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

2. Purpose of device: To make quantifying mechanical properties of cells quick and easy. Specific structures to quantify: 1. cell-cell linkage 2. adhesion protein linker system 3. cytoskeleton

3. Mechanical Signaling Pathways Modulate cellular function –Differentiation –Migration –Gene expression –Protein synthesis Directly affect form & function of tissue Abnormalities result in dramatic effects –Metastasis

4. 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 (Adapted from Meiners et al, 1998 and Hirohashi, 1998). Mechanical Signaling via E-cadherins in Cancer

5. p120 Bound p120 retains/stabilizes cadherin at the cell surface, and promotes adhesion Free p120 inhibits Rho, and promotes motility by reducing ASF’s Rho Signal-1Signal-2 Cadherin Integrin’s RTKs LPA Mechanical signaling through Cadherins GPCR’s ECM GF’s Rho

6. Strategy Property measured: Viscosity & stiffness of E-cadherin adhesion protein linker system (static & dynamic responses) with and without the P120 protein Strategy: Use a magnetic tweezer – based device to perturb, image, and analyze linker system mechanics…

7. F T=0 s T=1.5 s Fit to Mechanical Analog Extract Model parameter Force 1 nN Single Cell Instrumentation!!!

8. Microfluidic channels Cell type 1 Cell type 2 To CCD PDMS

9. 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

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

11. Viscosity (Pa-s-m) N = 10

12. Elasticity (Pa-m) N = 10

13. Relaxation Time (s) N = 10

14. Viscosity (Pa-s-m) 0.00200.0044Wild-type MDCK 0.00190.0038P120 Knockout MDCK Standard Deviation Mean p = 0.364 Elasticity (Pa-m) 0.00880.0217Wild-type MDCK 0.01060.0118P120 Knockout MDCK Standard Deviation Mean p = 0.0028 Relaxation Time (s) 0.00060.0421Wild-type MDCK 0.00060.0725P120 Knockout MDCK Standard Deviation Mean p = 0.0010 No significant difference in WT & KO Viscosities WT Elasticity is significantly larger than KO WT Relaxation Time is significantly faster than KO N = 10 cells

15. Conclusions 1.The stiffness and relaxation time constants are significantly different in p120 knockout and wild-type MDCK cells. 2. The stiffness decreases ~50% & relaxation time increases ~75% when p120 expression is reduced in MDCK cells. 3. The magnetic tweezer system can quantify the difference in cadherin-catenin adhesion mechanics following cancer-like alterations in P120 expression.

16. Strain Hardening The stiffness of the adhesion protein linker system increases when stress is repeatedly applied. Pull Wild-type Cell 10.0132 20.0152 30.0193 40.0249 Knockout Cell 10.0051 20.0063 30.0081 Stiffness (Pa-m)

17. Status Past: –Validated system theory –Finished prototype Present –Tests & performance evaluation –Prototype refinement –Documentation