CISMM: Computer Integrated Systems for Microscopy and Manipulation

Collaborators: Dr. R. E. Cheney, Dept of Cell and Molecular Physiology

Project Lead: Dr. R. Superfine Investigators: Sreeja B. Asokan, Dr. R. Lloyd Carroll

http://cs.unc.edu/Research/nano/cismm/motors

12/05/2003

Motivation

Fabrication of microfluidic channels

Flow Induced Forces on Molecular Motor/Filament Motility

The response of cytoskeletal filaments and associated motor proteins to external forces not only has relevance to biology within the cell, but is important for biomolecular integration with nanoelectronics. In vivo, Brownian fluctuations and viscous drag are common forces that affect the motility and self-organization of these cytoskeletal components. Observing their response to environments and external flow forces similar to what they experience in vivo will improve our understanding of the way they function and will help exploit physiological forces for nanotechnology applications.

Laminar flow

NegativePhotoresist

Photomask

Photolithography

Si Wafer

Master

Pour PDMS on

Master

Punch reservoirs

The channels were made out of PDMS by steps of rapid prototyping, lithography, replica molding and sealing.(Analytical Chemistry 1998 v70, p4974). The PDMS is then connected to a syringe pump through Teflon tubing

We performed gliding motility assays of actin filaments on myosin V in microfluidic channels. We were able to achieve controlled delivery of proteins and buffers, regulate velocity of fluid flow, exercise control over direction of flow and perform detailed analysis of force imparted by the fluid flow. Motility of actin filaments was successfully directed at varying flow rates. The flow exerted drag force and torque on the filaments, suppressed Brownian fluctuations and oriented them in the direction of flow. By reversing the flow direction, the direction of filament motility was reversed.

Cure and Peel

X

Y

U0=0

Uh=0

3 different colors of fluid flowing in the microfluidic channel do not mix except by diffusion

Bright field image of the center of the channel

yhyh

UU avgy

2

6

Flow in the channel has a parabolic velocity profile. The velocity is given by

External flow was applied to actin filaments undergoing gliding motility assay in microfluidic channels. The velocity in the channel and drag on actin filaments were calculated at a height of h=70nm above the surface which is the height of myosin from AFM experiments. The drag coefficients parallel and perpendicular to flow are

Drag is equal to Drag coefficient * Velocity of fluid. The velocity and drag are

r

h

lF

2ln

2||

r

h

lF

2ln

4

Velocity (/s) Drag force || (pN) Drag force (pN)

28 0.049 0.099

56 0.099 0.198

84 0.148 0.296

112 0.198 0.394

140 0.246 0.492

-150 -100 -50 0 50 100 1500

10

20

30

40

50

60

70

Dis

trib

uti

on

fre

qu

ency

Angle in degrees-150 -100 -50 0 50 100 150

0

10

20

30

40

50

Dis

trib

uti

on

fre

qu

ency

Angle in degrees-150 -100 -50 0 50 100 150

0

10

20

30

40

50

Dis

trib

uti

on

fre

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ency

Angle in degrees

The direction of motility of the gliding actin filaments were measured for different flow rates and histograms of angular distribution frequency in the presence and absence of flow were generated.

The above histograms are the frequency of angular distribution of filaments in the absence of flow, flow towards zero degrees and flow towards 180 degrees

Flow is an aligning and directing force for actin filaments during gliding assay

The work done(1) by a filament to align at an angle theta from the flow direction (zero) was equated to energy of filament. The most probable distribution of angular alignment of a set of actin filaments is the Maxwell Boltzmann distribution(2). This theoretical distribution was fitted to the histograms of angular distribution from data.

Last bound m5 acts as a frictionless pivot

)2

ln(

)cos1(2 2

r

hvl

W

Actin was modeled as a rigid rod rotating on a frictionless pivot under the influence of drag and thermal motion only.

-200 -150 -100 -50 0 50 100 150 200

0

50

100

150

200

250

Dis

trib

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Angle in degrees

140microns/s

-200 -150 -100 -50 0 50 100 150 2000

5

10

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20

25

30

35

Dis

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Angle in degrees

28microns/s

-200 -150 -100 -50 0 50 100 150 200-0.010.000.010.020.030.040.050.060.070.080.090.100.11

28um/s 56um/s 84um/s 112um/s 140um/s

Dis

trib

uti

on

fre

qu

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Angle in degrees

The black squares are experimental data points and the red curve is the theoretical fit at maximum and minimum flow rate(28 &140 /s). Also shown are the theoretical fits at different flow rates.

No flow Forward flow at 112/s

Reverse flow at 112/s

)cos1()( BAeP)

2ln(

2 2

r

hTk

vlB

B

0 20 40 60 80 100 120 1400.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Fit

tin

g p

ara

me

ter

B

velocity (microns/s)

The figure on the left is a plot of fitting parameter B Vs velocity. The slope of this line gives the value of

From B’, l was calculated to be 0.27±0.04µ

m

s

rh

Tk

lB

B

25760)

2ln(

2 2

(1) (2)

The theoretical model for the system fits the experimental data and a reasonable value for the length of actin subunit beyond the last bound myosin(l = 0.27±0.04µ) was obtained.