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Organism and Tissue Growth
Thomas Lecuitchaire: Dynamiques du vivant
Course 5: Growth control and mechanics
1
• Control of growth arrest at target size
Thomas LECUIT 2019-2020
— What is measured? Intrinsic « ruler » of growth: « size-meter » Organ intrinsic feedback of target organ size on cell growth/proliferation
— Organ and organism size may be regulated by: growth duration and/or growth rate.— Need to regulate growth arrest at the appropriate target size: process vs endpoint regulation.
arrest
time
mass/size
target size
2
1. Energy and mass/volume2. Pattern and mass/volume3. Mechanics and mass/volume
—Need to consider scaling between factors that promote growth and size itself
—Need to consider feedback mechanisms operating across scales (organ/tissue to cell)
Thomas LECUIT 2019-2020
• Conclusions
— Cell growth is controlled intrinsically— What are the mechanisms of organ specific tissue size sensing/measurement?
• Organ-size dependent negative feedback on cell growth/proliferation (chalone)• « Size-meter »: scaling of chalone concentration and tissue size
distance between activator and feedback inhibitor mechanical signal that scales with size
patterning cue: morphogen gradient
• Organ-size dependent growth inducer: Morphogen and growth arrest: Spatial model: gradient scaling and gradient slope. Temporal model: gradient scaling and rate of signal increase
— What is measured? Intrinsic « ruler » of growth: « size-meter », scaling.
• Importance of considering robustness of mechanisms: e.g. integral feedback
3
• Energy delivery/demand unbalance.
Programmed vs Self-organised regulation of Growth
• hierarchical• modular• deterministic rules (ie. genetically encoded)
Thomas LECUIT 2019-2020
• no hierarchy• feedbacks• statistical rules
4
Thomas LECUIT 2018-2019
Mechanical regulation of tissue size?
5
• Organs grow until they reach a fixed size characteristic of each organ/species• Growth is often uniform in the face of non uniform growth factor distribution
(morphogens)• Tissues can compensate for growth heterogeneities and reach a normal final
size arguing that cells assess their environment
—How do cells compare their growth rates?—How is growth uniform on average in a tissue?—How is tissue size determined?
• Cell-cell mechanical interactions
6Thomas LECUIT 2018-2019
Tissue Growth and Homeostasis
Thomas LECUIT 2018-2019 7
Tissue Growth and Homeostasis
A B
�-tubulin Myosin II Phosphorylated Moesin
Cytokinetic ring
Supracellularcable
Myosin II
Supracellular ring
• cell division
• cell death/extrusion
Tissue growth
Thomas LECUIT 2018-2019 8
Tissue Growth and Homeostasis
—Tissue growth depends on the balance between:• Rate of cell division• Rate of cell death and cell extrusion
—Tissue growth leads to homeostasis at a fixed size where cell growth/division and death/extrusion balance each other (unless growth is arrested): eg. gut.
• How is such a balance achieved?• Feedback interactions• Does growth impact on extrusion?• Does extrusion lead to compensatory growth?
—What are the consequence of differential tissue growth rates?• Cell and tissue competition (eg. tumours)• Tissue buckling (morphogenesis)
Biomechanical signalling
Thomas LECUIT 2019-2020
Biochemistry Mechanics
Information(mechano-chemistry)
9
driving tissue growth
Thomas LECUIT 2018-2019 10ASG. Curtis and GM Seehar, Nature 274, 52-53 (1978)
Cells in culture are subjected to low frequency stretching (60μm) for 1 hour
Control of cell division by mechanics: tension
Thomas LECUIT 2018-2019
Control of cell division by mechanics: geometry
11
• Increased cell-substrate adhesion causes reduction of cell height and increased cell diameter
• Cell proliferation increases as cells spread/adhere more to the substrate
• Is it adhesion per se or cell shape per se?
• Density dependence of cell proliferation and contact inhibition are characteristic of cells in culture
J. M. Folkman and A. Moscona, Nature 273, 345 (1978)
Thomas LECUIT 2018-2019
Geometric Control of Cell Life and DeathChristopher S. Chen, Milan Mrksich, Sui Huang,
George M. Whitesides, Donald E. Ingber*
12
Control of cell division by mechanics: geometry or adhesion?
—The role of surface of adhesion:• Cell division increases as the adhesive
island surface increases• Cell death by apoptosis decreases in
parallel
C. Chen et al., and D. Ingber. Science (1997):Vol. 276, Issue 5317, pp. 1425-1428DOI: 10.1126/science.276.5317.1425
—Disentangling cell geometry and substrate adhesion using a controlled micro fabricated substrate• The projected surface area, but not the
surface of adhesion to the ECM impacts on cell proliferation and cell death.
• Does this reflect cell shape per se or cell stresses?
ECM contact area
Thomas LECUIT 2019-2020
Control of cell growth by mechanics
13
—Disentangling the role of cell stress and cell shape • using micro fabricated substrates on colony island
of multiple cells (rather than single cells)• Cell division patterns depend on the geometry
colony islands
endothelial cells on fibronectin BrdU incorporation density maps
BrdU Proliferation map
C. Nelson et al. C. Chen. (2005) P.N.A.S. 102: 11594–11599 www.pnas.orgcgidoi10.1073pnas.0502575102
nN
traction force microscopy
• Traction Force microscopy confirms the deduced mechanical stress pattern.
Stress from FEM
• Use of finite element modelling (FEM) to deduce stress patterns in colony islands.
• Cell division patterns correlate with stress patterns that emerge from cell contractility and connectedness between cells and tissue geometry.
—Mapping stress pattern
Thomas LECUIT 2019-2020 14
—Disentangling the role of cell stress and cell shape
• Mechanical forces generated by the actomyosin cytoskeleton control cell proliferation patterns
Rho1 GDP Rho1 GTPGEF
GAP
MLCP
ROCKMRLC MRLC PDia
F-actin MyoIIminifilament
actinfilaments
passivecrosslinker
myosinIImotors
cell cortex
BrdUdensity
map
Y-27632
RhoV14*
dominant negative VE-cadherin (blocks mechanical coupling at cell junctions)
C. Nelson et al. C. Chen. (2005) P.N.A.S. 102: 11594–11599 www.pnas.orgcgidoi10.1073pnas.0502575102
Control of cell growth by mechanics
Thomas LECUIT 2018-2019
Control of cell growth by mechanics
15S. Dupont et al., and S. Piccolo. Nature (2011). 474::179-183
—Identification of transcription factors that mediate mechanically induced cell proliferation
a b
100
50
0si
Co.si
YZ1si
YZ240
kPa0.7kPa
mR
NA
exp
ress
ion Luciferase activity
90
60
30
0
CTGF ANKRD1 4xGTIIC-lux
10050
040 0.7
40 kPa 0.7 kPaNuclear
YAP/TAZ (%)
YAP
/TA
ZTO
TO3** ** **
β
β
β
• YAP/TAZ are known components of theHippo/LATS growth pathway that translocates in the nucleus once activated.• Substrate stiffness induces YAP activation
transcriptional targets of YAP
c
Micropillars
d
10,000 300 μm2
10,0
002,
025
1,02
430
0
Unpat
t.
100755025
0
Nuclear YAP/TAZ (%)
10,000 2,025 1,024 300 μm2Unpatt.
e
6,0003,000
0
300
μm2
Unpat
t.M
icrop
illars
Cell area(μm2)
10050
0
ECM area(μm2)
**
6,0003,000
0
YAP
/TA
ZYA
P/T
AZ
YAP
/TA
Z
NuclearYAP/TAZ (%)
• Cell substrate adhesion/geometry affects YAP/TAZ activation
• Cell projected area, but not adhesion per se activates YAP//TAZ
**
Thomas LECUIT 2018-2019
Control of cell growth by mechanics
16
YAP
/TA
Z
Control C3
Noco
Lat.A
NSC23766
mR
NA
exp
ress
ion
CTGF
100806040200
C3La
t.ANSCNoc
o.Co.
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Nuc
lear
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YAP
/TA
Z ** **
ANKRD1
S. Dupont et al., and S. Piccolo. Nature (2011). 474::179-183
—Identification of transcription factors that mediate mechanically induced cell proliferation
• The actomyosin cytoskeleton (but not the microtubule network) is required for YAP/TAZ activation.
Rho1 GDP Rho1 GTPGEF
GAP
MLCP
ROCKMRLC MRLC PDia
F-actin MyoIIminifilament
C3 Transferase
Y-27632
BlebbistatinLatrunculin A no actin polymerisation
c
125100
755025
0
Luci
fera
se a
ctiv
ity
4xGTIIC-lux
Co.
Lat.A
Actin R62D
d
Luci
fera
se a
ctiv
ity
4xGTIIC-lux
e
YAP
/TA
Z
Rigidpillars
Elasticpillars
Elastic
Rigid
100
50
0
Nuc
lear
YAP
/TA
Z (%
)
f Y27
632
Blebbist
.
Co.
Y27632 Blebbist. Control
YAP
/TA
Z
Nuc
lear
YAP
/TA
Z (%
)
100
50
0
Y2763
2Bleb
bist.
Co.
403020100
actinfilaments
passivecrosslinker
myosinIImotors
cell cortex
Thomas LECUIT 2018-2019
Control of cell growth by mechanics
17
30
20
10
0
30
20
10
0
60
40
20
0
60
40
20
0Pro
lifer
atio
n (%
) A
pop
tosi
s (%
)
ss s
d e
fAdhesive island area (μm2)
10,0
002,
025
1,02
430
010
,000
2,02
51,
024
300
10,0
002,
025
1,02
430
0
10,0
002,
025
1,02
430
010
,000
2,02
51,
024
300
Adhesive island area (μm2)
Pro
lifer
atio
n (%
) A
pop
tosi
s (%
)
Control +5SA-YAP siControl siYAP/TAZ siCo. + C3
n.s. n.s.
S. Dupont et al., and S. Piccolo. Nature (2011). 474::179-183
—Identification of transcription factors that mediate mechanically induced cell proliferation
• Cell stretching induces activation of cell proliferation and inhibition of apoptosis
• YAP/TAZ are both required for this process
• Constitutively active YAP expression induces cell proliferation and inhibits apoptosis
• This effect is independent of upstream Hippo/LATS pathway activity (not shown)
YAP/TAZ — YAP/TAZ * (C.A.)
a
c b
40 kPa 40 kPa
siYZ1siCo. + C3
40 kPa1 kPa40 kPa40 kPa
siYZ2
Ad
ipog
enes
is(A
.U.)
siCo.
siYZ1
1 40 40 (kPa)
60
40
20
0Ost
eoge
nesi
s(A
.U.)
40302010
0
siCo.
siYZ1
40 40 40 (kPa)40 1 40
siYZ2
+C3
siCo.
[see also A. Engler et al, D. Discher. (2006) Cell 126, 677–689]
• YAP/TAZ similarly mediates the cell differentiation of stem cells dependent on substrate stiffness
• On soft substrates adipocytes, on stiff substrates osteocytes.
substrate stiffness
Thomas LECUIT 2018-2019
Control of cell growth by mechanics
18
—Cell spreading affects cell volume via water efflux
M. Guo et al. J. Lippincott-Schwartz and D. Weitz. PNAS. (2017) E8618–E8627 www.pnas.org/cgi/doi/10.1073/pnas.1705179114
• Cells spread more on stiffer substrates and reduce concomitantly their volume
• Restricting cell area using smaller adhesive islands, increases cell volume
• Dynamic cell spreading is associated with dynamic change in cell volume
• The volume dependence on spread area is the same irrespective of how area is attained (substrate stiffness, adhesion area, dynamics of spreading)
substrate stiffness
size of adhesive
• Volume could be changed by protein concentration changes or water efflux: no [protein] change
• Volume change caused by water efflux
Thomas LECUIT 2018-2019
Control of cell growth by mechanics
19
—Volume changes induced by cell spreading or osmotic stress affect nuclear volume and molecular crowding in the nucleus.
M. Guo et al. J. Lippincott-Schwartz and D. Weitz. PNAS. (2017) E8618–E8627 www.pnas.org/cgi/doi/10.1073/pnas.1705179114
• Hyper-osmotic stress (with high PEG concentration in the medium) causes a reduction in cell volume through water efflux
irrespective of substrate stiffness.• Blocking Cl ion channels (NPPB) blocks
volume change as a function of substrate stiffness so volume adjustment involves changes in osmolytes.
• Blocking cell contractility also affects stiffness dependent volume change.
• Histine H2::GFP mean square displacement is lower in compressed cells consistent with increased molecular crowding
reviewed in R. Phillips, T. Ursell, P. Wiggins and P. Sens (2009) Nature 459: 379-385
• Consistent with tension dependent regulation of mechanosensitive ion channels, affecting osmolyte concentration and water flux.
Hyper-osmotic stress
Blocking Cl ion channels (
g yBlocking cell contractility
• In all conditions nuclear volume adjusts to cell volume
Thomas LECUIT 2018-2019
Control of cell growth by mechanics
20
—Volume changed induced by osmotic stress affects differentiation of stem cells
M. Guo et al. J. Lippincott-Schwartz and D. Weitz. PNAS. (2017) E8618–E8627 www.pnas.org/cgi/doi/10.1073/pnas.1705179114
• Differentiation of stem cells into osteocytes on a stiff substrate
• Hypertonic medium rescues differentiation on soft substrate.
• This is associated with volume reduction of cell and nucleus and increased cell stiffness
# YAP forms liquid like nuclear condensates in response to molecular crowding induced by hyperosmotic stressD. Cai et al. and J. Lippincott-Schwartz. bioRxiv. http://dx.doi.org/10.1101/438416. (2019)
ie. stiff substrate
osteogenesis
now in press in Nature Cell Biology
• On soft substrates, conversely cells differentiate into adipocytes
• Hypotonic medium rescues adipogenesis if on stiff substrate
Nat Cell Biol. 2019 Dec;21(12):1578-1589. doi: 10.1038/s41556-019-0433-z.
Thomas LECUIT 2018-2019
Control of cell growth/division & death by mechanics
21
Geometry: spreading, volume reduction (but not adhesion per se)
Mechanics: tension
Mechanochemical signalling: ion channels, YAP, etc.
Cell division No cell division Apoptosis
compression
• Summary
Thomas LECUIT 2018-2019 22
Mechanics of Tissue Growth and Homeostasis
—Tissue growth depends on the balance between:• Rate of cell division• Rate of cell death and cell extrusion
—Tissue growth leads to homeostasis at a fixed size where cell growth/division and death/extrusion balance each other (unless growth is arrested): eg. gut.
• How is such a balance achieved?• Feedback interactions?• Does growth impact on extrusion?• Does extrusion lead to compensatory growth?
—What are the consequence of differential tissue growth rates?• Cell and tissue competition (eg. tumours)• Tissue buckling (morphogenesis)
Thomas LECUIT 2018-2019
Control of cell growth/division by mechanics
23
A B
C
—Changes in cell behaviour during collective tissue growth in vitro: contact inhibition
A. Puliafito et al. and B. Shraiman. (2012) PNAS 109:739–744
• Growth of isolated colonies to disentangle cell contact from mechanics
• « free growth » regime is associated with exponential growth while cells are in contact
cell density
A BC
• Colony growth slows down, limited by constrained velocity of edge cells (ie. friction).
• This is associated with a transition to increased cell density and slower motility which are mechanically induced
cell density and size cell motilitycell division
• Increased cell density is associated with gradual increase of division time
Thomas LECUIT 2018-2019 24A. Puliafito et al. and B. Shraiman. (2012) PNAS 109:739–744
—1D vertex mechanical model of self-limiting growth • Short time elastic response of cells• Friction coupling to the substrate (elastic
restoring force)• Active random cell motion (Langevin driving
force) averages to zero in colony bulk but not at colony edges
• Cell growth is modelled by increasing the rest length of a cell edge when neighbouring cells stretch it.
• Cell division halves rest length• Cell division rate depends on cell size
—Changes in cell behaviour during collective tissue growth in vitro: contact inhibition
—Simulation results• 2 phases: exponential and linear growth• Cell division in the bulk and gradual arrests due to
increased cell density• Cell division persists at colony edges as cells are stretched.
time
cell size
time
time
trac
tion
forc
e
position
Control of cell growth/division by mechanics
Thomas LECUIT 2018-2019 25
Balancing cell growth and extrusion by mechanics
14 h
AP
26 h
AP
a
OM
OM
M
OM
OM
M
14–26 h AP
Cells that delaminate before division
Cells that delaminate after dividing
Cells that divide and only one daughter cell delaminates
Daughter cell that delaminates
b
Marinari, E., et al, and Baum, B. (2012). Nature 484, 542–545
—Compression induced cell extrusion
• Cells in the midline of a growing tissue delaminate.
–40 min –15 min –10 min
E-cad–GFP
0 min
0
0.4
0.2
0.6
0.8
1.0
0246
Nor
mal
ized
are
a
Junction number
• Cell delamination is associated with apical area reduction and junction remodelling (loss).
junction number
PTEN
control overgrowth undergrowth
Del
amin
atio
n (%
)
0
20
40
60
80
pnr-G
AL4pnr
-GAL4
> p11
0pnr
-GAL4
> PTE
N iRpnr
-GAL4
> Tsc1
> Ts
c2pnr
-GAL4
> PI(3
)K iR
pnr-G
AL4pnr
-GAL4
> p11
0pnr
-GAL4
> PTE
N iRpnr
-GAL4
> Tsc1
> Ts
c2pnr
-GAL4
> PI(3
)K iR
Midline
Outside midline
n = 3
P < 0.01P < 0.05
P < 0.05 P < 0.01 P < 0.05P < 0.05 P < 0.05
pnr-GAL4 > p110 pnr-GAL4 > Tsc1,Tsc2
a
bpnr-GAL4
Insulin
Pi3K
mTOR TSC
Growth/Cell division
• Tissue overgrowth (induced by activation of the Pi3K pathway) increases cell delamination
• Reducing tissue growth reduces cell delamination/extrusion
• Tissue crowding induces cell delamination
Thomas LECUIT 2018-2019 26
a
Compressibility Line tension Contractility
lij
ji
Aα
W = (Aα – A0)2K2
+ +lij 22lij
cells α junctions lj junctions lj
b c
d e
Initial state Final state
0
10
20
30
40
50
Time
Del
amin
atio
n (%
)
Time0
10
20
0
20
10
Del
amin
atio
n (%
)
High crowding
Wild type
No crowding
Anisotropic tissue
Isotropic tissue
M OM
T1 T2
Myosin II
Balancing cell growth and extrusion by mechanics
Marinari, E., et al, and Baum, B. (2012). Nature 484, 542–545
• Mechanical vertex model of junction remodelling underlying compression induced cell delamination.
—Compression induced cell extrusion
Thomas LECUIT 2018-2019 27
2 cm
2 cmStretch
28%
Release
Seed membrane with MDCK cells
Silicone membraneT0 1 h 2 h 3 h
Time After Release
Control
a
b c
jF-actin
FibronectinDAPI
d e2 h0.5 h 6 h
DAPIZO-1β-cat
hg Control 2 h0.5 h i******
***
Contro
l0.
5 1
1.5 2 3 4 6
Time (h)
f 150
140
130
120
110
100
Cel
ls p
er 1
00 μ
m2
Balancing cell growth and extrusion by mechanics
Eisenhoffer, G.T., et al., and Rosenblatt, J. (2012). Nature 484, 546–549.
• A tissue monolayer under compression adjusts its cell density in two phases:
• Phase 1: Density first increases rapidly due to compression.
• Phase 2: Cell density then slowly decreases until reaching initial steady state.
—Compression induced cell extrusion
b
Inhibitors against
***
***
**
**
* * ** *****
*
* *** * *
**
*Con
trol
0.5 1
1.5 2 3 4 6
Time post-overcrowding (h)
80
60
40
20
0Ext
rud
ing
cells
per
1,0
00 c
ells Non-apoptotic Apoptotic 80
60
40
20
0Ext
rud
ing
cells
per
1,0
00 c
ells
Contro
l
ROCK SK
S1P2
Non-apoptoticApoptoticApoptotic blocked
a
• Adjustment of cell density is associated with cell extrusion by apoptosis and independent of apoptosis.
• Cell extrusion requires ROCK, the sphingosine kinase SK and sphingosine phosphate (SIP2).
Thomas LECUIT 2018-2019 28
Dev
elop
ing
zeb
rafis
h ep
ider
mis
Non-apoptotic cell extrusion Apoptotic cell extrusion
Hum
an c
olon
tis
sue
Cel
l cul
ture
a b
DAPICasp3F-actin
d e
g h
c
Extruding cell
Proliferating cell
Actin ring in neighbouring cells
f
i
F-actin/DNAd
Bright-field (DIC)
Unt
reat
ed
hhh
Cell migrationc e
*37 h 49 h 56 h
6
4
2
0
8
10
*
**
***
***
No.
of e
xtru
din
g ce
lls p
er fi
sh
No.
of H
3P-p
ositi
ve c
ells
per
100
μm
2
g
Gd
3+p
iezo
1 p
hoto
-MO
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhf
i j k
Control Gd3+ piezo1 photo-MO
6
4
No.
of e
xtru
din
g ce
lls p
er fi
sh
2
0*** ***
Stretch-activated signals
Sphingosine-1-phosphate
Extrusion
Gd3+
Homeostatic cell death
Rho
Anoikis
e
Piezo1
Balancing cell growth and extrusion by mechanics
Eisenhoffer, G.T., et al., and Rosenblatt, J. (2012). Nature 484, 546–549.
• In whole organisms: gut villi and fins in zebrafish, cell crowding causes cell extrusion.
• This requires the mechanonosensitive receptors Piezo.
—Compression induced cell extrusion
Thomas LECUIT 2018-2019 29
Balancing cell growth and extrusion by mechanics
Gudipaty, S.A., et al., and Rosenblatt, J. (2017). Nature 543, 118–121.
—Stretch induced cell divisions
• Stretching of cell cultures (MDCK cells)induces cell division.• Wound healing in a cell monolayer causes
stretching of cells after closure and induces a burst of cell division after closure
Time after stretch (h)Ctrl 0.5 1 2 4 6
12
14
16
18
20
22
Cel
l len
gth
(μm
)
* *
b
Ctrl 0.5 1 2 4 6 0
50
100
150
H3P
+ c
ells
per
10,0
00 c
ells
Time after stretch (h)
***
**
a
c
12 (closure) 18 5
0
2
4
6
8
Time after wounding (h)
Div
idin
g ce
lls p
er fr
ame
5 10 15 20
closure
• Piezo activation gives rise to calcium signalling, ERK activation and Cyclin B induction
Ca2+
Piezo1 Microtubules Cell–cell junctions
ERK1/2 Cyclin B
Wound or stretch
Epithelia at steady state Stretch-induced mitosis
30 60 90 1200
200
400
600
Time after stretch (min)
Cyc
lin B
+ c
ells
per
10,
000
cells
Gd3+α-amanitin
No stretch
Control
Control Piezo1CRISPR
0
20
40
60
H3P
+ c
ells
per
fish
****
e f Piezo1 CRISPRControl
DNA H3P
Ctrl
Rosco
.Gd
3+
Piezo1
KD
0
50
100
150
H3P
+ c
ells
per
10,
000
cells
ControlStretch
***********
+Piezo1
aControl
0
1
2
3
4
5
6
4 8 12 16 20 24
Wound closed
Div
idin
g ce
lls p
er fr
ame
Time after wounding (h)
Piezo1 KD
0
1
2
3
4
5
6
Wound closed
4 8 12 16 20 24
Div
idin
g ce
lls p
er fr
ame
Time after wounding (h)
c
Pi
1A
ti
• The mechanosensitive receptor Piezo 1 is required for cell division in MDCK culture cells and in zebrafish fins
Thomas LECUIT 2018-2019 30
Balancing cell growth and extrusion by mechanics
—Stretch induced cell divisions
Regulation of cell cycle progression by cell–cell and cell–matrix forces
M. Uroz et al. and X. Trepat. (2018) Nature Cell Biology 20:646–654
g h iControl G1/S arrest
0 4 8 12 16 20 24 28 32 36
100200300400500600700
Time (h)
Num
ber
of n
ucle
i
Control G1/S arrest
0 4 8 12 16 20 24 28 32 36
8001,2001,6002,0002,4002,800
Time (h)
Cel
l are
a (μ
m2 )
Control G1/S arrest
Time (h)
0 4 8 12 16 20 24 28 32 36
0.00.10.20.30.40.50.60.70.8
Mon
olay
er a
rea
(mm
2 )
• MDCK cell culture expand at a constant cell density
• When cell division is blocked (G1/S arrest), cell area increases
• Cell division is sustained while the colony expands and thereby maintains a constant density.
Cdt1::RFP: G1-SGeminin::GFP: S-G2-M
G1/S arrest
control
Thomas LECUIT 2018-2019 31
Balancing cell growth and extrusion by mechanics
—Stretch induced cell divisions
M. Uroz et al. and X. Trepat. (2018) Nature Cell Biology 20:646–654
d e f
Time (h)
0 3 6 9 12 15 18 21 24 27 30
100
200
300
400
500
600Cdt1GemininG1 S–G2–M
0 3 6 9 12 15 18 21 24 27 30
200
300
400
500
600
700
800
Time (h)
0 2 4 6 8 10 12 14 16 18 20
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Time (h)
Tra
ctio
n ∣T
∣ (P
a)
Ten
sion
σ (
Pa)
Nuc
lear
inte
nsity
(no
rm.)
Traction Tx (on substrate)
Cdt1::RFP: G1-SGeminin::GFP: S-G2-M
Intercellular tension σ
σ
Bay
es fa
ctor
G1 104
103
102
101
100
A.
σ τc A τ
cσ τ
c A 0c
σ τc A τ
n
σ τc
τc
σ τc A τ
nT τ
c A τc
T τc A τ
nT τ
c A 0c
T τc A τ
cσ τ
c A 0n
Eτc (μJ)
G1
dura
tion
(h)
q
0.0 0.1 0.2 0.3
8
12
16
20
24
• Cell tension and surface energy E ( . A) are the best predictors of cell cycle arrest (over cell geometry).
• High surface energy correlates with a short G1 duration: cells with same surface should enter G1 earlier if tension is higher.
Large scale cross-correlation analysis
E ( . σ σ A)
A τc
σ τc A 0
n
Eτc (μJ)
Thomas LECUIT 2018-2019 32Gudipaty, S.A., et al., and Rosenblatt, J. (2017). Nature 543, 118–121.
Balancing cell growth and extrusion by mechanics
• Cells sense compression and stretch via the same mechanosensitive receptor Piezo and adjust their density depending on their mechanical state.
• Cell density evolves until the population reaches a steady state X where cell death/extrusion and cell division balance each other
—in 2D, balance between Stretch induced cell divisions and compression induced cell extrusion
Control Stretch0
50
100
Eve
nts
per
10,
000
cells
a
0
20
40
60
80
Eve
nts
per
10,
000
cells
b
**** ****
Control Crowd
DivisionExtrusion/death
DivisionExtrusion/death
X: steady-state density where cell death/extrusion and cell division balance each other
Thomas LECUIT 2018-2019 33
Balancing cell growth and death by mechanics: tumours
—In 3D, homeostatic pressure: a tissue property where cell growth/division balances apoptosis
Markus Basan , Thomas Risler , Jean-François Joanny , Xavier Sastre-Garau & Jacques Prost (2009) HFSP Journal, 3:4, 265-272, DOI: 10.2976/1.3086732
Tissue grows against wall resisted by a spring. Growth proceeds until internal pressure p = ph where growth and apoptosis balance each other.
The spring position is stable, since further growth increases the pressure above this value ph and induces apoptosis, whereas recession of the piston decreases the pressure and causes cell division.
P = P h. loserP < P h. winner P = P h. winnerP > P h. loserP < P h. winner
Net growth > 0 Net growth > 0Net growth = 0 Net growth < 0 Net growth = 0Current Biology
A. Matamoro-Vidaland R. LevayerCurrent Biology (2019)29, R762–R774,
• Cell competition
(see 26 Nov 2019)
pH,hpT,h >
As cells divide, the pressure rises and first reaches the homeostatic pressure pH,h. At this point, tissue H stops growing while tissue T continues to proliferate and drives the pressure above pH,h. As a result, the apoptosis rate of H becomes larger than its division rate, resulting in its recession. The process continues until tissue H completely disappears
Tissue HTissue T• Tumour growth
• Homeostatic pressure
Thomas LECUIT 2018-2019 34
Levayer, R., Hauert, B., and Moreno, E. (2015). Nature 524, 476–480.
—Differential growth by cell competition requires cell intercalation
• Differential cortical tension between winner-winner cell interfaces and loser-loser cell interfaces causes cell intercalation at the winner/loser interface, cell isolation and cell death.
• Mechanics (differential cortical tension) drives cell competition and differential growth.
Balancing cell growth vs death by mechanics: competition
Winner–loser mixingWinner expansionIncreased loser
apoptosis
w–w > w–l , w–l = l-l
Filamentous actin and tension modification
Winner LoserDifferential PIP3
0.3
0.9
1.5
clone
n =
40
n =
40
n =
27
WT in tub-dmyc pi3kDN in WT
n =
39
n =
35
n =
36
P = 0.03
P = 0.013
P = 0.89
P = 0.03
P = 0.007
P = 0.48
clone
clone
f
Vm
ax (μm
s–1
)
E-cad::GFP
t = –2 s
t = 12.4 s
1.5
2
2.5
3
3.5
4
4.5
–2.4 0.0
2.4
4.8
7.1
9.5
11.9
Vert
ex d
ista
nce
(μm
)
Time (s)
Vmax
1
3
1
313
2 4 2 4 2 4
370 min335 min225 min
RFP (WT loser in tub-dmyc)E-cad::GFP
l-l l-lw-w w-w
Thomas LECUIT 2018-2019 35
Moreno, E. et al and Levayer, R. (2019). Current Biology 29, 23–34
Balancing cell growth vs death by mechanics: competition
—Differential growth by cell competition requires tissue compaction induced cell delamination
Thomas LECUIT 2018-2019 36
Mechanical cues affects cell competition in 3 ways:
• Modulating the geometry of cell signalling leading to loser cell elimination,
• Inducing cell death by compression,
• Stimulating the compensatory growth of winner cells around extruded/dying loser cells via cell stretching.
Pressure
Loca
tion
Zone of death
Zone of death
* *
*
*
*
Cell intercalation (T1 transition)
Cell sorting Cell mixing
Winner cell (WT)
Loser cell
Apoptotic cell
Junction shrinking
Newly formed junction
A
Reviewed in:
A. Matamoro-Vidal and R. Levayer
Current Biology (2019) 29, R762–R774, https://doi.org/10.1016/j.cub.2019.06.030
Balancing cell growth vs death by mechanics: competition
see B. Shraiman PNAS, (2005) 102:3318–3323
Thomas LECUIT 2018-2019
Mechanical regulation of tissue size
37
• Organs grow until they reach a fixed size characteristic of each organ/species• Growth is often uniform in the face of non uniform growth factor distribution
(morphogens)• Tissues can compensate for growth heterogeneities and reach a normal final
size arguing that cells assess their environment
—How do cells compare their growth rates?—How is growth uniform on average in a tissue?—How is tissue size determined?
Thomas LECUIT 2019-2020
—How do cells compare their growth rates?—How is growth uniform on average in a tissue?• Mechanical Feedback
>0, then p increases until p0 where =0 ��r, t� � ���t��
��r, t� � ���t�� <0, then p decreases until p0 where =0
>0��r, t� � ���t��
��r, t� � ���t�� <0
If
If
• Inhomogeneous growth is smoothened through the mechanical negative feedback
��r, t� � ���t��
��r, t� � ���t����r, t���r, t� or until cells die ( <0)
38
Mechanical regulation of tissue size
B. Shraiman PNAS, (2005) 102:3318–3323
• Mechanics of inhomogeneous growth dp�r, t�dt
��K
1 � ���r, t� � ���t�� �
1�
p�r, t�,
growth rate
pressure
shear-rigidity relaxation time(ie. cell rearrangements)
bulk modulus
00
00
1
Stress p
Gro
wth
rat
e
ΓM
M
0
stronger feedback• Negative feedback of pressure
on growth
Thomas LECUIT 2019-2020
— Regulation as an integral feedback:
• Compression or stretching of a group of cells is dependent on integral over the history of the clone of its growth rate with respect to average surrounding growth.
• Cell rearrangements limits the « memory » of this feedback so adaptation of growth rate is not perfect.
• At later stages, the rate of growth is reduced to match that of the background, resulting in a ratio of mutant to background clone sizes (the overgrowth ratio) independent of time
• Initial rate of growth is faster than that of a background clone.
Ln (#cells)
time
39
Mechanical regulation of tissue size
B. Shraiman PNAS, (2005) 102:3318–3323
dp�r, t�dt
��K
1 � ���r, t� � ���t�� �
1�
p�r, t�,
Initial rate of growth i
At later stages
Thomas LECUIT 2019-2020
— Non-autonomous effect: The mechanical feedback as a possible mechanisms underlying cell competition
40
Mechanical regulation of tissue size
B. Shraiman PNAS, (2005) 102:3318–3323
• Non-autonomous cell death around fast growing clone
• Dynamics of a fast-growing clone in a background of slow growing cells
• Fast growing cells increase their pressure p to p0 and reduce their growth rate until they reach the growth rate of the surrounding
• Neighbouring slow growing cells may die as a consequence of high pressure
p0
p
Thomas LECUIT 2019-2020
— Integration of morphogen and mechanical feedback
41
Mechanical regulation of tissue size
E�ri, � � ��� � a�V � V0�
2 � b ��� ��
� � ��2 � c� � 1�2�perimeter
volumeheight
cost for cell heightmismatch deviation from
rest statedeviation from
rest stateline tension
00
0 1 20
1
Stress p
Gro
wth
rat
e
M
ΓM
M
0
��r, t� � ��M�r, t�, p�r, t��
1) growth rate goes down with increasing stressie. negative mechanical feedback.
2) growth rate has a maximum 3) the maximum depends on morphogen
concentration above a threshold value M0
M(r) � me�r/�M(r)A B C
D E F
Stress p is proportional to � 1 � 1 >0 � 1 <0
:compression:stretch:stretch:compression
Increased pressure reduces perimeter and increases height
cell divisions
L. Hufnagel et al. and B. Shraiman (2007). PNAS. 104:3835–3840 www.pnas.orgcgidoi10.1073pnas.0607134104
4) the morphogen M does not scale
Thomas LECUIT 2019-202042
Mechanical regulation of tissue size
L. Hufnagel et al. and B. Shraiman (2007). PNAS. 104:3835–3840 www.pnas.orgcgidoi10.1073pnas.0607134104
— Integration of morphogen and mechanical feedback
00
0 1 20
1
Stress p
Gro
wth
rat
e
M
ΓM
M
0
• Growth arrest: when M falls below threshold at edges, growth arrests there and pressure increases in the bulk thereby causing growth arrest.
0 4 810
1
102
103
0 50
50
t
N(t)J
Growth arrests at fixed size
0 2 40
10
20
30
d
λ• Tissue size depends on morphogen levels m, and length scale
but also on feedback strength q
size
length scaleM(r) � me�r/�
(m, q)(m, 2q)
(2m, q)
λ
p
γ = 0
γ = 0.01
γ = 0.08
γ = 0.5
M
p
γ = 0
γ = 0.01
γ = 0.08
γ = 0.5
M
p
γ = 0
γ = 0.01
γ = 0.08
γ = 0.5
0.7 0.9 1.1
1
0.1
0.01
M
IHG
0.7 0.9 1.1
1
0.1
0.01
0.7 0.9 1.1
1
0.1
0.01
D E F
Division follows iso-growth lines in the M, p phase plane
• Growth homogeneity: The mechanical feedback suppresses the effect of the high concentration of the morphogen at the center of the tissue and ensures uniform growth
Thomas LECUIT 2019-202043
— Integration of morphogen and mechanical feedback
Mechanical regulation of tissue size
• Two morphogens that scale with tissue size• Cell stretching induces growth above a threshold• No growth if both stretch and morphogen are too low
• Initially growth higher in center due to higher growth factors GF
• As a result cells are stretched at periphery, which promotes growth.
• Growth lowers stretching but cells remain stretched and compress the center, thereby reducing growth.
• The wider the periphery the greater the compression.• Growth arrests when compression overcomes the effect
of growth factors
Assumptions:
Results:
T. Aegerter-Wilmsen et al. Mechanisms of Development 124 (2007) 318–326
Thomas LECUIT 2019-202044
�
�
L. Le Goff et al . and T. Lecuit Development 140, 4051-4059 (2013) doi:10.1242/dev.090878
Y. Mao et al. and N. Tapon. The EMBO Journal (2013) 32, 2790–2803. doi:10.1038/ emboj.2013.197
— Pattern of tissue deformations in growing imaginal discs
Mechanical regulation of tissue size
• Gradient of cell area consistent with compression at the centre.
• Evidence of tissue anisotropy in a growing epithelium at tissue periphery
Δ Δ ΔΔ Δ Δ
Texture tensor
Graner, F., et al. (2008). Eur. Phys. J. E 25, 349-369.
Thomas LECUIT 2019-202045
�
L. Le Goff et al . and T. Lecuit Development 140, 4051-4059 (2013) doi:10.1242/dev.090878
Y. Mao et al. and N. Tapon. The EMBO Journal (2013) 32, 2790–2803. doi:10.1038/ emboj.2013.197
Distal
Proximal
P/D junctions
Lateraljunctions
Time (s)
D –
D0
(μm
)
–5 0 5 10 15
0
0.4
0.8
1.2
D –
D0
(μm
)
0 5–5 10 15
0
0.4
0.8
1.2
Time (s)
P/D junctions Lateral junctions
Proximaledge
Proximaledge
Distal centre Distal centre
— Stress patterns in growing imaginal discs
Mechanical regulation of tissue size
• Cells are stretched tangentially in the growing tissue
-laser ablation experiments and relaxation kinetics (related to tension over friction)
• Planar polarised distribution of actomyosin contractile network
Thomas LECUIT 2019-202046
�
�
�
�
L. Le Goff et al . and T. Lecuit Development 140, 4051-4059 (2013) doi:10.1242/dev.090878
Y. Mao et al. and N. Tapon. The EMBO Journal (2013) 32, 2790–2803. doi:10.1038/ emboj.2013.197
— Stress patterns in growing imaginal discs
Mechanical regulation of tissue size
G
Anisotropy alignment average
distance from clone edge (micron)
tang
entia
l alig
nmen
t S
color = anisotropy
Segmented ban-expressing clone
H
Y. Pan et al and B. Shraiman and K. Irvine. (2016) PNAS www.pnas.org/cgi/doi/10.1073/pnas.1615012113
• The induction of a fast-growing clone causes non-autonomous tissue deformation (anisotropy)
e.g. activation of the Hippo pathway (Yorkie++ or Expanded mutant) or of targets of Hippo pathway (mRNA bantam)
Thomas LECUIT 2019-2020
— Cytoskeletal actomyosin tension modulates tissue size
47
Mechanical regulation of tissue size
C. Rauskolb et al and K. Irvine. (2014) Cell 158, 143–156
Rho1 GDP Rho1 GTPGEF
GAP
MLCP
ROCKMRLC MRLC PDia
F-actin MyoIIminifilament
ExpandedBantam
• Activation of Rho1/Rok affects wing size
• Modulates Yorkie (YAP) pathway activity (Expanded expression)
• and Adjuba recruitment at cell junctions
Thomas LECUIT 2019-202048
Mechanical regulation of tissue size
C. Rauskolb et al and K. Irvine. (2014) Cell 158, 143–156
— Cytoskeletal actomyosin tension increases tissue size via activation of the Hippo growth pathway
— Tension at E-cadherin complexes recruits Adjuba and the kinase Warts at cell junctions and inhibits its activity via « sequestration » at cell cortex.—This releaves Yorkie repression by Warts and causes Yorkie nuclear translocation, hence activation of the pathway.
Thomas LECUIT 2019-2020
— Tension is reduced in fast growing clones
49
Mechanical regulation of tissue size
Y. Pan et al and B. Shraiman and K. Irvine. (2016) PNAS www.pnas.org/cgi/doi/10.1073/pnas.1615012113
Growth
Compression
1
23
Transcription
F
Compression
2
1. overgrowth compresses cells; 2. compression decreases cytoskeletal tension (conversely, stretching increases actomyosin network and tension)3. decrease in cytoskeletal tension triggers reduction in the cortical recruitment of Jub and Wts, and in Yki activity hence on growth.
• Mechanical Feedback
I
Cell shape reflects balance between edge tension and internal pressure
— This emerges from the mechanical feedback model where mechanical cell response adapts to external mechanical stress pattern.
Thomas LECUIT 2019-2020
— Adjuba and Warts localization at cell junction are decreased in overgrowing clone of cells
50
Mechanical regulation of tissue size
Y. Pan et al and B. Shraiman and K. Irvine. (2016) PNAS www.pnas.org/cgi/doi/10.1073/pnas.1615012113
J K
A
E
Growth
Compression
1
23
Transcription
3
and rescued when Myosin2 is constitutively active (and tension increased in these overgrowing cells)
Thomas LECUIT 2019-202051
Mechanical regulation of tissue size
A
G
Growth
Compression
1
23
Transcription Growth
1
Trranscription
Y. Pan et al and B. Shraiman and K. Irvine. (2016) PNAS www.pnas.org/cgi/doi/10.1073/pnas.1615012113
— Yorkie induced overgrowth downregulates Yorkie activity (evidence of negative feedback)
— Blocking the feedback affects causes growth heterogeneity
D F
J
H
A
• Forcing Bantam expression independent of mechanical feedback• Removing Warts so it cannot be subjected to feedback regulation• Forcing Myosin2 constitutively
Thomas LECUIT 2019-2020
— Integration of chemical and mechanical signalling
52
Mechanical regulation of tissue size
Feedback mechanisms responsible for uniform growth:
ABuild-up of compression during growth
Center Pouch edge
Com
pres
sion
Center Pouch edgeCenter Pouch edge
0
Compression > threshold
Compression slope < threshold
1
2
Growth termination when:
Dpp Wg
Arm
Yki
Fj
Ds
Notch Brk
Growth
Vg
MechanicalCompression
Fj
D
Ds
Hypotheticalvg quadrant enhancervg boundary enhancer
A P
DV
B
DppWgNotch
A
Tinri Aegerter-Wilmsen et al. and C. Aegerter and K. Basler. Development 139, 3221-3231 (2012) doi:10.1242/dev.082800
• vertex model with cell shape governed by
• Growth rate
Center
YkiNotch Brk
Growth
Vg
MechanicalCompression
YkiNotch Brk
Growth
Vg
MechanicalCompression
1
Termination
-increased growth leads to increased compression, which decreases growth,
Compression slope read-out
Yki
Fj
Ds
Vg
MechanicalCompression
Fj
D
Ds
Growth
D Yki Vg
Growth
MechanicalCompression
Large compression difference:
2
-it leads to a compression gradient that induces growth in regions with lower growth rates
Thomas LECUIT 2019-202053
Mechanical regulation of tissue size
— Integration of chemical and mechanical signalling
Tinri Aegerter-Wilmsen et al. and C. Aegerter and K. Basler. Development 139, 3221-3231 (2012) doi:10.1242/dev.082800
Non-trivial model predictions:
L M L
D
L M L
wt uniform Dpp
M L M L0
5
10
15
Ave
rage
cel
l cyc
le p
rogr
essi
on (
%/h
r)
wt uniform DPP
**
0 20 40 60 80 100101
102
103
104
Time (hours)
Num
ber
of c
ells
tkvQD clone; BrdU staining
+ 38 hrs
A A’
�
�
Thomas LECUIT 2019-202054
Mechanical regulation of tissue size
Mechanical feedback: - corrects the consequence of non uniform growth rates, and
smoothens out heterogeneities: growth homogeneity.- may be involved in compensatory mechanisms (eg. cell
competition) in the context of tissue growth and homeostasis.
- part of growth arrest mechanisms at the scale of organs.
Yet, tissue growth can be highly heterogeneous. - normal development: growth zone (limb bud, beak
morphogenesis), tissue folding. - disease: tumour evolution.
Thomas LECUIT 2019-2020 T. Tallinen et al and L. Mahadevan. PNAS (2014) 111:12667
physical mimic of brain
GI: gyration index (total area/exposed area)
brain
simulations
physical model of brain like instability
swelling of outer layer (growth)
Differential growth and tissue folding
• Cortex convolutions
Thomas LECUIT 2019-2020
E6 E8 E10 E12 E13 E15 E16 E17
No muscle + Circ. muscle + Long. muscle + Long. muscle
up until E6Smooth
E8 - E12Ridges
E13 - E15Zigzags
E16 onwardVilli
αS
MA
DA
PI
αS
MA
DA
PI
• Epithelium and mesenchyme form annealed surfaces ensheathed by sequential layers of smooth muscles
• Formation of Ridges in epithelium and mesenchyme parallel formation of circumferential smooth muscles
• Formation of Zigzags parallels formation of longitudinal muscles
• Formation of Villi formation of longitudinal muscles
Ridges Zigzag Villi
A. Shyer et al, C. Tabin and L. Mahadevan. Science 342: 212-218 (2013)
Differential growth and tissue folding
• Gut vilification:
Thomas LECUIT 2018-2019
—Computation model
—Theoretical studies:
A. Shyer et al, C. Tabin and L. Mahadevan. Science 342: 212-218 (2013)
M. Ben Amar and F. Jia. PNAS 110: 10525–10530 (2013)
E. Hannezo J. Prost and J-F. Joanny PRL 107, 078104 (2011)
Growth induced mechanical instabilities: Gut vilification
• Gut vilification
Thomas LECUIT 2019-202058
Tissue growth and mechanical feedback: - corrects the consequence of non uniform growth rates, and smoothens
out heterogeneities: growth homogeneity.- may be involved in compensatory mechanisms (eg. cell competition) in
the context of tissue growth and homeostasis.- required for growth arrest at the scale of organs.
Summary
Mechanical regulation of cell division and cell death/extrusion
- Sensing: mechanosensitive receptors (Piezo), cortical tension and YAP pathway- Cellular response: mechanics of junction remodelling (see Courses 2018)
- Cells respond differently to compressive and tensile stresses: - Cell growth, cell division, cell extrusion or simple cell shape changes, cell
intercalation.- These processes underlie viscoelastic properties of tissues: energy
dissipation in homeostatic conditions- Cell mechanics underlies tissue homeostatic pressure.- Not well understood what factors determine these properties
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