Tensegrity-Based Mechanical Control of

Mammalian Cell Fate Switching and Morphogenesis

Don Ingber, M.D.,Ph.D.

Judah Folkman Professor of Vascular Biology
Depts. of Pathology & Surgery, Harvard Medical School

Interim Co-Director, Vascular Biology Program, Children’s Hospital Boston

Interim Co-Director, Harvard Institute for Biologically Inspired Engineering

How are living cells
and tissues constructed?
A Linear View of Tissue Development
(Tumor Angiogenesis)
Local Control during Angiogenesis

Branching Patterns
Cells Exert Tension on their Matrix Adhesions

Extracellular Matrix
Micromechanical Control of Morphogenesis

Cell Stretching

Underlying Hypothesis:

• ECM remodeling changes LOCAL MECHANICS

• Increasing ECM flexibility promotes cell stretching

Cell Stretching
• Tension on adhesion receptors &
distortion of the cytoskeleton
alters cellular biochemistry

Localized Growth & Motility

(Ingber et al., PNAS 78:3901-5, 1981; Ingber & Jamieson, In: Gene Expression During
Normal & Malig. Differ. ,1985; Huang and Ingber, Nature Cell Biol. ,1999)
Making Cell Distortion an Independent Variable

GF
GF GF

GF GF
GF
GF
GF
GF
GF GF

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

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

(Singhvi et al., Science 1994; Chen et al. Science 1997;
UV Mask Chen et al. Methods Mol. Biol. 2000; 139: 209-219)
Photoresist
Silicon
PDMS
RESISTS Protein Adsorption
(polydimethyl-siloxane) =Stamp
SUPPORTS
Adsorption
Flexible
PDMS Stamp

Molecular
Self-Assembly
Adhesive alkanethiol
Au Silicon, Glass (methyl-terminated) on the NANOSCALE
+ ECM Protein

NON-adhesive alkanethiol ECM protein

(PEG-terminated)
SAM
 Stretching Cells Makes Them Grow
And Rounded Cells Die

Growth

Death Growth

Cell Function

Cell Distortion
(Singhvi et al. Science 1994; Chen et al. Science 1997)
Capillary Blood Vessel Formation In A Dish

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

Stretch-Dependent Control of Directional Motility

(Parker et al. FASEB J 2002)

 Cell Distortion Redirects Molecular Self-Assembly
In the Cytoskeleton & Extracellular Matrix

Actin Stress Fibers Focal Adhesions Fibronectin

(AFM) (F-Actin) (Vinculin) Fibrils

Guided by Localized Tension Application in Cell Corners

TRACTION FORCE MICROSCOPY:

Cell Morphology Strain Map Stress Map

(Parker et al. FASEB J 2002; Wang et al., Cell Cytosk. Motil. 2002; Brock et al. Langmuir 2003)
 Physical ECM Pattern Governs Directional Motility
+ PDGF (NO CHEMICAL GRADIENT!) (Xia et al., FASEB J 2008)

Migration Paths:

100um

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

Local Rules & Physical Determinants
Govern Pattern Formation

(Brangwynne et al. In Vitro Cell Dev Biol 2000;

Huang et al., Cell Cytosk Motil 2005)

(“Symmetry Breaking” in Mammalian Cells)

 Patterning Predicted by Whole Cell Behaviors

Experimental Results Computational Model

Capillary Endothelial Cell (High Persistence - Yin Yang)

Fibroblast (Low Persistence - No Yin Yang)

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

Cell Fate Switching Depends on Physicality of Microenvironment

Morphogenesis
Soluble
growth factors

Wound Healing

die differentiate
divide

 Spatial heterogeneity of
cell fates drives morphogenesis
How are Cells Constructed so that they can Sense Force?

Old View: Hypothesis:

Cells are like Water Balloons Cells are Built Like Tents
The Molecular Networks
of the Cytoskeleton
Cellular Tensegrity Model

(Ingber et al., PNAS 78:3901-5, 1981; Ingber & Jamieson,1985;

Wang et al. Science 1993, PNAS 2001; Ingber J. Cell Sci 1993, 2003)
 Resting Tension (Prestress) in Living Stress Fibers
Revealed using Laser Nano-Surgery (“tensed cables”)
(with Eric Mazur, Dept. Physics, Harvard U.)

Retraction of a single actin

stress fiber in a living cell
300 nm

(Kumar et al., Biophys J. 2006)

Mechanical Continuity in the Cytoskeleton and ECM
Flexible ECM Substrate

GFP-Actin

Rigid
Dish

Before Cut: Green

After Cut: Magenta
T ECM
(Kumar et al, Biophys J 2006)
Microtubules are Semi- Flexible Compression Struts

Live Beating Heart Cell

(Brangwynne et al, J Cell Biol 2006 with

D. Weitz & K. Parker, Harvard U. & F.
Macintosh, Amsterdam)

Constrained Buckling
Theory

Curved (Buckled) Microtubules in a Fixed Cell

 Tensegrity predicts
Adhesion Receptors
act as Mechanoreceptors

(Ingber and Jamieson, 1985;

Ingber, Curr Opin Cell Biol 1991)
Magnetic Twisting & Pulling
Cytometry
 Mathematical Tensegrity Model of the Cell
A Priori Predictions now Confirmed:
D. Stamenovic (Boston U.)
•Linear relation between Stiffness and Applied Stress
(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 to

Cell Mechanics
(Wang and Stamenovic, Am J Physiol Cell Physiol, 2000)
Stamenovic et al. J. Theor. Biol.1996
Coughlin & Stamenovic, J App Mech 1997,1998
Stamenovic & Coughlin, J Theor Biol. 1999
• Microtubules Bear Compression
(Keach et al., 1996; Wang et al., 2001; Hu et al., Bioscience, 2004;
Stamenovic & Coughlin, J. Biomech. Engin. 2000
Brangwynne et al., J Cell Biol 2006)
Wang & Stamenovic, Am J. Physiol Cell Physiol. 2000
Stamenovic,J. Biomech.,2005.
‘Hierarchical’ Tensegrity Model
(Cell & Nucleus Connected by Tension Elements)
A local stress can produce DISTANT responses

(Wang et al. PNAS 2001)

Intermediate Filaments are Suspensory Cables
(Maniotis et al. PNAS 1997; Eckes et al. JCS1998)

Link other filaments

& membrane to the
nucleus
From Nickerson and Penman

From R. Goldman
Mechanical Control of the Mitotic Spindle Axis

Spindle Axis:

(Maniotis et al., PNAS 1997)

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

Stiff Actin Protofilament

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

TE

(Ingber, FASEB J 2006)

Tensegrity Focuses Force on Molecules in the
Extracellular Matrix and Cytoskeleton
Cytoskeleton is
-
More than a Mechanical Scaffold
Solid-State Biochemistry on Cytoskeletal Scaffolds
(Structure = Catalyst)

‘Channeling’ of Chemical Reactions:

DNA replication
RNA Processing
Transcription
Translation
Glycolysis
Signal
Transduction
1
2 3 4

A
B
C
Cytoskeletal filament

(Ingber, Cell 1993)

 Surface Integrins Mediate
Mechano-Chemical Transduction

Pulling on “Integrins”
Activates Signaling &
Gene Transcription
Pulling on Integrins Specifically Activates Ca+2 Influx
(Time scale < 10 milliseconds) [Funded by DARPA & NIH]
Cytoskeletal Strain Calcium (FLUO4)

BEAD

Δ Calcium (F/Fo)
RGD
AcLDL
0.1 HDL
RGD+Gd3+

Magnetic 0.075
Pulses
0.05

0.025

0
100 450 850 2000
Force (pN)
Force Dependence Force & Integrin Dependence
(Matthews et al., in review)
 Mechanical Control of Gene Transcription

Activation of Gα proteins and cAMP Signaling by Mechanical Force

Transmitted Across Integrin Receptors

cAMP Levels
+ TWIST PKA Translocation

Gene Transcription

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

Focal Adhesion is a Nanoscale Mechanochemical Machine

Stress
 Mechanochemical Transduction
Cellular Tensegrity Model

Tensegrity provides a mechanism to INTEGRATE

both LOCAL and DISTANTAlter CSK Organization
structural responses
when forces are transmitted
through the cytoskeleton
Signal Integration through Cell Distortion:
Cells Act Locally, but “Think” Globally

Growth
The Small GTPase Rho Mediates Shape Control of Growth

Filamin
p190RhoGAP

Rho

ROCK TENSION

(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)
Micromechanical Control of Morphogenesis

Cell Stretching

Underlying Hypothesis:

• ECM remodeling changes LOCAL MECHANICS

• Increasing ECM flexibility promotes cell stretching

Cell Stretching
• Tension on adhesion receptors &
distortion of the cytoskeleton
alters cellular biochemistry

Localized Growth & Motility

(Ingber et al., PNAS 78:3901-5, 1981; Ingber & Jamieson, In: Gene Expression During
Normal & Malig. Differ. ,1985; Huang and Ingber, Nature Cell Biol. ,1999)
Developmental Control Requires a Fine Balance of Forces

Bud Inducing Activity

200

% Bud Increase
180
160
140
120
100
80
60
40
20
0
0 2 20 200
CNF-1 (ng/ml)

Control

CNF-1 Tension
(2-20 ng/ml)

CNF-1 Tension
(200 ng/ml)
Time (hrs): 0 48
Epitheliogenesis & Angiogenesis in Embryonic Lung
m
c
c

can be Controlled by Altering Cytoskeletal Tension

m
c
c
e
e
c
c
c
c

Epi

Epi

Epi

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

TENSION TENSION

(Moore et al, Dev. Dynamics 2005)

Dissipation of CSK Tension Prevents ECM Thinning
And Inhibits Morphogenesis
(Laminin Staining)

Control Y27632 Rescue CNF-1

(Moore et al, Dev. Dynamics 2005)

Extracellular Matrix & Cytoskeleton not just structural supports
they also are KEY DEVELOPMENTAL REGULATORS
Because they mediate MECHANICAL SIGNALING
But Where is the Specificity? [SOFTWARE Challenge]
Extracellular signals

FAK G a-actinin
Shc Ca2+ Jak1 Jak2
FADD PTEN GRB src PLC APC
RAIDD TRAF Sos PKC Stat1 IRS-1
Pyk2 cAMP
PIP3 Cdc42
β -catenin
Ras Rac
caspase 2 PKA
PDK PI3K Smad7
Rho α-catenin
caspase 8 Rap1
c-raf
FRAP Akt PAK
mitochodrial MLC
cyt C release B-Raf CaMKII
Bad MEK1/2
Bcl-2 TAK1
p70S6 NIK MEKK1 Smad2
Apaf-1

IKK MAPK(erk1/2)
caspase 9 IkB MKK3/6 Sek1(MKK4)

p90rsk p38=HOG
caspase 3,6,7 NFkB SAPK=JNK
Stat3

TCF Elk CREB myc Fos Jun TCF-Lef

p21 INK ATF2
p27 Smad4
cdk4,6 pRb
apoptosis
cyclin D E2F
cdk2
cyclin E
motilitycyclin A

growth differentiation
Cell Fate Switching: A Biological “Phase Transition”

Apoptosis Differentiation Growth

Cell Distortion
 Complex Behaviors are EMERGENT
(Distributed vs. Deterministic Regulation)

Pathway view Network view

Signal Multiple
Simultaneous
Signals

Effect Effect of Collective behavior

( constrained by network interactions)
 Stable Functional States Emerge from Dynamic Information Networks
(work from “Complexity Field” by Stuart Kauffman)

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

“mini DNA chip”
A B A B
A B Stable
A B A B A B C D Attractor
… 2 4 = 16 C D
C D state
C D
C C D
C D
All expression configurations
are equally possible. A B A B
C D C D
Gene activity State Space (for 6-gene network): Regulatory interactions CONSTRAIN paths!

Attractor Basin of attraction

state
Boolean Network of Cell Cycle Control

(Huang & Ingber,

Exp Cell Res 2000)
 Complex Systems Biology:
From Linear Paths to Dynamic Trajectories
mitogenic
[Sui Huang, M.D.,Ph.D] CELL FATE SWITCH:
survival
factor factor Requires changes in
multiple gene activities

Proliferation Differentiation

Plasma membrane

D G
B Apoptosis
E H
C
F J

Proliferation
Apoptosis
Differentiation
Survival
(Huang & Ingber, Exp. Ce l Res. 2000; Ingber, J Cell Sci. Part II 2003)

OLD VIEW: NEW VIEW:

Linear pathways Dynamic Networks
No conceptual link to phenotype Emergent Attractors = Phenotype
(‘High Dimensional’ Attractors)
 Simultaneous Dynamic Analysis of Whole Gene Array
with Genome Expression Dynamics Inspector (GEDI)

Existing Method: New Method:

Time 1

Time 2

Time 3

HELA Cell Cycle

(data from Whitfield et al., Mol. Biol. Cell 2002)

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

(Eichler, Huang & Ingber, Bioinformatics 2003)
 Dictyostelium Development
(data from Van Driessche et al., Development 2002)
Evidence that Cell Fates are High Dimensional Attractors
(work of Sui Huang and Hannah Chang)

HL60 cells =
Promyelocytic
Precursor (Stem)
Cells

Differentiated Precursor
neutrophil (HL60)

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 Lett 2005; Chang et al. BMC Bioinform 2006)
 Cell Fates are High Dimensional Attractors
mean of intertrajectory
gene
1.3
expression differences, < D
1.2
>
1.1

<D>
1

0.9 <D>
0.8
c
0.7

0.6
0 20 40 60 80 100 120 140 160

Time (hr)

Initial divergence and

terminal convergence of the
two trajectories in > 70%
Differentiated Precursor (2773 genes) of the state
neutrophil (HL60)
space dimensions

GEDI Analysis:
PCA Analysis:
40

30

20 24h
8h
10

0
168h
PC2

0h
-10

-20

-30
24h
8h
-40

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

PC1 (Huang et al., Phys Rev Lett 2005; Chang et al. BMC Bioinform 2006)
 Dynamic Networks: Simplicity in Complexity

PLCg Shc PKCsrcFAK G Ca2+

Jak1 Jak2Tyk
FGF-R PDGF-R
GRB Stat1
PI3K Sos Pyk2
Ras rac cdc42 rho
PDK
FRAP(TOR)c-raf cAMP PAK MLC
Akt rap MEKK1
MEK1/2 PKA CaMKII
p70S6 MKK3/6Sek1(MKK4)
Bad MAPK(erk1/2) RafB

IKK p38=HOGSAPK=JNK
IkB
p90rsk
NFkB Stat3
g e n o m e
Elk c-myc CREB Fos Jun ATF2

2 different GF RECEPTORS activate

same set of 60 genes to induce growth
1 CYTOKINE activates > 100 of genes (Fambrough et al., 1999)

Apoptosis Differen. Growth

Cell Distortion (Waddington, 1956)

SPECIFIC & NON-SPECIFIC STIMULI MUTUAL EXCLUSIVITY

produce same cell FATE switches of cell fates in Embryogenesis
 Micromechanics and Collective Behavior
Govern Pattern Formation

Normal Fractal Patterns

Morphogenesis Tumor
Soluble Disorganization
growth factors

Wound Healing

die differentiate
divide

 Spatial heterogeneity of
cell fates drives morphogenesis

Cell fate switching depends on physicality of microenvironment

 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)
Ingber Lab (Harvard/CH) Sui Huang (U. Calgary)
Francis Alenghat (resident BWH)
Cliff Brangwynne (grad. stud. HU)
Amy Brock
Ning Wang (U. Illinois)
Hannah Chang
Chris Chen (U. Penn)
Dimitrije Stamenovic (BU)
Sanjay Kumar (U.C. Berkeley)
George Whitesides (HU)
Tanmay Lele (U. Florida)
Akiko Mammoto
Eric Mazur (HU)
Bob Mannix
Ben Matthews
David Weitz (HU)
Chris Meyer (Dental Practice)
Martin Montoya
Darryl Overby (Tulane) Radhika Nagpal (HU)
Kevin Kit Parker (Harvard)
Jay Pendse David Mooney (HU)
Tom Polte HIBIE Interim Co-Director
Julia Sero
Charles Thodeti
WEBSITE:
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