Magnetic Tweezer System Development

Jason SherfeySenior BME, Vanderbilt University

Probing mechanical properties across multiple scales

Advisor: Dr. Franz Baudenbacher

• Purpose of device:

To make quantifying cell-cell adhesion quick and easy.

(i.e., finding mechanical properties)

• Specific structures to quantify:

1. cell-cell linkage

2. adhesion protein linker system

3. cytoskeleton

Motivation: - Cell-cell adhesion is essential to establishing and

maintaining cell and tissue morphology and in cellular migration

- Specific issue - alterations in cell morphology and migration are essential to tumor growth and metastasis- Idea. Quantify cell-cell adhesion Better understand

cellular morphology and migration Improve diagnostics and treatments for cancer

Principle Components of the Design Process:1. System development2. Model testing (E-cadherin system in p120 KO vs WT

mdck) for error analysis and concept testing & validation

1. Tight Junctions:

*permeability barriers

2. Adherens Junctions

*Classical cadherins (E-Cadherins)

*coordinate actin cytoskeleton

Four Major Types of Junctions

4. Focal Adhesions

* integrins

*coordinate actin cytoskeleton

3. Desmosomes

*desmosomal cadherins

*coordinate intermediate filaments

Actin

Normal epithelium

1st mutation

Tumor Progression

Mutation,Time, Probability

90% of human cancer is Epithelial (E-Cadherins) in origin = carcinoma*

*

Morphological changes

Adherens junctions

Differentiated

- E-cadherin- E-cadherin

+ E-cadherin+ E-cadherin

Dedifferentiated/EMT

Differentiated(Polarized/Adhesive)

- E-cadherin- E-cadherin

+ E-cadherin+ E-cadherinDedifferentiated/Permanent EMT(Reduced or mutated E-cadherin or catenins)

Normal Tissue

Transient EMT

Morphogenesis Cancer Tissue

Invasion and Metastasis

(dissociation from tumor)

Reduced adhesiveness

(Adapted from Meiners et al, 1998 and Hirohashi, 1998).

What is the best way to objectivelymeasure changes in cell-cell adhesion relevant to metastasis?

Is it sufficient to directly quantifyE-cadherin activity?

Cytoplasm

Monomer

LateralDimer

Ca++

JMD/p120RhoA

Phosphorylation?

CBDRac/Cdc42

VASPMenaVinculin

AdhesiveDimer(weak adhesion)

CadherinClustering(strong adhesion)

ActinCrosslinking(compaction)

Actin

E-cadherin is a Ca2+ -dependent adhesion protein

CadherinClustering

P

P

p120 induces cadherin clustering

1. The amount of E-cadherin is directly relevant toadhesive strength (all things being equal).

2. The amount of E-cadherin does not necessarily reflect adhesive activity. (eg, Rac, rho experiments)

Where does p120 fit in to all of this?

Cadherin Stability vs. Motility/Invasion

p120

Extracellular Space

-catenin

actinfilaments

-catenin

Kaiso

Wnt 11MatrilysinMetastasin

SRF AP-1

E-Cad.Increased E-cad

stability and adhesion

tumor suppressor?

JNK, p38, p38

RhoA

Rac1

Cdc42

Vav2?Lamellipodia

Filopodia

Stress FibersFocal Contacts

Cell cycle, proteases, etc.

Increasedmotility

andinvasion

metastasispromoter?

p120

E-c

adhe

rin

p120 is rate limiting for E-cadherin expression

p120 is essential for cadherin stability

Measure the mechanical properties of the E-cadherin adhesion system in cells with and without p120.

There should be big differences!

Target Systems

1. E-cadherin activity: linker system mechanics(magnetic tweezer)

2. Mechanics of underlying actin and the homophilic E-cadherin binding junction(fluorescent beads, inversed microgrippers?)

Magnetic Bead based Rheometry

Force vs. distance to tip (~0.1A)

0.00E+00

5.00E-10

1.00E-09

1.50E-09

2.00E-09

2.50E-09

0 100 200 300 400 500 600

Distance to tip (μm)

Fo

rce

(N)

vDF 6

Force Calibration

Forces up to 1.5nN

Protein-A

bead

Fc-Ecad

Ca++

Ecad-Fc Beads Bind Specificallyto E-cadherin Expressing Cells

MDA-231

MDA-231+ E-cad

Accomplishments

• Implemented particle tracking software

• Fabricated magnetic tweezer

• Protocol to quantify the elastic properties of E-cadherins using magnetic bead based microrheology– Validation of the linker system

F

T=0 s

T=1.5 s

0

1 2 30

1

2

3

dis

pla

cem

en

t [

m]

Time [s]

Fit to Mechanical Analog

Extract Model parameter

Force1 nN

Force displacement measurements on magnetic beads linked to the cell surface through E-Cadherin

Force-induced Displacement

0 10 20 30 40 50 600

10

20

30

40

50

60

50 100 150 200 250

50

100

150

200

250

0 10 20 30 40 50 600

10

20

30

40

50

60

Implementation Protocol• 1. Before initiating cell pulling, cultured MDCK cells are: 1. Before initiating cell pulling, cultured MDCK cells are:

– - Trypsinized - Trypsinized – - Seeded in a PDMS cell chamber- Seeded in a PDMS cell chamber– - Mixed w/ E-cadherin coated paramagnetic beads- Mixed w/ E-cadherin coated paramagnetic beads– - Mounted on the stage of the magnetic tweezer - Mounted on the stage of the magnetic tweezer

microrheometer. microrheometer.

• 2. After locating a suitable bead-bound cell, an automated 2. After locating a suitable bead-bound cell, an automated LabView/C++ routine acquires 3 seconds of images at 122Hz: 0-1s, LabView/C++ routine acquires 3 seconds of images at 122Hz: 0-1s, steady-state; 1-2s, power supply triggered to initiate the force; 2-3s, steady-state; 1-2s, power supply triggered to initiate the force; 2-3s, cell relaxation. This sequence is repeated several times for each cell relaxation. This sequence is repeated several times for each bead.bead.

• 3. The cell pulling videos are then analyzed using a particle tracking 3. The cell pulling videos are then analyzed using a particle tracking program in Matlab that allows high resolution quantification of bead program in Matlab that allows high resolution quantification of bead displacement for each bead pulled. This data is then fit to a displacement for each bead pulled. This data is then fit to a mechanical model that characterizes E-cadherin mechanics.mechanical model that characterizes E-cadherin mechanics.

Particle Tracking Algorithm

Spatial Bandpass Filter

Find coordinates of peak intensities in the current frame

Average around peaks to obtain particle centroid

Final Frame?

NO

YES

Analyze bead trajectories through all frames

Fit bead (i.e., membrane) displacements to a viscoelastic model.

{k, γ, τ}

(x(t),y(t),r(t),v(t),…)

(Peak intensity = beads)

Pre-Processing

Optimize parameters forparticle identification

Invert (if necessary) &normalize the images

Video images acquired from the CCD camera using LabView 7.1

(Raw video data)

,)/exp(1)(010

1

0 t

tkk

k

k

Ftx

where 10

101 )(

kk

kk

k = = Elastic constant10 kk

= Viscosity0 = Relaxation Time

00.005

0.010.0150.02

0.025

0.030.0350.04

0.0450.05

0 2 4 6 8 10 12 14 16 18 20

Experiment #

Ela

stic

ity (

Pa

m)

0

0.02

0.04

0.06

0.08

0.1

0.12

0 2 4 6 8 10 12 14 16 18 20

Re

laxa

tion

Tim

e (s

)

[1] Local Measurements of Viscoelastic Parameters of Adherent Cell Surfaces by Magnetic Bead MicrorheometryAndreas R. Bausch et. al., Biophysical Journal Volume 75 October 1998 2038–2049

0

0.002

0.004

0.006

0.008

0.01

0 2 4 6 8 10 12 14 16 18 20

Vis

cosi

ty (

Pa

s m

)

skk

kk024.0042.0

)(

10

101

msPa 0020.00044.00

mPakkk 0090.0020.0)( 10

Analysis of viscoeleastic response curves based on three observables [1]

Number of different cells = 7

0 0.5 1 1.5 2 2.5 30

0.5

1

1.5

2

2.5

3

3.5

time (s)

disp

lace

men

t (m

icro

ns)

0 0.5 1 1.5 2 2.5 30

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

time (s)

disp

lace

men

t (m

icro

ns)

0 0.2 0.4 0.6 0.8 1 1.2 1.4-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

time (s)

disp

lace

men

t (m

icro

ns)

k=-0.036244Pa m,n0=0.0025302Pa s m,tau=0.053513s

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

0.5

1

1.5

2

2.5

time (s)

disp

lace

men

t (m

icro

ns)

F~1nN, k=22.2696, n0=7.1283, tau=0.055134

Wild-type P120 Knockout

Force-Displacement Curves MDCK cells

What next?

How to quantify mechanical properties at different scales?

Measurements across multiple scales combined with finite element models

Larger Forces?

Local Measurements of Viscoelastic Parameters of Adherent Cell Surfaces by Magnetic Bead MicrorheometryAndreas R. Bausch, Biophysical Journal Volume 75 October 1998 2038–2049

Intracellular and membrane heterogeneity?

Imaging Brownian Motion

Mechanical deformation of neutrophils into narrow channels induces pseudopod projection and changes in biomechanical properties, Belinda Yap and Roger D. Kamm, J Appl Physiol 98: 1930–1939, 2005.

Capture heterogeneity!

- Membrane

- Cytoskeleton

Larger forces (>100nN) for increased spatial scales?

Dual Pipette Assay

prF 2

Force measurements in E-cadherin–mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42Yeh-Shiu Chu et. Al., JCB • VOLUME 167 • NUMBER 6 • 2004

Inversed Mircogrippers

Overview of Techniques

• Brownian motion – cytoskeletal and membrane heterogeneity

• Magnetic tweezer – adhesion protein linker system mechanics

• Dual pipette (large forces) – cadherin-cadherin separation force

• Inversed microgrippers (large forces?) – cadherin-cadherin separation force

Single device for multi-scale measurements of cell-cell adhesion

Now that the tracking and analysis software works and bead-based microrheology has been validated, how can a device be designed that characterizes the mechanical properties of the adhesion system of any cell line over multiple spatial and temporal scales?

CadherinActivatedSignals

E-cadherin

Signaling from Cadherins

adhesion molecules,receptors, etc. Cadherin

DependentSignals

Integration and Miniaturization!Fluorescent microbeads or quantum dots?

- imaging brownian motion

- tracking force-induced changes in heterogeneities

On chip electrical components (e.g., CMOS)?

- signal conditioning

- increase signal-to-noise ratio

High density GMR sensor array substrate with multiplexing?

- removes need for expensive microscope & CCD

- directly senses XY-position

Microfabricated electromagnets?

- removes need for expensive micromanipulators

Microfluidics with cell traps?- fixed cell positions (don’t have to search for good cells)

Micropatterning? …etc…