**2. Endocytosis and signalling**

Through endocytosis, active, signalling receptors - such as receptor tyrosine kinases (RTKs) – are removed from the plasma membrane (PM) and destined for degradation and this is crucial to achieve signal extinction and long-term attenuation. Endocytosis is able to remodel the composition of the plasma membrane (PM), thus allowing plasticity in the cellular responses to the microenvironment. Recent evidence, however, has demonstrated that endocytosis has a broader impact on signalling than simply signal extinction (Scita & Di Fiore, 2010; Sorkin & von Zastrow, 2009). Indeed, internalized receptors (and sometimes their ligands) are not only routed to the lysosome for degradation, but, in some cases, can be recycled to specific regions of the PM where polarized signalling is needed for events such as cell migration. Furthermore, signalling might not only occur from the PM, but also could persist along the endocytic pathway as, in the endosomal compartments, signalling receptors are often still bound to their ligands, and continue to be active. More interestingly, signalling receptors in the endosomal compartment could potentially interact with substrates that are not present at the PM. Under this scenario, endocytosis would be a mechanism to sustain signalling and to achieve signal diversification and specificity.

#### **2.1 Signalling elicited by the endocytic compartments**

The concept that signalling continues along the endocytic pathway was shown in the case of several signalling receptors, including RTKs and the TGFβR (tumor growth factor β receptor) (Sorkin & von Zastrow, 2009). In all cases, receptors remain bound to their ligand and active once internalized within endosomes, thus sustaining signalling from the intracellular compartments (Burke et al, 2001; Di Guglielmo et al, 1994; Grimes et al, 1996; Haugh et al, 1999; Hayes et al, 2002; Howe et al, 2001; Lai et al, 1989; Pennock & Wang, 2003;

aspect played by traffic in the dynamic control of cell-to-cell and cell-to-extracellular matrix

We, therefore, propose to illustrate the state of the art together with most recent discoveries

1. The signaling endosome: a modality to finely tune persistence of extracellular stimuli inside the cell and to control their re-distribution and compartmentalization. The latter aspect is of extreme relevance for the role of endo-exo membrane trafficking in the

2. Involvement of endocytosis and exocytosis in the formation and turnover of cell-to-cell and cell-to-extracellular matrix adhesion. We will review the major findings showing the relevance of membrane trafficking of adhesive receptors, namely cadherins and integrins, and describing the molecular machinery involved that has been identified so far. We will also address recent work indicating that distinct molecular machineries are

Trafficking molecules also participates to cell cycle progression and to the correct execution of mitosis. We will review the knowledge raised on this issue and discuss how the function of these molecules is related to their established role in membrane

Through endocytosis, active, signalling receptors - such as receptor tyrosine kinases (RTKs) – are removed from the plasma membrane (PM) and destined for degradation and this is crucial to achieve signal extinction and long-term attenuation. Endocytosis is able to remodel the composition of the plasma membrane (PM), thus allowing plasticity in the cellular responses to the microenvironment. Recent evidence, however, has demonstrated that endocytosis has a broader impact on signalling than simply signal extinction (Scita & Di Fiore, 2010; Sorkin & von Zastrow, 2009). Indeed, internalized receptors (and sometimes their ligands) are not only routed to the lysosome for degradation, but, in some cases, can be recycled to specific regions of the PM where polarized signalling is needed for events such as cell migration. Furthermore, signalling might not only occur from the PM, but also could persist along the endocytic pathway as, in the endosomal compartments, signalling receptors are often still bound to their ligands, and continue to be active. More interestingly, signalling receptors in the endosomal compartment could potentially interact with substrates that are not present at the PM. Under this scenario, endocytosis would be a

mechanism to sustain signalling and to achieve signal diversification and specificity.

The concept that signalling continues along the endocytic pathway was shown in the case of several signalling receptors, including RTKs and the TGFβR (tumor growth factor β receptor) (Sorkin & von Zastrow, 2009). In all cases, receptors remain bound to their ligand and active once internalized within endosomes, thus sustaining signalling from the intracellular compartments (Burke et al, 2001; Di Guglielmo et al, 1994; Grimes et al, 1996; Haugh et al, 1999; Hayes et al, 2002; Howe et al, 2001; Lai et al, 1989; Pennock & Wang, 2003;

**2.1 Signalling elicited by the endocytic compartments** 

required for trafficking integrins in active and inactive conformation. 3. Unconventional function of membrane trafficking proteins in mitosis.

contacts.

on the following issues:

trafficking.

**2. Endocytosis and signalling** 

execution of cell polarity programs.

Wada et al, 1992; Wang et al, 2004; Wang et al, 1996). In agreement with this, all the components of the MAPK (mitogen-activated protein kinase) activation cascade can be found in endosomes (Pol et al, 1998; Roy et al, 2002), showing that RTKs signalling persist also after internalization. In this way, sufficient duration and amplitude to signalling is allowed. Furthermore, endosomal-specific proteins have been identified and shown to be required to sustain signalling. One example is represented by P18, which works at the endosomal membrane as an anchor for an ERK-activating scaffold and is required to achieve maximal activation of ERK1/2 (Nada et al, 2009). A similar mechanism occurs in the case of GPCR (G protein-coupled receptor) signalling, where β-arrestin, similarly to P18, acts as a specific scaffold to anchor ERK1/2 to the endosome (DeWire et al, 2007) thus allowing proper signal duration.

A series of genetic evidence support a role for endocytosis in the sustaining of the signalling. Historically, the first proof was provided by the use of a dominant-negative mutant of dynamin that blocks EGF internalization and causes the inhibition of EGF-induced activation of PI3K and ERKs (extracellular signal-regulated kinases) (Vieira et al, 1996). This initial evidence was then reinforced by experiments with siRNAs (small interfering RNAs) targeting proteins involved in internalization, which show that endocytosis is required for ERK activation by several receptor kinases [reviewed in (Sorkin & von Zastrow, 2009)]. Not only endocytosis is crucial to sustain signalling, but it is also required to determine signal specificity and diversification. Indeed, endosomes can support signalling cascades that cannot happen at the PM. The existence of endosomespecific signalling cascades has been shown for different receptor systems, including RTKs, GPCRs and Notch (reviewed in (Scita & Di Fiore, 2010; Sorkin & von Zastrow, 2009)). In the TGFβR pathway, specific signalling proteins are recruited to endosomes through their binding to PI3P (phosphatidylinositol 3-phosphate, which is enriched in endosomal membrane compared to PM) and this allows intracellular-specific signalling. Indeed, the activated TGFβR receptor interacts with the adaptor protein SARA (smad anchor for receptor activation) in early endosomes. SARA is associated with the receptor target SMAD2, and this allows the efficient phosphorylation of SMAD2 by TGFβR in endosomes (Chen et al, 2007; Hayes et al, 2002; Tsukazaki et al, 1998). Once phosphorylated, SMAD2 forms a complex with SMAD4, which translocates to the nucleus to regulate gene transcription.

Importantly, early endosomes are a morphologically and functionally heterogeneous population, characterized by the presence of biochemically distinct membrane subdomains (Lakadamyali et al, 2006; Miaczynska et al, 2004; Sonnichsen et al, 2000; Zoncu et al, 2009).

At the molecular level, small GTPases play a crucial role in determining the different sorting fates of cargoes at these stations, which ultimately impact on the final signalling response [reviewed in (Stenmark, 2009)]. For instance, APPL1-containing endosomes are precursors of early endosomes specifically enriched in Rab5 but lacking EEA1. It has been proposed that the progressive accumulation of PI3P species (through association and activity of phosphatidylinositol 3-kinase, PI3KC3/Vps34) causes the recruitment of EEA1, which competes with APPL1 for Rab5 binding, displacing it from the maturing early endosomes (Zoncu et al, 2009). Importantly, APPL1- but not EEA1-positive endosomes are competent for AKT signalling (Zoncu et al, 2009). This "phosphoinositide switch" is responsible for the maturation of endosomes and it is involved in signalling specification.

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 161

back to the PM, ready to undergo an additional round of activation (Decker, 1990; Ebner & Derynck, 1991; French et al, 1995; Longva et al, 2002). In agreement with this, TGFα is a more potent mitogen than EGF (Waterman et al, 1998). The idea that endosome sorting regulates signalling output as a function of ligand type was shown also in the case of KGFR (keratinocyte growth factor receptor). Indeed, stimulation with two different ligands, KGF or FGF10, targets the receptor to two distinct trafficking routes, degradation vs. recycling, respectively, and this correlates with the higher mitogenic activity exerted by FGF10 on

The central role of endocytosis in cellular signalling raises the possibility that alteration of this process might contribute to pathological phenotypes in which aberrant signalling is central, such as development and progression of cancer. Several lines of indirect evidence support a role of endocytosis in cancer [reviewed in (Lanzetti & Di Fiore, 2008; Mosesson et al, 2008)]. However, solid proof for a causative role of endocytosis in tumourigenesis is missing. A recent advance in this direction came from a study by Kermorgant's group (Joffre et al, 2011), who investigated the mechanism leading to tumourigenesis of two oncogenic Met mutants (M1268T and D1246N). These mutations cause constitutive Met kinase activity that was originally considered at the basis of their oncogenic potential. By using a combination of *in vitro* and *vivo* approaches, Kermorgant's group showed that endocytosis and intracellular trafficking of these mutants play a crucial role in determining their tumorigenic activity, besides their basal kinase activation. Indeed, these mutants are constitutively internalized and recycled back to the PM at a higher rate compared to WT receptor, and they also show impaired degradation. Importantly, inhibition of internalization with different genetic and pharmaceuticals tools is able to significantly reduce the ability of these mutants of induce transformation *in vitro* and to generate tumours in *ex vivo xenograft* experiments, without altering their activation status. Although the endocytic mechanism used by these mutant receptors is far to be clear (they seem to enter a constitutive pathway that depends on Cbl, Grb2, Clathrin and dynamin and that is independent from receptor kinase activity and ubiquitination), this is the first evidence for a

direct involvement of endocytosis and endosome sorting in cancer development.

Different internalization pathways are often associated to distinct intracellular fates. Several signalling receptors, including RTKs, GPCRs, TGFβR, NOTCH and WNT undergo both clathrin-mediated endocytosis (CME) and non-clathrin endocytosis (NCE) and this influences the final signalling output (Le Roy & Wrana, 2005). A mechanism of this kind takes place during internalization and signalling of the EGFR (Sigismund et al, 2005). At low doses of EGF, the EGFR is almost exclusively internalized through CME, which leads to recycling of the receptor and sustains signalling, with only a minor fraction of EGFRs targeted to degradation (Sigismund et al, 2008). At higher doses, about half of the ligandengaged receptors are then internalized through NCE, a pathway that targets EGFRs to lysosomal degradation causing rapid signal extinction (Sigismund et al, 2008). This dual mechanism perfectly couples with the different EGF concentrations found in body fluids [ranging from 1 to hundreds of ng/ml, reviewed in (Sigismund et al, 2005)]. Indeed, under scarce ligand availability, endocytosis (through CME) preserves the activated receptors from degradation, maximizing the signalling response; at high EGF, the NCE pathway destines

**2.3 Different trafficking routes determine signalling outputs** 

epithelial cells (Belleudi et al, 2007).

A non-canonical example of endosome-specific signalling is provided by the TNFR (tumor necrosis factor receptor) signalling cascade (Schutze et al, 2008) that promotes pro-apoptotic signalling. The components of this pathways are recruited to the ligand-bound TNFR at the plasma membrane (Micheau & Tschopp, 2003). In order for apoptosis to be achieved, the cysteine protease caspase-8 has to be activated by its proteolytic cleavage and this occurs on endosomes (Schneider-Brachert et al, 2004). Although, the mechanisms that prevent caspase-8 recruitment and activation at the PM are not yet known, this represents another example of how endocytosis contributes to signal specificity.

#### **2.2 Regulation of signalling by endosome sorting**

Once internalized and sorted to the early endosomes, cargoes can be routed to degradative pathways, terminating signalling, or recycled back to PM, allowing further rounds of activation. Both these mechanisms contribute to regulate signalling in space and time [reviewed in (Marchese et al, 2008; Sorkin & von Zastrow, 2009)].

Transfer of activated receptors to late endosomes/multivesicular bodies (MVB) terminates signalling, either by sequestering receptors in intraluminal vesicles, thus preventing their interaction with signal transducers, or by promoting their lysosomal degradation. Receptor ubiquitination plays a critical role in this process. Indeed, several protein complexes harbouring ubiquitin (Ub)-binding domains recognize ubiquitinated cargoes and escort them along the degradative route to the lysosome (Dikic et al, 2009). These complexes called ESCRT (endosomal sorting complex required for transport) act sequentially at various stations of the degradative route and are involved in MVB inward vesicles budding and cargo sequestration in the intraluminal vesicles of MVBs [for reviews see (Hurley & Hanson, 2010; Raiborg & Stenmark, 2009)].

On the other hand, recycling of internalized receptors to the PM allows the recovery of unoccupied/free receptors to the cell surface and restores receptor sensitivity to extracellular ligands, as is the case for GPCRs. One classical example is represented by β2AR (β2 adrenergic receptor). This class of receptors signals though interaction with PM-resident trimeric G proteins, which transduce signalling from the PM. Upon agonist stimulation, coupling of β2AR trimeric G proteins is inhibited by receptor phosphorylation events [see, for instance, (Benovic et al, 1985; Benovic et al, 1986; Pitcher et al, 1992), reviewed in (Kelly et al, 2008)], which cause functional desensitization of signalling in the absence of endocytosis. However, β-arrestins are recruited to the phosphorylated receptors, triggering their internalization and sorting into a rapid recycling pathway. This step promotes receptor dephosphorylation by an endosome-associated PP2A protein phosphatase, thus ensuring the return of intact receptor for successive rounds of signalling (Pitcher et al, 1995; Vasudevan et al 2011; Yang et al, 1988), a process called "resensitization".

A related example, where the differential trafficking fate determines the duration of the signal, is represented by the EGFR system. When stimulated with TGFα or EGF, EGFR is rapidly internalized. However, while EGF binding to EGFR remains stable at the pH of endosomes, TGFα rapidly dissociates from the receptor. This results in different signalling outputs: EGFR/EGF complex remains stable and active at the endosomal station and is then transported to lysosomes for degradation, allowing signal termination; in contrast, in the case of TGFα, the receptor detaches from ligand at the endosomal station and it is recycled

A non-canonical example of endosome-specific signalling is provided by the TNFR (tumor necrosis factor receptor) signalling cascade (Schutze et al, 2008) that promotes pro-apoptotic signalling. The components of this pathways are recruited to the ligand-bound TNFR at the plasma membrane (Micheau & Tschopp, 2003). In order for apoptosis to be achieved, the cysteine protease caspase-8 has to be activated by its proteolytic cleavage and this occurs on endosomes (Schneider-Brachert et al, 2004). Although, the mechanisms that prevent caspase-8 recruitment and activation at the PM are not yet known, this represents another example

Once internalized and sorted to the early endosomes, cargoes can be routed to degradative pathways, terminating signalling, or recycled back to PM, allowing further rounds of activation. Both these mechanisms contribute to regulate signalling in space and time

Transfer of activated receptors to late endosomes/multivesicular bodies (MVB) terminates signalling, either by sequestering receptors in intraluminal vesicles, thus preventing their interaction with signal transducers, or by promoting their lysosomal degradation. Receptor ubiquitination plays a critical role in this process. Indeed, several protein complexes harbouring ubiquitin (Ub)-binding domains recognize ubiquitinated cargoes and escort them along the degradative route to the lysosome (Dikic et al, 2009). These complexes called ESCRT (endosomal sorting complex required for transport) act sequentially at various stations of the degradative route and are involved in MVB inward vesicles budding and cargo sequestration in the intraluminal vesicles of MVBs [for reviews see (Hurley & Hanson,

On the other hand, recycling of internalized receptors to the PM allows the recovery of unoccupied/free receptors to the cell surface and restores receptor sensitivity to extracellular ligands, as is the case for GPCRs. One classical example is represented by β2AR (β2 adrenergic receptor). This class of receptors signals though interaction with PM-resident trimeric G proteins, which transduce signalling from the PM. Upon agonist stimulation, coupling of β2AR trimeric G proteins is inhibited by receptor phosphorylation events [see, for instance, (Benovic et al, 1985; Benovic et al, 1986; Pitcher et al, 1992), reviewed in (Kelly et al, 2008)], which cause functional desensitization of signalling in the absence of endocytosis. However, β-arrestins are recruited to the phosphorylated receptors, triggering their internalization and sorting into a rapid recycling pathway. This step promotes receptor dephosphorylation by an endosome-associated PP2A protein phosphatase, thus ensuring the return of intact receptor for successive rounds of signalling (Pitcher et al, 1995;

A related example, where the differential trafficking fate determines the duration of the signal, is represented by the EGFR system. When stimulated with TGFα or EGF, EGFR is rapidly internalized. However, while EGF binding to EGFR remains stable at the pH of endosomes, TGFα rapidly dissociates from the receptor. This results in different signalling outputs: EGFR/EGF complex remains stable and active at the endosomal station and is then transported to lysosomes for degradation, allowing signal termination; in contrast, in the case of TGFα, the receptor detaches from ligand at the endosomal station and it is recycled

Vasudevan et al 2011; Yang et al, 1988), a process called "resensitization".

of how endocytosis contributes to signal specificity.

**2.2 Regulation of signalling by endosome sorting** 

2010; Raiborg & Stenmark, 2009)].

[reviewed in (Marchese et al, 2008; Sorkin & von Zastrow, 2009)].

back to the PM, ready to undergo an additional round of activation (Decker, 1990; Ebner & Derynck, 1991; French et al, 1995; Longva et al, 2002). In agreement with this, TGFα is a more potent mitogen than EGF (Waterman et al, 1998). The idea that endosome sorting regulates signalling output as a function of ligand type was shown also in the case of KGFR (keratinocyte growth factor receptor). Indeed, stimulation with two different ligands, KGF or FGF10, targets the receptor to two distinct trafficking routes, degradation vs. recycling, respectively, and this correlates with the higher mitogenic activity exerted by FGF10 on epithelial cells (Belleudi et al, 2007).

The central role of endocytosis in cellular signalling raises the possibility that alteration of this process might contribute to pathological phenotypes in which aberrant signalling is central, such as development and progression of cancer. Several lines of indirect evidence support a role of endocytosis in cancer [reviewed in (Lanzetti & Di Fiore, 2008; Mosesson et al, 2008)]. However, solid proof for a causative role of endocytosis in tumourigenesis is missing. A recent advance in this direction came from a study by Kermorgant's group (Joffre et al, 2011), who investigated the mechanism leading to tumourigenesis of two oncogenic Met mutants (M1268T and D1246N). These mutations cause constitutive Met kinase activity that was originally considered at the basis of their oncogenic potential. By using a combination of *in vitro* and *vivo* approaches, Kermorgant's group showed that endocytosis and intracellular trafficking of these mutants play a crucial role in determining their tumorigenic activity, besides their basal kinase activation. Indeed, these mutants are constitutively internalized and recycled back to the PM at a higher rate compared to WT receptor, and they also show impaired degradation. Importantly, inhibition of internalization with different genetic and pharmaceuticals tools is able to significantly reduce the ability of these mutants of induce transformation *in vitro* and to generate tumours in *ex vivo xenograft* experiments, without altering their activation status. Although the endocytic mechanism used by these mutant receptors is far to be clear (they seem to enter a constitutive pathway that depends on Cbl, Grb2, Clathrin and dynamin and that is independent from receptor kinase activity and ubiquitination), this is the first evidence for a direct involvement of endocytosis and endosome sorting in cancer development.

#### **2.3 Different trafficking routes determine signalling outputs**

Different internalization pathways are often associated to distinct intracellular fates. Several signalling receptors, including RTKs, GPCRs, TGFβR, NOTCH and WNT undergo both clathrin-mediated endocytosis (CME) and non-clathrin endocytosis (NCE) and this influences the final signalling output (Le Roy & Wrana, 2005). A mechanism of this kind takes place during internalization and signalling of the EGFR (Sigismund et al, 2005). At low doses of EGF, the EGFR is almost exclusively internalized through CME, which leads to recycling of the receptor and sustains signalling, with only a minor fraction of EGFRs targeted to degradation (Sigismund et al, 2008). At higher doses, about half of the ligandengaged receptors are then internalized through NCE, a pathway that targets EGFRs to lysosomal degradation causing rapid signal extinction (Sigismund et al, 2008). This dual mechanism perfectly couples with the different EGF concentrations found in body fluids [ranging from 1 to hundreds of ng/ml, reviewed in (Sigismund et al, 2005)]. Indeed, under scarce ligand availability, endocytosis (through CME) preserves the activated receptors from degradation, maximizing the signalling response; at high EGF, the NCE pathway destines

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 163

implications in particular in the establishment of cell polarity, a process that largely relies on the correct localization of protein complexes and signalling platforms at cell-to-cell and cellto–extracellular matrix contacts. In this regards, a key role in the controlled distribution of signal transducers in restricted areas of the plasma membrane, in response to extracellular

Rab5 is a master regulator of endocytosis and actin remodelling (Lanzetti et al, 2004; Lanzetti et al, 2000; Palamidessi et al, 2008; Zerial & McBride, 2001). It controls the internalization of a variety of distinct receptors, including the adhesive molecules integrins and cadherins (Palacios et al, 2005; Pellinen et al, 2006), as detailed in paragraph 3, thus participating to the processes of cell-to-cell and cell-to-extracellular matrix adhesion. Importantly, in *Drosophila melanogaster* deletion of Rab5 or disruption of the endocytic protein Syntaxin/Avalanche affects the polarized, restricted apical distribution of the fatedecision receptor Notch and of the polarity determinant Crumbs (Lu & Bilder, 2005). Failure in internalization of Notch and Crumbs causes their accumulation and results in the expansion of the apical membrane domain. Impaired Notch internalization severely impacts on its degradation and signalling and, in turn, this leads to overgrowth of imaginal epithelial tissues (Lu & Bilder, 2005) indicating that endocytosis may also control epithelial

Rab8 participates in polarized transport of molecules to the basolateral membrane (Huber et al, 1993) and also in cilia (Nachury et al, 2007). Genetic deletion of Rab8 in mice has been found to affect the distribution of apical proteins to the surface of intestinal epithelial cells resulting in accumulation of vacuoles containing apical hydrolases and microvilli with the final outcome of animal death by starvation (Sato et al, 2007). Thus, Rab8 has been proposed to play a crucial role in the biogenesis of the apical membrane, a process that is profoundly influenced also by another Rab protein involved in recycling routes: Rab11 [reviewed in (Hoekstra et al, 2004)]. Indeed trafficking *via* the recycling endosomes is required for the establishment or rearrangement of cell polarity in various settings including cellularization, cell–to-cell boundary rearrangement, asymmetric cell division, and cell migration (Assaker et al, 2010; Bryant et al, 2010; Emery et al, 2005; Xu et al, 2011). Furthermore, it provides a very efficient mechanism to reinforce polarity by feedback loops (Assaker et al, 2010).

In addition to these GTPases, the endocytic protein Numb has also been implicated in the establishment of apical-basolateral polarity. Numb participates to cadherin endocytosis by interacting with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induces mislocalization of E-cadherin from the lateral to the apical membrane. This function of Numb appears to rely on its phosphorylation by Atypical protein kinase C (aPKC), a member of the PAR complex, as it prevents association of phosphorylated Numb with p120 and α-adaptin thereby attenuating E-cadherin endocytosis

Beside the involvement of endo-exocytosis in apical-basolateral polarity, these trafficking routes are also required in the establishment of planar cell polarity (PCP) [for a detailed reviews on membrane trafficking in cell polarity see (Nelson, 2009)]. Intracellular membrane trafficking has emerged as a crucial regulator of PCP in the *Drosophila* wing where inhibition of dynamin or Rab11 disrupts PCP-dependent hexagonal repacking (Classen et al, 2005). More recently, Rab5 has been found to bind to Go and to participates in PCP and in Wingless signal transduction, pathways initiated by G-protein coupled receptors of the

cues, is played by small GTPases of the Rab family like Rab5, Rab8 and Rab11.

tissue proliferation.

(Sato et al, 2011).

the excess of activated EGFR/EGF complex to degradation, protecting cells from overstimulation. This concept has been challenged in other studies, where EGFR was reported to be internalized exclusively through CME at all concentrations of EGF (Kazazic et al, 2006; Rappoport & Simon, 2009). The discrepancy may be due to the different cellular systems used in these studies. It still remains to establish the nature of the NCE pathway used by the EGFR and the molecular mechanism involved [which is still poorly characterized, although it has been shown to be caveolin-independent and to require receptor ubiquitination (Sigismund et al, 2008; Sigismund et al, 2005)].

A similar scenario was previously reported in the case of TGFβR. This receptor is internalized both by CME and NCE and this has profound impact on the final signalling output (Di Guglielmo et al, 2003). Proteins of the TGFβ superfamily signal through the transmembrane Ser-Thr kinase TGFβR type I and type II heteromeric complex (TβRI and TβRII). Ligand-induced assembly of the heteromeric receptor complex activates TβRI, which initiates Smad signalling by phosphorylating the receptor-regulated Smads. The Smad adaptor protein SARA plays a crucial role at this step. Indeed, SARA binds the receptor and contains a FYVE (Fab1p, YOTB, Vac1p and EEA1) domain, which also binds to membranes through specific interactions with phosphatidyl inositol 3′ phosphate (PI3P). Receptor internalization through the clathrin pathway is essential for signalling and SARA has been found in the PI(3)P-enriched EEA1-positive endosomes that are downstream of this route (Di Guglielmo et al, 2003). Conversely, receptors that enter cells through NCE are associated with Smad7 and the E3 Ub ligase SMURF; they are ubiquitinated and subjected to degradation (Di Guglielmo et al, 2003).

It is important to note that CME is not always associated to signalling and NCE to degradation, but the opposite is also true, as it was shown in the case of LRP6 [WNT3aactivated low-density receptor-related protein 6, (Yamamoto et al, 2008)]. In the presence of Wnt3a, LPR6 is phosphorylated and internalized into caveolin-positive vesicles, where it can stabilize β-catenin and activates signalling via the CK1g kinase. If LRP6 binds the Wnt3a antagonist Dkk (Dickkopf), it is targeted to the clathrin pathway, which is not competent for signalling but rather directs LRP6 to degradation.

Other examples on how the route of internalization influences the final signalling output have been recently provided in the case of IGF-1R (Martins et al, 2011; Sehat et al, 2008) and PDGFR (De Donatis et al, 2008). In both cases, it has been proposed that they can enter through both clathrin-dependent and -independent pathways depending on the amount of ligand used to stimulate cells, similarly to what has been shown for the EGFR system. This again impacts on the final biological response. For instance, in the case of PDGFR, cells switch from a migrating to a proliferating phenotype in response to an increasing PDGF gradient. It was proposed that the decision to proliferate or migrate relies on the distinct endocytic route followed by the receptor in response to ligand concentration (De Donatis et al, 2008). Although these studies remain at the phenomenological level with no mechanistic insights, they confirm the idea that integration of different internalization pathways is crucial to decode signal information and to specify the signalling response.

#### **2.4 Role of endo-exo membrane trafficking in the execution of cell polarity programs**

Endo and exocytosis not only control the persistence and the nature of signals as highlighted above, but also the restricted compartmentalization of the signals. This has profound

the excess of activated EGFR/EGF complex to degradation, protecting cells from overstimulation. This concept has been challenged in other studies, where EGFR was reported to be internalized exclusively through CME at all concentrations of EGF (Kazazic et al, 2006; Rappoport & Simon, 2009). The discrepancy may be due to the different cellular systems used in these studies. It still remains to establish the nature of the NCE pathway used by the EGFR and the molecular mechanism involved [which is still poorly characterized, although it has been shown to be caveolin-independent and to require

A similar scenario was previously reported in the case of TGFβR. This receptor is internalized both by CME and NCE and this has profound impact on the final signalling output (Di Guglielmo et al, 2003). Proteins of the TGFβ superfamily signal through the transmembrane Ser-Thr kinase TGFβR type I and type II heteromeric complex (TβRI and TβRII). Ligand-induced assembly of the heteromeric receptor complex activates TβRI, which initiates Smad signalling by phosphorylating the receptor-regulated Smads. The Smad adaptor protein SARA plays a crucial role at this step. Indeed, SARA binds the receptor and contains a FYVE (Fab1p, YOTB, Vac1p and EEA1) domain, which also binds to membranes through specific interactions with phosphatidyl inositol 3′ phosphate (PI3P). Receptor internalization through the clathrin pathway is essential for signalling and SARA has been found in the PI(3)P-enriched EEA1-positive endosomes that are downstream of this route (Di Guglielmo et al, 2003). Conversely, receptors that enter cells through NCE are associated with Smad7 and the E3 Ub ligase SMURF; they are ubiquitinated and subjected to

It is important to note that CME is not always associated to signalling and NCE to degradation, but the opposite is also true, as it was shown in the case of LRP6 [WNT3aactivated low-density receptor-related protein 6, (Yamamoto et al, 2008)]. In the presence of Wnt3a, LPR6 is phosphorylated and internalized into caveolin-positive vesicles, where it can stabilize β-catenin and activates signalling via the CK1g kinase. If LRP6 binds the Wnt3a antagonist Dkk (Dickkopf), it is targeted to the clathrin pathway, which is not competent for

Other examples on how the route of internalization influences the final signalling output have been recently provided in the case of IGF-1R (Martins et al, 2011; Sehat et al, 2008) and PDGFR (De Donatis et al, 2008). In both cases, it has been proposed that they can enter through both clathrin-dependent and -independent pathways depending on the amount of ligand used to stimulate cells, similarly to what has been shown for the EGFR system. This again impacts on the final biological response. For instance, in the case of PDGFR, cells switch from a migrating to a proliferating phenotype in response to an increasing PDGF gradient. It was proposed that the decision to proliferate or migrate relies on the distinct endocytic route followed by the receptor in response to ligand concentration (De Donatis et al, 2008). Although these studies remain at the phenomenological level with no mechanistic insights, they confirm the idea that integration of different internalization pathways is

**2.4 Role of endo-exo membrane trafficking in the execution of cell polarity programs**  Endo and exocytosis not only control the persistence and the nature of signals as highlighted above, but also the restricted compartmentalization of the signals. This has profound

crucial to decode signal information and to specify the signalling response.

receptor ubiquitination (Sigismund et al, 2008; Sigismund et al, 2005)].

degradation (Di Guglielmo et al, 2003).

signalling but rather directs LRP6 to degradation.

implications in particular in the establishment of cell polarity, a process that largely relies on the correct localization of protein complexes and signalling platforms at cell-to-cell and cellto–extracellular matrix contacts. In this regards, a key role in the controlled distribution of signal transducers in restricted areas of the plasma membrane, in response to extracellular cues, is played by small GTPases of the Rab family like Rab5, Rab8 and Rab11.

Rab5 is a master regulator of endocytosis and actin remodelling (Lanzetti et al, 2004; Lanzetti et al, 2000; Palamidessi et al, 2008; Zerial & McBride, 2001). It controls the internalization of a variety of distinct receptors, including the adhesive molecules integrins and cadherins (Palacios et al, 2005; Pellinen et al, 2006), as detailed in paragraph 3, thus participating to the processes of cell-to-cell and cell-to-extracellular matrix adhesion. Importantly, in *Drosophila melanogaster* deletion of Rab5 or disruption of the endocytic protein Syntaxin/Avalanche affects the polarized, restricted apical distribution of the fatedecision receptor Notch and of the polarity determinant Crumbs (Lu & Bilder, 2005). Failure in internalization of Notch and Crumbs causes their accumulation and results in the expansion of the apical membrane domain. Impaired Notch internalization severely impacts on its degradation and signalling and, in turn, this leads to overgrowth of imaginal epithelial tissues (Lu & Bilder, 2005) indicating that endocytosis may also control epithelial tissue proliferation.

Rab8 participates in polarized transport of molecules to the basolateral membrane (Huber et al, 1993) and also in cilia (Nachury et al, 2007). Genetic deletion of Rab8 in mice has been found to affect the distribution of apical proteins to the surface of intestinal epithelial cells resulting in accumulation of vacuoles containing apical hydrolases and microvilli with the final outcome of animal death by starvation (Sato et al, 2007). Thus, Rab8 has been proposed to play a crucial role in the biogenesis of the apical membrane, a process that is profoundly influenced also by another Rab protein involved in recycling routes: Rab11 [reviewed in (Hoekstra et al, 2004)]. Indeed trafficking *via* the recycling endosomes is required for the establishment or rearrangement of cell polarity in various settings including cellularization, cell–to-cell boundary rearrangement, asymmetric cell division, and cell migration (Assaker et al, 2010; Bryant et al, 2010; Emery et al, 2005; Xu et al, 2011). Furthermore, it provides a very efficient mechanism to reinforce polarity by feedback loops (Assaker et al, 2010).

In addition to these GTPases, the endocytic protein Numb has also been implicated in the establishment of apical-basolateral polarity. Numb participates to cadherin endocytosis by interacting with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induces mislocalization of E-cadherin from the lateral to the apical membrane. This function of Numb appears to rely on its phosphorylation by Atypical protein kinase C (aPKC), a member of the PAR complex, as it prevents association of phosphorylated Numb with p120 and α-adaptin thereby attenuating E-cadherin endocytosis (Sato et al, 2011).

Beside the involvement of endo-exocytosis in apical-basolateral polarity, these trafficking routes are also required in the establishment of planar cell polarity (PCP) [for a detailed reviews on membrane trafficking in cell polarity see (Nelson, 2009)]. Intracellular membrane trafficking has emerged as a crucial regulator of PCP in the *Drosophila* wing where inhibition of dynamin or Rab11 disrupts PCP-dependent hexagonal repacking (Classen et al, 2005). More recently, Rab5 has been found to bind to Go and to participates in PCP and in Wingless signal transduction, pathways initiated by G-protein coupled receptors of the

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 165

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** 

epithelia (Rodriguez-Boulan et al, 2005).

Leibfried et al, 2008).

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

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

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;

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

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,

the PM, hence favoring the recycling of the adhesion receptor (Langevin et al, 2005).

(Goldstein & Macara, 2007; Iden & Collard, 2008; McCaffrey & Macara, 2009).

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 (Mottola et al, 2010).

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

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 al, 2005).

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 protrusions (Palamidessi et al, 2008).
