1. A Biomechanical Comparison of Cancerous and Normal Cell Plasma Membranes Olivia Beane Syracuse University BRITE 2009

2. Introduction Plasma Membranes consist of neutral phospholipids, creating a bilayer.

3. Introduction Normal cells internalize anionic phospholipids Cancerous cells externalize anionic phospholipids Cancer Res. 2002;62:6132–6140.

4. The externalization of anionic phospholipids in cancerous cells may cause a variance from the biomechanical properties of normal cell plasma membranes. =\

5. Objective To test this theory, compare the biomechanical properties of normal Human Bronchial Epithelial cell plasma membranes (HBE4) and cancerous Human Bronchial Epithelial cell plasma membranes (H460), using optical tweezers.

6. Experimental Setup Diode Pump Laser Beam Expanders Mirror Micromanipulator Stage Piezo Stage Piezo Controller Objective Lens Adapted from Journal of Biomechanics, 40, 2007, 476- 480

7. Experimental Setup Diode Pump Laser –λ=1064 nm Objective Lens – 100x resolution and 1.47 NA to cause higher gradient for electrical field to form optical trap. Piezo Stage –Controls fine movement.

8. Piezo Stage Cell Dish Objective Laser Bead The laser provides enough force to create an optical trap where the sulfate-modified bead is unable to escape.

9. Cell Bead - - + + The negatively charged bead attaches to the positively charged exterior of the cell when they have been moved into contact with each other.

10. Piezo Stage Cell Bead Tether After 5-10 seconds, the Piezo stage moves the cell a controlled distance away (10 μm, 15 μm, and 20 μm) from the bead and a tether, nanotubule, is formed.

11. Piezo Stage Cell The tether has elastic properties and relaxation tendencies. Tested tether forces after no-delay, 1 minute delay, and 2 minute delay. The current at which the force of the tether exceeds that of the optical trap, portrays the force of the tether.

12. Piezo Stage Bead Static Calibration and Force Measurements A static calibration is done with each individual media, due to viscosity variances.

13. Piezo Stage Bead The piezo staged is moved a set distance at chosen velocities until it escapes the optical trap. Static Calibration and Force Measurements

14. Piezo Stage Bead This is known as the “escaping velocity”. The calibration continues at different currents to determine their respective escaping velocities. Static Calibration and Force Measurements

15. Stoke’s Law Implement Stoke’s Law to convert the escaping velocities to Viscous Drag Force. This calculates the force of the optical trap at each current value. η= Dynamic Viscosity (known) ν = Escaping Velocity r = Bead Radius (known) F = Viscous Drag Force F= 6πηνr

16. Power Measurements To calibrate power values from specific currents, a power meter was used. Power Meter Measurement Power vs Current

17. Static Calibration and Force Measurements Each media’s viscosity differed, thus resulting in varying calibration graphs. RPMI Cancer Cell MediaNormal Cell Media LHC-9 Output Power (W)

18. No Delay Force Measurements The forces of cancerous cell tethers are larger than those of normal cell tethers.

19. 1 Minute Delay Force Measurements The forces of normal cell tethers are larger than those of the cancerous cell tethers.

20. 2 Minute Delay Force Measurements

21. Conclusions The no-delay forces generated by cancerous cell tethers are larger than those generated by normal cell tethers suggesting carcinoma cell plasma membranes have higher elasticity than normal cells. The opposite was true after 1 minute delay. This suggests that the viscous properties of carcinoma and normal cell membranes differ. No conclusions can yet be determined from the 2 minute delay results.

22. Future Directions Perform same experiment with dynamic force measurements to obtain time- resolved force of plasma membrane. Use standing wave microscopy to measure diameter of tether.

23. Acknowledgements Thank you to… Professor Anvari The Anvari Lab Jun Wang Dr. Victor Rodgers