The 5th Korean-US NanoForum: Nanobiotechnology. Jeju Island, April 17th-19th, 2008

Focal Adhesion Mechanotransduction:Focal Adhesion Mechanotransduction:Cellular Response to Nanoscale Cellular Response to Nanoscale

Mechanical FactorsMechanical Factors

Nathan J. Sniadecki, Ph.D.

Mechanical Engineering DepartmentUniversity of Washington

Seattle, Washington

Mechanotransduction at Focal AdhesionsMechanotransduction at Focal Adhesions

Focal adhesions are force sensitive integrin-receptor complexes (100 nm – 1 μm).

Extracellular Matrix (ECM)

CellActin

Myosin

Extracellular matrix (ECM)

Focal Adhesion (FA)

Structural proteinsStructural proteins link integrins to actin to develop traction force for cellular migration or contraction

Signaling proteinsSignaling proteins activated with integrin-matrix binding and force vinvin

taltal

FAK

tal

p130

Actin

Integrins Integrins

α-ActFAK

Srcpax

Traction Force

External Force

Traction forces or external forces are transduced into biological signals that can regulate cell function

Traction Force Sensor ArrayTraction Force Sensor Array

20 nN

DAPI Actin PDMS Vinculin10 μm

Arrays of flexible microposts to measure traction forces

Made with soft lithography of polydimethlysiloxane (PDMS)

3-μm diameter, 10-μm heightmicroposts

Magnetic MicropostsMagnetic Microposts

Sensor and Actuator SystemSensor and Actuator System• Measure traction forces with microposts

• Apply forces with magnetic nanowires

Cobalt NanowiresCobalt Nanowires• 300 nm diameter, 5-7 μm long• High magnetic moment (µ) along long axis

Nanowire

NanowiresN.J. Sniadecki, et al., PNAS, 2007, 104:114553-8

N.J. Sniadecki, et al., Rev Sci Instr, 2008, 79, 044302

2 2

3 ( )2( )

wMag

w w

L LFL L L Lτ +

=+ +

d

3

3 Eδ δ 64LtrF K π

= =

Bµ ⋅= ⊥τ

Magnetic NanowiresMagnetic Nanowires

2e-

Co2+

Co

Co2+

Co2+

Co2+

Co2+(aq) + 2e- → Co(s)

2 µm

Template

Cobalt Electrodeposition Vibrating Sample Magnetometer

N.J. Sniadecki, et al., PNAS, 2007, 104:114553-8N.J. Sniadecki, et al., Rev Sci Instr, 2008, 79, 044302

FabricationFabrication

1. Lithography

Nanowires magnetically embedded into PDMS mold

4. Deposit Nanowires

2. Develop 5. Encase in PDMS

3. Cast in PDMS 6. Release

SEM Image

EDS Measurement

N.J. Sniadecki, et al., PNAS, 2007, 104:114553-8N.J. Sniadecki, et al., Rev Sci Instr, 2008, 79, 044302

Magnetic StimulationMagnetic Stimulation

N.J. Sniadecki, et al., PNAS, 2007, 104:114553-8

Magnetic actuation for external force

Focal adhesion protein recruitment

Stimulation relaxes traction forces

Spatial ResponseSpatial Response

JumpJump GradualGradual

ControlControl

Magnetic Post

Relaxation occurs at remote sites along cell periphery but not at interior posts

Binomial Analysis:Edge posts have significant loss versus interior posts (p<0.05)Post local to magnetic posts have no significant difference to distal posts (p>0.25)

N.J. Sniadecki, et al., PNAS, 2007, 104:114553-8

NanopostsNanoposts

Sloped-wall cantilever

High Spatial Resolution of Traction Forces

830 nm dia, 3.3 μm tal16 nN/μm

830 nm dia, 3.0 μm tall28 nN/μm

670 nm dia, 3.3 μm tal8 nN/μm

M.T. Yang, N.J. Sniadecki, C.S. Chen, Adv Mat, 2007, 104:114553-8

SUPPORT: National Institutes of Health National Science FoundationThe Hartwell FoundationUW Royalty Research Fund

Nathan J. SniadeckiSangyoon HanAimi Ahmad Shukri

Collaborators:Christopher S. Chen (UPenn)Daniel H. Reich (JHU)

AcknowledgementsAcknowledgements

J. L. Tan, et al., Proc. Nat. Acad. Sci., 2003, 100:1484-1489C. A. Lemmon, et al., Mech. & Chem. Biosystems., 2005, 2(1) 1-16

Micropost Force SensorsMicropost Force Sensors

4

3

3 EdF 64L

K πδ δ

= =

F … Cellular ForceK… Spring Stiffness (32 nN/μm)δ … DisplacementE … Elasticity Modulus of PDMSd … Post diameter (3 μm dia)L … Post Length (10 μm)

Posts deflects as simple cantilever springs:

Microcontact printing:No adhesion blocking: Adhesion only on tips:Post Array:

Fibronectin

Measurements:

PEO

PPO