IntroductionThis study deals with the effects of cholesterol on human embryonic kidney (HEK293) cells’ plasma membrane mechanics using an optical tweezers setup.

Cholesterol regulates trans-membrane protein movement and plasma membrane-to-cytoskeleton attachment mechanics. Testing the effect of differing cholesterol concentrations on the biomechanics of HEK cells is integral to our understanding of the role cholesterol plays. Using the optical tweezers setup (Fig. 1), we extract nanotubes (tethers) from the plasma membrane under cholesterol-enriched and cholesterol-depleted conditions to determine the tether force. We are assuming that a second-order Maxwellian spring – dash plot model (Fig. 2) of viscoelasticity holds for the mechanics of these tethers2.

Materials and MethodsStatic Force Calibration:The trapping force was calibrated by passing DMEM Complete (Dulbecco’s Modified Eagle Medium with FBS and Penicillin/Strep.) Serum-enriched through a trapped bead using a piezoelectric stage at various velocities (in µm/s) and measuring the velocity when the bead is dislodged from the trap. From these, we use a modified version of Stokes’ Law to calculate the Escaping Force (visible in Fig. 3).

Static Tether Force Measurement:Using the Optical Tweezers setup, we brought a single cell in contact with a trapped bead and held the two together for a short time. After this time, the cell was moved away (at 1 µm/s) to pull a tether of length 10µm, 15 µm, or 20 µm. At this time (depending on the time-resolution – no delay, one minute, or two minutes delay), the laser power was lowered until the bead jumps out of the trap and returns rapidly to the cell (indicating a tether had formed). The final diode current was recorded to calculate the Tether Force (pN).Cholesterol Manipulation: HEK 293 cells are incubated in cholesterol-enriched and -depleted medias with the following concentrations: 3 mM, and 5 mM. Cholesterol is depleted using M-β-CD (Methyl-Beta-Cyclodextrin), and cholesterol-enrichment is done using water-soluble cholesterol from Sigma-Aldrich™. The prepared media are vortexed for 3-4 minutes, followed by incubation for 30 minutes (37°C at 5% CO2) before experimentation.

AcknowledgmentsI would like to offer special thanks to N. Khatibzadeh and Sharad Gupta for all of their assistance and advice through the course of this study. I would also like to thank O.S. Beane for her moral support. Additionally, I would like to thank J. Wang and the National Science Foundation for providing the BRITE REU grant. Further thanks to B. Anvari and the rest of theDepartment of Bioengineering at the University of California, Riverside for all of their help and expertise.

ResultsThe static calibration results are presented first, to show the result of our The static calibration results are presented first, to show the result of our Stokes’ Law calculation.Stokes’ Law calculation.

Conclusion and Future Work

We have determined that depleting cholesterol through the use of Methyl-β-Cyclodextrin causes the tether force to increase significantly over the untreated value, while enriching the plasma membrane with cholesterol causes a decrease in the tether force. With both cholesterol-enrichment and –depletion, a greater decrease or increase, respectively, is visible as the tether length is increased from 10 µm to 20 µm. We have also determined that the presence of a time-resolved study inherently highlights the viscous component of the second-order Maxwellian spring – dash plot model2. 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.

In the future, work involving different tether pulling velocities must be tested to further examine the time-resolution effect. Also, dynamic force measurements must be taken into account. Finally, a quantification schema for the amount of cholesterol present in the membrane must be devised.

A. Kesavaraju1; Advised by: N. Khatibzadeh2, S. Gupta3 and B. Anvari3

1Department of Bioengineering, University of California, Berkeley, CA

2Department of Mechanical Engineering, University of California, Riverside, CA

3Department of Bioengineering, University of California, Riverside, CA

References1Ermilov, S.A., et al. "Studies of plasma membrane mechanics

and plasma membrane-cytoskeleton interactions using optical tweezers and fluorescence imaging." Journal of Biomechanics 40(2007): 476-480. Print.

2Murdock, David R., et al. "Effects of Chlorpromazine on Mechanical Properties of the Outer Hail Cell Plasma Membrane." Biophysical Journal 89(2005): 4090-4095. Print.

3Li, Zhiwei, Et al. "Membrane Tether Formation from Outer Hair Cells with Optical Tweezers." Biophysical Journal 82(2002): 1386-1395. Print.

The modified Stokes’ Law expressionWhere η = viscosity (in Pa∙s), r = radius of the fluorescent bead, and v = the escaping velocity of the bead 3

Figure 3: Static Calibration with DMEM Complete Media + Fluorescent beads. Graph shows Escaping Force (in pN) vs. the Output Power (in W) along with the quadratic fit of the data. An r2 value of 0.99218 was obtained.

For further informationPlease contact anand.k@berkeley.edu. More information on this and related projects can be obtained at www.engr.ucr.edu/faculty/bio/anvari.shtml

Figure 5. The untreated values (in Black) are exactly in the middle of the other two variations. As the concentrations increase, the values become either higher or lower, respectively with cholesterol-depletion and –enrichment.

Figure 6a: After one minute, there is still a positive trend, but it is no longer linear and correlated.

Effect of Cholesterol on Plasma Membrane Biomechanics of HEK293 Cells

Figure 2: Second-Order Maxwellian spring – dash plot model of tether pulling2

Due to this, we also study the tether force with a degree of time resolution - so as to better understand how the viscoelastic properties change. Thus, we study the force after a relaxation period of one minute and two minutes.

Figure 4: This shows the final results in a column graph, along with the error bars.

The following graphs (Figs. 6a and 6b) show the time-resolved effect of cholesterol enrichment and depletion. Figure 6a is after a one minute delay, and Figure 6b is after a two minute delay .

Figure 1: This graphic shows the Optical Tweezers setup1. The laser(1 & 2) is in red, the piezoelectric stage (11) is in black, and the microscope apparatus and micromanipulators (13 & 23) are in turquoise.

Figure 6b: After two minutes, the pattern degrades further – there is no clear trend, positive or otherwise in the data.

The above graph shows that the tether forces increase as the cholesterol is depleted. This is a very significant result, and can be backed up with statistical t-tests that show that the means are statistically different. There seems to be a clear, positive, and linear trend of forces for the concentrations studied – with no delay. The order of tether forces is:

Cholesterol-Depleted > Untreated > Cholesterol-Enriched

Also, the higher the concentration, the stronger the effect in either direction is. This becomes more evident in Fig. 5.

M-β-CD structure

Cholesterol structure