Anand Kesavaraju Department of Bioengineering, University of California, Berkeley BRITE REU, University of California, Riverside

Importance in cell membrane integrity and signaling. Regulates trans-membrane protein movement and plasma membrane-to- cytoskeleton attachment mechanics. Encyclopedia Britannica. 19 Aug 2009.

We are testing the effect of various concentrations of cholesterol on plasma membrane biomechanics by pulling nanotubes (tethers) from the membrane and calculating the tether force.

 We are using optical tweezers to study plasma membrane biomechanics.  Our setup consists of a Solid-state diode pump laser (λ = 1064 nm), various optical components (100x objective), and a piezoelectric stage (nm resolution for both {x,y,z} movement and velocity).

 The plasma membrane can be represented by a viscoelastic model.  The mechanics of the tethers are explained by a second-order Maxwellian spring – dash plot model of viscoelasticity.  The time-resolved effect on the tether force will be tested (more on this in the Methodology). Biophysical Journal 89(2005):

II. Methodology

 Trapping Force Calibration Materials: 1. DMEM Complete Media (Dulbecco’s Modified Eagle Medium with FBS and Penicillin/Strep.) Serum-enriched 2. Invitrogen™ Fluorescent Sulfate-Modified Beads (2 µm radius) 3. Piezoelectric stage 4. Power Meter  Calibration procedure: 1. Pass DMEM media through a trapped bead using a piezoelectric stage at various velocities (in µm/s) at various output power measurements (W) 2. Measure the velocity when the bead is dislodged from the trap. 3. Use Stokes’ Law to calculate the Escaping Force (pN) F d = 6πηRV Where F d = Viscous Drag Force, η = Viscosity, R = Radius of the Bead, and V = Escaping Velocity

Here, the diagram illustrates the bead becoming dislodged from the trap.

This calibration graph will be used to convert the diode current found in the tether pulling experiments into output power.

This calibration graph will be used to convert the output power into the tether force.

 HEK 293 cells should be passaged when the flask/plate is ~80% confluenced.  DMEM Complete Medium and trypsin should be heated in a water bath for ~30 minutes before use to prevent thermal shock for the cells.  To passage:  Old media should be removed from the flask/plate  Cells should be washed with FBS to remove all of the old media, then FBS should be removed  500 µL to 1 mL of trypsin should be added to the plate/flask, and should then be incubated for 1-2 minutes  Clusters of cells should be broken apart using both physical taps as well as rapid sucking in-and-out of 5 mL of new medium from the plate/flask.  Medium and trypsin in the plate/flask should be pipetted into another container, then distributed in different concentrations for different types of containers.

 3 mM and 5 mM concentrations.  Cholesterol depleted using M-β-CD (Methyl-Beta-Cyclodextrin)  Cholesterol enriched using water- soluble cholesterol obtained from Sigma-Aldrich™ in the form of cholesterol carrier – 51 mg cholesterol / 1 g of material.  The prepared media are vortexed for 3-4 minutes, followed by incubation for 30 minutes (37°C at 5% CO 2 ) before experimentation.

Step 1 Step 2 Step 3 Step 4

III. Results

Very clear correlation: As cholesterol is depleted, the tether force increases, and as cholesterol is enriched, the tether force decreases. The means are also statistically significantly different.

Still somewhat linear, but less correlated

No clear correlation visible.

IV. Conclusions

 The tether forces increase as the cholesterol is depleted, and vice versa.  The order of tether forces is: Cholesterol-Depleted > Untreated > Cholesterol-Enriched  Statistically significant results  Also, the higher the concentration, the stronger the effect in either direction is – the elastic regime becomes more dominant as concentration increases.

 With no delay, the elastic regime more accurately portrays the peak tether forces.  As the viscous regime takes over, the tether forces are much lower, and are much less correlated.  This means that the viscous component of force is more dominant over time.

 Testing different tether pulling velocities.  Dynamic Force measurement.  Quantifying amount of cholesterol present.

VI. Acknowledgements

 N. Khatibzadeh  Dr. Sharad Gupta  O.S. Beane  Professor B. Anvari  Anvari Lab  J. Wang  National Science Foundation Thanking:

Thanks for your time.