**Endocytosis and Exocytosis in Signal Transduction and in Cell Migration**

Guido Serini1,2, Sara Sigismund3 and Letizia Lanzetti2,4 *1Cell Adhesion Dynamics Laboratory-IRCC, Candiolo 2Department of Oncological Sciences University of Torino 31IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan 4Membrane Trafficking Laboratory- IRCC, Candiolo Italy* 

#### **1. Introduction**

156 Crosstalk and Integration of Membrane Trafficking Pathways

[92] Wösten HAB, Moukha SM, Sietsma JH, Wessels JGH. Localization of growth and

Endocytosis is a complex process that is used by eukaryotic cells to internalize fragments of plasma membrane, cell-surface receptors, and various soluble molecules. Many different mechanisms have been developed to achieve internalization of membrane-bound receptors and their ligands and they can be distinguished in clathrin-mediated endocytosis and nonclathrin internalization routes. In the clathrin-mediated endocytosis, receptors bind to the adaptor protein AP2 that, in turn, recruits clathrin to coat the invaginating pits at the plasma membrane. Coated pits are pinched off by the large GTPase dynamin to generate vesicles that traffic from the plasma membrane, undergo uncoating and fuse to the early endosomal compartment. Of note, dynamin is also required in non-clathrin-mediated endocytosis [for detailed recent reviews see (Doherty & McMahon, 2009; Loerke et al, 2009; Mettlen et al, 2009; Traub, 2009)].

From early endosomes vesicles can be re-delivered to the plasma membrane through the exocytic pathway (Grant & Donaldson, 2009). Vesicle budding, uncoating, motility and fusion are controlled by the large family of Rab small GTPases. Rab proteins, in their active GTP-bound form, recruit downstream effectors that, in turn, are responsible for distinct aspects of endosomes function from signal transduction to selection and transport of cargoes. Furthermore, they control vesicular movements on microtubules thus supporting polarized distribution of internalized receptors and signalling molecules [reviewed in (Stenmark, 2009; Zerial & McBride, 2001)]. In this regards, the endo-exocytic processes are profoundly linked with the ability of the cell to elicit receptor-mediated signaling cascades.

Endocytosis has long been considered as an attenuator of signaling as it downregulates receptors at the plasma membrane. However, the ability of internalized receptors to signal from the endosomal compartment and to be recycled to specific regions of the plasma membrane allows signal modulation both in time and in space. Indeed, endocytosismediated recycling of receptors is also a major mechanism in the execution of spatially restricted functions, such as cell motility. Moreover, the endo-exocytic cycle of adhesive receptors back and forth from the plasma membrane represents another crucial regulatory

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 159

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

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

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.

proper signal duration.

to regulate gene transcription.

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

We, therefore, propose to illustrate the state of the art together with most recent discoveries on the following issues:

