Transcript Anand Kesavaraju - University of California, Riverside

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-tocytoskeleton attachment mechanics.
Encyclopedia Britannica. 19 Aug 2009 <http://media-2.web.britannica.com/eb-media/74/53074-004-9F65D813.jpg>.
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): 4090-4095.
II. Methodology

1.
2.
3.
4.
Trapping Force Calibration Materials:
DMEM Complete Media (Dulbecco’s Modified Eagle Medium with FBS and
Penicillin/Strep.) Serum-enriched
Invitrogen™ Fluorescent Sulfate-Modified Beads (2 µm radius)
Piezoelectric stage
Power Meter

1.
2.
3.
Calibration procedure:
Pass DMEM media through a trapped bead using a piezoelectric stage at
various velocities (in µm/s) at various output power measurements (W)
Measure the velocity when the bead is dislodged from the trap.
Use Stokes’ Law to calculate the Escaping Force (pN)
Fd= 6πηRV
Where Fd = 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 watersoluble 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% CO2) 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
Thanking:
N. Khatibzadeh
 Dr. Sharad Gupta
 O.S. Beane
 Professor B. Anvari
 Anvari Lab
 J. Wang
 National Science Foundation

Thanks for your time.