Local 3D matrix microenvironment

regulates cell migration through

spatiotemporal dynamics of
contractility-dependent adhesions
by
Andrew D. Doyle, Nicole Carvajal,
Albert Jin, Kazue Matsumoto &
Kenneth M. Yamada

Abstract
The physical properties of two-dimensional (2D)
extracellular matrices (ECMs) modulate cell adhesion
dynamics and motility, but little is known about the roles of
local microenvironmental differences in three-dimensional
(3D) ECMs. Here we generate 3D collagen gels of varying
matrix microarchitectures to characterize their regulation of
3D adhesion dynamics and cell migration. ECMs containing
bundled fibrils demonstrate enhanced local adhesion scale
stiffness and increased adhesion stability through balanced
ECM/adhesion coupling, whereas highly pliable reticular
matrices promote adhesion retraction. 3D adhesion
dynamics are locally regulated by ECM rigidity together
with integrin/ECM association and myosin II contractility.
Unlike 2D migration, abrogating contractility stalls 3D
migration regardless of ECM pore size. We find force is not
required for clustering of activated integrins on 3D native
collagen fibrils. We propose that efficient 3D migration

INTRODUCTION
Cell interactions with the surrounding microenvironment
regulate key intracellular processes, including signalling
cascades, gene regulation, and cell fate. This type of
outside-in signalling includes physical signals from the
extracellular matrix (ECM) that can profoundly affect cell
migration. The physical characteristics of an ECM, including
stiffness, composition, and topography, can regulate
migration. While cell migration is considered a cyclic
process consisting of (1) protrusion, (2) adhesion, (3)
translocation and (4) retraction, it is the direct coupling
between the cell and the ECM at integrin-based adhesion
sites that permits cells to mechanically sense their physical
surroundings and adjust mechanisms of migration.

Beyond the ECM physical regulators known for 2D

migration (stiffness, ligand density and composition), a 3D
matrix adds ECM microarchitecture, porosity, and elastic
behavior.
However, it is not clear how local differences in 3D matrix
stiffness and other ECM physical factors affect the
mechanical behavior of 3D adhesions and cell migration.

In this study, we examine how the

physical attributes of 3D matrix
regulate the mechanics of cell
adhesions and their role in 3D cell
migration. By changing the local
physical characteristics of type I
collagen gels to mimic a more in vivolike environment

Methodology
Collagen gel formation: Rat tail monomeric collagen was used.
Briefly, rat tail tendons were dissected out with special care given to
remove fragments of bone, cartilage, blood vessels and even the
tendon sheaths, all of which can contribute contaminating collagens
(II, III, IV and so on) and other ECM components (fibronectin,
proteoglycans) which may cause lot-to-lot variations.
Tendon fibers were then suspended in 0.5 M acetic acid and stirred at
4C over a three-day period. Protein concentration was determined.
HR(Highly Reticular) Gels were polymerized at 37C in a tissue
culture incubator, LR(Loose Reticular) and FB16(Fibrillar-bundled)
were polymerized on an Echotherm chilling/heating dry bath at 21
and 16 C, respectively. FB4 gels were polymerized at 4 C without
cells in a refrigerator after sealing the dishes with parafilm.
After FB4 gel polymerization, cells were added to the 3D lattice and
allowed to invade for 48 h before imaging.

Polymerization of rat
tail collagen gels at
different temp. (37,
21, 16 and 4C then
structured
illumination
microscopy

Live-cell
spinning disk
microscopy of
EGFP-Lifeact

Immuno
staining
for
activate
d 1
integrin

Immunostaining
for flourescent
protein-tagged
adhesion proteins

Treated HFFs within

HR and FB16 gels
with mAb13 to
reduce 1 integrin
binding together with
25 M blebbistanin.

Immunostaini
ng for type 1
collagen

Timelapse
microscopy
and cell
tracking

Examination of protein
turnover via flourescence
recovery after
photobleaching (FRAP) in
EGFP-zyxin containing
adhesions in the different
3D ECMs and on 2D
collagen covalently linked
to the coverslip

Integrin/ligand
interactions
were altered.

Performing of atomic force

microscopy(AFM) using
conical-tipped pyramidal
cantilevers at 1m resolution
then Raster scanning of
collagen gel

AFM
measurement
using a 38m diameter
bead

Imaging of HFFs
expressing EYFPpaxillin and used
automated adhesion
tracking to determine
adhesion lifetime

Adhesion
dynamics via
adhesion
vector
mapping

RESULTS

It showed that
Fibril bundling
Immunostaini
these
fibril
and not fibre
ng for type 1
architectures
thickening was
werecollagen
present
resposible for
in vivo
the structural
differences.
Confirmed
Timelapse
Live-cell
Altered
Different matrix
microscopy
that cell
spinning
disk
protrusions
and cell
migration
microscopy
ofarchitectures can
in the
tracking
influence cell
presence of
rates
rose
EGFP-Lifeact
bundled
migration rateswith
fibrils
Polymerization of rat
tail collagen gels at
different temp. (37,
21, 16 and 4C then
structured
illumination
microscopy

increasing
ECM
porosity
Examination
of protein

Immuno
turnover via flourescence
staining
Immunostaining
recovery
Kinetics
data suggest that
rigidafter
for Adhesion for
flourescent
photobleaching (FRAP) in
the efficiency
of the containing
protein-tagged
EGFP-zyxin
activate fibrils promote
adhesions
in the different
adhesion
clutchproteins
within adhesions,
while
d 1 molecular
3D ECMs and on 2D
complaint ECMs result in increased
adhesion
integrin
collagen
covalently linked
to
the
slipping with inefficient coupling coverslip
Treated HFFs within
Datassuggested
a
HR and FB16 gels
requirement for active
with mAb13 to
contraction
reduce 1 to
integrin
physically
detach with
binding together
clustered
25 M integrins
blebbistanin.
during fibroblast 3D

Addition of FN
Integrin/ligand
substantially
interactions
increased adhesion
were altered.
lifetime in HR gels
by reducing
nascent/retracting

cell: No significant
differences
in gel
Performing
of atomic
force
stiffness
microscopy(AFM) using

conical-tipped
pyramidal
At the scale
of
cantilevers at 1m resolution
CellRaster
adhesions:
then
scanning of
collagen
gel
bundle

Couldand
be
thickness
attributed
overall
fiber to
AFM
differing
heterogeneity
measurement
of
FBporosities
collagen
betweer
ECM
using
a 38was
associated
types
m
diameter
with
large
beadin
variations
local fibre
rigidity
Imaging of HFFs

Local fibril
stiffness
expressing
EYFP-can
determine
paxillin
adhesion
and used
lifetime
automated
by altering
theadhesion
relative
tracking to determine
proportion
of nascent
versus
adhesion
lifetime
mature adhesions

Adhesion
Adhesion
displacement
dynamics via
was substantially lower
adhesion
within ECMs
containing
vector
stiff bundled
mappingfibrils

DISCUSSION