules,
receptors, etc.
Cadherin
Dependent
Signals

36. 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…