Tuning Collective Cell Migration by Cell–Cell
Junction Regulation
Peter Friedl
1,2,3
and Roberto Mayor
4
1
Department of Cell Biology, Radboud University Medical Centre, Nijmegen 6525GA, The Netherlands
2
David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas
MD Anderson Cancer Center, Houston, Texas 77030
3
Cancer Genomics Center, 3584 CG Utrecht, The Netherlands
4
Department of Cell and Developmental Biology, University College London, London WC1E 6BT,
United Kingdom
Correspondence: peter.friedl@radboudumc.nl; r.mayor@ucl.ac.uk
Collective cell migration critically depends on cell–cell interactions coupled to a dynamic
actin cytoskeleton. Important cell–cell adhesion receptor systems implicated in controlling
collective movements include cadherins, immunoglobulin superfamily members (L1CAM,
NCAM, ALCAM), Ephrin/Eph receptors, Slit/Robo, connexins and integrins, and an adap-
tive array of intracellular adapter and signaling proteins. Depending on molecular compo-
sition and signaling context, cell–cell junctions adapt their shape and stability, and this
gradual junction plasticity enables different types of collective cell movements such as
epithelial sheet and cluster migration, branching morphogenesis and sprouting, collective
network migration, as well as coordinated individual-cell migration and streaming.
Thereby, plasticity of cell–cell junction composition and turnover defines the type of col-
lective movements in epithelial, mesenchymal, neuronal, and immune cells, and defines
migration coordination, anchorage, and cell dissociation. We here review cell–cell adhe-
sion systems and their functions in different types of collective cell migration as key regu-
lators of collective plasticity.
M
ulticellular organisms form and maintain
their bodies through the ability of individ-
ual cells to adhere to neighbor cells by cell –cell
junctions, which are mechanically both stable
and flexible and secure cell position and func-
tion over time. Stable junctions anchor cells in
their tissue niche and define cell–cell coopera-
tion and mechanical function such as contrac-
tion or cell–cell signaling. These junctions are
the basis of all polarized epithelia, vessels, mus-
cle, neuronal tissue, as well as cell organization
in connective tissue. Dynamic cell–cell junc-
tions enable cells to change position relative to
their neighbors or as multicellular groups; they
are relevant during morphogenesis and phases
of tissue activation, for example, in response to
injury or inflammation (Collins and Nelson
2015). By regulating junction “fluidity,” the ag-
Editors: Carien M. Niessen and Alpha S. Yap
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gregate state and dynamics of cells can change
remarkably and, accordingly, alter collective
functions (Collins and Nelson 2015; Park et al.
2016).
Depending on the cell ty pe and activation
state, a range of adhesion receptor and cytoskel-
etal adaptor systems are involved in securing
short- or long-lived, dynamic or stable cell–
cell interactions. These include cadherins and
protocadherins, immunoglobulin (Ig) super-
family members, desmosomal and tight junc-
tion (TJ) proteins, as well as integrins, selectins,
ephrin/eph receptors, and, likely, connexins,
which all directly or indirectly couple to the
intracellular cytoskeleton and mediate distinct
cell–cell adhesion types (Theveneau and Mayor
2012a; Collins and Nelson 2015). Controlled by
upstream signaling, each receptor type can un-
dergo context-dependent alteration in surface
expression, ligand interaction, and cytoskeletal
coupling, and mediate a range of dedicated
types of cell–cell coupling.
Many types of collective cell–cell behaviors
depend on stable cell–cell anchorage to form
layered cell sheets or complex forms of tissue
organization, including barrier function medi-
ated by epithelia and endothelia toward the
extra- and intracorporal spaces, intercellular
signaling network functions as in neuronal net-
works, or large-scale contraction and force gen-
eration as in muscle or purse –string contrac-
tion of epithelia (Tada and Heisenberg 2012;
Sunyer et al. 2016). Most dynamic multicellular
functions, which depend on long-lived cell–cell
junctions lasting hours to days or weeks, can be
categorized as collective movements in which
clusters, sheets, or strands of cells move as a
multicellular unit across or through tissue for
developing and maintaining epithelial struc-
tures (Friedl and Gilmour 2009; Shamir and
Ewald 2015). More dynamic cell–cell junctions
lasting in the range of minutes are critical in
mediating multicellular crowd behaviors in
which groups of cells move individually, but
coordinate their directionality and speed by
less stable and comparably short-lived adhe-
sions and cell–cell sensing ( Theveneau and
Mayor 2013). Last, immune cells use even
more short-lived cell– cell junctions for coordi-
nating their migration and transient clustering
with other leukocytes for signal exchange,
which depends on very dynamic physical and
chemical cell–cell interactions (Malet-Engra
et al. 2015).
By combining different adhesion systems in
a modular manner in time and space, cells re-
spond to extracellular triggers and tune their
levels of cell– cell cooperation. We here summa-
rize the range of cell–cell junction types ex-
pressed by different cell types, their morpholo-
gies and kinetics, and implications for collective
migration, anchorage, and cell dissociation. We
further review how different types of cell–cell-
interaction-based dynamics and collective cell
migration are “tunable” and allow for adaptive
strategies of cell movements for different phys-
iological and pathological contexts and discuss
their implications for classifying collective and
single-cell behaviors.
CELL–CELL ADHESION SYSTEMS
Common to all adhesion systems is the require-
ment for an initial interaction between trans-
membrane cell-surface receptors on adjacent
cells, which usually are followed by the recruit-
ment of intracellular adaptor and cytoskeletal
proteins. This complex regulates the shape and
mechanical stability of the adhesion junction,
its interaction with intracellular effectors, and
adhesion-mediated activation of downstream
signaling pathways. Typically, cells use several
complementary adhesion systems in parallel,
resulting in a cell– cell interactome (Porterfield
and Prescher 2015).
Adherens junctions (AJs). AJs are protein
complexes found at cell–cell junctions of epi-
thelial and endothelial tissues that connect the
actin cytoskeleton of adjacent cells (Shapiro and
Weis 2009). AJs depend on the homophilic
binding of calcium-dependent cadherins, which
interact via their intracellular domains with sev-
eral regulatory and cytoskeletal proteins such as
p120-, a-, b-, g-catenin, and vinculin, among
others (Harris and Tepass 2010). Although AJs
are usually associated with epithelial and endo-
thelial tissues, it has been shown that mesenchy-
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mal cells form transient adhesion complexes in
which ty pe I N-cadherin, together with the full
repertoire of intracellular adhesion proteins
(p120, a-, b-, g-catenin, and vinculin), are en-
gaged (Theveneau and Mayor 2012a). Both E-
and N-cadherin-based AJs control apicobasal,
as well as front–rear, polarity of interacting cells
(Venhuizen and Zegers 2016).
The main functional difference between ep-
ithelial and mesenchymal AJs is their stability:
epithelial junctions tend to be more stable (in
the range of hours to days), whereas mesenchy-
mal junctions are transient (minutes to hours)
(Scarpa et al. 2015). The stability of AJs is con-
trolled by several mechanisms, including endo-
cytosis and cytoskeletal regulation. Endocytosis
of AJ receptors and adapters occurs both by
clathrin-dependent and -independent mecha-
nisms (Delva and Kowalczyk 2009; Schill
and Anderson 2009), which cooperate with reg-
ulation by Rho family GTPases. For example,
Cdc42 works upstream of Par6/aPKC and
Cdc42-interacting protein 4 (CIP4), which con-
trol actin dynamics at the internalization site
(Harris and Tepass 2010). Besides controlling
the stability of cell–cell interactions, Rho
GTPases, via PAK and bPIX, are reciprocally
controlled by AJs in which they play an essential
role on actin dynamics (Zegers et al. 2003;
Zegers and Friedl 2014). Interaction between
cadherin–catenin clusters leads to the recruit-
ment of the Rac guanine nucleotide exchange
factor (GEF) TIAM1, which activates the Rho
GTPase Rac1, and the activation of Rac1 in lead-
er cells is, in turn, required for the formation of
cell protrusions and traction forces observed at
the edge of a cell cluster during collective cell
migration (Hordijk et al. 1997; Kovacs et al.
2002; Mertens et al. 2005). Another activator
of Rho GTPases within the AJ is Nectin, and
Nectin-like proteins, a family of Ig-like cell ad-
hesion molecules (CAMs) (Takai et al. 2008). To
aid the formation of AJ, nectin recruits afadin
and ponsin, which lead to the activation of
Cdc42 and Rac and cytoskeletal remodeling at
the site of cell– cell contact (Fukuyama et al.
2006). The interaction between AJs and actin
is mutual, leading to an increase in the stability
of cortical actin toward the maturing AJ com-
plex (Baum and Georgiou 2011). Consequently,
AJ are central hubs controlling cell–cell cohe-
sion and collective cell migration underlying
tissue dynamics and remodeling.
Tight junctions (TJs). TJs are adhesion com-
plexes in which the plasma membranes of adja-
cent cells become closely associated, forming an
impermeable barrier within the tissue. TJs are
indispensable for creating a barrier between dif-
ferent regions of the body, and their main role is
to function as paracellular gates that restrict dif-
fusion on the basis of size and charge. TJs are
composed of transmembrane proteins (claudin,
occludin, tricellulin, and marveld3) that seem
sufficient to trigger at least some of the aspects
required in TJ formation, including mechanical
junction stability and apicobasal polarity of
connected cells (Zihni et al. 2016). Other TJ
transmembrane adhesion proteins comprise
the junctional adhesion molecules (JAMs),
which enhance TJ stability and turnover (Ebnet
et al. 2004; Luissint et al. 2014). The intracellu-
lar function of TJs depends on a dense network
of proteins, composed of ZO1, ZO2, ZO3, plus
a large number of other adaptor proteins (Van
Itallie and Anderson 2014). By binding several
transmembrane and adaptor proteins, TJs con-
trol various signaling pathways involved in actin
organization, cell polarity, as well as transcrip-
tional regulation (Gonzalez-Mariscal et al.
2014). The interaction of TJ proteins with the
actin cy toskeleton seems to be essential for
junction formation and turnover. For example,
myosin light chain kinase stimulates TJ remod-
eling and occludin internalization during
inflammation (Herrmann and Turner 2016).
Rho GTPase signaling is also controlled by TJ-
associated proteins: RhoA, Cdc42, and Rac are
regulated by GEFs recruited to cingulin, ZO1,
and tricellulin (Otani et al. 2006; Terry et al.
2011; Oda et al. 2014). Thereby, TJs form a cen-
tral hub between cell–cell interactions and actin
dynamics (Balda and Matter 2016).
Gap junctions (GJs). GJs are intercellular
membrane channels made up of a multigene
family, called connexins in vertebrates (Willecke
et al. 2002). GJs are specialized junctions char-
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acterized by close apposition of the plasma
membranes between neighboring cells and con-
tain a hydrophilic channel that mediates the
intercellular passage of molecules .1 kDa in
size. The extracellular domains of connexins
form a tight connection between adjacent cells
contributing to cell-cell adhesion. Connexins
interact w ith several proteins to form multipro-
tein complexes, which are important in cell-cell
junction stability and function. For example,
Cx43 interacts with N-cadherin and other
members of the AJ complex (Xu et al. 2001),
as well as cytoskeletal proteins such as microfil-
aments and microtubules (Wei et al. 2004).
Phosphorylation of the cytoplasmic domain
of connexin is critical in regulating GJ assembly,
trafficking, channel gating, and turnover. GJs
contribute to cell migration during develop-
ment and in homeostatic processes such as
wound healing (Kotini and Mayor 2015), and
it has been proposed that their channel activity
could be important for cell coupling and coor-
dination during migration (Lorraine et al.
2015).
IgCAMs. IgCAMs correspond to immuno-
globulin-like cell-adhesion molecules contain-
ing one or more Ig-like domains in their extra-
cellular regions. IgCAMs are expressed in a wide
variety of cell types, such as cells of the nervous
system, leukocytes, and epithelial and endothe -
lial cells (Cavallaro and Christofori 2004). By
homophilic and heterophilic interactions of
their Ig-like domains IgCAMs mediate adaptive
cell–cell interactions and play an important role
in cell migration (Cavallaro and Christofori
2004). IgCAM adhesion is regulated by lateral
oligomerization, which in turn depends on
phosphorylation of their Ankyrin-binding do-
main (Garver et al. 1997). A second mechanism
that controls IgCAMs-mediated adhesion is
based on their internalization or recycling
from the plasma membrane; for example, the
internalization of aplysia cell adhesion molecule
(apCAM) is controlled by phosphorylation by
mitogen-associated protein (MAP) kinase (Bai-
ley et al. 1997). A third mechanism that regu-
lates IgCAM-based cell adhesion is their proteo-
lytic cleavage. For example, the leukocyte
adhesion molecule L-selectin is cleaved by shed-
dases of the metalloprotease and ADAM fami-
lies, and is protected from this cleavage by in-
tracellular regulators, which engage with its
cytoplasmic domain, including calmodulin
and moesin (Kahn et al. 1998; Ivetic et al.
2002). IgCAMs have been reported to associate
with a range of other proteins at the cell mem-
brane, including growth-factor receptors, integ-
rins, and cadherins, and with intracellular pro-
teins, such as effectors of signal transduction
pathways and cytoskeletal proteins (Juliano
2002), and thus contribute to a range of signal-
ing programs involved in cell adhesion and mi-
gration.
Slit/Robo. Slit/Robo corresponds to the
Roundabout receptors (Robo) and their Slit li-
gand. Robo receptors belong to the superfamily
of IgCAMs and engage in both homophilic and
heterophilic interactions (Hivert et al. 2002).
Slits are the principal ligands for the Robo re-
ceptors (Kidd et al. 1999), which, together with
heparan sulphates, form a ternary complex re-
quired for signaling (Ypsilanti et al. 2010). The
cytoplasmic domains of Robo do not possess
catalytic activities and, therefore, interact w ith
different signaling molecules to exert their spe-
cific effects; these include netrin and several
GTPase activating proteins (GAPs) and GEFs
that control actin cytoskeletal dynamics by reg-
ulating the activity of RhoA, Rac1, and Cdc42
(Ypsilanti et al. 2010). The activity of Slit/Robo,
including adhesion, is controlled by transcrip-
tional regulation and endocytosis and degrada-
tion (Keleman et al. 2005). Slit–Robo interac-
tion t ypically mediates cell repulsion, but in
some cases also supports cell–cell adhesion.
The formation of cranial ganglia requires the
adhesion to different cell ty pes, and increased
adhesion between neural crest and placodes is
promoted by an interaction between Robo2/
Slit1, which increases the N-cadherin-depen-
dent adhesion between these cells (Shiau and
Bronner-Fraser 2009). Slit/Robo interactions
are also involved in collective cell migration
of neural crest cells during development and
endothelial cells in angiogenesis (Legg et al.
2008).
P. Friedl and R. Mayor
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Ephrin/Eph receptor. Ephrin/Eph receptor
corresponds to a pair of ligands and receptors
involved in short-distance cell–cell signaling.
Eph are Tyr kinase receptors and ephrins are
membrane-tethered ligands, which can elicit
signaling that affects the cytoskeleton, mediat-
ing primarily cell repulsion but, dependin g on
context, also cell–cell adhesion (Kania and
Klein 2016). Phosphorylation of the intracellu-
lar domains of Ephs regulates the recruitment of
effector proteins, such as the noncatalytic re-
gions of Tyr kinase adaptor protein 1 (Nck1)
and Nck2, Vav2 and Vav3, Src, a2-chimerin,
and ephexins, which directly regulate actin or
modulate the activity of the small GTPases
RhoA and Rac1 (Kania and Klein 2016). Nor-
mal morphogenesis of the neural tube and neu-
ral progenitors requires ephrin-dependent cell–
cell adhesion (Arvanitis et al. 2013), and alter-
native usage of different splice forms of Eph
receptor was implicated in mediating cell–cell
coupling during embryonic development. Eph
signaling promotes the formation of AJ through
interaction with E-cadherin and TJs via the in-
teraction with claudin (Dravis and Henkemeyer
2011). Likely, the consequences of Ephrin/Eph
interactions for cell– cell contact stability de-
pend on the overall junction protein repertoire
expressed by the cell. Ephrin/Eph has been
shown to be important for collective movement;
for example, the formation of the thymus anlage
requires EphB /ephrin B, which seems to sup-
port collective mobility by a collective separa-
tion mechanism (Foster et al. 2010).
Integrins. Integrins are transmembrane
proteins that connect the cytoskeleton with
the extracellular matrix (ECM). ECM ligands
for integrins include fibronectin, vitronectin,
collagen, and laminin, among others, which,
beyond their well-established function as
structural connective tissue scaffolds, also
may be located between cells and contribute
to cell–cell interactions (Barczyk et al. 2010).
Integrins interact with F-actin and intermedi-
ate filaments allowing a mechanical coupling
between the cytoskeleton and the ECM, and
act as important transducers of mechanical
forces (Fagerholm et al. 2005). Integrin en-
gagement results in the formation of focal ad-
hesion complexes of varying sizes and func-
tions, which interact with F-actin and recruit
FAK and Src, leading to the activation of sig-
naling pathways involving extracellular signal–
regulated kinase (ERK), c-Jun N-terminal ki-
nase ( JNK), and small GTPases (Bouvard et al.
2013). Interaction of integrins with cadherins
and selectins has been proposed to be required
for the par ticipation of integrins in cell–cell
adhesion (Bouvard et al. 2013).
CELL–CELL ADHESION STATES AND
DYNAMICS
The type and durability of cell– cell adhesion
and cytoskeletal interaction systems that are en-
gaged by stationary and moving cells provide a
range of adhesion strategies between cells,
which jointly define the level of collective adhe-
sion and polarity, junction dynamics, and the
typ e of collective migration. The spectrum of
tissue fluidity can be found to vary, in a cell-
and context-dependent manner, from fully
immobilized, hig hly contractile to loosely con-
nected but highly mobile collective cell– cell or-
ganizations and kinetics (Fig. 1).
Myoblast fusion and myofiber formation.
Myofibers are multicellular syncytia that de-
velop by the fusion of individual myoblasts.
Rather than forming a collectively migrating
group, myoblasts remain stably anchored to
the substrate while establishing stable cell– cell
junctions that enable contractility across many
cells but show little junction dynamics. Myo-
blast interactions engage multiple receptor
systems in parallel, including focalized high-
density accumulation of M- and N-cadherin,
neural cell adhesion molecule (NCAM), vascu-
lar cell adhesion molecule (VCAM-1), meltrin,
and integrins (Fig. 1A) (Charrasse et al. 2006;
Abmayr and Pavlath 2012; Ozawa 2015). Once
myoblasts connect with each other, individual
mobility is largely disabled, whereas collective
contractility and force transmission across
cell– cell junctions are gained, par ticularly
throug h the actomyosin cy toskeleton, which
develops prominent stress fibers under the
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