Tensegrity-Based Mechanical Control of Mammalian Cell Fate Switching and Morphogenesis by Ingber

8/9/2019 Tensegrity-Based Mechanical Control of Mammalian Cell Fate Switching and Morphogenesis by Ingber

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Don Ingber, M.D.,Ph.D.Judah Folkman Professor of Vascular BiologyDepts. of Pathology & Surgery, Harvard Medical School

Interim Co-Director, Vascular Biology Program, Childrens Hospital Boston

Interim Co-Director, Harvard Institute for Biologically Inspired Engineering

Tensegrity-Based Mechanical Control ofMammalian Cell Fate Switching and Morphogenesis


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How are living cellsand tissues constructed?


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A Linear View of Tissue Development(Tumor Angiogenesis)


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Local Control during Angiogenesis

Branching Patterns


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Extracellular Matrix

Cells Exert Tension on their Matrix Adhesions


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MicromechanicalControl of Morphogenesis

Underlying Hypothesis:

ECM remodeling changes LOCAL MECHANICS

Increasing ECM flexibility promotes cell stretching

Tension on adhesion receptors &

distortion of the cytoskeleton

alters cellular biochemistry

Localized Growth & Motility(Ingber et al., PNAS78:3901-5, 1981; Ingber & Jamieson, In: Gene Expression During

Normal & Malig. Differ. ,1985; Huang and Ingber, Nature Cell Biol. ,1999)

Cell Stretching

Cell Stretching


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Making Cell Distortion an Independent Variable

GFGF

GF

GF

GF

GF

GFGF

GF

GF

GF

GF


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(with George Whitesides, Chemistry Dept. Harvard U.)

Nanotechnology-Based Microfabrication(Soft Lithography + Self Assembling Monolayers)

PDMS(polydimethyl-siloxane)

Photoresist

Adhesive alkanethiol(methyl-terminated)

NON-adhesive alkanethiol(PEG-terminated)

Silicon

Mask

ECM protein

SUPPORTSAdsorption

RESISTS Protein Adsorption

Au Silicon, Glass

=Stamp

+ ECM Protein

(Singhvi et al., Science1994; Chen et al. Science1997;Chen et al. Methods Mol. Biol. 2000;139: 209-219)

UV

MolecularSelf-Assembly

FlexiblePDMS Stamp

SAM

on the NANOSCALE


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Stretching Cells Makes Them Grow

And Rounded Cells Die

Growth

Cell Distortion

CellF

unctio

n

Death Growth

(Singhvi et al. Science1994; Chen et al. Science1997)


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Capillary Blood Vessel Formation In A Dish

(Dike et al. In Vitro Cell Dev Biol 1999)


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(Parker et al. FASEB J 2002)

Stretch-Dependent Control of Directional Motility


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Actin Stress Fibers(AFM) (F-Actin)

FibronectinFibrils

Focal Adhesions(Vinculin)

Cell Distortion Redirects Molecular Self-AssemblyIn the Cytoskeleton & Extracellular Matrix

(Parker et al. FASEB J2002; Wang et al., Cell Cytosk. Motil. 2002; Brock et al. Langmuir 2003)

TRACTION FORCE MICROSCOPY:

Cell Morphology Stress Map

Guided by Localized Tension Application in Cell Corners

Strain Map


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100um

Physical ECM Pattern Governs Directional Motility

1C-3,3 3L-3,3 8L-3,3

+ PDGF (NO CHEMICAL GRADIENT!) (Xia et al., FASEB J2008)

Migration Paths:


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(Brangwynne et al. In Vitro Cell Dev Biol2000;Huang et al., Cell Cytosk Motil2005)

Local Rules & Physical Determinants

Govern Pattern Formation

(Symmetry Breaking in Mammalian Cells)


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(Huang et al., Cell Motil. Cytosk. 2005)

Patterning Predicted by Whole CellBehaviors

Experimental Results Computational Model

Fibroblast (Low Persistence - No Yin Yang)

Capillary Endothelial Cell (High Persistence - Yin Yang)


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Solublegrowth factors

Morphogenesis

Wound Healing

Spatial heterogeneity ofcell fates drives morphogenesis

die differentiatedivide

Cell Fate Switching Depends on Physicality of Microenvironment


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How are Cells Constructed so that they can Sense Force?

Hypothesis:

Cells are Built Like Tents

Old View:

Cells are like Water Balloons


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he Molecular Networksof the Cytoskeleton


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(Ingber et al., PNAS78:3901-5, 1981; Ingber & Jamieson,1985;

Wang et al. Science1993, PNAS2001; Ingber J. Cell Sci1993, 2003)

Cellular Tensegrity Model


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Resting Tension (Prestress) in Living Stress FibersRevealed using Laser Nano-Surgery (tensed cables)

(Kumar et al., Biophys J. 2006)

300 nm

Retraction of a single actinstress fiber in a living cell

(with Eric Mazur, Dept. Physics, Harvard U.)


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Mechanical Continuity in the Cytoskeleton and ECM

RigidDish

Flexible ECM Substrate

T ECM

(Kumar et al, Biophys J 2006)

GFP-Actin

Before Cut: GreenAfter Cut: Magenta


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Microtubules are Semi- Flexible Compression Struts

Curved (Buckled) Microtubules in a Fixed Cell

Live Beating Heart Cell

(Brangwynne et al, J Cell Biol 2006 with

D. Weitz & K. Parker, Harvard U. & F.

Macintosh, Amsterdam)

Constrained BucklingTheory


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Tensegrity predictsAdhesion Receptors

act as Mechanoreceptors

(Ingber and Jamieson, 1985;

Ingber, Curr Opin Cell Biol 1991)


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Magnetic Twisting & Pulling

Cytometry


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D. Stamenovic (Boston U.)A PrioriPredictions now Confirmed:

Stamenovic et al. J. Theor. Biol.1996Coughlin & Stamenovic, J App Mech 1997,1998Stamenovic & Coughlin, J Theor Biol. 1999Stamenovic & Coughlin, J. Biomech. Engin. 2000

Wang & Stamenovic, Am J. Physiol Cell Physiol. 2000Stamenovic,J. Biomech.,2005.

Linear relation between Stiffness and AppliedStress(Wang et al., Science 1993; Wang and Ingber, Biophys. J. 1994))

Cell Mechanics depends on Prestress(Wang & Ingber, Biophys. J. 1994; Lee et al., Am. J.Physiol. 1997)

Linear relation between Stiffness and Prestress(Wang et al., PNAS 2001; Wang & Stamenovic, Am. J. Physiol 2002)

Hysteresivity independent of prestress(Maksym et al.,Am. J. Phys. 2000;Wang et al., PNAS 2001)

Quantitative Prediction of Cellular Elasticity(Stamenovic and Coughlin, J. Biomech. Engineer. 2000)

Prediction of Dynamic Mechanical Behavior(Sultan et al., Ann Biomed Engin. 2004)

Mechanical Contribution of Intermediate Filaments toCell Mechanics(Wang and Stamenovic, Am J Physiol Cell Physiol, 2000)

Microtubules Bear Compression(Keach et al., 1996; Wang et al., 2001; Hu et al., Bioscience, 2004;Brangwynne et al., J Cell Biol 2006)

Mathematical Tensegrity Model of the Cell


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Hierarchical Tensegrity Model(Cell & Nucleus Connected by Tension Elements)


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(Wang et al. PNAS2001)

A local stress can produce DISTANT responses


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Intermediate Filaments are Suspensory Cables

Link other filaments& membrane to thenucleus

From Nickerson and Penman

From R. Goldman

(Maniotis et al. PNAS 1997; Eckes et al. JCS1998)


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Mechanical Control of the Mitotic Spindle Axis

(Maniotis et al., PNAS 1997)

Spindle Axis:


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Tensed Spectrin

Stiff Actin Protofilament

Cortical Membrane as a Prestressed Tensegrity(Vera et al. Annals Biomed. Engin. 2005; with Bob Skelton, UCSD)

www.jacobsschool.ucsd.edu/news_events/releases/release.sfe?id=484


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TE

Tensegrity-Based Hierarchical Integration(computer images by Eddy Xuan, U. Toronto)

(Ingber, FASEB J2006)


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Tensegrity Focuses Force on Molecules in theExtracellular Matrix and Cytoskeleton


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-Cytoskeleton is More than a Mechanical Scaffold


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1 2 3

AB

C

Solid-State Biochemistry on Cytoskeletal Scaffolds(Structure = Catalyst)

4

Cytoskeletal filament

Channeling of Chemical Reactions:DNA replicationRNA ProcessingTranscriptionTranslationGlycolysisSignalTransduction

(Ingber, Cell1993)


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Pulling on IntegrinsActivates Signaling &Gene Transcription

Surface Integrins Mediate

Mechano-Chemical Transduction


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Pulling on Integrins Specifically Activates Ca+2 Influx(Time scale < 10 milliseconds) [Funded by DARPA & NIH]

MagneticPulses

Force Dependence Force & Integrin Dependence

(Matthews et al., in review)

0

0.025

0.05

0.075

0.1

RGD

AcLDL

HDL

RGD+Gd3+

C

alcium(

F/Fo)

Force (pN)

100 450 850 2000

Cytoskeletal Strain Calcium (FLUO4)

BEAD


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Activation of G proteins and cAMP Signaling by Mechanical ForceTransmitted Across Integrin Receptors

Gene Transcription

Mechanical Control of Gene Transcription

cAMP LevelsPKA Translocation

CREB Activation

+ TWIST

(Meyer et al., Nature Cell Biol. 2000)


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Focal Adhesion is a Nanoscale Mechanochemical Machine

Stress


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Mechanochemical Transduction

Alter CSK Organization

Cellular Tensegrity Model

Tensegrity provides a mechanism to INTEGRATEboth LOCAL and DISTANT structural responses

when forces are transmittedthrough the cytoskeleton


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Signal Integration through Cell Distortion:Cells Act Locally, but Think Globally

Growth


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The Small GTPase Rho Mediates Shape Control of Growth

TENSION

Rho

ROCK

(Huang et al., Mol. Biol. Cell, 1998; Huang & Ingber, Exp Cell Res. 2002; Numaguchi et al.,

Angiogenesis 2003; Mammoto et al., J. Biol. Chem. 2004;Mammoto et al., J. Cell Sci. 2007)

Filaminp190RhoGAP


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MicromechanicalControl of Morphogenesis

Underlying Hypothesis:

ECM remodeling changes LOCAL MECHANICS

Increasing ECM flexibility promotes cell stretching

Tension on adhesion receptors &

distortion of the cytoskeletonalters cellular biochemistry

Localized Growth & Motility(Ingber et al., PNAS78:3901-5, 1981; Ingber & Jamieson, In: Gene Expression DuringNormal & Malig. Differ. ,1985; Huang and Ingber, Nature Cell Biol. ,1999)

Cell Stretching

Cell Stretching


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CNF-1

(200 ng/ml)

Control

CNF-1(2-20 ng/ml)

Time (hrs): 0 48

0

20

40

60

80

100

120

140

160

180

200

0 2 20 200

%BudIncrease

CNF-1 (ng/ml)

Bud Inducing Activity

Developmental Control Requires a Fine Balance of Forces

Tension

Tension


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c

mc

c

ce

c

mc

c

ce

Control CNF-1 (20 ng/ml) Y27632 (40 uM)

Epitheliogenesis & Angiogenesis in Embryonic Lungcan be Controlled by Altering Cytoskeletal Tension

(Moore et al, Dev. Dynamics2005)

TENSION TENSION

Epi

Epi

Epi


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Extracellular Matrix & Cytoskeleton not just structural supportsthey also are KEY DEVELOPMENTAL REGULATORS

Because they mediate MECHANICAL SIGNALING


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GRB

Sos

Ras

c-raf

MEK1/2

MAPK(erk1/2)

MEKK1

NFkB

Sek1(MKK4)

SAPK=JNK

ATF2

CREB

Rac

CaMKII

MKK3/6

p38=HOG

Akt

PIP3

p70S6

PLC

mycElk

IKK

FAK

src

Rho

Cdc42

PAK

IkB

Shc

p90rsk

JunFos

Stat3

Ca2+

Bad

MLC

G

FRAP

PDK

Rap1

Jak1

Stat1

Jak2

Bcl-2

caspase 9

caspase 2

FADD

caspase 8

mitochodrialcyt C release

PKC

PKA

Apaf-1

PTEN

TCF

APC

-catenin

TCF-Lef

PI3K

RAIDD

NIK

TRAF

TAK1Smad2

Smad4

Smad7

cyclin E

cyclin DpRb E2F

p27cdk4,6

cdk2

-catenin

a-actinin

cyclin A

INKp21

caspase 3,6,7

Extracellular signals

IRS-1Pyk2 cAMP

B-Raf

But Where is the Specificity? [SOFTWARE Challenge]

growthapoptosis

differentiationmotility


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Cell Distortion

Apoptosis Differentiation Growth

Cell Fate Switching: A Biological Phase Transition


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C

Stable Functional States Emerge from Dynamic Information Networks(work from Complexity Field by Stuart Kauffman)

A B

C D 2 4 = 16

A B

D

A B

C D

mini DNA chip

If genes were independent:

All expression configurations

are equally possible.

A B

C D

If genes are interacting via Logic F(x)s:

A B

C DA B

C D

Stable

Attractor

state

Regulatory interactions CONSTRAIN paths!

A B

C D

A B

C D

Attractor

stateBasin of attraction

Gene activity State Space (for 6-gene network):

C


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Boolean Network of Cell Cycle Control

(Huang & Ingber,

Exp Cell Res 2000)


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HELA Cell Cycle(data from Whitfield et al., Mol. Biol. Cell2002)

Simultaneous Dynamic Analysis of Whole Gene Arraywith Genome Expression Dynamics Inspector (GEDI)

(Eichler, Huang & Ingber, Bioinformatics2003)

Available at: www.childrenshospital.org/research/ingber/

Existing Method:

Time 1

Time 2

Time 3

New Method:


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Dictyostelium Development(data from Van Driessche et al., Development2002)


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Evidence that Cell Fates are High Dimensional Attractors

Precursor

(HL60)

Differentiated

neutrophil

HL60 cells =Promyelocytic

Precursor (Stem)

Cells

Neutrophil Differentiation was induced by:

1. all-trans Retinoic Acid (AtRA)[SPECIFIC HORMONE]

2. Dimethyl Sulfoxide (DMSO)[NON-SPECIFIC SOLVENT]

(Huang et al., Phys Rev Lett2005; Chang et al. BMC Bioinform 2006)

(work of Sui Huang and Hannah Chang)


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Cell Fates are High Dimensional Attractors

Precursor

(HL60)

Differentiated

neutrophil

Initial divergence and

terminal convergence of thetwo trajectories in > 70%

(2773 genes) of the state

space dimensions

< D >

c

0 20 40 60 80 100 120 140 1600.6

0.7

0.8

0.9

1

1.1

1.2

1.3

mean of intertrajectory

gene expression differences, < D

>

Time (hr)

GEDI Analysis:

-50 -40 -30 -20 -10 0 10 20 30 40 50-50

-40

-30

-20

-10

0

10

20

30

40

PC1

PC2

0h

8h

24h

168h

24h

8h

PCA Analysis:

(Huan et al. Ph s Rev Lett2005 Chan et al. BMC Bioinform 2006)


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Dynamic Networks: Simplicity in Complexity

GRBSos

Ras

c-raf

MEK1/2

MAPK(erk1/2)

MEKK1

NFkB

Sek1(MKK4)

SAPK=JNK

ATF2

cAMP

PKA

CREB

rac

CaMKII

MKK3/6

p38=HOG

Akt

PI3K

p70S6

PLCg

c-mycElk

IKK

FAKsrc

rhocdc42

PAK

IkB

Shc

p90rsk

JunFos

PKC

Stat3

Ca2+

Pyk2

Bad

MLC

G

FRAP(TOR)PDK

rap

RafB

Jak1TykStat1

Jak2

1 CYTOKINE activates > 100 of genes

FGF-R PDGF-R

g e n o m e

2 different GF RECEPTORS activatesame set of 60 genes to induce growth

(Fambrough et al., 1999)

(Waddington, 1956)

SPECIFIC & NON-SPECIFIC STIMULI

produce same cell FATE switches

Cell Distortion

Apoptosis Differen. Growth

MUTUAL EXCLUSIVITY

of cell fates in Embryogenesis


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Solublegrowth factors

Micromechanics and Collective Behavior

Govern Pattern Formation

Morphogenesis

Wound Healing

Spatial heterogeneity ofcell fates drives morphogenesis

die differentiatedivide

Cell fate switching depends on physicality of microenvironment

Normal Fractal Patterns TumorDisorganization


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Biomimetic Multicellular Robot Swarms(Radhika Nagpal, SEAS; D. Ingber, HMS)[NSF Funded]

Bioinspired Algorithms for robust and complex

shape formation using modular (multicellular) robots

Distributed Homeostasis Desired shape is described relative to the environment

Individual robots use local sensing to adapt their behavior

Inspired by homeostasis in biology and tissue remodeling

in response to environment needs

(Yu et al., IEEE Intl. Conf. on Intelligent Robots and Systems 2007)


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Sui Huang (U. Calgary)

Ning Wang (U. Illinois)

Dimitrije Stamenovic (BU)

George Whitesides (HU)

Eric Mazur (HU)

David Weitz (HU)

Radhika Nagpal (HU)

David Mooney (HU) HIBIE Interim Co-Director

Ingber Lab (Harvard/CH)Francis Alenghat (resident BWH)

Cliff Brangwynne (grad. stud. HU)

Amy BrockHannah Chang

Chris Chen (U. Penn)

Sanjay Kumar (U.C. Berkeley)Tanmay Lele (U. Florida)

Akiko Mammoto

Bob MannixBen Matthews

Chris Meyer (Dental Practice)

Martin MontoyaDarryl Overby (Tulane)

Kevin Kit Parker (Harvard)

Jay PendseTom Polte

Julia Sero

Charles ThodetiWEBSITE:Google search Ingber Lab

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Tensegrity-Based Mechanical Control of Mammalian Cell Fate Switching and Morphogenesis by Ingber

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