Endocytosis and spatial restriction of cell signaling

Abstract

Endocytosis and recycling are essential components of the wiring enabling cells to perceive extracellular signals and transduce them in a temporally and spatially controlled fashion, directly influencing not only the duration and intensity of the signaling output, but also their correct location. Here, we will discuss key experimental evidence that support how different internalization routes, the generation of diverse endomembrane platforms, and cycles of internalization and recycling ensure polarized compartmentalization of signals, regulating a number of physiological and pathologically-relevant processes in which the resolution of spatial information is vital for their execution.

Figure 1

Endosomal vesicles, trafficking routes and their regulators. Plasma membrane (PM) and membrane‐bound proteins are internalized through various routes. Here, are depicted Clathrin‐dependent (CME) and Clathrin‐independent (NCE) endocytosis. The latter one includes also raft‐mediated pathways. Both routes converge into Rab5‐positive early endosomes (EE), which represent the first endosomal sorting station. From EE cargos can be redirected through a fast recycling, Rab4‐dependent routes back to the plasma membrane, or enter, via Rab8, into a Rab11‐endocytic recycling compartment (ERC) before being retargeted to the PM (Zerial and McBride, 2001). Alternatively, cargo can traffic to a Rab7‐dependent lysosomal degradative route, by entering into multivesicular body/late endosome (MVB/LE) before being degraded into lysosome (Dautry‐Varsat, 1986). In addition to these “canonical routes”, cargos, such as MHC‐I or the interleukin receptor that do not enter through CME, can be sorted into Arf6‐positive recycling pathways (Arf6‐RE) and redelivered back to the PM (D'Souza‐Schorey and Chavrier, 2006; Donaldson, 2005). It is unclear what are the precise regulatory factors and intermediate endomembrane(s) that may connect EE and ERC to Arf6‐RE.

Figure 2

The internalization routes influence signaling output. (A) CME promotes sustained signaling, while NCE target cargos to degradation. EGFR and TGF‐β receptors enter cells either via CME or raft‐dependent, NCE pathways. EGFR and TGF‐β entering through CME are efficiently recycled. This trafficking route is coupled to sustained and spatially restricted signaling. Raft‐mediated internalization mainly targets these receptors to degradation leading to signal attenuation (Le Roy and Wrana, 2005). Ubiquitination of the receptors mediated by the E3 ligases Smad7 (Di Guglielmo et al., 2003) or CBL (Sorkin and Goh, 2009) for TGF‐β and EGFR, respectively preferentially direct these cargos toward degradative pathways, indicating that this posttranslational modification might be critical to determine receptor fate and signaling outcome, albeit the underlying molecular mechanisms responsible for this routing are ill‐defined. The cycle of internalization and recycling associated to CME is thought to be essential to spatially restrict signaling leading to directional migration and chemotaxis. (B) NCE promotes signal activation, while CME signal attenuation. The Wnt family of secreted ligands binds to the seven transmembrane‐spanning receptors of the Frizzled‐type and to the Low Density Receptor‐related protein (LRPs) (Kikuchi et al., 2007). Among Wnt ligands, Wnt3a can bind to its co‐receptor LRP6, promoting its raft‐mediated internalization and signaling. In the presence of Wnt3a, LRP6 is phosphorylated and internalized into a raft‐dependent, caveolin‐positive vesicular compartment, where it can stabilize β‐catenin and transduce the signal. Phosphorylation does not require the trafficking of the receptor to the caveolin‐1 positive compartment (where its kinase, CK1 g, is found) (Yamamoto et al., 2008). Conversely, signal transduction (i.e., β‐catenin stabilization) requires both phosphorylation and endocytosis, suggesting that endosomes may become critical signaling platform. LRP6 can also bind a Wnt3a antagonist, Dickkopf (Dkk), which diverts it from the caveolin to the Clathrin pathway, preventing the encounter of the receptor with its kinase, its phosphorylation and β‐catenin stabilization.

Figure 3

Endocytic trafficking of Rac and integrin is required for spatial restriction of signaling in the control of migratory programs. (A) Hepatocyte growth factor (HGF) receptor activation induces the formation of circular dorsal ruffles (CDR, arrow) and peripheral lamellipodia (PL, arrowhead). This is accompanied by Clathrin‐mediated internalization of the cognate receptor tyrosine kinase, c‐Met. Clathrin‐ and Rab5‐dependent endocytosis is required for Tiam‐1‐mediated, a guanine‐nucleotide exchange factor, Rac activation in endosomal vesicles. Recycling of Rac back to plasma membrane areas is essential to promote the reorganization of actin into CDR. The small GTPase Arf6 targets activated Rac to specialized plasma membrane areas (such as ruffles) with high actin activity. The integrin α5β1 can associate with the small GTPase Rab25, a member of the Rab11 family, involved in vesicle recycling (Caswell et al., 2007). Both Rab25 and α5β1 integrin co‐localize in intracellular vesicular compartments within the distal tips of pseudopods (the lamellipodia equivalent in a 3D setting) (Caswell et al., 2007). Integrin α5β1 can traffic bidirectionally between intracellular Rab25 vesicles and the plasma membrane within the confines of the pseudopodial tips, this promotes the compartmentalization of a spatially restricted subpopulation of cycling α5β1 within the tip regions of extending pseudopods (Caswell et al., 2007). A HeLa cell stimulated with HGF and stained with phalloidin to detect F‐actin (white) is shown. (B) Mode of motility of individual cells in 3D. Two modes of single‐cell 3D migration in extracellular matrices have been described (Wolf and Friedl, 2006). The elongated‐lamellipodia movement (mesenchymal mode) begins with the formation of flat, adherent protrusions driving directional motility. Conversely, amoeboid migration depends on Rho/Rock‐dependent actomyosin contractility, driving blebbing‐like movements of loosely adherent cells. Conversion between these migratory modes, amoeboid‐to‐mesenchymal (AMT) and mesenchymal‐to‐amoeboid (MAT) transition, confers plasticity to metastatic tumor cell migration (Wolf and Friedl, 2006). The endocytic/recycling of Rac and α5β1 may control the migration into 3D matrices by promoting a mesenchymal mode of migration. GFP‐expressing, HeLa cells embedded in matrigel and treated (top) or not (bottom) with the Rock inhibitor Y27632 or expressing Rab5 (not shown) undergo an amoeboid‐to‐mesenchymal‐like cell shape transition in 3D. [Images are reprinted from Palamidessi et al. (2008) Copyright (2008), with permission from Elsevier].