Dynamic Regulation of the Structure
and Functions of Integrin Adhesions
Haguy Wolfenson,1,2Irena Lavelin,1and Benjamin Geiger1,*
1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
2Current address: Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Integrin-mediated cell adhesions to the extracellular matrix (ECM) contribute to tissue morphogenesis and
coherence and provide cells with vital environmental cues. These apparently static structures display
remarkable plasticity and dynamic properties: they exist in multiple, interconvertible forms that are
constantly remodeled in response to changes in ECM properties, cytoskeletal organization, cell migration,
and signaling processes. Thus, integrin-mediated environmental sensing enables cells to adapt to chemical
and physical properties of the surrounding matrix by modulating their proliferation, differentiation, and
dynamic properties is the focus of this article.
The History of Integrin Adhesion Research: From Early
Structural Studies to Contemporary Functional
Since the very early days of cell culture research more than
100 years ago, researchers have recognized the importance of
cell adhesion to the extracellular environment and its essential
role in cell survival, growth, and migration. As early as 1911,
Ross G. Harrison noted that cells ‘‘require some form of solid
support in order to carry out the growth process’’ (Harrison,
1911). A decade later, Warren H. Lewis wrote, ‘‘Cells that
migrate out on the under surface of the cover-glass. are sticky
for glass. not only the [cell] bodies but the cell processes as
well possess this adhesive quality’’; and, he added, ‘‘we may
in time be able to measure the force of the adhesions in some
way’’ (Lewis, 1922).
The observation that adhesions are located at the edges of
lamellae was first made by Hubert B. Goodrich (Goodrich,
1924) and later corroborated by others (Chambers and Fell,
1931; Algard, 1953; Rappaport and Rappaport, 1968). However,
as Albert Harris wrote in 1973, ‘‘perhaps because of the
oddness of this observation, or perhaps because it was not
the principal conclusion of any of the papers cited, the phenom-
enon has not become generally recognized, and its conse-
quences and likely significance have never been fully explored’’
(Harris, 1973). Meanwhile, renewed interest during the 1950s
in the discoveries of Francis Peyton Rous on the viral cause of
cancer (Rous, 1911) led researchers to produce malignancies
in cell culture (Temin and Rubin, 1958; Sanford et al., 1961)
and highlighted the importance of cell adhesion in the so-called
‘‘contact inhibition’’ (Abercrombie and Heaysman, 1954) and
‘‘anchorage dependence’’ (Stoker et al., 1968) of nonmalignant
It was only in the mid-1960s and early 1970s that researchers
were able to view the focal nature of matrix adhesions and their
precise locations, using interference reflection microscopy (IRM)
(Curtis, 1964; Abercrombie and Dunn, 1975) and transmission
electron microscopy (TEM) (Abercrombie et al., 1971; Revel
and Wolken, 1973). These studies led to several important
observations, including the distinction between focal adhesions
(or focal contacts, as they were often called), which are located
under the lamella, and close contacts, which are somewhat
less tight and are broadly associated with the lamellipodium
(Izzard and Lochner, 1976). These experiments also provided
the earliest evidence that focal adhesions are connected to
the cell’s cytoskeleton (Izzard and Lochner, 1976; Heath and
Dunn, 1978; Kreis et al., 1979) via actin stress fibers that take
an active role in regulating adhesion (Rees et al., 1977).
Around the same time, fibronectin emerged as the major
extracellular protein participating in the formation of focal adhe-
sions (Hynes and Destree, 1978; Thom et al., 1979). Additional
evidence further demonstrated that the two sets of fibrils—actin
inside the cell and fibronectin on the outside—are physically
connected (Heggeness et al., 1978; Hynes and Destree, 1978;
Singer, 1979). These findings led to the conclusion that a trans-
membrane linker protein (a ‘‘fibronectin receptor’’) must exist,
but it was not until 1987 that integrins were ultimately identified
as the elusive receptors (Hynes, 1987). It was also recognized
then that integrins operate as heterodimers composed of
a and b subunits.
The molecular era of integrin adhesions began in the late
1970s and early 1980s, when vinculin and tyrosine-phosphory-
lated proteins were first shown to reside in these extracellular
matrix (ECM) adhesions (Geiger, 1979; Burridge and Feramisco,
1980; Rohrschneider, 1980). These were followed by further
discoveries of adhesion-related proteins, including structural
proteins (such as paxillin, zyxin, a-actinin, and tensin), as well
as signaling molecules (kinases such as FAK, Abl, and PKC,
phosphatases such as SHP-2 and LAR-PTP, and other enzymes
such as PI3-kinase and calpain II). The functional and molecular
diversity of integrin adhesions was initially clarified in 2000
(Zamir et al., 2000), with distinctions drawn among focal
adhesions, focal complexes, fibrillar adhesions, and podosomes
(see examples in Figure 1). This body of knowledge was further
expanded in 2007, when the ensemble of focal adhesion-
associated proteins was defined as the ‘‘integrin adhesome’’
(Zaidel-Bar et al., 2007a) (see following section).
Developmental Cell 24, March 11, 2013 ª2013 Elsevier Inc.
The Integrin Adhesome: Molecular Diversity versus
During the past several decades, attempts to characterize the
molecular components of integrin adhesions have yielded
a long list of molecules that are known to be directly associated
with the formation and regulation of focal adhesions and related
structures. Efforts to place these molecules within a unifying
functional framework resulted in some literature-based publi-
cations describing the complexity, order, and switchability of
the integrin adhesome (Zaidel-Bar et al., 2007a; Zaidel-Bar and
Geiger, 2010). These were complemented by detailed proteomic
studies directly mapping the components of integrin adhesions
and their interconnections (Humphries et al., 2009; Kuo et al.,
2011; Schiller et al., 2011; for further reading, see Geiger and
Generally, the molecular components of the integrin adhe-
some (about 180 to date) can be subdivided into two major
groups: namely, scaffolding molecules (adhesion receptors,
adaptor proteins, cytoskeletal proteins) and signaling/regulatory
molecules (kinases, phosphatases, GTPases and their regula-
tors, and proteases, among others) (Figure 2). However, some
overlap exists between the groups, as certain multidomain
proteins apparently display both docking andsignaling functions
(e.g., FAK [Carragher et al., 2003]). The assembly of the
adhesome network is believed to be triggered by the binding
of the extracellular domain of integrins to the ECM ligand.
Figure 1. Diversity of Integrin-Mediated Cell
Matrix Adhesion Structures as Viewed by
Different Microscopy Techniques
of chicken lens cells in culture. The black arrow
indicates a focal adhesion. (B) A cryo-EM image of
chicken gizzard smooth muscle. Arrows indicate
focal adhesions sites. (C) Focal adhesions (arrows)
and focal complexes (arrowheads) formed by a
human foreskin fibroblast stained for paxillin (red)
and actin (green). (D) Fibrillar adhesions (arrows)
tensin (red) and fibronectin (green). (E) Podosomes
forming a ‘‘sealing zone’’ in a cultured osteoclast
derived from murine bone marrow, stained for pax-
illin (red) and actin (green). (F) Invadopodia (arrows)
formed by an A375 metastatic melanoma cell
(A) and (B) were provided by Ilana Sabanay; images
was provided by Or-Yam Shoshana.
This interaction induces conformational
b subunits, thus exposing binding sites
for cytoplasmic proteins within integrins’
cytoplasmic tails (Kim et al., 2003). So
far, no direct connection between integ-
rins and actin has been found; rather,
the integrin-actin interaction is mediated
by a group of ?30 adaptor proteins.
Some (e.g., paxillin) bind directly to integ-
rins (Liu et al., 1999), others (e.g., vinculin)
bind directly to actin (Johnson and Craig,
1995), and still others (e.g., talin) bind to
both actin and integrins (Horwitz et al., 1986; McCann and Craig,
1999). Secondary adaptors (e.g., p130Cas) also exist that can
bind to other adaptors, reinforcing the adhesion protein network
(Sakai et al., 1994; Barrett et al., 2012). These interconnections
between the different adaptors, together with additional
signaling molecules, form the ‘‘focal adhesion plaque’’ that is
connected to the actin stress fibers. This interaction is actually
mediated via short, tangential actin fibers, as recently indicated
by cryoelectron tomography studies (Patla et al., 2010; see
below for further details). The formin mDia1 seems to play
a central role in regulating stress fiber formation, because its
knockdown slows the elongation rate of the actin filaments
and alters the morphology of the stress fibers (Hotulainen and
Lappalainen, 2006). In addition, Ena/VASP proteins tether actin
filaments at sites of active assembly, thereby supporting actin
filament elongation (Breitsprecher et al., 2008).
Recruitment of adaptor and signaling molecules to the devel-
oping adhesions appears to occur in a sequential manner,
whereby certain proteins must be present at the adhesion site
in order to recruit others (Zervas et al., 2011). A comprehensive
understanding of the assembly hierarchy is still missing, yet
the very early stages of adhesion assembly involve recruitment
of talin to integrins, followed by paxillin and ILK, and subse-
quently vinculin, a-actinin, FAK, and, possibly, VASP (Zaidel-
Bar et al., 2004). Actomyosin-generated forces subsequently
applied to the adhesions may pull on several tension-sensitive
Developmental Cell 24, March 11, 2013 ª2013 Elsevier Inc.
molecules (e.g., p130Cas, talin, vinculin [Sawada et al., 2006;
del Rio et al., 2009; Grashoff et al., 2010]), thereby exposing
additional binding sites and enabling further recruitment of pro-
teins (e.g., zyxin) to the adhesion sites. Details of this process
are still lacking; however, it is clear that it is driven by a finely
tuned interplay between self-assembly and force-dependent
assembly within adhesion-associated, multiprotein complexes.
Adhesion-Mediated Scaffolding and Signaling: Integrin
Adhesions as Integrators of Environmental Sensing
interactions were primarily viewed vis-a `-vis their mechanical,
tissue-scaffolding functions (Abercrombie and Dunn, 1975;
Rees et al., 1977). In recent years, however, it has become
Figure 2. Molecular Complexity of Focal
(A) Interactions between functional families of
adhesome molecules. The various components
of the adhesome form two major groups: scaf-
groups have been further subdivided, according to
the known biological activities of the different
components. The dominant interactions between
families (red arrows, activating interactions; blue
arrows, inhibiting interactions; black lines, binding
interactions) are shown (modified from Zaidel-Bar
et al., 2007a). (B) To illustrate the complex molec-
ular composition of focal adhesions, a HeLa cell
was immunolabeled for three different adhesion
components: ILK, zyxin, and actin. The merged
image highlights the distinct localization of the
different proteins within the same structures.
increasingly clear that they also act as
sensing and signaling cellular ‘‘devices’’
capable of processing complex informa-
tion induced either by the external micro-
(andnano-)environment or,locally, bythe
cytoskeleton (Chandrasekar et al., 2005;
Choi et al., 2008; Na et al., 2008). It is
noteworthy that unlike classical signal-
ing receptors (e.g., receptor tyrosine
kinases), integrins do not possess enzy-
properties are based on their capacity to
recruit specific, ‘‘classical’’ adhesome
signaling components to the adhesion
site, thereby activating a wide variety
of signaling networks (Avizienyte and
Frame, 2005; Dupont et al., 2011).
Due to the great molecular complexity
and diversity of the adhesome, as well
as to their apparent cooperation with
other transmembrane receptors (Wor-
thington et al., 2011), integrins are able
to sense and respond to different kinds
of extracellular cues, including the chem-
ical, physical, and topographical proper-
ties of the cell’s microenvironment. The
most critical chemical signal transmitted
via integrins is the specific molecular composition of the ECM,
including the relative contents of such molecules as fibronectin,
vitronectin, collagen, and laminin (Humphries et al., 2006). To
discriminate between these (and other) ECM ligands, integrins
can form a wide variety of different a and b heterodimers.
Mammals express 18 a subunits and eight b subunits and form
up to 24 different functional heterodimers whose binding affini-
ties differ, depending on the nature of the ECM ligand (Hynes,
2002). Cells can express and form different heterodimers at
the same time, potentially increasing their ability to fine-tune
the sensing of ECM composition. This process can be even
more sophisticated, as the expression patterns of the different
integrin subunits or the associated adhesome molecules
(as well as of other associated transmembrane receptors) may
Developmental Cell 24, March 11, 2013 ª2013 Elsevier Inc.
be modulated over time in response to changes in ECM compo-
sition, thereby altering the cellular response to the adhesive
interactions (Yarwood and Woodgett, 2001; Kass et al., 2007).
Integrin adhesions can also sense the physical properties of
the ECM, including its rigidity, ligand density, isotropy, and
topography (Jiang et al., 2006; The ´ry et al., 2006; Cavalcanti-
Adam et al., 2007). The specific molecular mechanisms whereby
cells sense such physical cues are unclear, but a crucial step in
these processes is the local response of integrin adhesions to
mechanical forces applied to the adhesion sites, either by the
matrix or by the contractile actomyosin machinery (Chen,
2008; Geiger et al., 2009). It was shown, for example, that
changes in matrix rigidity can trigger the formation of adhesions
with distinct morphologies, compositions, andsignaling capabil-
ities (Schlunck et al., 2008; Prager-Khoutorsky et al., 2011).
Hence, on stiff substrates, cells typically produce larger and
more stable adhesions whose molecular properties are clearly
distinct from those formed on more compliant substrates
of adhesions affect cytoskeletal organization and overall cell
morphology, leading to the formation of elongated and polarized
cells when on stiff substrates, compared to round, nonpolarized
cells when on soft surfaces (Prager-Khoutorsky et al., 2011).
Beyond these morphological changes, different ECM rigidities
can lead to long-term physiological changes in the transcrip-
tional program and in the regulation of protein expression and
stability that can, eventually, affect cell behavior and fate (Engler
et al., 2006; Dupont et al., 2011). Several pathways have been
cells to sense rigidity, including conformational changes in
proteins (del Rio et al., 2009), strengthening of integrin-ligand
bonds (Choquet et al., 1997), and induction of specific phos-
phorylation events (Sawada et al., 2006) (for a detailed review,
see Moore etal., 2010).However, the basic mechanism whereby
physical cues (rigidity, force) are ‘‘translated’’ into chemical cues
(e.g., phosphorylation, protein-protein interactions) is still not
These examples of possible mechanisms responsible for
adhesion-mediated signaling are just the tip of an exciting,
poorly understood iceberg. The main challenges lying ahead
involve not only characterization of the individual sensing
processes ofdistinct physical properties, but alsotheintegration
of multiple environmental signals and the development of
of this article, although additional, still limited, information may
be found in several recent review articles (Vogel and Sheetz,
2009; Geiger and Yamada, 2011; Bershadsky, 2012).
The Life Cycle of Integrin Adhesions: Formation,
Transfiguration, and Dissociation
Live-cell microscopy studies have revealed four main stages in
the ‘‘life cycle’’ of integrin adhesions; these include nascent
adhesions, focal complexes, focal adhesions, and fibrillar adhe-
sions. Nascent adhesions are submicron-sized structures that
are usually ‘‘born’’ under the lamellipodium and are barely visible
once the lamellipodium forms the first contact with the matrix,
initial interaction of integrins with talin and kindlin takes place
(‘‘inside-out activation’’), enhancing integrin activation and
stabilizing its grip on the ECM (Ma et al., 2008; Montanez et al.,
2008). This is followed by integrin clustering and generation of
the initial complexes: namely, nascent adhesions. This process
seems to occur very rapidly, on a timescale of seconds (Yu
et al., 2011), and involves only a small number of integrin mole-
cules. Moreover, assembly of nascent adhesions is seemingly
independent of myosin II activity but does induce actin polymer-
ization (Yuetal.,2011).This finding suggestsanessential rolefor
actin-nucleating protein(s) in these early adhesions; however, to
date, such information is still lacking.
The subsequent pull of myosin on these clusters reinforces
the strength of the adhesions (Roca-Cusachs et al., 2009) and
is believed to lead to the recruitment of additional adhesome
into the somewhat larger focal complexes. Nascent adhesions
and focal complexes display similar molecular compositions
and seem to differ mainly in their myosin dependence and size
(Choi et al., 2008). Focal complexes are small (?1 mm diameter),
punctate, and short-lived structures (a lifetime of ?1–2 min) that
are located close to the edge of the lamellipodium (Choi et al.,
2008). These transient entities either disassemble by an as-yet
unknown mechanism or rapidly grow centripetally and evolve
into larger, elongated focal adhesions. The growth process
clearly depends on forces generated by actomyosin-based
stress fibers (Riveline et al., 2001; Choi et al., 2008), but it also
requires that the stress fibers serve as physical contractile
anchors (Oakes et al., 2012).
Focal adhesions are considerably larger and more elongated
(commonly ?1 mm wide, 3–5 mm long) than focal complexes,
with typical lifetimes of up to several tens of minutes (Zaidel-
Bar et al., 2007b). The most prominent protein marker for mature
focal adhesions is zyxin, as it is recruited to the adhesions at
laterstages in their development and doesnotappear innascent
adhesions and focal complexes (Zaidel-Bar et al., 2003). As they
mature, the growing focal adhesions define a new boundary
between the lamellipodium and the ‘‘lamella proper’’ (Alexan-
drova et al., 2008; Burnette et al., 2011) and are believed to
playakey roleinsupporting theforwardprotrusion oftheleading
edge of migrating cells (Ponti et al., 2004). The disassembly of
focal adhesions often occurs at the rear of the cell when
the ‘‘tail’’ of the cell is retracting. This process involves
microtubule-mediated destabilization of the adhesions and
plays an important role during persistent cell migration (Kaverina
et al., 1999).
Another mechanism leading to loss of focal adhesions
involves the transformation of these adhesion sites into another
type of integrin-mediated contact: fibrillar adhesions. These
structures are considerably more elongated than most focal
adhesions and are located (mostly in fibroblasts) around the
cell center, where they are associated with fibronectin fibrils
(Pankov et al., 2000). In fact, a characteristic property of fibrillar
adhesions is their capacity to remodel the ECM and induce
fibrillogenesis (Pankov et al., 2000). The transformation requires
mechanical force, but the maintenance of these adhesions is
far less sensitive to inhibition of actomyosin contractility
compared to classical focal adhesions (Zaidel-Bar et al., 2007b).
The transformation of one form of adhesion into another is
tightly regulated by the cellular signaling system, primarily by
small GTPases, including Rac1, cdc42, and RhoA (Jaffe and
Developmental Cell 24, March 11, 2013 ª2013 Elsevier Inc.
Hall, 2005). Rac1 and Cdc42 have long been implicated in the
formation of focal complexes and actin polymerization, whereas
RhoA is associated with focal adhesion maturation (Nobes and
Hall, 1995; Rottner et al., 1999). Notably, Cdc42 can activate
Rac1 (Nobes and Hall, 1995), and Rac1 and RhoA suppress
each other’s activity (Yamaguchi et al., 2001; Nimnual et al.,
2003). Thus, as focal adhesions grow and mature, Cdc42 and
Rac1 activities are suppressed, and RhoA activates myosin
pulling on actin fibers by phosphorylating myosin light-chain
(MLC) via Rho kinase (ROCK). Transition of focal adhesions
into fibrillar adhesions requires further RhoA-mediated contrac-
tility and the centripetal translocation of ligated a5b1integrins
from the focal adhesions, accompanied by the stretching of
soluble fibronectin dimers (Pankov et al., 2000). A recent study
using FRET biosensors indicated that RhoA is activated at the
cell edge about 40 s prior to Cdc42 and Rac1, which are acti-
vated 2 mm behind RhoA (Machacek et al., 2009). This finding
indicates that RhoA may play a role in the early formation of
nascent adhesions, and, subsequently, the coordinated action
of Rac1 and Cdc42 leads to growth into slightly larger focal
complexes. For additional reading on the transitions between
the different forms of adhesions, see recent reviews (Wolfenson
et al., 2009a; Vicente-Manzanares and Horwitz, 2011; Bershad-
Structural and Dynamic Characterization of Integrin
Adhesions: Stability versus Turnover
One of the intriguingaspects of integrin adhesions isthatdespite
their capacity to persist for relatively long periods of time with
minimal change (Figure 3A), they are able to rapidly undergo
radical alterations (e.g., disassembly upon reduction of actomy-
osin contractility). To address the mechanism underlying such
variations in focal adhesions, several studies have focused on
the molecular kinetics of integrin adhesions, mainly using
fluorescence recovery after photobleaching (FRAP) or fluores-
cence correlation approaches. These studies revealed that
even in apparently stable focal adhesions, there is a constant
turnover of proteins on timescales of milliseconds to seconds
(Wolfenson et al., 2009a). Moreover, within the adhesions
themselves, different adhesome proteins (mainly adaptors)
Figure 3. Molecular and Structural Kinetics of Focal Adhesions
(A) Structural dynamics of focal adhesions. A HeLa cell expressing GFP-vinculin was recorded for 12 min by time-lapse fluorescence microscopy. The resulting
images were analyzed by temporal ratio imaging (bottom right image), enabling comparison of the same structures at different time points. A red shift denotes
structures with decreased intensity; a blue shift denotes an increase in intensity; green and yellow hues mark unchanged pixels. (B) Different focal adhesion
proteins display varying turnover rates. Normalized averaged curves of FRAP experiments performed in HeLa cells on different GFP-tagged adhesion-related
recovery (T1/2) values.
Developmental Cell 24, March 11, 2013 ª2013 Elsevier Inc.
display vastly different turnover rates (Figure 3B). These rates
depend on the state of the adhesion (e.g., growing, at steady
state, disassembling, or ‘‘sliding’’) (Digman et al., 2008), and
most likely reflect differences in the available binding sites for
On average, each molecule within the adhesome has ?8–10
potential binding partners (Zaidel-Bar et al., 2007a). Although
this number does not necessarily reflect the actual situation in
individual cells at any given time point, it demonstrates the
molecular complexity of integrin adhesions and the difficulty in
determining the factors that regulate adhesion dynamics at the
molecular level. In general, it appears likely that over time, as
adhesions develop, more and more binding sites are exposed
to cytoplasmic proteins at or near existing adhesion sites,
leading to an apparently more avid interaction of these proteins
with the adhesions. Several examples support this hypothesis,
including force-induced exposure of cryptic vinculin binding
sites within the talin rod (del Rio et al., 2009), very rapid accu-
mulation of vinculin into growing adhesions following application
of force (Riveline et al., 2001), and an increase in focal adhesion
area upon expression of vinculin mutants with exposed binding
sites (Humphries et al., 2007), among others.
Examination of the dynamic properties of diverse focal adhe-
sion-associated proteins points to considerable variability. In
stable and mature adhesions, most plaque proteins display
both dynamic and stable subpopulations of components
(the latter is demonstrated by a 20%–40% immobile fraction in
the FRAP studies) (Wolfenson et al., 2009b). Furthermore, stable
adhesions also display varying molecular exchange rates, de-
pending on their location—e.g., distal or proximal regions—
within individual adhesions (Digman et al., 2008; Wolfenson
et al., 2009b). This phenomenon has been implicated with differ-
ential force distribution within focal adhesions (Shemesh et al.,
2005; Besser and Safran, 2006) or with different phosphorylation
states of specific proteins (e.g., paxillin; Zaidel-Bar et al.,
2007a)—two phenomena that are possibly linked. In fact,
mechanical forces are major regulators of the kinetics of plaque
proteins, as demonstrated by studies utilizing inhibitors of acto-
myosin contractility (Pasapera et al., 2010; Wolfenson et al.,
2011). Interestingly, different plaque proteins vary in their
response to force reduction, as some (e.g., vinculin) display
bition (Wolfenson et al., 2011). This phenomenon is not entirely
understood, but additional studies indicate that the stability of
focal adhesions is regulated by the binding and unbinding rates
of the exchanging proteins with the adhesions: once there is
a reduction in the degree of force applied to the adhesion, these
rates change such that there is a lower binding rate and a higher
unbinding rate (H.W. and B.G., unpublished data).
Taken together, these dynamic results suggest that focal
adhesions are not uniform entities; rather, they contain different
subpopulations of plaque proteins within them. This finding is
particularly interesting in view of recent nanoscale structural
data generated using cryoelectron tomography (Medalia and
Geiger, 2010; Patla et al., 2010) and the superresolution fluores-
cence technique, iPALM (Kanchanawong et al., 2010). The EM
studies revealed that the transmembrane interactions at focal
adhesions are mediated via discrete particles of ?25 nm in
Developmental Cell 24, March 11, 2013 ª2013 Elsevier Inc.
size that are attached to both the inner surface of the plasma
membrane and the actin fibers. These results not only suggest
that focal adhesions contain multiple ‘‘hot spots’’ of cytoskeletal
anchorage (rather than a uniform protein mesh), but they also
raise the possibility that differences in composition and molec-
ular turnover may exist between the various particles within
individual adhesions (Wolfenson et al., 2009b). In the superreso-
lution iPALM studies (Kanchanawong et al., 2010), the z axis
ing that focal adhesions are laminated structures and that the
integrins and actin inside focal adhesions are vertically bridged
by a layer of plaque proteins composed of several subregions.
These results imply a hierarchical distribution of different plaque
proteins within focal adhesions, whereby integrin-binding
proteins are located close to the plasma membrane and actin-
binding proteins are located close to the actin filaments,
?40 nm above the integrins. Such superresolution techniques,
capable of reaching xy resolution of a few tens of nanometers,
hold promise for future research in determining the nanoscale
composition of integrin adhesions and may help to determine
the nature of the nanostructures observed in the cryo-EM
studies. Moreover, by using sophisticated fluorescent probes
and constructs such as FRET sensors (Grashoff et al., 2010) or
double-tagged molecules (Margadant et al., 2011) in combina-
tion with superresolution microscopy, it may be possible to
clarify the molecular mechanisms underlying the regulation of
TheStructure andFunction ofIntegrinAdhesionsInVivo
Widespread interest in cell adhesion, originating in the 1970s,
was largely initiated and motivated by developmental biologists
(Hay, 1981; Sanes, 1983; Thiery et al., 1985). As a consequence,
most of the research on cell-cell and cell-matrix adhesions was
initially directed toward understanding the roles of adhesion
processesduring embryogenesis andorganogenesis. Neverthe-
less, shortly thereafter, cultured cells growing in a monolayer
became the primary models for studying integrin adhesions.
The reasons for this choice included the convenience of working
with such cells, the availability of excellent imaging systems
and biophysical tools for studying the adhesion structures, the
amenability of such cellular systems to genetic perturbation
(e.g., using small interfering RNA technology) or live-cell imaging
(e.g., using intrinsically fluorescent proteins), and the possibility
of engineering specific adhesive surfaces that could stimulate
cell growth and differentiation.
Naturally, the question of whether ‘‘classical focal adhesions’’
viewed in experiments on two-dimensional (2D) surfaces indeed
constitute suitable models for integrin adhesions was frequently
asked over the years, as these structures are certainly much
harder to detect in vivo (Burridge et al., 1988). It is certainly
possible that, for certain cell types, a hard, flat surface imposes
an artificial phenotype, resulting in adhesion structures that do
not exist in vivo in the same cells. For example, focal adhesion
proteins are diffusely localized in salivary gland epithelial cells
in vivo but are concentrated in focal adhesions when the cells
are plated on 2D surfaces (Sequeira et al., 2012). On the other
hand, many indications from other cell types imply that similar
adhesions do exist in vivo. A classic example is that of smooth
muscle cells, in which most of the early studies on cell-matrix
adhesions were performed (Burridge et al., 1988). These cells
produce large, stable adhesions with the ECM that are required
for cell stabilization during generation of force within the tissue.
The molecular characteristics of these adhesions are similar to
focal adhesions that form in vitro.
While there is probably no definitive answer to the concerns
described above, certain obvious differences between matrix
adhesions in the body and those on a coverslip motivated
researchers to engineer ‘‘bio-inspired’’ adhesive surfaces, which
moreclosely resemble physiological adhesion conditions. These
include the formation of more compliant matrices, the use of
three-dimensional (3D) matrices, and the modulation of the
adhesive epitopes and their density, among others (reviewed in
Geiger et al., 2009). Importantly, through the use of such
surfaces, it immediately became apparent that the adhesion
structures are altered in various ways. For instance, cells grown
on 2D soft gels form focal adhesions that are much smaller and
moreunstable thanthose formedon stiffersurfaces (Pelhamand
Wang, 1997). The molecular mechanisms underlying this obser-
vation are still unclear, but researchers have begun to address
this issue (Prager-Khoutorsky et al., 2011; Ghassemi et al.,
2012; Trappmann et al., 2012). Recently, this phenomenon
was also observed in studies of cells grown in 3D collagen
matrices (Fraley et al., 2010): cells located close to the edge of
the collagen gel formed focal adhesions at their lamellipodia,
they presumably sense lower tensile forces), focal adhesion
proteins were distributed diffusely throughout the cytoplasm,
rather than in adhesions. Notably, these results were challenged
by the claim that reducing the level of background fluorescence
reveals focal adhesions even in deeply embedded cells (Kubow
and Horwitz, 2011). The differences between the two groups’
aration of the 3D matrices. However, these seemingly contradic-
tory results can give us insights into the reasons for the difficul-
ties in detecting focal adhesions in vivo: cells that are located at
less-rigid locations may produce adhesions so small that they
cannot be visualized using traditional fluorescence microscopy.
Importantly, in the aforementioned study, even when focal adhe-
sions were not noticeable in deeply embedded cells, focal adhe-
sion proteins did play a major role in cellular motility in the 3D
environment(Fraley etal.,2010).Certainly,studiesoffocal adhe-
sions in 3D cultures are only in their initial stages, and the proper
technical conditions for these studies are still being resolved.
These issues should also be addressed in the future using novel
superresolution fluorescence microscopy techniques.
to study integrin adhesions include modulations of the distances
between integrin ligands (Cavalcanti-Adam et al., 2007;
Schvartzman et al., 2011), surfaces with several types of ligands
(Holst et al., 2010), and micropatterned surfaces with specific
ligand organization (The ´ry et al., 2006). All of these specialized
surfaces altered the adhesions in various ways, lending support
to the view that integrins are, indeed, classical surface sensors.
In view of these (and other) results, it is clear that tissue culture
powerful models for the study of matrix adhesions. Because
integrin adhesions function to integrate various types of environ-
mental signals, dissecting these signals in vivo, as well as the
molecular mechanisms that guide them, is extremely difficult,
let alone at high temporal and spatial resolution. In this sense,
‘‘bio-inspired’’ artificial surfaces can yield new, exciting informa-
tion as to the role of matrix dimensionality, compliance, and
anisotropy, for example, in scaling and modulating the adhe-
sions, affecting both their structure and signaling activities. The
advances in surface chemistry and surface engineering during
the last decade provide novel, powerful tools to study these
processes in unprecedented detail and in a highly quantitative
Functions of Integrin Adhesions in Development
has attracted the attention of many developmental biologists
wishingto clarifytheir rolein embryogenesisand organogenesis.
In a series of studies beginning in the early 1990s, researchers
systematically knocked out the different a- and b-integrin
subunits in mice to study their roles at various developmental
stages (reviewed in Bouvard et al., 2001). These knockouts
resulted in various developmental phenotypes, from minor
apparent effects to early embryonic lethality. For instance,
knockout of b1, the most abundantly expressed integrin subunit,
capable of forming up to 12 types of dimers with different
a subunits (Hynes, 2002), led to very early lethality due to failure
of the embryo to develop the inner cell mass (Fa ¨ssler and Meyer,
1995). Mice that lacked b5-integrin showed no apparent change
in phenotype at first (Huang et al., 2000), but further investiga-
tions have shown defects in the adult mice, such as reduced
vascular permeability in response to VEGF (Eliceiri et al., 2002)
or defective retinal phagocytosis (Nandrot et al., 2004), among
others. Other types of integrins have more distinct roles in
specialized biological systems such as immune cells; as a result,
their ablation does not induce early lethality, but instead leads to
impaired immune function only under specific circumstances.
For example, in the absence of aL-integrin (more commonly
known as lymphocyte function-associated antigen, or LFA-1),
which is expressed on the surface of leukocytes, mice are viable
and display a normal immune response against systemic viral
infections; however, their response to injected immunogenic
tumor cells is impaired, and they cannot reject the tumors
(Schmits et al., 1996). Other developmental studies involved
knockouts or mutations of central adhesion plaque proteins, as
well as of ECM genes (e.g., fibronectin, vitronectin, or collagen).
These, too, resulted in various phenotypes, including lethality or
impaired development of critical organs such as the cardiovas-
cular system, kidney, brain, and muscle (Gustafsson and Fa ¨ss-
ler, 2000; Bo ¨kel and Brown, 2002).
Studies that involved phenotypic analyses of mutated
Drosophila melanogaster have proven to be very valuable in
elucidating not only developmental processes but also the
fundamental components that play roles in regulating integrin-
mediated adhesion. The vast knowledge of Drosophila develop-
adhesion-related genes, has allowed screening for mutants
with similar phenotypes and, thus, the construction of maps of
pathways downstream of integrins. For example, as early as
1918, a Drosophila mutant was identified that had blisters in its
Developmental Cell 24, March 11, 2013 ª2013 Elsevier Inc.
which is required for the proper connection of the two opposing
epithelial sheets that make up the adult wing (Brower and Jaffe,
1989). In subsequent genetic screens, researchers identified
several mutants that had similar phenotypes to the inflated
mutant, as well as other defects in wing and muscle develop-
ment (Volk et al., 1990; Wilcox, 1990). Among others, some of
the central genes that were identified as critical were the ortho-
logs for talin (Brown et al., 2002), ILK (Zervas et al., 2001), PINCH
latter three are referred to as the IPP complex, which is appar-
ently formed in the cytoplasm and then interacts with b-integrins
to link them to the actin cytoskeleton. Interestingly, in muscles,
the stability and localization of the IPP complex depends on
ILK alone, but in the wing epithelium all three proteins are mutu-
ally dependent on each other (Vakaloglou et al., 2012). This
suggests that the proper regulation of integrin adhesions may
differ between different tissues during development.
The phenotypic analysis of different mutants also supports
the view that a hierarchy of function exists between the
proteins involved in adhesion regulation. Thus, integrin is acti-
vated by talin, which subsequently recruits the IPP triplet (of
which ILK’s scaffolding function seems to be central), as well
as paxillin and tensin. In recent years, a new level of complexity
arose when the kindlin family (which was first identified in
Caenorhabditis elegans [Rogalski et al., 2000]) was shown to
coregulate integrin activation together with talin. Moreover,
kindlins, too, can act as scaffolding proteins, recruiting adhe-
sion-related proteins (including ILK) to the adhesion sites
(Karako ¨se et al., 2010). Kindlin deletion in mice results in major
adhesion-related defects, depending on the expression pattern
of the specific kindlin isoform—from severe bleeding (kindlin 3)
(Moser et al., 2008), to skin atrophy and a detached colon
epithelium (kindlin 1) (Siegel et al., 2003; Ussar et al., 2008),
or death at implantation (kindlin 2) (Montanez et al., 2008)—all
due to defective integrin function.
Recent improvements in fluorescence microscopy have
enabled time-lapse imaging of single cells or groups of cells in
living organisms. These advances paved the way toward in vivo
studies of integrin adhesions during embryonic development.
Thus, it was shown that disrupting or enhancing cell-ECM
adhesions in Drosophila results in altered cytoskeletal forces
and, as a consequence, altered tissue elongation and shape
mediate actomyosin-generated cues that lead to increased
tissue stiffness and proliferation (Samuel et al., 2011). In a recent
study, FRAP measurements were performed on Drosophila
embryos to study the effect of tensile force on integrin adhesion
turnover in vivo (Pines et al., 2012). Using temperature-sensitive
mutations that caused muscle contraction, Pines et al. showed
that, under increased tension, the rate of integrin turnover
(measured as the ratio between integrin endocytosis and
delivery to the membrane) is low and the adhesions are stable,
whereas under reduced tension integrin turnover is high and
the adhesions are less stable. These results corroborate the
in vitro findings on the role of forces in regulating adhesion
dynamics (see above), and also provide a link between forces,
adhesion stability, and tissue development.
Taken together, the above-mentioned studies demonstrated
the diverse roles of integrin adhesions in developing tissues
and organs, including scaffolding processes involved in tissue
and organ morphogenesis, and environmental sensing pro-
cesses whereby integrin-mediated interactions activate specific
signaling networks. They also highlighted the importance of
using model organisms in which the number of integrin-related
adhesion components is smaller, thus enabling one to distin-
in evolution, and therefore exist in Drosophila and C. elegans,
and more ‘‘sophisticated’’ components, which might be critical
in more specific processes.
Future Prospects and Open Questions Relevant to the
Involvement of Integrin Adhesions in Development
The apparent complexity and diversity of the adhesome offer
major challenges in analyzing the structure-function relationship
of integrin adhesions. The huge diversity of the adhesome
provides important versatility whereby the cellular adhesion
matrices it encounters. Moreover, the dynamic nature of these
adhesions enables them to sense and respond to temporal
processes such as matrix remodeling and transcellular stress.
The mechanisms underlying the complex crosstalk between
the microenvironment and the integrin adhesome network
remain poorly characterized, and novel experimental ap-
proaches are needed to clarify, at the molecular level, processes
such as matrix-induced assembly of the adhesion sites and
matrix mechanosensing, both of which take place in integrin
adhesions. Encouraging results from a variety of cell bio-
logical models, using novel microscopic technologies, powerful
computational approaches, and nano-engineered adhesive
surfaces, offer promising insights into adhesion biology that
might prove relevant to important processes taking place within
the developing organism. Among these novel technologies, we
would highlight, in particular, the in vivo characterization of the
dynamics of adhesions, using multiple, intrinsically fluorescent
adhesome components, and the accurate measurement of their
molecular exchange rates and turnover. Emerging cutting-edge
technologies such as superresolution microscopy and cryoelec-
tron tomography (and their possible use in tandem) can shed
further light on the inner molecular architecture of adhesion sites
at the nanoscale level. The major future challenge lies in the
integration of this dynamic, complex, and multidimensional in-
formation with a mechanistic understanding of adhesion and
adhesion-mediated signaling processes.
Studies carried out in our laboratory addressing adhesion formation and
dynamics were supported by the Israel Science Foundation, the National Insti-
tutes of Health Cell Migration Consortium (grant U546M64346), the National
Institutes of Health Nanomedicine Development Center Network (grant PN2
EY016586), andtheEuropeanUnion’s SeventhFrameworkProgramme,under
grant agreement 258068 (NoE Systems Microscopy). B.G. is the incumbent of
the Erwin Neter Professorial Chair in Cell and Tumor Biology. The authors wish
to express their gratitude to Talila Volk for helpful comments and to Barbara
Morgenstern for expert editorial assistance.
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