Supplementary Information for

Spatial coordination between cell and nuclear shape

within micropatterned endothelial cells.

Marie Versaevel, Thomas Grevesse and Sylvain Gabriele*

Mechanobiology & Soft Matter Group, Laboratoire Interfaces et Fluides Complexes,

Université de Mons, Place du Parc, 20, B-7000, Mons, Belgium

*Corresponding author:

Sylvain Gabriele, Ph.D.

Mechanobiology & Soft Matter Group

Laboratoire Interfaces et Fluides Complexes

Université de Mons

Place du Parc, 20

B-7000 Mons, Belgium

Phone +32 65 37 38 24, Fax +32 65 37 38 33

Email: sylvain.gabriele@umons.ac.be

Supplementary Figure S1. Spreading area is a key component of nuclear shape

remodeling. (a) The nuclear shape index, NSI, decreases linearly with increasing the

spreading area, SA, of similar shaped cells (CSI ~ 0.26). Data are given as Mean ±

SD with 9 ≤ n ≤ 12. (b) DIC images of elongated cells (CSI ~ 0.26) plated on

fibronectin-coated micropatterns with a surface area ranging from 600 to 2000 µm2.

The nucleus (in blue) of cells plated on small spreading areas was rounded and

extended out of the adhesive pattern (delineated by white rectangles), whereas the

nucleus of cells plated on large spreading areas was elongated and significantly

narrower than the width of the fibronectin pattern. The scale bars correspond to : 8

µm (SA = 600 µm2, in red), 9 µm (SA = 800 µm

2, in blue), 11 µm (SA = 1200 µm

2, in

green), 13 µm (SA = 1600 µm2, in black) and 14 µm (SA = 2000 µm

2, in purple).

Supplementary Figure S2. Actin filaments are involved in nuclear orientation.

DIC images of (a) a single endothelial cell and (b) a latrunculin A-treated endothelial

cell, both plated on 1600 µm2 rectangular micopatterns. The nucleus (in blue) was

stained with DAPI and superimposed on the DIC image. White rectangles correspond

to the delineation of the FN-coated micropatterns. (a) Control cells were

characterized by a well-oriented nuclei (0 ≤ θ ≤ 10°) with respect to the long-cell axis

(dashed black line) whereas (b) latrunculin A-treated cell shown large nuclear

orientations (θ = 59°). The scale bars correspond to 10 µm.

Supplementary Figure S3. Spatial distribution of apical actin filaments in

response to cell shape changes. Confocal microscopy sections of the actin network

organization in the apical zone of endothelial cells plated on 1600 µm2 adhesive

micropatterns for 1 ≥ CSI ≥ 0.26. A weak density of filamentous actin was observed

in the apical zone for CSI ~ 1.0, whereas apical actin filaments became denser and

oriented with respect to the long cell axis as the cell elongated. The scale bar

corresponds to 12 µm.

Supplementary Figure S4. Focal adhesions in response to cell shape changes.

Quantification of the total area of vinculin-containing focal adhesions per cell plotted

against the cell shape index, CSI, for a similar spreading area (SA = 1600 µm2). The

red line indicates a linear increase of the total area of vinculin-containing focal

adhesions for 0.78 ≥ CSI ≥ 0.26. Note the large value of the total focal adhesion area

observed for CSI ~ 1.0. Data are given as Mean ± SD with 12 ≤ n ≤ 15.

Supplementary Figure S5. Organization of central actin filaments of elongated

cells as a function of the spreading area. Confocal microscopy sections of the

spatial organization of actomyosin filaments (in green) in the central zone for similar

shaped endothelial cells (CSI ≈ 0.26) plated on (a) 600, (b) 800, (c) 1200, (d) 1600

and (e) 2000 µm2 elongated micropatterns. White rectangles correspond to the

delineation of the FN-coated micropatterns. The nucleus is stained in blue with

DAPI. As the spreading area increased, central actomyosin filaments initially curved

on small spreading areas became straight on large spreading areas due to an

accumulation of tension. The scale bars correspond to (a) 8 µm, (b) 9 µm, (c) 11 µm,

(d) 13 µm and (e) 14 µm.

Supplementary Figure S6 – Influence of myosin II activity on the nuclear

volume. Evolution of the nuclear volume for 1600 µm2 circular-shaped cells (CSI ~

1, in dark grey) and 1600 µm2 rectangular-shaped cells (CSI ~ 0.26, in white) treated

with blebbistatin (hatched bar). Data are given as Mean ± SD with 9 ≤ n ≤ 12.

Supplementary Figure S7 – Influence of the intermediate filament network on

the nuclear volume. Evolution of the nuclear volume for 1600 µm2 elongated cells

(CSI ~ 0.26 in dark grey) treated with calyculin A (light grey, hatched bar) and

acrylamide (light grey, cross-hatched bar). Data are given as Mean ± SD with n = 12.

Supplementary Figure S8 – Chromatin compaction and DNA synthesis. The

linear evolution of the DNA synthesis as a function of the nuclear average spatial

density for 1600 µm2 micropatterned cells (1 ≥ CSI ≥ 0.26) suggested a relation

between cell proliferation and chromatin condensation. Data are given as Mean ± SD

with n = 12.