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A Biomechanical Comparison of Cancerous and Normal Cell Plasma Membranes Olivia Beane Syracuse University BRITE 2009 Introduction • Plasma Membranes consist of neutral phospholipids, creating a bilayer. Introduction • Normal cells internalize anionic phospholipids Cancer Res. 2002;62:6132–6140. • Cancerous cells externalize anionic phospholipids • The externalization of anionic phospholipids in cancerous cells may cause a variance from the biomechanical properties of normal cell plasma membranes. =\ 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. Experimental Setup Beam Expanders Diode Pump Laser Piezo Stage Piezo Controller Objective Lens Mirror Adapted from Journal of Biomechanics, 40, 2007, 476- 480 Micromanipulator Stage Mirror 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. Bead Dish Cell Piezo Stage Laser Objective The laser provides enough force to create an optical trap where the sulfate-modified bead is unable to escape. 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. Bead Tether Cell Piezo Stage 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. Cell Piezo Stage • 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. Static Calibration and Force Measurements Bead Piezo Stage A static calibration is done with each individual media, due to viscosity variances. Static Calibration and Force Measurements Bead Piezo Stage The piezo staged is moved a set distance at chosen velocities until it escapes the optical trap. Static Calibration and Force Measurements Bead Piezo Stage • This is known as the “escaping velocity”. • The calibration continues at different currents to determine their respective escaping velocities. Stoke’s Law F= 6πηνr η= Dynamic Viscosity (known) ν = Escaping Velocity r = Bead Radius (known) F = Viscous Drag Force • 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. Power Measurements Power vsPower Current August 10 RPMI vs Current 0.25 2 Y =0.43118-0.09915 X+0.00493 X B Power Meter Measurement Polynomial Fit of Data1_B Power (W) 0.20 0.15 0.10 0.05 0.00 13 14 15 16 17 18 Diode Current (A) • To calibrate power values from specific currents, a power meter was used. Static Calibration and Force Measurements Cancer Cell Media Normal Cell Media LHC-9 RPMI Output Power (W) Output Power (W) • Each media’s viscosity differed, thus resulting in varying calibration graphs. No Delay Force Measurements • The forces of cancerous cell tethers are larger than those of normal cell tethers. 1 Minute Delay Force Measurements Tether Force (pN) August 11 HBE4/H460 1 Minute Delay Comparison 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 HBE4 H460 10 12 14 16 18 20 Tether Length (m) • The forces of normal cell tethers are larger than those of the cancerous cell tethers. 2 Minute Delay Force Measurements August 11 HBE4/H460 2 Minute Delay Comparison 14 HBE4 H460 12 Tether Force (pN) 10 8 6 4 2 0 -2 10 12 14 16 Tether Length (m) 18 20 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. Future Directions • Perform same experiment with dynamic force measurements to obtain timeresolved force of plasma membrane. • Use standing wave microscopy to measure diameter of tether. Acknowledgements Thank you to… Professor Anvari The Anvari Lab Jun Wang Dr. Victor Rodgers