Polarity reversal primes cell scattering during epithelial to mesenchymal transition

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Polarity reversal primes cell scattering during epithelial to mesenchymal transition

Mithila Burute, M. Prioux, G. Blin, Sandrine Truchet, T. Bessy, G. Letort, Q. Tseng, J. Young, Odile Filhol, Manuel Théry

To cite this version:

Mithila Burute, M. Prioux, G. Blin, Sandrine Truchet, T. Bessy, et al.. Polarity reversal primes cell scattering during epithelial to mesenchymal transition. 2015 Cell Biology ASCB annual meeting, 2015, San Diego, United States. 2015. �hal-01606992�

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Polarity reversal primes cell scattering during epithelial to mesenchymal transition

Epithelial to mesenchymal transition (EMT) is a morphogenetic event that takes place during specific stages

of embryo development and early stages of tumour dissemination. We used minimal model of tissue

compri-sing two cells to understand reorientation of internal polarity of cells during EMT. We found that after EMT

induction, cells undergo polarity reversal by repositioning their centrosome and thus reorientating nucleus

-centrosome axis. This polarity reversal involves redistribution of cellular forces which primes them for cell

se-partion after EMT. We also found evidence of polarity reversal at different stages of developing mouse

emb-ryo and this reversal was essential for scattering of resulting mesenchymal cells.

Abstract

γ-Tubulin

/

Par-3

/

Dapi

0 _1.0 _1.2 _1.4 _1.6 _1.8

10 20 30 WT r = 0.27 ns_{TGFβ + SB431542 r = -0.30 *}TGFβ r = -0.56 **** WT TGF TGF SB431542+ Inter -centr osome distance ( μm) WT TGFβ

Results

Par-3

EMT

Mouse E8 embryo Gastrulation A P Endoderm Epiblast Primitive Streak Mesoderm movement Laminin 1 2 2 1 1 2 c

c

γ-Tubulin/Laminin/Dapi/T-Brachyury

γ-Tubulin

/

Dapi

c c cavity Giantin / Actin /Ninein/ Dapi n: 230 200 Zoom in α BM WT TGFβ α ( degree) 0 60 120 180 0 10 20 Disor ganisat

ion Index (um)

****

c

Polarity inversion during mouse development and in 3D acini

d

Polarity reversal is sensitive matrix rigidity

Par3 and MT reorganization for centrosome positioning during EMT

Polarity reversal initiates cell scattering during EMT

Proposed mechanism for EMT

Working Model

Cellular force redistribution of cell-matrix and cell-adhesion forces

WT TGFβ WT TGFβ RGD RGD RGD Glass slide Azide-Poly-l-Lysine PEG Glass slide RGD-Poly-l-Lysine PEG

BCN-RGD

Cell confinement

_{Release of Cell confinement}

N3 N3 N3 N3

0 30 60 90 120

Time (min) after addition of BCN-RGD

0 40 80 % se p era te d ce ll-doub le ts TGFβ Treated cells 0 20 40 60 15% 53 % WT TGFβ WT TGFβ % cel l doublet separat ion N3 N3 N3 N3 RGD RGD WT TGFβ T0 T120 T0 T120 Primitive Streak NN axis γ-T ub / DAPI / FNG WT

MCF10A

TGFβ

MDCK

_γ-T ub / Dapi / FNG Cx, Cy Nx,Ny NN axis NCx, NCy NR x y

Polarity reversal occurs during EMT

-2 -1 21 -2 -1 1 2 2 -2 -1 1 2 -2 -1 1 2 Y -2 -2 2 2 -1 1 -2 -2 2 2 -1 1 -1 1 -2 -2 2 2 HGF WT 28% 72% 68% 32% _43% _57% _57% 43% EB1 Total Microtubules WT TGFβ 15 Density (A.U) 10 5 0 Microtubules at CCJ WT TGFβ 4 2 0 Density (A.U) WT TGFβ WT TGFβ 0 1000 2000 **** ****

EB1 density (AU) _WT

Nucleation at Centrosome EB1 count TGFβ **** γ-Tubulin/Dapi WT TGFβ α-Tubulin 8 4 0 WT Par3 at CCJ

Polarity Index towards CCJ

40 20 0 60 80 TGFβ WT TGFβ

Total EB1 count

EB1 at CCJ WT_10Kpa WT_Glass TGFβ_Glass

EPH4

WT_1kPa WT_10kPa WT_Glass TGFβ_Glass

NMuMG

Giantin/Actin/Dapi

Giantin/Dapi

γ-Tub/E-cad/Dapi γ-Tub/E-cad/Dapi γ-Tub/Actin/Dapi

WT_Glass TGFβ_Glass WT_10kPa _{WT_10kPa} _{WT_1kPa}

-0.6 -0.3 0.0 0.3 0.6 -0.6 -0.3 0.0 0.3 0.6 Polarity Index towards CCJ Apical

Basal

Epithelial Polarity

Centrosome acts as Microtubule organizing center (MTOC) and is often located at the geometric center of the single cell. In epithelial tissue, centrosome is preferrentially located close to apical domain and thus defines Nucleus-Centrosome

axis that is directed towards the lumen of the organ.

Hypothesis

Burute M, Prioux M, Blin G, Truchet S, Bessy T, Letort G, Tseng Q, Young J, Filhol O, Th

é

ry M

**** ns **** **** 0 10 20 30 40 Distance ( μm) 0 10 20 30 40 Distance ( μm) NN distance -0.8 -0.4 0.0 0.4 0.8 TGFβ + Par3b

Introduction

WT TGFβ **** n: 709, N=7 n: 665, N=8

WT HGF

****

n: 494, N=5

-0.6 -0.3 0.0 0.3 0.6 -0.6 -0.3 0.0 0.3 0.6 n: 458, N=5 CC distance NN distance CC distance Avg n= 20

Actin p-Myosin II Paxillin

WT TGFβ WT Fc(i) = -ΣF(i) || Fc || -|Σ_F(i)| ΣІ_F(i)І

|| Fc ||

/

Σ |

F(i)

|

_****

WT TGFβ HGF

MCF10A

MDCK

0.0 0.5 1.0 1.5 WT Cell(i) Cell(j) -Fc F Fx Fy Fc Total Traction =

0 10 20 30 DMSO Blebbistatin NN CC NN CC -0.4 0.0 0.4 WT+DMSO HGF+DMSOHGF+BB WT+BB WT HGF Distance ( μm ) **** **** * ns WT TGFβ

Traction force Microscopy

EMT involves active reorganization of polarity by repositioning the centrosome and hence microtubule organization.

Polarity reversal was observed in mammary gland (MCF10A, EpH4, NMuMG) and kidney (MDCK) cells as response

to EMT inducers.

Polarity reversal could be initiated in the absense of external EMT inducer, by only increasing the matrix rigidity.

Reduction of MT number and nucleation after EMT suggest centrosome position can be affected by MT number.

Polarity reversal by centrosome repositioning is required for cell scattering after EMT, in metastatic-like events.

Par-3 accumulation at CCJ controls centrosome

offcentering towards cell-cell Junction. After EMT,

Par-3 loss from CCJ causes centrosome

repositioning to the center of the cell, which results

in polarity reversal after EMT

***

537 430 322 215 107 0 pa

****

Conclusions

L L ?

Luminal Polarity Loss of Polarity

Polarity reversal Invasion L L

Current view

Hypothesis

EMT

mithila.burute@cea.fr, manuel.thery@cea.fr, cytomorpholab.com

1, 2, 3 1 ₄ ₅ ₂ 1 ₁ ₃ 6 _{1, 2}

1. Cytomorpholab, CEA, Université Grenoble Alpes, Grenoble, France 2. Unit Thérapie Cellulaire, Hôpital Saint Louis, Paris, France 3. CYTOO SA, Grenoble, France 4. Medical Research Council, UK 5. INRA, Jouy-en-Josas, France 6. iRTSV, CEA, Grenoble, France

Abbreviations: MT- Microtubles, CCJ- Cell cell Junction, BB-Blebbistatin, NN- Intenuclear Distance, CC- Intercentrosomal distance NN axis- Nucleus-nucleus axis, BM- Basement Membrane