**3. Regulation of cell adhesion dynamics by trafficking adhesive receptors**

The acquisition of key molecular strategies that support social cell functions, such as intercellular communication and adhesion either to other cells or to the surrounding environment, represented a tenet in the evolution from simple unicellular to complex multicellular organisms on the Earth (Rokas, 2008). Indeed, the appearance of genes encoding for adhesion receptors is likely to have represented a major driving force of the so called Cambrian explosion during which, around 500 million years ago, the appearance on our planet of multicellular organisms, aka metazoans, and an astonishingly wide exploration of most of their possible morphological organizations took place (Abedin & King, 2010). In mammalians, the ability of dynamically regulating cell adhesion in space and time is crucial for several physiological and pathological phenomena, such as embryonic development (Hynes, 2007), tissue and organ morphogenesis and repair (Insall & Machesky,

Frizzled (Fz) family. Additionally, Rab4 and Rab11 function in Fz- and Go-mediated signaling to favor PCP over canonical Wingless signaling (Purvanov et al, 2010). Furthermore, the Rab5-effector Rabenosyn-5 is required for the polarized distribution of PCP proteins at the apical cell boundaries aiding the establishment of planar polarity

The requirement for regulation of clathrin-mediated endocytosis in planar cell polarity also emerges from the study showing that the planar polarized RhoGEF2 controls the function of Dia and Myosin II which, in turn, are responsible for the initiation of E-cadherin endocytosis

Another relevant instance of the involvement of endo/exocytosis in the execution of polarized function is directed cell migration. Also in this case important lessons come from the *Drosophila* model. In the fruit fly, endocytosis of motogenic receptors and their recycling to the plasma membrane serve to maintain their polarized distribution at the leading edge of migrating cells, thus promoting directional motility (Jekely et al, 2005; McDonald et al, 2006; McDonald et al, 2003; Montell, 2003; Wang et al, 2006). This is achieved *via* a tight control of endocytosis and recycling in restricted areas of the cell membrane through the regulation of a subset of molecules such as the endocytic E3 ligase Cbl, or the Rab5 GEF Sprint (Jekely et

Collectively, these observations provide genetic evidence that one physiological role of endocytosis is to ensure localized intracellular responses to extracellular cues, i.e. the spatial restriction of signalling. Similar circuitries are also exploited in mammalian cells to achieve and maintain cell polarity and also to execute polarized functions such as directed cell migration (Balasubramanian et al, 2007; Caswell & Norman, 2008; Jones et al, 2006; Palamidessi et al, 2008; Riley et al, 2003; Schlunck et al, 2004). Of note, directed cell migration in mammalian cells has been found to require Rab proteins like Rab25 and Rab5 (Caswell et al, 2007; Palamidessi et al, 2008). Rab25 promotes the extension of long pseudopodia in 3D matrices, by regulating the recycling of a pool of �5�1 (Caswell et al., 2007; detailed in paragraph 3), Instead, Rab5-dependent endocytosis allows for the activation of Rac, induced by motogenic stimuli, on early endosomes. Subsequent recycling of Rac to the plasma membrane ensures localized formation of actin-based migratory

**3. Regulation of cell adhesion dynamics by trafficking adhesive receptors** 

The acquisition of key molecular strategies that support social cell functions, such as intercellular communication and adhesion either to other cells or to the surrounding environment, represented a tenet in the evolution from simple unicellular to complex multicellular organisms on the Earth (Rokas, 2008). Indeed, the appearance of genes encoding for adhesion receptors is likely to have represented a major driving force of the so called Cambrian explosion during which, around 500 million years ago, the appearance on our planet of multicellular organisms, aka metazoans, and an astonishingly wide exploration of most of their possible morphological organizations took place (Abedin & King, 2010). In mammalians, the ability of dynamically regulating cell adhesion in space and time is crucial for several physiological and pathological phenomena, such as embryonic development (Hynes, 2007), tissue and organ morphogenesis and repair (Insall & Machesky,

by regulating their lateral clustering (Levayer et al, 2011).

(Mottola et al, 2010).

al, 2005).

protrusions (Palamidessi et al, 2008).

2009), leukocyte extravasation (Hogg et al, 2011), platelet aggregation (Tao et al, 2010), and cancer cell metastatic dissemination throughout the body (Roussos et al, 2011). Cadherins (Takeichi, 2011) and integrins represent the main classes of transmembrane receptors respectively mediating cell-to-cell and cell-to-extracellular matrix (ECM) adhesion in mammals. A dynamic control of cell adhesion can be accomplished by regulation of either conformation or endo-exocytic traffic of adhesion receptors. Cadherin and integrin conformational activation can be triggered by the binding of either extracellular divalent cations, e.g. Ca2+ for cadherins (Takeichi, 2011) or Mg2+ for integrins (Tiwari et al, 2011), or cytosolic proteins, such as talin and kindlin in the case of integrins (Moser et al, 2009). The mechanisms that directly supersede to the control of cadherin (Gumbiner, 2005; Niessen et al, 2011; Takeichi, 2011) and integrin conformation (Moser et al, 2009; Shattil et al, 2010) have been extensively described elsewhere. Here, we will instead review the emerging evidence of how cell adhesion and migration critically depends on cadherin and integrin traffic.

### **3.1 Role of E-cadherin traffic in adherens junction maintenance and remodeling**

Normal epithelial tissues are hold together by adherens junctions (AJs), *i.e.* cell-to-cell adhesion sites that originate after the dimerization *in trans* of epithelial (E)-cadherin molecules (Gumbiner, 2005; Niessen et al, 2011; Takeichi, 2011). E-cadherin-dependent assembly of AJs is required to assemble and maintain the apico-basal polarity of functional epithelia (Rodriguez-Boulan et al, 2005).

In *Drosophila* and in mammals, the maintenance of both AJs and epithelial polarity depends on a complex formed by the small GTPase Cdc42 and its partner PAR6 that binds aPKC (Goldstein & Macara, 2007; Iden & Collard, 2008; McCaffrey & Macara, 2009).

Interestingly, Cdc42, PAR6, and aPKC are required for the activation of a signaling pathway responsible for the dynamin-driven pinch-off of vesicles during E-cadherin endocytosis from *Drosophila* AJs (Baum & Georgiou, 2011; Georgiou et al, 2008; Leibfried et al, 2008) and a genome wide siRNA screen in *C. elegans* also identified Cdc42, PAR6, and aPKC as key regulators of endocytosis (Balklava et al, 2007). In addition, pharmacological inhibition of dynamin coupled to two-photon FRAP microscopy demonstrated that in mammalian cells E-cadherin engaged at mature stationary AJs turns over by endocytosis and not by free diffusion through the PM (de Beco et al, 2009). *Drosophila* Cdc42 interacting protein 4 (Cip4), aka transducer of Cdc42-dependent actin assembly 1 (TOCA-1) in mammals, displays both an FCH-Bin–Amphiphysin–Rvs (F-BAR) and a Src homology 3 (SH3) domains that respectively bind curved membranes and dynamin (Fricke et al, 2009). Of note, Cip4 knockdown causes AJ and E-cadherin endocytosis defects identical to those caused by the lack of components of the Cdc42/PAR6/aPKC apical complex (Baum & Georgiou, 2011; Leibfried et al, 2008).

Once internalized, E-cadherin is first trafficked to Rab5 containing early endosomes and from there to a Rab11-positive recycling compartment (Emery & Knoblich, 2006; Harris & Tepass, 2010; Wirtz-Peitz & Zallen, 2009). Sec10 and Sec15 proteins then directly bind and interconnect the β-catenin-bound endosomal E-cadherin to the exocyst complex located at the PM, hence favoring the recycling of the adhesion receptor (Langevin et al, 2005).

There is now a mounting consensus that the maintenance of stable AJs requires the continuous and local traffic of E-cadherin back and forth from the PM (Baum & Georgiou,

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 167

migrating in 3D matrices a spatially restricted subpopulation of α5β1 integrin is instead internalized from the PM of ECM-adhesions located at the cell front and quickly recycled back to the same or proximal adhesive structures (Caswell & Norman, 2008; Caswell et al, 2007; Caswell et al, 2009). The Rab11 subfamily member Rab25, which resides in a vesicular compartment located in close proximity of the tips of invading pseudopods, physically interacts with the β1 subunit of the internalized integrins and promotes tumor cell invasion, likely by favoring the localized recycling of α5β1 integrin (Caswell & Norman, 2008; Caswell et al, 2007; Caswell et al, 2009). Another key regulator of integrin traffic in motile cells is the Rab11 effector Rab-coupling protein (RCP), which binds with β3 integrin and, when αvβ3 integrin is inhibited, switches to the cytodomain of β1 integrin, connecting α5β1 integrin with Rab11 and thus favoring its recycling to the PM (Caswell et al, 2008; Caswell et al, 2009). Of note, RCP also associates with EGFR and, upon αvβ3 inhibition, the recycling to the PM of endocytosed EGFR is enhanced in coordination with that of α5β1, finally resulting in an increased EGFR auto-phosphorylation and downstream activation of AKT

In the last couple of years, the new concept that endocytosis of active and inactive integrins could be mediated by different sorting machineries started emerging. Neuropilin 1 (Nrp1) is a transmembrane protein, initially identified in neurons, that is also expressed in ECs, where it works as a co-receptor for both pro- and anti-angiogenic factors, such as vascular endothelial growth factor (VEGF)-A165 and semaphorin 3A (SEMA3A) respectively (Bussolino et al, 2006; Neufeld & Kessler, 2008; Serini & Bussolino, 2004). The very Cterminal SEA motif of Nrp1 cytodomain binds the endocytic adaptor GAIP interacting protein C terminus 1 (GIPC1)/synectin (Cai & Reed, 1999) that can also bind to the motor Myosin VI (Myo6) (Reed et al, 2005). Nrp1, *via* its cytodomain, controls EC adhesion to FN in a way that does not depend on its function as co-receptor for either VEGF-A or SEMA3A, but rather on its ability to promote the GIPC1/synectin- and Myo6-dependent endocytosis of the active, but not inactive conformation of α5β1 integrin from ECM adhesions (Valdembri et al, 2009). Remarkably, Nrp1 silencing does not affect the ratio between active and inactive α5β1 integrin, indicating that not only the conformational switch of integrins, but also the regulation of active integrin traffic and distribution constitutes an equally crucial parameter in the control of EC adhesion to the ECM (Valdembri et al, 2009). It has hence been proposed a model in which, upon FN binding, active α5β1 integrin associates with Nrp1 at the PM. GIPC1/synectin and Myo6 then favor the rapid internalization of the active α5β1/Nrp1 complex into Rab5-positive early endosomes, from which (active) α5β1 is

then recycled back to the PM, likely in newly forming ECM-adhesion sites.

The described endo-exocytic cycle of active integrins back and forth from ECM adhesions is remarkably similar to the traffic dependent E-cadherin dynamics observed in AJs of epithelial cells (*see above*). It is therefore tempting to speculate that also an ECM adhesion site could result from a rapid sequence of localized and exceptionally brief adhesive events, during which traffic could be crucial either to endocytose and then immediately recycle active integrins or to generate the force that has to be applied on ECM-bound active integrins to allow cell adhesion. Likely because GIPC1/synectin also binds the C-terminal SDA motif of the α5 integrin subunit cytodomain (Tani & Mercurio, 2001), while in ECs Nrp1 and Myo6 are specifically dedicated to the endocytosis of active α5β1 integrin, GIPC1 controls inactive α5β1 internalization as well (Valdembri et al, 2009). The different molecular

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

2011; Emery & Knoblich, 2006; Harris & Tepass, 2010; Wirtz-Peitz & Zallen, 2009). Therefore, endless cycles of polarized endocytosis and recycling of E-cadherin are responsible for the existence in space and time of AJs that warrant an efficient intercellular adhesion in stable epithelia.

This would suggest that in living cells, because of the intrinsic physical and biochemical properties of its molecular components, what appears as a stable adhesion site is nothing but an almost continuous and swift spatio-temporal succession of short-lived adhesive events. In this framework, endocytosis could be required either to remove and then replenish *via* recycling the adhesive material or to provide a substantial fraction of the force required to maintain adhesion.

Moreover, the incessant turnover of E-cadherins would allow cells to rapidly adapt the structure of their AJs in response to extracellular signals during tissue reshaping. Indeed, during embryonic development, cancer cell metastatization, and tissue fibrosis epithelial cells activate the epithelial-mesenchymal transition (EMT) program during which they lose their AJs and become motile (Kalluri & Weinberg, 2009; Thiery & Sleeman, 2006). For example, in epithelial cells, hepatocyte growth factor (HGF), acting through the MET tyrosine kinase receptor, activates H-Ras that, by stimulating the Rab5 guanosine exchange factor Ras and Rab interactor 2 (RIN2), induces E-cadherin endocytosis (Kimura et al, 2006). In addition, HGF signals *via* Src and generates a tyrosine phosphosite on E-cadherin where the E3-ubiquitin ligase Hakai docks to trigger the ubiquitination and lysosomal degradation of E-cadherin (Fujita et al, 2002; Palacios et al, 2005).

#### **3.2 Combined regulation of integrin function by conformation and traffic**

Integrin heterodimers can switch from low (inactive) to high affinity (active) conformation for their ECM ligands (Hynes, 2002). Conformational activation of integrins can be due to the interaction of their cytoplasmic tails with different proteins acting as positive (e.g. talin and kindlin) (Moser et al, 2009; Shattil et al, 2010) or negative (e.g. mammary-derived growth inhibitor, MDGI) modulators (Nevo et al, 2010). Due to their ability to mechanosense the surrounding ECM environment and mediate the interactions that support cell adhesion and migration (Parsons et al, 2011), active integrins are key regulators of several important adhesion dependent functions, such as assembly and morphogenetic movements of tissues and organs or migration of isolated/clustered cells through the body (e.g. immune or cancer cells). For example, the remodeling of immature vascular networks that occurs during embryonic, but not tumor angiogenesis, depends on the ability of endothelial cells (ECs) to instantaneously mechanotransduce variations in fluid shear stress (Hahn & Schwartz, 2009).

Integrin traffic is increasingly recognized as a key determinant in the dynamic control of cell adhesion to the ECM (Caswell et al, 2009; Pellinen et al, 2006). Integrins can be internalized in a clathrin-dependent as well as in a clathrin-independent way. For example, α5β1 integrin, the major fibronectin (FN) receptor, can be endocytosed into clathrin-coated vesicles (CCVs) (Pellinen et al, 2008) or by a caveolin-mediated pathway (Shi & Sottile, 2008). It was initially hypothesized that in migrating cells integrins can be preferentially endocytosed in ECM-adhesion sites located at the trailing edge and then recycled back *en masse* toward the leading edge (Bretscher, 1989). More recently, such a theoretical long range model has been challenged by an experimental short range model that showed how in cells

2011; Emery & Knoblich, 2006; Harris & Tepass, 2010; Wirtz-Peitz & Zallen, 2009). Therefore, endless cycles of polarized endocytosis and recycling of E-cadherin are responsible for the existence in space and time of AJs that warrant an efficient intercellular adhesion in stable

This would suggest that in living cells, because of the intrinsic physical and biochemical properties of its molecular components, what appears as a stable adhesion site is nothing but an almost continuous and swift spatio-temporal succession of short-lived adhesive events. In this framework, endocytosis could be required either to remove and then replenish *via* recycling the adhesive material or to provide a substantial fraction of the force

Moreover, the incessant turnover of E-cadherins would allow cells to rapidly adapt the structure of their AJs in response to extracellular signals during tissue reshaping. Indeed, during embryonic development, cancer cell metastatization, and tissue fibrosis epithelial cells activate the epithelial-mesenchymal transition (EMT) program during which they lose their AJs and become motile (Kalluri & Weinberg, 2009; Thiery & Sleeman, 2006). For example, in epithelial cells, hepatocyte growth factor (HGF), acting through the MET tyrosine kinase receptor, activates H-Ras that, by stimulating the Rab5 guanosine exchange factor Ras and Rab interactor 2 (RIN2), induces E-cadherin endocytosis (Kimura et al, 2006). In addition, HGF signals *via* Src and generates a tyrosine phosphosite on E-cadherin where the E3-ubiquitin ligase Hakai docks to trigger the ubiquitination and lysosomal degradation

Integrin heterodimers can switch from low (inactive) to high affinity (active) conformation for their ECM ligands (Hynes, 2002). Conformational activation of integrins can be due to the interaction of their cytoplasmic tails with different proteins acting as positive (e.g. talin and kindlin) (Moser et al, 2009; Shattil et al, 2010) or negative (e.g. mammary-derived growth inhibitor, MDGI) modulators (Nevo et al, 2010). Due to their ability to mechanosense the surrounding ECM environment and mediate the interactions that support cell adhesion and migration (Parsons et al, 2011), active integrins are key regulators of several important adhesion dependent functions, such as assembly and morphogenetic movements of tissues and organs or migration of isolated/clustered cells through the body (e.g. immune or cancer cells). For example, the remodeling of immature vascular networks that occurs during embryonic, but not tumor angiogenesis, depends on the ability of endothelial cells (ECs) to instantaneously mechanotransduce variations in fluid shear stress (Hahn & Schwartz, 2009). Integrin traffic is increasingly recognized as a key determinant in the dynamic control of cell adhesion to the ECM (Caswell et al, 2009; Pellinen et al, 2006). Integrins can be internalized in a clathrin-dependent as well as in a clathrin-independent way. For example, α5β1 integrin, the major fibronectin (FN) receptor, can be endocytosed into clathrin-coated vesicles (CCVs) (Pellinen et al, 2008) or by a caveolin-mediated pathway (Shi & Sottile, 2008). It was initially hypothesized that in migrating cells integrins can be preferentially endocytosed in ECM-adhesion sites located at the trailing edge and then recycled back *en masse* toward the leading edge (Bretscher, 1989). More recently, such a theoretical long range model has been challenged by an experimental short range model that showed how in cells

epithelia.

required to maintain adhesion.

of E-cadherin (Fujita et al, 2002; Palacios et al, 2005).

**3.2 Combined regulation of integrin function by conformation and traffic** 

migrating in 3D matrices a spatially restricted subpopulation of α5β1 integrin is instead internalized from the PM of ECM-adhesions located at the cell front and quickly recycled back to the same or proximal adhesive structures (Caswell & Norman, 2008; Caswell et al, 2007; Caswell et al, 2009). The Rab11 subfamily member Rab25, which resides in a vesicular compartment located in close proximity of the tips of invading pseudopods, physically interacts with the β1 subunit of the internalized integrins and promotes tumor cell invasion, likely by favoring the localized recycling of α5β1 integrin (Caswell & Norman, 2008; Caswell et al, 2007; Caswell et al, 2009). Another key regulator of integrin traffic in motile cells is the Rab11 effector Rab-coupling protein (RCP), which binds with β3 integrin and, when αvβ3 integrin is inhibited, switches to the cytodomain of β1 integrin, connecting α5β1 integrin with Rab11 and thus favoring its recycling to the PM (Caswell et al, 2008; Caswell et al, 2009). Of note, RCP also associates with EGFR and, upon αvβ3 inhibition, the recycling to the PM of endocytosed EGFR is enhanced in coordination with that of α5β1, finally resulting in an increased EGFR auto-phosphorylation and downstream activation of AKT (Caswell et al, 2008; Caswell et al, 2009).

In the last couple of years, the new concept that endocytosis of active and inactive integrins could be mediated by different sorting machineries started emerging. Neuropilin 1 (Nrp1) is a transmembrane protein, initially identified in neurons, that is also expressed in ECs, where it works as a co-receptor for both pro- and anti-angiogenic factors, such as vascular endothelial growth factor (VEGF)-A165 and semaphorin 3A (SEMA3A) respectively (Bussolino et al, 2006; Neufeld & Kessler, 2008; Serini & Bussolino, 2004). The very Cterminal SEA motif of Nrp1 cytodomain binds the endocytic adaptor GAIP interacting protein C terminus 1 (GIPC1)/synectin (Cai & Reed, 1999) that can also bind to the motor Myosin VI (Myo6) (Reed et al, 2005). Nrp1, *via* its cytodomain, controls EC adhesion to FN in a way that does not depend on its function as co-receptor for either VEGF-A or SEMA3A, but rather on its ability to promote the GIPC1/synectin- and Myo6-dependent endocytosis of the active, but not inactive conformation of α5β1 integrin from ECM adhesions (Valdembri et al, 2009). Remarkably, Nrp1 silencing does not affect the ratio between active and inactive α5β1 integrin, indicating that not only the conformational switch of integrins, but also the regulation of active integrin traffic and distribution constitutes an equally crucial parameter in the control of EC adhesion to the ECM (Valdembri et al, 2009). It has hence been proposed a model in which, upon FN binding, active α5β1 integrin associates with Nrp1 at the PM. GIPC1/synectin and Myo6 then favor the rapid internalization of the active α5β1/Nrp1 complex into Rab5-positive early endosomes, from which (active) α5β1 is then recycled back to the PM, likely in newly forming ECM-adhesion sites.

The described endo-exocytic cycle of active integrins back and forth from ECM adhesions is remarkably similar to the traffic dependent E-cadherin dynamics observed in AJs of epithelial cells (*see above*). It is therefore tempting to speculate that also an ECM adhesion site could result from a rapid sequence of localized and exceptionally brief adhesive events, during which traffic could be crucial either to endocytose and then immediately recycle active integrins or to generate the force that has to be applied on ECM-bound active integrins to allow cell adhesion. Likely because GIPC1/synectin also binds the C-terminal SDA motif of the α5 integrin subunit cytodomain (Tani & Mercurio, 2001), while in ECs Nrp1 and Myo6 are specifically dedicated to the endocytosis of active α5β1 integrin, GIPC1 controls inactive α5β1 internalization as well (Valdembri et al, 2009). The different molecular

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 169

endosomal membranes (Ayad et al, 1997). During mitosis, phosphorylated Rab4 is in the cytosol complexed with the peptidyl-prolyl isomerase Pin1 and it is no longer able to recruit downstream effectors on endosomes (Gerez et al, 2000). Thus an appealing possibility is that Rab4 phosphorylation might participates in the inhibition of the recycling pathway

Of note, fusion of early endosomes in mitosis is blocked via cdc2-dependent phosphorylation events (Tuomikoski et al, 1989). This might represent an additional mechanism to inhibit vesicles recycling at the plasma membrane by altering the homeostasis of the endosomal compartment and affecting the generation of exocytic vesicles. Inhibition of homotypic fusion of early endosomes at mitosis is also caused by decreased residence time of the early endosome-tethering molecule EEA1 on endosomal membranes (Bergeland et al, 2008). It would be interesting to define how the acceleration of the EEA1 cycle between

Endocytic/trafficking proteins are also emerging as important factors required for the proper execution of cell division. Beside the involvement of trafficking molecules in membrane delivery to the cleavage furrow at cytokinesis [for recent reviews see (McKay & Burgess, 2011; Montagnac et al, 2008)], some of these proteins also display specific functions

One of the best-characterized endocytic molecules showing a distinct role in mitosis is the clathrin heavy chain. The clathrin complex is organized in a triskelion made of three heavy chains each with an associated light chain (ter Haar et al, 1998). At metaphase, clathrin also localizes to kinetochore fibers (spindle microtubules connecting kinetochores to spindle poles) of the spindle apparatus (Royle et al, 2005). Here it stabilizes spindle microtubules aiding congression of chromosomes on the metaphase plate. Depletion of clathrin heavy chain by RNA interference causes failure in the correct attachment of chromosomes to kinetochore fibers resulting in misaligned chromosomes and in persistent activation of the mitotic checkpoint thus prolonging mitosis (Royle et al, 2005). More recently, some advances in understanding clathrin function at the spindle have been made. Clathrin heavy chain has been found to bind to TACC3, phosphorylated on serine 558 by Aurora A, and to recruit it to the spindle. In turn, TACC3 is responsible for localization of ch-TOG, a protein that promotes microtubule assembly and spindle stability, to spindles (Lin et al, 2010). In agreement, functional ablation of clathrin heavy chain causes loss of ch-TOG from spindles and destabilizes kinetochore fibers affecting chromosome congression. Based on electron microscopy data, it has been proposed that TACC3/ch-TOG/clathrin heavy chain complex works as an inter-microtubules bridge that stabilizes kinetochore fibers by physical

measured by Boucrot and Kirchhausen during the early steps of mitosis.

cytosol and membranes is achieved in mitotic cells.

in mitosis. Here we will review knowledge rising on this issue.

crosslinking reducing the rate of microtubule catastrophe (Booth et al, 2011).

spindles (Liu & Zheng, 2009).

Another important endocytic player is epsin, an adaptor molecule that binds and deforms membranes driving curvature of clathrin-coated pits (Ford et al, 2002). At mitosis, epsin participates in spindle morphogenesis indirectly through its ability to regulate mitotic membrane organization (Liu & Zheng, 2009). In cells depleted of epsin, by RNAi-mediated silencing, the membrane network that uniformly surrounds the chromosomes is distorted with uneven membrane distribution frequently showing layers of membrane whorls. This, in turn, alters spindle morphology resulting in splayed spindle poles and multipolar

composition of the machineries that control active *vs.* inactive integrin traffic could imply that higher amounts of endocytic proteins are required to effectively internalize ECM-bound integrins. Accordingly, the force-generating retrograde motor Myo6 (Spudich & Sivaramakrishnan, 2010) participates to endocytosis, transport of endosomal vesicles along F-actin (Hasson, 2003), and active integrin internalization (Valdembri et al, 2009) as well.

Clathrin coats exist either as classical curved clathrin-coated pits or as flat clathrin-coated plaques that depend on the presence of the actin cytoskeleton and occur only at ECMadherent surfaces, indicating that integrin-mediated adhesion of cells to the ECM likely control the organization of the different clathrin-based endocytic structures (Kirchhausen, 2009; Saffarian et al, 2009). The potential role of cell-to-ECM adhesion in regulating clathrinmediated endocytosis is further supported by the recent experimental observation that the closer clathrin-coated pits are to integrin-containing adhesion sites the slower are their internalization dynamics (Batchelder & Yarar, 2010). It is indeed possible that the binding of integrins to the ECM could give rise to forces that counteract the pulling forces required to deform and curve the PM to finally allow clathrin-based internalization. Such a hypothesis could also account for the requirement of different molecular complexes for active *vs.* inactive integrin internalization.

To date, only few proteins have been selectively involved in inactive, but not active, integrin traffic and the degree of specificity for the bent/inactive integrin conformation is still matter of debate. A prominent example is represented by the endocytic adaptor protein disabled 2 (DAB2), that is able to directly bind the cytodomain of integrin β subunits (Calderwood et al, 2003), and was recently found to selectively promote the internalization of inactive β1 integrins (Teckchandani et al, 2009). However, during ECM-adhesion disassembly experiments, Chao and Kunz, by incubating living cells with the anti-active β1 integrin monoclonal antibody 12G10, found that active β1 integrins could be endocytosed in a DAB2-dependent manner as well (Chao & Kunz, 2009). However, since incubation of living cells with function activating or blocking antibodies represents a significant bias in the study of integrin activation physiology, further work is needed to better characterize the role of DAB2.
