Endocytic Trafficking of Integrins in Cell Migration.

Current Biology

Review Endocytic Trafficking of Integrins in Cell Migration Nikki R. Paul1,2, Guillaume Jacquemet1,3, and Patrick T. Caswell1,* 1Wellcome

Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, M13 9PT, UK address: CRUK Beatson Institute, Glasgow, G61 1BD, UK 3Present address: Turku Centre for Biotechnology, FIN-20521, Turku, Finland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.09.049 2Present

Integrins are a family of heterodimeric receptors that bind to components of the extracellular matrix and influence cellular processes as varied as proliferation and migration. These effects are achieved by tight spatiotemporal control over intracellular signalling pathways, including those that mediate cytoskeletal reorganisation. The ability of integrins to bind to ligands is governed by integrin conformation, or activity, and this is widely acknowledged to be an important route to the regulation of integrin function. Over the last 15 years, however, the pathways that regulate endocytosis and recycling of integrins have emerged as major players in controlling integrin action, and studying integrin trafficking has revealed fresh insight into the function of this fascinating class of extracellular matrix receptors, in particular in the context of cell migration and invasion. Here, we review our current understanding of the contribution of integrin trafficking to cell motility. Introduction Integrins are a family of receptors for ligands found in the extracellular space, including extracellular matrix (ECM) ligands found in basement membranes (collagen IV and laminins), interstitial matrix (collagen I and fibronectin) and coagulating blood (fibrinogen and thrombospondin) [1]. Heterodimerisation of 18 a-integrin and 8 b-integrin subunits within the endoplasmic reticulum leads to the assembly of 24 distinct integrin receptors, which undergo further post-translational modification in the Golgi apparatus before they reach the cell surface and are able to perform their myriad functions. Integrins can be broadly classified into RGD receptors (which bind the peptide motif Arg-Gly-Asp), collagen receptors, laminin receptors and leukocyte-specific receptors; most integrins can interact with more than one ligand, and in fact different integrins can bind to the same ligands in many instances [1,2]. Genetic ablation of specific integrins has given insight into their critical role in development, and interactions between integrins and their ligands are known to control processes including cell proliferation, death, differentiation and migration [2]. Adhesion signalling via integrins is therefore a key contributor to both health and disease. Integrins are bi-directional signallers. Ligand binding and clustering of integrins provides a platform for the assembly of multimeric complexes that provoke signalling responses downstream of integrin-mediated adhesion (‘outside-in’ signalling). Different types of adhesion complex (nascent adhesions, focal contacts, focal adhesions and fibrillar adhesions) are formed in migrating cells, and they are characterised by maturation stage, subcellular distribution and protein composition (Figure 1). Binding of intracellular factors, such as talins and kindlins, to the cytoplasmic integrin tail regulates the activation status of integrins by inducing a conformational change to stabilise an extended conformation of integrin that has high affinity for ligand (‘insideout’ signalling) [1–3]. The mechanisms regulating integrin activation have been the focus of intense research activity over the last three decades and are therefore relatively well understood. Over the last 15 years it has become apparent that receptor trafficking through the endocytic system can also regulate

integrin function [4–7]. In simple terms, cycles of endocytosis and re-exocytosis (recycling) can control the availability of integrins at the plasma membrane and, for some integrins, almost all of the surface pool of integrin is cleared within a time period of around 30 minutes. The degradative turnover of integrins is slow (the half-life of surface-labelled integrins is 12–24 hours [8–11]), and hence the majority of internalised integrins are recycled [6]. Clearly, this tight regulation of the surface integrin population will impact upon integrin function, and compelling evidence implicates the various endocytic and recycling routes that control integrin trafficking as critical regulators of cell migration, including in the context of haptotaxis, chemotaxis and invasive cell migration in 3D matrix. GTPases of the Rab and Arf family are key regulators of transit through the endolysosomal system and are themselves controlled by cycles of GTP binding, catalysed by guanine nucleotide exchange factors (GEFs), and GTP hydrolysis, catalysed by GTPase-activating proteins (GAPs). Rab GTPases recruit effector proteins to regulate cargo sorting, motor protein binding, tethering, docking and fusion events [12], whilst the Arfs are associated with recruitment of coat proteins that control vesicle budding in addition to linking to motor proteins and regulating phospholipid signalling [13,14]. Some Rab and Arf GTPase effectors, and indeed some Rab proteins themselves, can interact directly with integrins, providing exquisite levels of control for a specified integrin and its co-cargoes. The molecular mechanisms that control integrin trafficking in general have been reviewed in depth elsewhere [4–7]; here, we will focus on the pathways that control integrin internalisation and recycling in migrating cells. In the late 1980s and early 1990s, Mark Bretscher first demonstrated, using biochemical techniques, that certain integrins are internalised from the cell surface and recycled back to the plasma membrane [15,16]. The hypothesis that cycles of internalisation followed by recycling might serve to redistribute the pools of integrin from one part of the cell to another emerged from these studies and led to the idea that migrating cells must remove their integrin ‘feet’ from the rear of the cell for re-introduction towards the front of the cell to permit forward movement

R1092 Current Biology 25, R1092–R1105, November 16, 2015 ª2015 Elsevier Ltd All rights reserved

Current Biology

Review [17]. Subsequent studies reported that integrins were indeed endocytosed at the rear of neutrophils and recycled at the leading edge, as integrin-containing vesicles were observed at the cell rear and the cell front; however, upon disruption of Ca2+ signalling, the pool of integrin vesicles at the cell front was lost [18,19]. In fibroblasts, integrin-containing vesicles were indeed found to internalise from the plasma membrane before delivery to the perinuclear recycling compartment, but long-range movement of recycling vesicles to the leading edge was not observed, and in fact endocytosis was seen to occur at membrane protrusions as well as at the cell rear [20–22]. The hypothesis that integrins are internalised at the cell rear for recycling at the leading edge is attractive and remains popular despite a lack of clear evidence. In this review, we will present recent evidence indicating that trafficking of integrins does occur directionally, but in surprising ways — from the cell front to the cell front, from the cell front to the cell rear, and from the perinuclear region to the cell rear — all of which contribute to cell motility. We will also discuss how internalisation of integrins regulates the turnover of focal adhesions and promotes adhesion formation at the front of migrating cells in 2D. Finally, we will summarise the role of integrin trafficking in cancer cell migration and invasion in more physiologically relevant 3D microenvironments. Endocytosis of Integrins Facilitates Cell Migration Members of the integrin family have been shown to be internalised by macropinocytosis, clathrin-dependent endocytosis and clathrin-independent endocytosis, which includes endocytosis mediated by caveolae and clathrin-independent carriers (CLICs) [6]. Both inactive and active (ligand-bound) conformations of integrin heterodimers can be internalised, and there is clear evidence that internalisation of integrins is an important step in the regulation of cell migration in both 2D and 3D ECM [23,24]. Cycles of adhesion assembly and disassembly are crucial to cell migration [25], and in many cases integrin endocytosis has been linked to cell migration through the turnover of focal adhesions/focal complexes (Figures 1 and 2). Clathrin-dependent Endocytosis of Integrins Regulates Adhesion Turnover Targeting of adhesion complexes by microtubules is important in the destabilisation of cell–matrix contacts leading to adhesion turnover because it provides the tracks for vesicular transport delivering both matrix metalloproteases (which cleave ECM and allow release of integrins from the immobile substrate) and the molecular machinery that promotes integrin endocytosis, including dynamin-2 (Dyn2) [26,27]. The GTPase Dyn2 polymerises around the neck of budding vesicles to execute scission events [28], such as during clathrin- and caveolar-mediated endocytosis, and binds directly to focal adhesion kinase (FAK) [29]. FAK recruits Dyn2 in a Src- and PI(4,5)P2-dependent manner at or near adhesion sites to facilitate turnover of large focal adhesions that form in the presence of the microtubule-depolymerising agent nocodazole and are induced to disassemble upon nocodazole washout [26,29–31]. The adaptors Dab2, Eps8 and Numb have all been implicated in the regulation of clathrindependent endocytosis, and interact directly via their phosphotyrosine-binding (PTB) domains with conserved NPxY/NxxY motifs within integrin b-subunit cytoplasmic tails [32]. Numb

and Dab2 are clathrin adaptors that have been shown to localise at adhesion sites together with clathrin and dynamin to directly regulate integrin endocytosis and control adhesion turnover [26,29,30,33,34]. However, the precise mechanistic details of clathrin-dependent endocytosis of integrins appear to depend upon the localisation of adhesions and adhesion type to a large extent (Figure 1), although cell or tissue type may also be a factor. Numb binds to b1 and b3 integrins and is found at the cell– substrate interface polarised towards the leading edge of migrating cells in a manner dependent on cycles of phosphorylation and dephosphorylation by Par-3–atypical protein kinase C (aPKC). Numb, together with the clathrin adaptor AP-2, regulates clathrin-dependent endocytosis of b1 and b3 integrins to promote turnover of adhesions at the leading edge and facilitate cell migration (but not cell spreading) on ligands for these integrins [33]. The kinesin family motor KIF15 is required for localisation of Dab2 to the ventral surface of the cell to regulate integrin internalisation [35], and Dab2 regulates microtubule-dependent adhesion turnover by promoting clathrin-dependent endocytosis of active b1 integrins in conjunction with the clathrin adaptor ARH and/or AP-2 [30,34]. In migrating cells Dab2 and ARH localise to large adhesions beneath the central region of the cell, rather than to focal complexes found at the leading edge (as is the case for Numb [33,36]) or adhesion structures toward the rear [34]. Clathrin-dependent endocytosis of integrins can also be regulated by Arf GTPases; for example, the Arf6 GAP ARAP2 (ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2) is required for b1-integrin internalisation and adhesion disassembly [37]. Furthermore, the Arf5/Arf6 GEF BRAG2 (brefeldin A-resistant ARF-GEF2) binds to clathrin and AP-2 and activates Arf5 to promote internalisation of a5b1 in cancer cells [38,39] and also regulates endothelial cell sprouting and angiogenesis [40]. The small GTPase Rab5 regulates vesicle formation and early endosome function, and is required for microtubule-dependent adhesion disassembly [41]. In endothelial cells, the Rab5 GEF Rin2 localises to adhesion complexes at the leading edge and within endosomes, and plays a clear role in the endocytosis of active b1 integrins. In addition to its function as a Rab5 activator, Rin2 recruits R-Ras to the endosomal Rin2–Rab5 complex to further promote integrin internalisation, activate Rac (via its exchange factor Tiam1), and coordinate cell adhesion and migration on fibronectin substrates [42]. RN-tre, a Rab5 GAP, localises to adhesion complexes across the whole cell–substratum interface and inactivates Rab5 to suppress the internalisation of b1 (but not b3) integrins to slow cell spreading and migration specifically on fibronectin. RN-tre also forms a complex with Eps8, an integrin interactor, and diverts Eps8 from its function as a Rac activator to a suppressor of epidermal growth factor receptor (EGFR) endocytosis [43]. It is therefore tempting to speculate that, in large adhesions, RN-tre is recruited by an integrin-associated protein, such as Eps8, to suppress Rab5 activity and slow adhesion turnover, whereas in nascent adhesion/focal complexes toward the cell front the presence of Rin2 provides an activation signal for Rab5 and promotes integrin endocytosis. This could also link Rab5-dependent integrin endocytosis to the suppression of Rac activity toward the central region of migrating cells by maintaining large focal/fibrillar adhesions, and to the activation of Rac (via the R-Ras–Rin2–Rab5 complex)

Current Biology 25, R1092–R1105, November 16, 2015 ª2015 Elsevier Ltd All rights reserved R1093

Current Biology

Review A Inactive α1/2/3β1 integrin Focal contacts/ nascent adhesions

Dorsal plasma membrane

B

A

B

Rab5 Rin2

Eps15 Dab2

Clathrin

R-Ras-GTP

Rab5

Tiam1

R-Ras-GTP

E C

Rac activity

α?β1 integrin

Ventral plasma membrane

D

Rin2

Focal adhesions

Nascent adhesions

C

D

E

KIF15 Rab5

Microtubules M

Numb

P

Numb

Dab2

FAK

? RN-Tre Eps8

Dynamin

Par-3–aPKC

Numb

α5β1 integrin α?β1 integrins

Focal adhesion

Early Plasma membrane endosomes and macropinosomes

Active integrin

αvβ3 integrin Focal complexes

α?β1 integrin focal adhesions

Inactive integrin Trafficking routes

Activation

Inhibition

α5β1 integrin

αvβ3 integrin

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Figure 1. Clathrin-dependent endocytosis of integrins and adhesion turnover in migrating cells. (A) Dab2-dependent clathrin-dependent endocytosis of inactive b1 integrins from the dorsal surface allows re-delivery to the ventral plasma membrane. (B) The R-Ras–Rin2 complex promotes clathrin-dependent endocytosis of b1 integrins by recruiting Rab5-GTP to adhesions. The Rab5–Rin2 complex subsequently acts to localise R-Ras-GTP to endosomes to activate Rac via Tiam1. (C) Microtubule-dependent adhesion turnover is mediated by the kinesin KIF15, which promotes recruitment of Dab2 to the ventral plasma membrane. Dynamin is recruited by FAK and is required for vesicle scission. (D) Eps8 binds the NPxY/NxxY motif of b integrins via its PTB domain. Eps8 also binds RN-tre, which inhibits Rab5-mediated internalisation of b1 (but not b3) integrins. (E) Numb undergoes cycles of Par-3–aPKC-mediated phosphorylation/dephosphorylation, which regulates endocytosis of b1 and b3 integrins at the leading edge of the cell.

at the leading edge to promote lamellipodial extension. Rab21, which is related to Rab5 and interacts directly with the a chain of b1 integrins [44], can promote the endocytosis of b1 integrins in a manner insensitive to monodansyl cadaverine (which inhibits clathrin-dependent endocytosis) [45], suggesting a form of clathrin-independent endocytosis. In addition, dominant-negative forms of Rab21 promote formation of large peripheral adhesions, indicative of a role in focal adhesion turnover [44]. Moreover, Rab21 promotes cell adhesion and migration on collagen, implying a role in trafficking of collagen-binding integrins (a1b1, a2b1, a10b1 and a11b1) [44,46]. In addition to controlling adhesion disassembly, clathrindependent endocytosis of integrins can, perhaps counterintuitively, contribute to focal adhesion formation. Dab2 recruits Eps15 homology domain (EHD) proteins, which are accessory proteins involved in clathrin-dependent endocytosis, to specifically regulate the internalisation of inactive a1b1, a2b1 and

a3b1 integrins (but not transferrin receptor or a5b1 integrin) from the dorsal surface of the cell, to generate an internal pool of integrin that is subsequently recycled to form adhesions at the leading edge [36,47]. In this way, integrin internalisation can lead not only to disassembly of adhesions, but also to the generation of a pool of integrin that can be recycled for adhesion reassembly. Clathrin-independent Routes of Integrin Internalisation in Cell Migration In recent years, the relevance of CLICs to bulk endocytosis has become increasingly apparent. Interestingly, CLICs act as endocytic carriers of integrins and the hyaluronan receptor CD44, and CLICs distribute toward the leading edge of migrating cells [48,49]. The biogenesis of CLICs at the plasma membrane requires glycosphingolipids, the N-glycan-binding protein galectin-3 and GRAF1 (GTPase regulator associated with FAK 1) [49,50]. GRAF1 is found in a complex with phosphorylated FAK,


Current Biology

Review Focal contacts/ nascent adhesions

A

Circular dorsal ruffles

B

PDGFR

B

A

? αvβ3 integrin

FAK P GRAF1

Macropinocytosis

E C Focal adhesions

D α?β1 integrin

α5β1 integrin

Fibrillar adhesions

Gal-3 Gal-3 Gal-3

C

D

E

mTORC1

Gαq/11 Rab25

Lysosome Arf4

Caveolin

RhoG

Tensin

Tensin

α5β1 integrin

LFA-1 FN ICAM-1

Early endosomes Plasma membrane and macropinosomes

Fibrillar adhesion

Lysosomes

Active integrin

Inactive integrin

Caveolin

PKCα

Scar/WAVE

α5β1 integrin Syndecan-4 FN

Focal adhesion

Trafficking routes

Activation

Inhibition α5β1 integrin

αvβ3 integrin

LFA-1 (αLβ2 integrin)

Current Biology

Figure 2. Clathrin-independent endocytosis of integrins and adhesion turnover in migrating cells. (A) Stimulation of the PDGF receptor (PDGFR) induces circular dorsal ruffles on the dorsal surface of the cell and promotes internalisation of integrins via macropinocytosis. (B) Clathrin-independent carriers (CLICs) that are distributed towards the leading edge of the cell permit b1-integrin internalisation. They require extracellular galectin-3 (Gal-3) and intracellular GRAF1, which forms a complex with phosphorylated FAK. (C) In the trailing uropod of leukocytes, endocytosis of LFA-1 (aLb2 integrin) is dependent upon caveolin and is regulated by GPCR signalling. (D) a5b1 integrin is internalised from sub-nuclear fibrillar adhesions via a clathrin- and dynamin-independent mechanism that requires Arf4 and Scar/WAVE. Ligand-bound integrins are delivered to lysosomes to activate mTOR complex 1 (mTORC1). (E) Syndecans are adhesion receptors that bind ECM ligands. Syndecan-4 binding to fibronectin (FN) activates PKCa and RhoG, and this promotes caveolar endocytosis of b1 integrins at the leading edge.

localises to podosome-like adhesions in HeLa cells, and is required for efficient cell migration [50]. Hence, it is tempting to speculate that CLICs regulate the internalisation of adhesion receptors at the leading edge of cells to facilitate cell migration, perhaps by controlling adhesion turnover and/or generating a pool of internal integrin for recycling (Figure 2). Caveolae are cholesterol- and sphingolipid-rich membrane microdomains that control clathrin-independent and dynamindependent internalisation of cargoes, including integrins. Caveolar function is associated with cell migration, although these endocytic structures often partition towards the rear of migrating cells [51]. PKCa regulates integrin internalisation and haptotactic cell migration [52] and has been linked to caveolar function [53]. Furthermore, PKCa can activate the formin FMNL2 to promote direct binding of this formin to b1 integrins and integrin internalisation, although the dependence of this specific pathway

on clathrin/caveolae is not known [54]. Integrin endocytosis via caveolae has been demonstrated [55–57], and in some cases this contributes to cell migration. In fibroblasts, the binding of Syndecan-4 to fibronectin (at a site distinct from the integrinbinding sites) triggers a signalling cascade, via PKCa and RhoG, which promotes caveolar endocytosis of b1 integrins [58]. This allows redeployment of the integrin elsewhere around the cell surface, and promotes 2D cell migration as well as wound healing in vivo [58]. Caveolar endocytosis of LFA-1 (aLb2 integrin) is thought to occur in the trailing uropod of leukocytes migrating on the LFA-1 ligand ICAM-1, and this requires the adhesion molecule JAM-A [59,60]. Interestingly, this trafficking pathway is regulated by G-protein-coupled receptor (GPCR) signalling, as Gaq/11 gene silencing abrogates LFA-1 internalisation, uropod detachment and leukocyte migration [61].


Current Biology

Review a5b1 integrin can also be internalised through a mechanism dependent on the SCAR/WAVE actin nucleation complex and Arf4 that does not require clathrin or dynamin and therefore represents a distinct route of internalisation for ligand-bound integrins [62]. In Rab25-expressing cancer cells, active integrins are rapidly internalised from sub-nuclear adhesions (originating from tensin-dependent translocation of peripheral adhesions) into late endosomes/lysosomes which contact adhesion sites in an Arf4-dependent manner. Arf4 is generally associated with Golgi transport and could therefore play an indirect role in delivery of cargos that mediate endocytosis [13]. However, tensin can associate with Arf4 [62], so it is possible that Arf4 functions outside of the secretory pathway in a defined endocytic capacity, as is the case for Arf5 (another class II Arf family member [39]). Delivery of ligand-bound integrins to lysosomes activates mammalian target of rapamycin (mTOR), links integrin trafficking to the nutrient status of cancer cells, and impacts on invasive migration and metastasis [62]. Interestingly, the putative oncogene tensin-4 does not localise to fibrillar adhesions, but suppresses b1-integrin endocytosis and provides a link to c-Met signalling and invasion [63]. Macropinocytosis can also play an important role in the rapid redistribution of integrins in growth-factor-stimulated fibroblasts. Platelet-derived growth factor (PDGF) stimulates the formation of circular dorsal ruffles, which accumulate b1 and b3 integrins from the dorsal surface of the cell before they are taken up by macropinocytosis. Again, an increase in integrin uptake creates an internal pool that can subsequently be recycled and promote formation of new adhesions at the leading edge [64]. From Endosomes to Plasma Membrane: Recycling Integrins Promote Migration Many studies over the last 15 years have linked integrin recycling to the ability of cells to move on 2D surfaces (Table 1) [7]. The first identifications of the molecular machinery involved in integrin trafficking directly implicated these components in the regulation of cell spreading and cell migration [65–69], and an indepth interrogation of the role of protein kinase D1 (PKD1) in avb3-integrin recycling showed that this kinase was required for the formation of focal contacts found at the leading edge of migrating cells [70]. Much of the recent work has hinged upon identifying key integrin-specific regulators of recycling pathways that form the basis for intervention. In a similar manner to endocytosis, integrin recycling is thought to regulate adhesive capacity, through maintenance of existing, and formation of new, cell– matrix adhesions. However, trafficking of integrins can also alter signalling downstream of other receptors to promote cell migration, which may be distinct from the adhesive function of integrins in adhesion complexes [71,72]. The pathways that regulate integrin recycling in migrating cells are summarised in Table 1. Like many other cargoes, internalised integrins enter early endosomes and the fate of the receptor depends upon sorting decisions that determine onward trafficking to late endosomes/lysosomes, or recycling back to the plasma membrane via recycling endosomes or alternative routes [14]. The binding of sorting nexins (SNX) 17 and 31 to a conserved motif in the b-integrin subunit is crucial for diverting integrins away from a degradative route toward recycling [11,73–75]. Similarly, the Arp2/3 activator WASH is required to direct a5b1 integrin away from a

late endosomal destination and toward a recycling route [76]. Whilst integrins can follow the late endosome/lysosomal route to destruction [8–10], autophagosomes may also play a role in degradation of b1 integrins [77]. The best characterised integrin-recycling routes are analogous to the fast (short-loop) and slow (long-loop) pathways that control trafficking of other receptors [78]. Importantly, integrins are often specifically recruited into these routes by virtue of protein–protein interactions with trafficking regulators, and some of these are specific sub-pathways that handle the recycling of integrins (and their co-cargoes) but not other cell-surface receptors. Recycling from Early Endosomes The Rab4 short-loop or fast recycling pathway selectively controls recycling of integrins in cells stimulated with growth factors or serum [6]. The mechanism through which specific integrin heterodimers are recruited is incompletely understood, but entry into this sorting compartment is controlled by Rabaptin-5, which binds the Rab5 GEF Rabex-5 to promote Rab5 activity on early endosomes [79,80]. Rabaptin-5 also acts to functionally link Rab5 and Rab4 [81] and is a substrate for PKD, with PKD-mediated phosphorylation controlling the formation of a Rab4–Rabaptin-5 complex and rapid recycling of avb3 integrin [79]. Interestingly, PKD1 binds directly to the b subunit of avb3 to control its recycling, and phosphorylation of Rabaptin-5 may control recruitment of Rab4 to subdomains of early endosomes that harbour avb3 to permit integrin recycling directly from this compartment [65,70,79,82]. This pathway is required for the incorporation of avb3 integrin into newly forming adhesions at the lamellipodial leading edge of cells, and drives directionally persistent migration of cells on 2D substrates and invasive migration in 3D matrices with low levels of fibronectin [70,79,82]. Studies in fibroblasts and endothelial cells showed that avb3, but not a5b1, follows this growth-factor-driven recycling itinerary [65,83,84], but more recent evidence suggests that other b1-containing integrins can also recycle in a Rab4-dependent fashion. For example, an imaging-based approach showed that inactive b1 integrins can follow this route [85], and others have shown that supervillin, which binds both to actin and to cholesterolrich lipid rafts, also regulates recycling of b1 and b3 integrins [86]. Rabenosyn-5, a Rab5 effector, and Vps45 (which interacts with the SNARE machinery) are also involved in the recycling of b1 integrins, likely via this route [87]. Furthermore, Rab5c promotes the formation of a complex between the Arf6 effector AMAP1, PKD2 and the b1 cytoplasmic tail of a2b1 and a3b1 (but not a5b1), which regulates recycling of these b1 integrins in cancer cell lines and promotes cell migration and invasion [88]. It therefore appears that aspects of the recycling of integrins from early endosomes — the requirement for Rab4/Rab5 and the involvement of PKD family members — are conserved in different cell types, but that the details —the PKD family member and the specific integrin heterodimer — may vary. Nevertheless, it is clear that the early endosomal recycling of integrins contributes to the directionally persistent migration of fibroblasts (by promoting formation of new adhesions at the leading lamellipodia) as well as the migratory and invasive properties of cancer cells. The Perinuclear Recycling Compartment The perinuclear recycling compartment is characterised by the presence of Rab11, although other GTPases (Rab8, Rab22a and Arf6) also play a role in this slow recycling route [78]. Several


Current Biology

Review Table 1. Integrin recycling pathways in migrating cells. Recycling route

Integrin(s)

Key regulators

Comments

References

Rab4

avb3

PKD1/2, Rabaptin-5, AMAP1, Rabip4, AAKL1, EHD3

Growth-factor-induced recycling in migrating fibroblasts, endothelial and cancer cells

[65,70,79,82–84,88,132,133]

a?b1

Bro1 (HDPTP), Supervillin, hVPS42, Rabenosyn-5

Serum-induced recycling, inactive integrin

[85,86,134]

a5b1/avb3

PKB/Akt-GSK3b, Syndecan-4, Syntaxin-6

Src phosphorylation of Syndecan-4 dictates recycling of avb3 versus a5b1; Syntaxin-6 localises to the perinuclear recycling compartment when trans-Golgi network cholesterol is depleted

[66,103,135]

a5b1

RCP, DGKa

Promoted by expression of gain-of-function mut-p53 or avb3 inhibition/loss

[71,92,101]

a?b1

PKB/Akt–ACAP1, Clathrin

PKB/Akt phosphorylates ACAP1 to recruit b1 integrins, ACAP1 and clathrin form a recycling coat

[69,94,95]

PKCε, vimentin

Phosphorylation of vimentin releases b1-integrin-containing vesicles from the perinuclear region

[67,68]

P120RasGAP

p120RasGAP displaces Rab21 from the a-subunit to promote recycling

[93]

ACAP4

EGF induces Grb2–ACAP4 interaction to control recycling

[136]

Rab35/Chloride intracellular channel 4 (CLIC4)

Rab35 suppresses Arf6-dependent recycling; CLIC4 inhibits Rab35 to promote recycling

[137,138]

EHD1, MICAL-L1, Rab8a

MICAL-L1 links Rab8a to EHD1

[96,97]

Rab11/Arf6

ARNO/Cytohesin-2

[139,140]

SNAREs, VAMP3, SNAP23/ Syntaxin-4

[141–144]

a6b4

Microtubules

Hypoxia stabilises microtubules to promote recycling and invasion

[112]

LFA-1

Lipid raft

Recycling promotes neutrophil migration

[59]

Rab25

a5b1

CLIC3, Neu3

Sorting of inactive and active integrin for recycling in invasive cancer cells

[10,98,145]

Rab13

LFA-1

Mst1, DENND1C

Mst1 activates DENND1C (Rab13 GEF) to regulate lymphocyte motility/trafficking

[146]

Rab1a

a?b1

Recycling to lipid rafts

[147]

Arf1

a5b1

Rap2

LFA-1

?

a3b1

?

a?b1

Phosphatase of regenerating liver-3

[148] Internalisation and recycling in T-lymphocytes

[149]

Syntaxin6/VAMP-3

Recycling via the trans-Golgi network

[150]

Clathrin, Hip1, actin

Gyrating (G-) clathrin controls exit of internalised integrin from endosomes

[151]


Current Biology

Review Figure 3. Directional trafficking of integrins.

A Neutrophils Directional migration

Focal contacts/ nascent adhesions

y x

z x

Focal adhesions

PNRC Ca2+/calcineurin ECM

B Rear-steering epithelial cells y x

Focal adhesions

Focal contacts/ nascent adhesions

Directional migration

(A) In neutrophils, integrins are internalised at the rear of the migrating cell and recycled to the leading edge via the perinuclear recycling compartment (PNRC), in a process that is dependent upon calcium signalling. (B) In rearsteering epithelial cells, the kinesin KIF1C drives the movement of integrin-containing vesicles from the perinuclear region along microtubules to the tip of the trailing cell tail. This allows maturation of adhesions at the rear of the cell, providing directionality towards guidance cues and promotes persistent migration. (C) In cancer cells migrating in 3D ECM, a5b1 integrins are internalised at the leading edge and sorted into Rab25-positive endosomes; inactive integrins return to the plasma membrane at the cell front, whereas active integrins are trafficked to chloride intracellular channel 3 (CLIC3)-positive lysosomes. Subsequent recycling of active integrins to the cell rear and an increase in Src activity drive forward movement.

z x

recycling and cell migration [93]. Similarly, PKB/Akt-mediated phosphorylation KIF1C Microtubules of the Arf6 GAP ACAP1 controls its inter+ action with b1 integrins to regulate Arf6– ECM Rab11-dependent recycling of integrins and migration towards fibronectin [69,94,95]. The Arf GEF ARNO/cytohesin C Cancer cells in 3D ECM: Rab25 and CLIC3 2 is also required for b1-integrin recycling to promote cell migration [96,97], and it is z Directional migration y x likely that Arf6 acts together with the pathways described above to form ECM distinct steps on a common route controlling integrin trafficking in motile cells. Directional Transport of Integrins in Migrating Cells Rab25 Rab25 CLIC3The attractive hypothesis that integrins positive lysosome are internalised at the cell rear and reSrc activity cycled at the leading edge has persisted despite a lack of convincing evidence Forward movement of rear z across multiple cell types. Whilst it is intux itive that this occurs to some extent in migrating cells, and evidence has been put forward that this occurs in neutrophils Active Inactive Recycling Lysosomes Trafficking Early Activation Inhibition α5β1 integrin αvβ3 integrin Plasma membrane integrin endosomes endosomes integrin routes and macropinosomes (Figure 3A) [18,19], it is clear that much of the turnover of adhesion complexes (and Current Biology hence internalisation of integrins) occurs at the front of migrating cells and that integrin heterodimers have been shown to traffic via this the recycling of integrins contributes to formation of adhesion compartment and contribute to cell migration (Table 1; reviewed complexes at the leading edge. In dividing cells, vesicular integin [4,5,89,90]). Some elements of the recycling machinery in this rins move from the cleavage furrow to the leading edge to facilcompartment are common between integrins and other cargoes, itate re-spreading and movement of daughter cells away from for example the EHD proteins [91], whilst other components each other [45], but evidence for this en masse movement to appear to show selectivity toward integrins. For instance, the the leading edge of conventionally migrating cells has not been Rab11 effector Rab-coupling protein (RCP) associates with forthcoming; in fact, spatially restricted cycles of internalisation a5b1 integrin and regulates its recycling in migrating cells and recycling have been observed (Figure 3C) [90,98]. Two [71,92]. PKCε phosphorylates vimentin to release collagen- recent studies have shown that, surprisingly, long-distance inand fibronectin-binding b1 integrins from perinuclear intermedi- tegrin trafficking can be directed towards the cell rear, and this ate filaments and control motility [67,68], and p120RasGAP dis- has important consequences for migration in both 2D and 3D places Rab21 from internalised integrins to control b1-integrin (Figure 3B,C). R1098 Current Biology 25, R1092–R1105, November 16, 2015 ª2015 Elsevier Ltd All rights reserved

Current Biology

Review In migrating epithelial cells, the kinesin KIF1C controls the movement of a5b1-integrin-containing vesicles along microtubules from the perinuclear region to the distal tip of the trailing cell tail, rather than forward to the leading edge. This rearward trafficking promotes the maturation of adhesions that anchor the tail, allowing the cell to orient towards guidance cues and move in a directionally persistent manner through this ‘rearsteering’ mechanism [99]. In invasive cancer cells migrating in 3D ECM, active a5b1 integrins are endocytosed at the cell front and sorted within Rab25 endosomes for retrograde trafficking to late endosomes/lysosomes in the cell body. Active integrins are subsequently recycled to the plasma membrane at the cell rear to facilitate forward movement of the trailing end [10]. Reciprocal Recycling: avb3 vs. a5b1 Integrins Many integrin recycling routes have been determined for b1 integrins, which can form 12 distinct heterodimer receptors for a variety of ligands. However, several studies have focused on the recycling of specific heterodimers (Table 1), and accumulating evidence has demonstrated an antagonistic relationship between the recycling of avb3 and a5b1 that influences cell migration in 2D and 3D microenvironments. The Rab4 short-loop recycling pathway controls recycling of avb3, but not a5b1 [65]. The transit of avb3 through the Rab4 recycling route requires direct interaction of PKD1 with the b3 subunit and PKD1-dependent phosphorylation of Rabaptin-5 [70,79,82]. Recycling through this route promotes formation of avb3-integrin-containing cell–matrix adhesions at the leading edge, which in turn supports Rac- and Arp2/3-mediated lamellipodial extension to maintain directionally persistent migration of fibroblasts and cancer cells in 2D [70,79,82,100]. Interestingly, disruption of Rab4–avb3 recycling, by direct inhibition of avb3 or abrogation of avb3 recycling (for example, via mutations in PKD1, b3 integrin or Rabaptin-5), causes lamellipodial collapse and promotes a rapid random mode of migration as cells move on 2D substrates [71,82,101]. This is due to an increase in the Rab11–RCP-dependent recycling of a5b1 integrin, which promotes activation of the RhoA–ROCK–cofilin pathway, generating ruffling protrusions that drive rapid random migration [71,82,101]. Syndecan-4 plays a key role in the trafficking of cell-surface receptors, including integrins [102]. Src-dependent phosphorylation of Syndecan-4 promotes the recruitment of the adaptor protein Syntenin to suppress Arf6 activity and consequently a5b1-integrin recycling via this route, whereas slow recycling of avb3 is maintained to promote directional migration. Nonphosphorylatable Syndecan-4 mutants (or a fast-cycling Arf6 mutant) promote recycling of a5b1, suppressing avb3 recycling and leading to rapid adhesion turnover but decreased cell migration [103]. These studies suggest that the relationships between integrin heterodimers are more complex than previously thought, and the reciprocity between avb3 and a5b1 defines the signalling that underpins migratory strategies in 2D (directionally persistent versus random). Integrin Trafficking in Migration and Invasion in 3D Cell migration in physiologically relevant 3D environments has become the focus of intense research in recent years. Interestingly, focal adhesions are sites of matrix degradation, and the FAK/dynamin adhesion disassembly factors targeted directly

by microtubules are also thought to promote delivery of proteases, such as membrane-type 1 matrix metalloproteinase (MT1-MMP) [27,104,105]. Trafficking of MT1-MMP is critically important in the context of 3D migration, and MT1-MMP is directed from late endosomes to the plasma membrane [106– 109]. Whilst the relationship between integrins and MT1-MMP is unclear in the context of a 3D ECM, it is tempting to speculate that some degree of cooperativity and coordination underlies the interrelated function of both ECM receptor and ECM degrader. Integrin endocytosis is an important event regulating the ability of cancer cells to invade; for example, HS1-associated protein X-1 (HAX-1), a ubiquitously expressed adaptor protein and regulator of cell migration, controls the clathrin-dependent endocytosis of avb6 integrin to facilitate organotypic invasion [110]. Furthermore, the formin FMNL2 interacts with the a chains of b1 integrins when activated by PKCa and RhoC, to promote integrin internalisation and invasion of melanoma cells [54], and a similar mechanism may also promote trafficking of a5b1 to increase invasion of pancreatic ductal adenocarcinoma cells [111]. Recycling pathways have also been shown to mediate invasive migration; for example, hypoxia promotes migration of cancer cells in Matrigel transwell assays by mobilising Rab11-dependent a6b4 recycling [112], and mobilisation of a3b1 integrin via Arf6–AMAP1 can similarly induce invasive migration of breast cancer cells [88]. avb3 and a5b1 integrins have both been implicated in invasive migration of cancer cells, but the inverse relationship between these heterodimers observed in 2D is also translated in 3D. avb3 recycling via the Rab4–Rabaptin-5–PKD1 pathway promotes migration and invasion in ECM when fibronectin levels are low or absent, by inducing lamellipodial-type migration and invasion (Figure 4A) [79]. Abrogation of avb3 recycling or gene silencing/inhibition of avb3 (using small-molecule inhibitors, e.g. cilengitide and cRGDfV, or soluble ligands copiously produced by tumours e.g. osteopontin) has a matrix-specific effect on invasive migration, inhibiting invasion into low-fibronectin environments but promoting invasion in fibronectin-rich ECM, given that avb3 integrin suppresses the recycling of the major receptor for fibronectin, a5b1 (Figure 4; discussed below) [71,79]. RCP-driven a5b1 Recycling in Cancer Cell Invasion and Metastasis Gain-of-function forms of mutant p53 (p53mut; e.g. 175H, 273H) are known to promote metastasis in mouse models [85,92,113– 115]. These forms of mutant p53 act to suppress expression of the ribonuclease Dicer, in some cases through p63 inhibition, and thereby lead to lowered levels of specific mature microRNAs that influence invasive migration and metastasis [116,117]. The p53mut–Dicer pathway controls a5b1 integrin recycling by regulating the recruitment of this integrin to RCP and, as a consequence, controls the migratory strategy of cancer cells in 2D and increases invasion into fibronectin-rich 3D ECM (Figure 4) [92,117,118]. Interestingly, a mechanistically similar pathway is controlled by avb3 integrin, which inhibits the recruitment of a5b1 to RCP, thus preventing recycling of this integrin. Surprisingly, cells that rapidly recycle a5b1 integrin through the RCP pathway do not show enhanced adhesion [71], and


Current Biology

Review Figure 4. Integrin recycling in cancer cell invasion.

A αvβ3 integrin recycling promotes invasion into low-fibronectin ECM α5β1 integrin Lamellipodial-like protrusions Rac activity

P

Dicer

PKD

Rabaptin-5

Rab4

RacGAP1

miRs RC

p63

P

αvβ3 integrin ECM B α5β1 integrin recycling promotes invasion into fibronectin-rich ECM Actin spikes and filopodial bundles

ECM α5β1 integrin Rac RacGAP1

Dicer RC

Mutant p53

P

p63

DGKα

DAG

RCP EGFR

P PKB/ Akt

IQGAP1 RacGAP1

PA

αvβ3 integrin Small molecules (cilengitide, cRGDfV) Soluble ligands (osteopontin)

RhoA activity FHOD3mediated actin polymerisation

(A) In 3D ECM with a low fibronectin content, trafficking of avb3 integrin promotes invasion, presumably through redistribution of this integrin to the leading edge resulting in high Rac activity and lamellipodial-like protrusions. However, avb3 (and avb3 recycling), or expression of miRNAs through Dicer, suppress the association of a5b1 with RCP. This results in low levels of invasion in the presence of the a5b1 ligand fibronectin. (B) In 3D ECM with a high fibronectin content, when avb3 integrin is inhibited with small cyclic peptides or soluble ligands such as osteopontin, or when mutant p53 inhibits p63 and Dicer, a5b1 integrin is recruited by RCP and rapidly recycled to the plasma membrane. This is coordinated by diacylglycerol kinase (DGK)-a which produces phosphatidic acid (PA) from diacylglycerol (DAG) to recruit RCP-containing vesicles to the pseudopodial tip. RCP and a5b1 integrin recruit RTKs, such as EGFR, and promote their trafficking and localised signalling. This drives PKB/Akt-mediated phosphorylation of RacGAP1, which is recruited to the scaffold IQGAP and inhibits Rac. This in turn allows localised RhoA activity, which drives the formation of actin spikes and bundles through ROCK-mediated activation of FHOD3.

Signalling gradient PKB/Akt and RhoA

Whilst integrin targeting to lysosomes can lead to degradation, integrin ligands such as fibronectin are targeted from late endosomes/lysosomes to the cell surface to promote migration and invaEarly Recycling α5β1 integrin Trafficking routes Activation Inhibition αvβ3 integrin Plasma membrane endosomes endosomes sion [122,123]. Additionally, signalling and macropinosomes coordinated by the scaffold protein p14Current Biology MP1 at late endosomes has been implicated in focal adhesion organisation in therefore changes in adhesive properties do not explain the 2D [124]. More evidence for the role of late endosomes/lysoenhanced invasive capacity of cells into fibronectin-rich ECM. somes in the positive regulation of integrin function has been The RCP–a5b1 complex is able to recruit receptor tyrosine ki- determined from studies of cancer cell invasion. Rab25 is a nases (RTKs), including EGFR, ErbB2 and c-Met ([71,92,118] Rab11 family member that binds directly to the b1-integrin cytoand P.T. Caswell unpublished data). Together, RCP and a5b1 in- plasmic tail to regulate trafficking of a5b1, and as a consetegrin coordinate the recycling of RTKs and potentiate down- quence of this can promote migration and invasion in fibrostream signalling [71,92,118]. In cells moving through 3D ECM, nectin-rich ECM [98]. Rab25 has been described as both an diacylglycerol kinase (DGK)-a produces phosphatidic acid to re- oncogene and tumour suppressor; its contribution to tumouricruit RCP-containing vesicles to the tips of invasive pseudopods genesis is cancer-type and context specific [125]. Rab25extending into the 3D matrix [101], and this creates a local signal containing vesicles localise to the tips of pseudopods as cells through Akt/PKB to promote the recruitment of RacGAP1 to the migrate in 3D ECM, and sort internalised inactive a5b1 for recycytoskeletal scaffold protein IQGAP1 [119]. RacGAP1 directly in- cling to the cell front, which re-engages the ECM to promote activates Rac and allows activation of RhoA specifically at the protrusion [10,98]. Interestingly, Rab25 sorts active a5b1 back front of cells moving in 3D ECM [119]. RhoA in turn promotes towards the cell body through late endosomes to chloride intraROCK-dependent activation of the formin FHOD3, which drives cellular channel 3 (CLIC3)-positive lysosomes, and CLIC3 prethe formation of filopodial actin spike protrusions, rather than la- vents degradation of this integrin by facilitating recycling back mellipodia, to mediate invasive migration in 3D ECM and in vivo to the plasma membrane [10]. This pathway activates Src signal(Figure 4) [120]. Interestingly, p53mut promotes the expression of ling and promotes forward movement of the rear of the cell to inmyosin X, a molecular motor that promotes filopodia formation crease invasive migration, whereas in the absence of CLIC3 and transports integrin to the tips of filopodia, suggesting that in- a5b1 is degraded and cell motility is abrogated; CLIC3 levels tegrin trafficking and filopodia formation may be directly coordi- may therefore dictate the contribution of Rab25 to tumourigenesis. Rab25-positive late endosomes collect internalised a5b1 nated in this context [121]. from tensin-positive adhesions for the delivery of integrin–ligand Late Endosomes, Lysosomes and Rescued Integrins The role of late endosomes and lysosomes in delivery of cargoes complexes to lysosomes, which recruits and activates to regulate cell migration is becoming increasingly apparent. mTORC1, whilst nutrient deprivation promotes internalisation R1100 Current Biology 25, R1092–R1105, November 16, 2015 ª2015 Elsevier Ltd All rights reserved

Current Biology

Review of active integrin–ligand complexes to lysosomes. This link to lysosomal signalling suggests that integrin trafficking supports a favourable bioenergetic profile in addition to controlling integrin trafficking in invasive cancer cells [62]. Future Perspectives Whilst studies of integrin trafficking have given new insight into both integrin function and mechanisms of cell motility, particularly in 3D ECM, several questions remain incompletely answered and recent studies have opened further avenues for investigation. The relationship between RTK signalling and integrin trafficking is fascinating yet complex; for example, growth factor stimulation can lead to changes in integrin internalisation and recycling [65,126], integrin–RTK co-trafficking potentiates signalling [71,118], and integrin–tensin-4 complexes suppress RTK endocytosis to perhaps localise and/or enhance signalling [63]. How these activities are coordinated will help to reveal how the complex spatiotemporal signalling of such pathways is integrated within a migrating cell. It is clear that trafficking of integrins influences adhesion formation/turnover and is likely to affect signalling from adhesion complexes. However, compartments within the endo-lysosomal system are known to act as signalling hubs [127], and how integrin trafficking might be involved in coordinating signalling from intracellular compartments is now beginning to be uncovered [62]. Furthermore, the activation status of trafficking integrins is still somewhat controversial, but a consensus is emerging that integrins can indeed be transported into and through the endo-lysosomal system in inactive (closed) as well as active (extended/primed) conformations [7,24]; however, whether integrins directly promote signals from endocytic compartments by virtue of their activation status is as yet unclear. Tools and techniques have been developed to precisely map localisation of trafficking events; for example, photoactivatable GFP has been used to monitor internalisation [62,128] and recycling [10,76,98,128]. These approaches need to be developed for use alongside biosensors, together with super-resolution microscopy, to reveal micro- and nanoscale organisation of cell systems through endocytic trafficking of specific cargoes, avoiding the caveats associated with biochemical techniques, which capture an average across populations. Furthermore, whilst we are beginning to understand the functional relevance of integrin trafficking in physiological 3D contexts, recent studies have revealed the dynamics of integrin trafficking in vivo within the Drosophila myotendinous junction [129–131]. The next challenge is to visualise integrin trafficking in cells migrating within in vivo contexts, including developmental processes and cancer cell invasion and metastasis, to fully comprehend both how the endocytic trafficking of these compelling cargoes controls cell motility within multicellular organisms, and how they can be targeted in the future for therapeutic benefit.

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Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1-Dependent Trafficking of Integrins.

Endocytic Rabs in membrane trafficking and signaling.

Endocytic pathways and endosomal trafficking: a primer.

Investigating signaling consequences of GPCR trafficking in the endocytic pathway.

Rerouting of plant late endocytic trafficking toward a pathogen interface.

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae.

Rab7b at the intersection of intracellular trafficking and cell migration.

Methods to study endocytic trafficking of the EGF receptor.

Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides.

Cadherin tales: Regulation of cadherin function by endocytic membrane trafficking.

Endocytic Trafficking at the Mature Podocyte Slit Diaphragm.

LMTK3 deficiency causes pronounced locomotor hyperactivity and impairs endocytic trafficking.

The calcium-sensing receptor and integrins modulate cerebellar granule cell precursor differentiation and migration.

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins.

Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation.

Endocytic trafficking of laminin is controlled by dystroglycan and is disrupted in cancers.

Involvement of microtubular network and its motors in productive endocytic trafficking of mouse polyomavirus.

The urokinase receptor takes control of cell migration by recruiting integrins and FPR1 on the cell surface.

Alterations in late endocytic trafficking related to the pathobiology of LRRK2-linked Parkinson's disease.

Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton.

Dynamin2 downregulation delays EGFR endocytic trafficking and promotes EGFR signaling and invasion in hepatocellular carcinoma.

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei.

Endocytic trafficking of membrane-bound cargo: a flotillin point of view.

Distribution of integrins and their ligands in the trunk of Xenopus laevis during neural crest cell migration.