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

A Biomechanical Comparison of Cancerous and Normal Cell

Plasma Membranes

Olivia BeaneSyracuse University

BRITE 2009

Introduction

• Plasma Membranes consist of neutral phospholipids, creating a bilayer.

Introduction• Normal cells internalize

anionic phospholipids• Cancerous cells externalize

anionic phospholipids

Cancer Res. 2002;62:6132–6140.

• 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

Diode Pump Laser

Beam Expanders

Mirror

Mirror

Micromanipulator Stage

Piezo Stage

Piezo Controller

Objective Lens

Adapted from Journal of Biomechanics, 40, 2007, 476- 480

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.

Piezo Stage

CellDish

Objective

Laser

Bead

The laser provides enough force to create an optical trap where the sulfate-modified bead is unable to escape.

CellBead

- -++

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

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.

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.

Piezo Stage

Bead

Static Calibration and Force Measurements

A static calibration is done with each individual media, due to viscosity variances.

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

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

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 Velocityr = Bead Radius (known)F = Viscous Drag Force

F= 6πηνr

Power Measurements

• To calibrate power values from specific currents, a power meter was used.

13 14 15 16 17 18

0.00

0.05

0.10

0.15

0.20

0.25

August 10 RPMI Power vs Current

Y =0.43118-0.09915 X+0.00493 X2P

ow

er

(W)

Diode Current (A)

B Polynomial Fit of Data1_BPower Meter Measurement

Power vs Current

Static Calibration and Force Measurements

• Each media’s viscosity differed, thus resulting in varying calibration graphs.

RPMI

Cancer Cell Media Normal Cell Media

LHC-9

Output Power (W) Output Power (W)

No Delay Force Measurements

• The forces of cancerous cell tethers are larger than those of normal cell tethers.

1 Minute Delay Force Measurements

• The forces of normal cell tethers are larger than those of the cancerous cell tethers.

10 12 14 16 18 201

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19August 11 HBE4/H460 1 Minute Delay Comparison

Te

the

r F

orc

e (

pN

)

Tether Length (m)

HBE4 H460

2 Minute Delay Force Measurements

10 12 14 16 18 20-2

0

2

4

6

8

10

12

14August 11 HBE4/H460 2 Minute Delay Comparison

Te

the

r F

orc

e (

pN

)

Tether Length (m)

HBE4 H460

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 time-resolved 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