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Integrin trafficking ata glance

Rebecca E. Bridgewater1, Jim C.Norman2 and Patrick T. Caswell1,*1Wellcome Trust Centre for Cell Matrix Research,Faculty of Life Sciences, University of Manchester,Manchester M13 9PT, UK2Beatson Institute for Cancer Research, GarscubeEstate, Glasgow G61 1BD, UK

*Author for correspondence

(patrick.caswell@manchester.ac.uk)

Journal of Cell Science 125, 3695–3701

� 2012. Published by The Company of Biologists Ltd

doi: 10.1242/jcs.095810

Integrins are heterodimeric transmembrane

receptors for extracellular matrix (ECM)

components and they link the intracellular

actin cytoskeleton to the cellular environment

(Humphries et al., 2006; Hynes, 2002). The

a- and b-subunits of integrin heterodimers

are type I membrane proteins that typically

have a large extracellular domain and a

short intracellular tail. Through different

combinations of the 18 a- and eight b-

subunits, 24 distinct integrin heterodimers

exist in mammals. The integrin family thus

comprises an array of cell surface receptors,

which are expressed in a cell- and tissue-

specific manner, for a plethora of soluble and

insoluble ECM ligands, including collagens,

laminins, fibronectin and vitronectin

(Humphries et al., 2006). As a result,

integrins are able to control diverse cellular

processes, including proliferation, apoptosis,

differentiation and cell migration and thus

have key roles in development, immune

responses and the progression of diseases

such as cancer (Legate et al., 2009). Integrin

heterodimers can adopt a bent or closed

conformation that has a low affinity for ligand

(‘inactive’) or an extended or open

conformation that has a high affinity for

ligand (‘active’). As a consequence of this

conformational switching, integrins are

able to signal bidirectionally across the

membrane: ligand binding elicits signalling

responses within the cell (‘outside-in’

signalling), but binding of intracellular

proteins such as talin and kindlins to

integrins regulates the activation of integrins

to promote ligand-binding (‘inside-out’

signalling) (Legate et al., 2009).

In addition, the control of integrin

availability at the plasma membrane is

key to their function. However, although

we have known for over 20 years that

integrins undergo an exocytic–endocytic

cycle, these pathways have only been

studied in detail relatively recently. In

this Cell Science at a Glance article, we

will describe the mechanisms that control

integrin endocytosis and recycling, and

discuss the insight that understanding these

mechanisms has provided into the role of

trafficking in integrin function and vice

versa. Furthermore, we draw attention to

two emerging features of integrin

trafficking: first, the specific nature of the

mechanisms that control aspects of integrin

trafficking, and, second, the role that

integrin trafficking has in intracellular

signalling.

(See poster insert)

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Mechanisms of integrin endocytosisIntegrin endocytosis has been studied

extensively in the context of viral entry

(Pellinen and Ivaska, 2006), but mechanisms

that regulate integrin internalisation in the

absence of pathogens have been identified.

Integrins can be internalised through the

major endocytic routes (Box 1), including

the formation of circular dorsal ruffles

(CDRs) during macropinocytosis (Gu

et al., 2011), and clathrin-dependent and

-independent endocytic pathways. Direct

recruitment of endocytic regulators to

integrins constitutes a general mechanism

that can drive integrin endocytosis.

Examples of this include the recruitment of

HS1 associated protein X-1 (HAX-1) to the

cyoplasmic tail of integrin b6 (Ramsay et al.,

2007) and the recruitment of Rab21 to the

cytoplasmic tail of integrin a subunits

(Pellinen et al., 2006). Protein kinase C

alpha (PKCa) controls caveolar endocytosis

(Smart et al., 1995), and can itself bind to b1

integrins (i.e. integrins that contain a b1

subunit and any a subunit) and regulate

integrin internalisation (Ng et al., 1999). In

endothelial cells, recruitment of active a5b1

integrin to the neuropilin–GIPC1 (for GAIP

C-terminus-interacting protein) complex

regulates endocytosis of this integrin

directly from fibrillar adhesions (Valdembri

et al., 2009). Xenopus GIPC1 (also known

as kermit2) regulates a5b1 integrin

internalisation into Rab21-positive vesicles

(Spicer et al., 2010), which suggests that

mechanisms that regulate integrin

internalisation are conserved.

Clathrin-dependent endocytosis

The clathrin adaptors disabled homologue 2

(DAB2) and NUMB interact, through their

phosphotyrosine-binding (PTB) domains,

with the NPxY-motifs in the b-integrin

cytoplasmic tails (Calderwood et al., 2003),and several lines of evidence support thenotion that clathrin-adaptors regulate

integrin endocytosis. Microtubule-inducedfocal adhesion (FA) disassembly hasbeen shown to require clathrin-dependentendocytosis (CDE) (Chao and Kunz, 2009;

Ezratty et al., 2009). Indeed clathrin andDAB2 localise to FAs in the mid-region ofmoving cells and regulate a5b1 integrin

internalisation to facilitate migration.NUMB binds to b1 and b3 integrins, andlocalises around focal contacts at the

leading edge of migrating cells togetherwith clathrin. Phosphorylation of NUMBdownstream of the PAR3 (also known as

PARD3)–atypical PKC (aPKC) complexregulates integrin internalisation at thesesites to promote migration (Nishimura andKaibuchi, 2007). Inactive integrins are also

internalised in a clathrin-dependent manner:DAB2 and AP2 regulate endocytosis ofa1b1, a2b1 and a3b1 (but not a5b1)

integrins from the dorsal surface of thecell, and the rate of DAP2–AP2-mediatedendocytosis correlates positively with

migration specifically towards ligands forthese integrins (Teckchandani et al., 2009).

Clathrin-independent endocytosis

Accumulating evidence indicates thatintegrins can follow clathrin-independentendocytic routes. Indeed, b1 integrins arefound within clathrin-independent carriers

(CLICs, see Box 1) (Howes et al.,2010a). Rab21 overexpression circumventsthe requirement for CDE to internalise b1

integrins in dividing cells by driving a formof clathrin-independent endocytosis (CIE)(Pellinen et al., 2008). Cholesterol depletion

experiments have shown that integrins canenter the cell in a raft-dependent fashion(Fabbri et al., 2005), and a5b1 and avb3

integrins are thought to associate withcaveolin-1 (Galvez et al., 2004; Wickstromet al., 2002). More direct evidence supports arole for caveolae in the internalisation of

a5b1 in myofibroblasts (Shi and Sottile,2008), a2b1 in an osteosarcoma cell line(Upla et al., 2004) and active b1 integrins in

bone marrow mesenchymal stem cells(BMMSCs; Du et al., 2011).

Regulation of integrin endocytosis bycell–matrix adhesion

Although integrin endocytosis can beconstitutive and ligand-independent, cell

adhesion to the ECM through otheradhesion receptors can promote integrinendocytosis. For instance, myelin-associated

Box 1. Endocytosis

Endocytosis regulates membrane lipid and protein composition, thereby allowing cellular

functions to be controlled. Many distinct routes of internalisation have been identified, and

these are typically characterised by the mechanism employed, the cargo transported and/or

the morphology of the internalising structure (Hansen and Nichols, 2009). Generally,

endocytic processes are classified as clathrin-dependent endocytosis (CDE) or clathrin-

independent endocytosis (CIE), whereby CIE describes several distinct endocytic routes

(Doherty and McMahon, 2009).

Clathrin-dependent endocytosis

CDE is the best-characterised internalisation route and involves the recruitment of clathrin

triskelia to the membrane, where they are assembled to form a clathrin-coated pit (CCP). This

is preceded by a nucleation step, wherein factors such as phosphatidylinositol 4,5-

diphosphate [PtdIns(4,5)P2], epidermal growth factor receptor pathway substrate 15

(EPS15), intersectins and FCH domain only (FCHO) proteins define the site of CCP

formation. This ‘nucleation module’ recruits the clathrin-adaptor AP-2, along with cargo-

specific adaptor proteins that mediate cargo selection, and, at the same time, clathrin

assembles to form the CCP. The GTPase dynamin is subsequently recruited to the neck of

the CCP, where it polymerises. GTP hydrolysis by dynamin finally leads to membrane

scission (McMahon and Boucrot, 2011).

Clathrin-independent endocytosis

In general, less is known about the mechanisms involved in CIE, and, in particular, how

cargoes are recruited. Caveolae are flask-shaped invaginations of the membrane that are

50–80 nm in diameter and enriched in cholesterol, sphingolipids and oligomeric caveolin-1.

Endocytosis through caveolae requires the function of dynamin (Mayor and Pagano, 2007). A

second type of lipid-raft-associated CIE involves clathrin- and dynamin-independent carriers

(CLICs) or glycosylphosphatidylinositol (GPI)-enriched early endosomal compartments

(GEECs), which are endomembrane compartments that are formed by clathrin- and

dynamin-independent endocytosis. It is thought that BAR domain proteins such as the Rho

GTPase activating protein 26 (ARHGAP26, also known as GRAF) confer membrane

curvature that might be sufficient for scission. The cargoes of CLIC and GEEC pathways

include GPI-anchored proteins, but given that many transmembrane proteins (including

flotillins) are found within CLIC and GEEC structures it is likely that these pathways constitute

a general internalisation route for a variety of proteins (Howes et al., 2010b; Hansen and

Nichols, 2009). Finally, macropinocytosis produces large endocytic vesicles (with a diameter

.500 nm), and this process is often associated with fluid uptake as well as internalisation of

membrane-associated proteins that are recruited into the macropinocytic cup. Growth-factor

stimulation promotes actin polymerisation and the formation of circular dorsal ruffles that is

associated with the initiation of macropinocytosis. However, little is known about the precise

mechanisms that govern scission and internalisation (Doherty and McMahon, 2009).

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glycoprotein (MAG) inhibits axonal growth

in neurons and repels growth cones by

increasing the local Ca2+ concentration,

thereby stimulating clathrin-dependent

endocytosis of b1 integrins to promote

growth cone turning (Hines et al., 2010).

Syndecan-4 can act as a co-receptor for

fibronectin (alongside integrins). In addition,

syndecan-4 engagement controls the

availability of a5b1 at the plasma

membrane in fibroblasts through a

mechanism that is consistent with caveolar

endocytosis of the integrin (Bass et al., 2011).

Post-endocytic trafficking andrecyclingInternalised integrins are rapidly trafficked

to early endosomes (EEs), where cargoes

are sorted for degradation or recycling

(Box 2; Caswell and Norman, 2006). In the

presence of fibronectin ligand, a5b1

integrin can be routed to late endosomes

and degraded (Dozynkiewicz et al., 2012;

Lobert et al., 2010; Tiwari et al., 2011), but

the majority of internalised integrins are

rapidly recycled back to the plasma

membrane (Bretscher, 1989; Bretscher,

1992). Integrins can follow the major

recycling routes of other cargoes, such as

the transferrin receptor (TfnR, also known

as TFRC) (Box 2) (Caswell and Norman,

2006; Caswell et al., 2009; Pellinen and

Ivaska, 2006). However, although there are

shared mechanisms between these two

functionally distinct cargoes, there are

also integrin-specific elements that

provide spatial and temporal control of

adhesion receptor availability at the plasma

membrane.

Rab4-dependent recycling

avb3 integrins recycle through the Rab4-

dependent ‘short-loop’ pathway (Box 2),

which returns the heterodimers from EEs

back to the plasma membrane without

transiting through the perinuclear recycling

compartment (PNRC) (Roberts et al., 2001;

Jones et al., 2009; di Blasio et al., 2010).

PKD1 (for protein kinase D1), when

autophosphorylated on Ser916 in response

to growth factor stimulation, interacts with

the extreme C-terminus of integrin b3, and

this interaction is required for Rab4-

dependent recycling of this integrin

(Woods et al., 2004; White et al., 2007; di

Blasio et al., 2010). Rab4-dependent

trafficking of avb3 is key to the control of

directionally persistent migration (di Blasio

et al., 2010; White et al., 2007; Woods et al.,2004) and branching morphogenesis of

endothelial vessels (Jones et al., 2009).Although a5b1 does not follow theRab4 recycling pathway in platelet-derivedgrowth factor (PDGF)-stimulated fibroblasts

(Roberts et al., 2001), recent studies haveindicated that b1 integrins can follow a Rab4-dependent route to the plasma membrane:

epidermal growth factor (EGF) promotes b1integrin recycling in HeLa cells in a mannerthat requires the raft-associated membrane

protein supervillin (Fang et al., 2010), andrecycling of inactive b1 integrins is Rab4-dependent in breast cancer cell lines (Arjonenet al., 2012).

Rab11- and Arf6-dependent recycling

Integrins can also follow a second, ‘long-

loop’ recycling pathway through the PNRC(Box 2), and this is dependent on theGTPases Rab11 and Arf6 and is linked to

cell migration (Powelka et al., 2004;Caswell and Norman, 2006; Roberts et al.,2004). For b1 integrins, many of themechanistic details are shared with other

cargoes. EHD1 (for EH-domain containing1), which is recruited to tubulesemanating from the PNRC by MICAL-like

1 (MICALL1) and phosphoinositides,regulates the recycling of both b1 integrinsand the TfnR (Jovic et al., 2007; Jovic et al.,

2009; Naslavsky and Caplan, 2011; Sharmaet al., 2009). In addition, Myotubularin, aphosphoinositide phosphatase, is required

for the exit of integrins from intracellularcompartments in Drosophila (Ribeiro et al.,2011). SNARE function is crucial indocking and fusion of vesicles throughout

the endosomal system, and several Q-SNAREs (e.g. syntaxin 6, SNAP29,syntaxin 4 and SNAP23) and R-SNAREs

(e.g. VAMP2 and VAMP3) have beenshown to regulate b1 integrin trafficking(Veale et al., 2010; Tiwari et al., 2011;

Skalski and Coppolino, 2005; Rapaportet al., 2010; Hasan and Hu, 2010).

Although integrins share much of thebasic machinery for Rab11-dependent

recycling with the TfnR, stages of thelong-loop recycling pathway that arespecifically required for integrin recycling

have been identified. Rab21 binds to aconserved motif within the a-subunits ofb1 integrin heterodimers and specifically

promotes their internalisation andtrafficking through early endosomes tothe PNRC (Pellinen et al., 2008; Pellinen

et al., 2006). Here, p120RasGAP (alsoknown as RASA1) displaces Rab21,thereby promoting the release of the

Box 2. Rab and Arf GTPases control endocytic recycling

Small GTPases are thought of as molecular switches that cycle between a GTP-bound ‘on’

state, and a GDP-bound ‘off’ state, in response to the activities of guanine nucleotide

exchange factors (GEFs) and GTPases activating-proteins (GAPs), respectively. This switch

controls the ability of the small GTPases to interact with their effector molecules. There are

more than 60 Rab proteins and six Arf proteins that localise to distinct membrane

compartments, and these families of GTPases control most of the known intracellular

trafficking events in eukaryotic cells (Grant and Donaldson, 2009; Stenmark, 2009).

The function of early endosomes, where internalised cargoes are sorted, is dependent on

Rab5. Early endosomal cargo can be directed to late endosomes through a maturation

process that involves the exchange of Rab5 for Rab7 on the endosomal membrane (Rink et

al., 2005). Late endosomal proteins are either lysosomally degraded or recycled through

alternative pathways [e.g. the Rab9–retromer pathway (Seaman et al., 1998; Lombardi et al.,

1993) or the Rab25–CLIC3 (for chloride intracellular channel 3) pathway (Dozynkiewicz et al.,

2012)]. Early endosomal cargoes can also be recycled through ‘short-loop’ (fast) or ‘long-

loop’ (slow) recycling pathways. The short-loop recycling pathway is regulated by Rab4 and

Rab35, and is characterised by cargoes that exit the early endosome to recycle to the plasma

membrane. Long-loop recycling involves trafficking of cargo through the perinuclear recycling

compartment (PNRC) and is thought to primarily require the activity of Rab11 and Arf6, but

roles for Rab8, Rab10 and Rab22a have also been described (Grant and Donaldson, 2009;

Stenmark, 2009).

Rab and Arf proteins act together to coordinate the main steps of membrane trafficking. For

example, Rab9 mediates vesicle budding by recruiting sorting adaptors to promote cargo

selection into budding recycling vesicles on late endosomes (Carroll et al., 2001). Rab

GTPases can also regulate tethering, docking and fusion events by recruiting tethering

factors, SNAREs and the exocyst complex, and they can promote vesicle transport by

recruiting both microtubule- and actin-based motor proteins directly or through additional

effectors (Grant and Donaldson, 2009; Stenmark, 2009). By contrast, Arf6 regulates

phospholipid signalling, which is important in many trafficking steps, but can also provide links

to microtubule motors kinesin and dynein through interactions with scaffold proteins and

provide a link to vesicle transport (Donaldson and Jackson, 2011).

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integrin-containing vesicles from theperinuclear region and their recycling to

the plasma membrane (Mai et al., 2011). Incarcinoma cells, Rab-coupling protein(RCP), a member of the Rab11-familyinteracting proteins (RAB11FIPs), which

act as Rab11 effectors, has a crucial role inregulating integrin recycling but isdispensable for TfnR recycling through

Rab11 recycling endosomes (Caswell et al.,2008; Muller et al., 2009). Both a5b1 andavb3 integrins follow a Rab11-dependent

route in unstimulated fibroblasts. Thisrequires the inactivation of glycogensynthase kinase 3 beta (GSK3b) throughphosphorylation by AKT (also known as

PKB) (Roberts et al., 2004). Serumstimulation can also promote traffickingof b1 integrins through this route, and

AKT activity is required to phosphorylateACAP1 (for ArfGAP with coiled-coil,ankyrin repeat and PH domains 1), which

recruits b1 integrins to a recycling coatcomplex that contains clathrin (Powelkaet al., 2004; Li et al., 2005; Li et al., 2007).

PKCe phosphorylates vimentin, a type IIIintermediate filament protein, and releasesb1-integrin-containing vesicles fromintermediate filaments in the perinuclear

region to allow recycling of b1 integrins(Ivaska et al., 2005; Ivaska et al., 2002).The events regulated by AKT and PKCeare specific to integrin trafficking, and arenot required for recycling of TfnR.

Cellular functions of integrintraffickingMany of the studies described above providecompelling evidence that integrin trafficking

contributes directly to cellular processessuch as cell migration. Endocyticmechanisms can contribute to the turnover

of adhesion complexes, and, in this way,directly regulate interactions with the ECMand proteins involved in adhesion signalling

[e.g. focal adhesion kinase (FAK)phosphorylation (Ezratty et al., 2005;Ezratty et al., 2009)]. It is unclear,however, whether trafficking pathways can

influence the activation status of integrins orthe ability of integrins to mediate adhesivecontacts with the substratum, and in some

cases this is evidently not the case (Caswellet al., 2007; Caswell et al., 2008). Trackingintegrin trafficking using antibody surface-

labelling approaches has indicated thatinternalised integrins can subsequentlylocalise to focal complexes and focal

adhesions, but only after extended periods(i.e. hours) following antibody exposure (Guet al., 2011). Given that the t1/2 of integrin

recycling is often ,15 minutes, this could

indicate that integrins do not recycle directly

to focal adhesions (Caswell and Norman,

2006). Accumulating evidence suggests that

instead integrin trafficking influences

intracellular signalling, either directly or by

influencing trafficking of other cargoes.

Integrin trafficking dictates Rho GTPase

signalling

Integrin trafficking pathways can directly

impact on Rho GTPase signalling. Rab21-

mediated recycling of a5b1 is required for

the activation of RhoA at the cleavage

furrow and, hence, the completion of

cytokinesis (Pellinen et al., 2008). Rho

GTPases are master regulators of the

cytoskeleton, and balancing RhoA and Rac

activities is crucial for controlling the

directional persistence of migrating cells

(Danen et al., 2005). In fibroblasts, Rab4-

dependent recycling of avb3, and the

activity of this integrin, suppresses

recycling of a5b1, thereby promoting

directionally persistent migration that is

characterised by a broad leading

lamellipodium (Caswell et al., 2008; White

et al., 2007). Inhibition of avb3 recycling or

ligand binding enhances recycling of a5b1

and signalling through the ROCK (RhoA–

Rho-associated, coiled-coil containing

protein kinase) pathway, which, in turn,

leads to phosphorylation of the downstream

effector cofilin and rapid random migration

(White et al., 2007).

Integrin trafficking promotes tumour cell

invasion and metastasis

Integrin trafficking influences the ability

of cells to move in three-dimensional

matrices, as well as on two-dimensional

substrates. Hypoxia-driven invasion

through a laminin-rich matrix requires

Rab11, and there is evidence that this

involves the Rab11-dependent recycling

of a6b4 (Yoon, 2005). avb6 promotes

invasion in squamous cell carcinoma, and

the interaction of this integrin with HAX1

allows its clathrin-dependent endocytosis,

which in turn promotes carcinoma cell

motility in organotypic culture (Ramsay

et al., 2007).

Inhibition of avb3, or expression of

cancer-associated mutant forms of p53,

promotes a5b1 recycling in carcinoma cell

lines (Caswell et al., 2008; Muller et al.,

2009). This requires the interaction of b1

integrin with RCP (Caswell et al., 2008),

which is itself upregulated in breast cancer

(Zhang et al., 2009). The actin nucleation

promoting factor (NPF) WASH (for WAS

protein family homologue) has a role in

a5b1 recycling through this pathway,

presumably by remodelling actin on

endosomes (Zech et al., 2011). RCP-

dependent a5b1 trafficking promotes

invasive migration in three-dimensional

matrices, which is characterised by the

extension of invasive pseudopods. The

production of phosphatidic acid within

the pseudopod tip is required to localise

RCP and permit integrin recycling

(Rainero et al., 2012). Rather than

influence a5b1 activity, a5b1 and RCP

recruit EGF receptor 1 (EGFR1) and

coordinate its recycling, which,

in turn, potentiates EGFR activation and

downstream signalling to promote invasion

(Caswell et al., 2008; Muller et al., 2009).

In this context, integrins and RCP act as

key components of a ‘recyclosome’

complex, which recruits receptor tyrosine

kinases (RTKs), including EGFR1, ErbB2

and Met, promotes their recycling and

potentiates signalling to induce metastasis

(Muller et al., 2009; Caswell et al.,

2008; Muller et al., 2012; P. T. Caswell,

unpublished observations). Interestingly, in

neuronal axons, Rab11 and RCP can

control trafficking of b1 integrins to

promote axonal extension (Eva et al.,

2010), indicating that this mechanism is

not unique to cancer cells.

Overexpression of Rab25 (also known

as Rab11c) is associated with aggressive

ovarian cancers (Cheng et al., 2004).

Rab25 specifically regulates trafficking of

a5b1 by directly interacting with b1

integrin (Caswell et al., 2007). Inactive

integrins that become internalised into

Rab25 vesicles at the front of invasive

cancer cells are recycled back to the

plasma membrane within this subcellular

region, thus promoting their spatial

restriction (Caswell et al., 2007; Caswell

and Norman, 2008; Caswell et al., 2009).

Those integrins that remain in the active

conformation following internalisation,

however, are sorted through Rab25-

positive late endosomes to lysosomes,

moving from the front of the cell towards

the rear. CLIC3 regulates the subsequent

recycling of active a5b1 from lysosomes

to the plasma membrane. This allows

Rab25 to control pseudopod extension at

the front of the cell and coordinate

retraction of the cell body by trafficking

active integrins towards the rear of the cell

to promote integrin signalling and forward

movement (Dozynkiewicz et al., 2012).

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Integrin trafficking and signallingregulates endocytic transport ofother cargoes

Integrin trafficking pathways can indirectlyinfluence intracellular signalling bycontrolling endocytic trafficking of other

cell surface receptors. In BMMSCs, softsubstrates promote endocytosis of b1integrins through caveolae, and this is

required for the internalisation of bonemarrow morphogenetic protein (BMP)receptor IA (BMPRIA). BMPRIA

colocalises with integrins in an intracellularcompartment, which implies that theseendocytic cargoes co-internalise, and thatthis, in turn, inhibits BMP-induced SMAD

signalling, which allows cells to differentiatealong the neuronal rather than osteogenic ormyogenic lineage (Du et al., 2011).

avb3 integrin controls receptortrafficking, suppressing the recycling ofa5b1 integrin, and thereby leads to changes

in Rho GTPase signalling in fibroblasts andpromotes receptor-tyrosine kinase traffickingand signalling in carcinoma cells asdiscussed above. Inhibiting avb3 with

small molecule inhibitors such asCilengitide in endothelial cells drives Rab4-dependent vascular endothelial growth factor

receptor 2 (VEGFR2) recycling, protectingthis receptor from degradation in thepresence of VEGF ligand and increasing its

levels on the cell surface (Reynolds et al.,2009). This promotes VEGF-drivenendothelial cell migration, sprouting of

aortic explants and tumour angiogenesis invivo, which ultimately leads to enhancedtumorigenesis.

Clearly, integrins are cargoes of

intracellular trafficking pathways;however, the relationship betweenintegrins and endocytic traffic is complex

because integrins themselves govern signalsthat control endocytic flux. In someinstances adhesion negatively regulates

endocytosis: engagement of integrin b1 byligand can slow the dynamics of clathrin-coated structures, and adhesion of cells tofibronectin decreases the rate of TfnR

endocytosis (Batchelder and Yarar, 2010).By contrast, b1 integrin engagementpromotes endocytosis of the platelet-

derived growth factor receptor (PDGFR)during fibroblast chemotaxis. In thisscenario, integrins are required to maintain

the stability of the actin NPF neuralWiscott–Aldrich syndrome protein (N-WASP), which controls the formation of

CDRs and the internalisation of PDGFthrough macropinocytosis (King et al.,2011). Signalling from b1 integrins is also

required for the fusion of vesicle-associated

membrane protein 7 (VAMP7) vesicleswith the plasma membrane in extendingneurites on laminin substrates (Gupton and

Gertler, 2010).

Integrin engagement also regulates thetrafficking of caveolae and/or lipid rafts.Loss of adhesion triggers internalisation of

lipid rafts, which is controlled by the shiftof phosphorylated caveolin from adhesioncomplexes to caveolae (del Pozo et al.,

2005; del Pozo et al., 2004). Following re-adhesion, lipid rafts recycle to the plasmamembrane from the PNRC in an Arf6-dependent manner (Balasubramanian et al.,

2007). RalA regulates exocytosis throughthe exocyst complex (Balasubramanianet al., 2010), which provides a platform

for the activation of Rac. Keratinocytesthat lack integrin b1, or the adhesionsignalling protein integrin-linked kinase

(ILK), also show a defect in themembrane targeting of caveolae. Caveolarendocytosis occurs independently of b1

integrins and ILK, but integrin signallingpromotes the recruitment of a complexcontaining IQGAP (IQ motif containingGTPase activating protein) and mDia1

(also known as DIAPH1) to the plasmamembrane, which in turn locally capturesand stabilises microtubules to permit re-

exocytosis of caveolin-1 (Wickstrom et al.,2010).

Future perspectivesIntegrin trafficking is a dynamic processthat is important for the regulation ofcellular processes, including migration

and cytokinesis. Technical limitations havehindered our understanding of integrintrafficking in vivo, but these are beingovercome in some model systems (Yuan

et al., 2010; Ribeiro et al., 2011), andfurthering these studies to directly visualisetrafficking events in vivo is a priority.

Trafficking pathways can promotespatial restriction or en masse movementof integrins, and, interestingly, some

regulators, such as Rab25, have a role inboth (Caswell et al., 2007; Dozynkiewiczet al., 2012). Further studies are necessary to

determine the sorting steps that controlthe decision to recycle integrins to themembrane from which they were initiallyinternalised or to more distal sites. It will

also be important to determine how thedirectional movement of integrins interfaceswith downstream signalling pathways.

Unlike most cargoes, integrins are almostuniquely positioned in that they are cargoesfor pathways that they themselves regulate.

Further investigation is needed to determinewhether integrins form central componentsof multi-cargo trafficking complexes that

are controlled by adhesion signalling. Thisalso raises an interesting question: areintegrins able to elicit signals when they

are transiting the endosomal system?The presence of active integrins onendomembrane compartments has nowbeen noted by several investigators, and

the recent identification of the integrininactivator sharpin (Rantala et al., 2011)suggests that integrins could remain active

on intracellular compartments untilinactivated. Endomembranes are surfacesthat provide a scaffold for signalling

pathways such as the AKT (Schenck et al.,2008) and ROCK–myosin light chain 2(MLC2) pathways (Sturge et al., 2006). This

opens up the exciting possibility thatintegrins could associate with downstreamsignalling components on endosomes topropagate signals from a lipid and protein

environment distinct from the adhesionplaque.

Funding

The work of our laboratories is supported by

the Wellcome Trust (P.T.C.) and Cancer

Research UK (J.C.N). R.E.B is supported by

a BBSRC studentship and a Research Impact

Scholarship from the ‘Your Manchester’

Fund.

A high-resolution version of the poster is available for

downloading in the online version of this article at

jcs.biologists.org. Individual poster panels are available

as JPEG files at http://jcs.biologists.org/lookup/suppl/

doi:10.1242/jcs.095810/-/DC1.

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