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Actin in membrane trafficking
Actin in membrane trafficking Letizia Lanzetti Actin cytoskeleton remodeling provides the forces required for a variety of cellular processes based on membrane dynamics, such as endocytosis, exocytosis, and vesicular trafficking at the Golgi. All these events are coordinated by networks of associated proteins, and some of them are functionally connected with cell migration. The site and the duration of actin polymerization, in connection with vesicle budding and fusion, are tightly controlled by both small GTPases and the large GTPase dynamin. Recent advances in the understanding of the mechanisms coupling actin dynamics with membrane trafficking at the cell surface have been brought by the combined studies of actin polymerizing factors and of the endocytic/exocytic machinery. Addresses Dipartimento di Scienze Oncologiche, Universita` degli Studi di Torino, Istituto per la Ricerca e la Cura del Cancro, Str. Provinciale 142, 10060 Candiolo, Torino, Italy Corresponding author: Lanzetti, Letizia ([email protected])
Current Opinion in Cell Biology 2007, 19:453–458 This review comes from a themed issue on Membranes and Orgaelles Edited by Peter McPherson and Thierry Galli Available online 5th July 2007 0955-0674/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
Actin polymerization in endocytosis
In mammalian cells, the requirement for actin polymerization in endocytosis has been addressed in particular in clathrin-mediated internalization. Relevant insights into the kinetics of actin remodeling, recruitment of endocytic proteins, and membrane invagination came from studies based on live cell imaging coupled to the use of epifluorescence and pH-sensitive cargoes. These revealed that actin polymerization at endocytic sites is an early event, already occurring during invagination of clathrin-coated pits [4]. Moreover, F-actin dynamics are needed at multiple stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and vesicle scission [5,6], suggesting that actin may play several distinct, and probably coordinated, functions in endocytosis. Vesicles scission depends on the activity of the large GTPase dynamin that is recruited early during clathrin-coated pit formation [4] and accumulates with a strong burst just before vesicles pinching off [7,8]. Concomitant with scission is the recruitment of cortactin [5], a protein that binds to dynamin and to F-actin [9] and activates the Arp2/3 complex, which in turn is responsible for nucleation of actin polymerization [10]. The timing of cortactin recruitment coincides with that of Arp2/3-dependent actin polymerization [5,11], suggesting that cortactin may link actin rearrangements with dynamindependent vesicle scission.
DOI 10.1016/j.ceb.2007.04.017
Introduction The cortical actin cytoskeleton undergoes tightly controlled remodeling concomitant with membrane budding or fusion events occurring at the cell surface. The complex molecular machinery involved is starting to be elucidated. By means of imaging technologies, actin dynamics [1] and vesicle generation and movement [2] can be followed in living cells, thereby providing relevant advances in the understanding of the underlying mechanisms. The involvement of the actin cytoskeleton in vesicular trafficking is not restricted to the plasma membrane. For instance, actin is also required during budding, fission, and transport of Golgi carriers. Since actin contribution to Golgi architecture and function has been recently reviewed [3], it will not be discussed here. I will focus instead on selected achievements over the last 2 years, which highlight the role of actin in endocytosis and exocytosis, and how these trafficking events participate in cell locomotion. www.sciencedirect.com
Recent findings show that the constricting activity of dynamin on membrane tubules results in scission only if tension is applied [12]. Thus an appealing hypothesis is that actin polymerization could promote membrane invagination and provide tension at the vesicle neck cooperating with dynamin in vesicle budding [12]. A number of other endocytic proteins including intersectin-1 and HIPs have been shown to interact directly or indirectly with cortical actin, adding complexity to the picture (reviewed in [13]). Notably, HIP1R, which stimulates the assembly of clathrin coats on the forming vesicles, binds to cortactin preventing its association with dynamin [14]. The HIP1R–cortactin complex works as an actincapping protein; it inhibits actin assembly by blocking actin filament- barbed end elongation [14]. Since HIP1R localizes to the surface of the clathrin-coated vesicles while dynamin is at the neck, it is tempting to suggest a spatial separation of the capping and nucleating activity associated with cortactin. Furthermore, the HIP1R–cortactin dependent local capping activity could limit the growth of the actin network to the neck. This would prevent the Current Opinion in Cell Biology 2007, 19:453–458
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network from surrounding the vesicles and from causing their inward movement into the cytoplasm [14]. An additional link between membrane invagination and actin polymerization has been brought by findings on the activity of membrane-deforming proteins, containing BAR and F-BAR domains [15,16]. The superfamily of BAR proteins shares common features: they participate in membrane bending during endocytosis, they bind to dynamin and to synaptojanin, and some of them also bind to the Arp2/3 activator N-WASP. According to a current model, BAR proteins sense membrane curvature and associate with the invaginating pit further bending it (reviewed in [17]). The ability of F-BAR proteins to induce membrane tubulation in living cells is counteracted by dynamin overexpression and enhanced by disruption of the actin cytoskeleton with latrunculin [15,16]. These observations suggest that the actin cytoskeleton might control the extent of membrane tubulation by regulating rigidity at the plasma membrane, which in turn would allow the constricting activity of dynamin, (as proposed by Roux et al. [12]) finally resulting in vesicle scission. Thus, F-BAR proteins appear to promote membrane invagination coupled to concomitant actin polymerization and dynamin recruitment. An interesting question is whether the requirement for actin remodeling also extends to other modalities of internalization, besides clathrin-mediated endocytosis. Indeed this seems to be the case, as shown by studies of N-WASP and WAVE proteins. In vivo, actin filaments are generated with two distinct modalities. One depends on the activity of formins, F-actin nucleating factors that, via a ‘processive-capping’ mechanism, promote the formation of linear F-actin arrays. The second is based on the N-WASP–Arp2/3 or WAVE–Arp2/3 complex that generate new filaments at the membrane, in a sitedirected fashion, by branching off existing filaments [18–20]. Notably, N-WASP participates in clathrin-dependent endocytosis, and its functional ablation results in the accumulation of receptors at the plasma membrane [21,22]. Conversely, the family of WAVE proteins appears to connect actin polymerization to some forms of clathrin-independent internalization. WAVE1 is required for the formation of dorsal or circular ruffles [23], which are sites of rapid receptor endocytosis [24], while WAVE2 has been localized to macropinosomes and its depletion affects this endocytic route [22,23]. A recent work proposes the involvement of N-WASP in the formation of circular ruffles [25]; whether this would implicate a function for this protein also in dorsal ruffle-dependent endocytosis is still not known. Interestingly, circular ruffles couple internalization of significant amount of activated receptors with extensive actin remodeling at the dorsal cell surface [24], further supporting the notion that the two mechanisms are strictly connected. Current Opinion in Cell Biology 2007, 19:453–458
In another setting, extensive actin remodeling has also been found in caveolae-mediated endocytosis [26]. Thus, the requirement for actin dynamics seems to be a common motif in most of the known internalization processes. Actin polymerization in exocytosis
Local actin assembly at the plasma membrane plays a role during exocytosis as well. Exocytosis controls cell surface expansion and protein secretion by fusing secretory vesicles with the plasma membrane. Live cell imaging studies have shown that F-actin is rapidly assembled around the secretory vesicle upon fusion, suggesting that dynamic F-actin coats might drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments [27]. Thus, the function of actin would be to stabilize the secretory compartment during docking with the plasma membrane and also to drive compensatory endocytosis. Local actin assembly appears to be triggered by compartment mixing between secretory vesicles and the plasma membrane [28]. This is achieved at least in part by incorporation of diacylglycerol from the plasma membrane to the secretory granule and recruitment of protein kinase Cb that activates the GTPase Cdc42 [28]. In turn, Cdc42 stimulates actin coat polymerization via its downstream effectors Toca1 and N-WASP [27,28,29]. Remarkably, Cdc42 coordinates actin remodeling and exocytosis also during neurite outgrowth as shown by the ability of its dominantactive mutant to promote exocytosis of TI-VAMP-containing vesicles in an actin-dependent manner at the growth cone [30]. An important molecular actor of exocytosis is the exocyst, an octameric protein complex conserved through evolution and essential for tethering secretory vesicles to specific domains of the plasma membrane [31]. Recently, one of the exocyst components, Exo70, has been found to directly bind to the Arp2/3 complex. Functional ablation of Exo70 prevents Cdc42-dependent or Rac-dependent actin cytoskeleton remodeling, recruitment of Arp2/3 at the leading edge of lamellipodia, and membrane protrusion, thereby affecting the migratory ability of the cell [32]. In conclusion, both membrane budding and fusion events require the interaction of actin polymerizing factors with the endocytic and exocytic machinery. These interactions appear to regulate actin polymerization at sites of active membrane dynamics. In turn, the remodeling of the actin cytoskeleton might participate in the execution of trafficking events in several ways, which also probably depend on the nature of the event, that include pushing of membrane invaginations, propelling of vesicles, creation of platforms for protein–protein interactions, or modulation of membrane plasticity (see also the following www.sciencedirect.com
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reviews addressing the role of actin in exocytosis and endocytosis [33,34]). Spatial and temporal control of actin polymerization in membrane trafficking
The Rho, Rab, and ARF families of small GTPases play a key role in determining the site and the duration of actin remodeling and membrane trafficking. Their localization at distinct membrane sites is tightly controlled via several mechanisms including post-translational modification by prenylation, association with GDIs (guanine nucleotide dissociation inhibitors), and phosphorylation. In an active GTP-bound form, these GTPases bind to downstream effectors, thus creating membrane-associated signaling platforms where membrane remodeling may occur and actin polymerization is stimulated ([35–37]; advances on the role of ARF proteins are extensively covered in a recent review [38]). An example of GTPase-driven formation of actin coats on vesicles is provided by studies on Rho proteins. Activated RhoB, by recruiting the Diaphanous-related formins mDia1 and mDia2, promotes the polymerization of actin around endosomes and the association of the latter with subcortical actin cables [39,40]. The prevailing model is
that RhoB binding to self-inhibited mDia relieves the inhibition resulting in the nucleation and processive elongation of nonbranched actin filaments at the site of activation. This in turn might generate the forces to propel vesicles in short-range movements. Indeed, treatment with actin depolymerizing or stabilizing drugs blocks the movement of endocytic vesicles, further supporting the requirement for actin dynamics in endosomes motility [40]. In addition, active RhoA has been found in association with PDGF-induced pinosomes, while they traffic through the cell, suggesting that RhoA stimulates actin polymerization thereby enabling pinosomes propulsion into the cytosol [1]. These data add to the previously identified function of another Rho family member, RhoD, which was found to regulate motility of early endosomes along actin fibers via its effector, hDia2C [41]. The coordination between actin cytoskeletal remodeling and membrane trafficking appears to be critical in particular for cell motility. In order to migrate, cells generate oriented lamellipodial extensions. Lamellipodia are formed by coupling strong actin polymerization, promoted by Rac, Cdc42, and Rho [1,42], and membrane protrusion, supported by vesicles delivery and recycling. While
Figure 1
A model for Rab GTPases function in integrin traffic. The migrating cell is extending lamellipodia where actin is polymerized as a meshwork of branched filaments. Integrin-containing structures (focal complexes) are formed at the base of the extending edge of lamellipodia and matured into focal adhesion. Integrins may internalize via Rab21-positive endosome and Rab5-positive endosome and recycle via the Rab4-recycling pathway or the Rab11-recycling pathway. www.sciencedirect.com
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a number of mechanistic studies have extensively addressed the role of actin dynamics in cell migration, the relevance of the exo–endocytic machinery and its link with the cytoskeleton is only starting to be appreciated ([43] and reviewed in [31,44,45]). The exo–endocytic processes have a dual function in cell locomotion: they provide new membranes for extension, and they also control cell spreading and adhesion by regulating the turnover of adhesive molecules at the plasma membrane. As the cell moves forward, adhesive structures, mainly the matrix-adhesive receptors integrins, are internalized and recycled to cell protrusive regions. Integrin internalization may occur, depending on the stimulus and on the type of receptor, via lipid rafts/caveolae [46,47]. Since caveolar trafficking requires actin cytoskeleton dynamics [26], an interesting question is whether actin remodeling also participates in integrin endocytosis. Furthermore, Rab21 and Rab5 have been recently found to bind to b1-integrins and to control their endosomal trafficking [48]. Notably, the motility of Rab21 vesicles close to the plasma membrane requires the actin cytoskeleton, and Rab21 mutants with impaired GTP binding or membrane localization induce the formation of exaggerated adhesion sites [48]. This is in agreement with the recent finding that integrins, in newly forming protrusions, travel on actin cables associated with the actin-based motor protein Myosin X [49]. Finally, integrins recycle via two routes: a Rab4-dependent early endosomes route, named ‘short loop,’ and a second path, which takes place from the perinuclear recycling Rab11-positive compartment, called ‘long loop’ [45]. The ‘long loop’ is regulated by the activity of both ARF6 and Rab11 and it also requires actin rearrangements [50]. Notably, Rab11-dependent endosomal recycling appears to be necessary in particular for polarized cell migration since disruption of its activity results in disorganized motility [51] (Figure 1).
Conclusions A growing body of evidence points to a requirement for actin dynamics in the majority of the membrane trafficking events investigated so far. In particular, future developments will certainly provide a high-definition mechanistic picture of how polarized exo–endocytic traffic of membranes and of adhesive cues is coupled to the actin cytoskeleton. This will be extremely relevant not only for physiology but also for a better understanding of those pathological states characterized by alterations of the normal migratory phenotypes, the first and foremost tumor cell invasion.
Acknowledgements Work in the author’s lab is supported by grants from Associazione Italiana per la Ricerca sul Cancro, the European Community (VI Framework), and the Association for International Cancer Research. The author thanks Giorgio Scita and Guido Serini for critically reading the manuscript. Current Opinion in Cell Biology 2007, 19:453–458
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Merrifield CJ, Perrais D, Zenisek D: Coupling between clathrincoated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 2005, 121:593-606. The authors analyze in vivo the minute inward movements made by clathrin-coated pits during internalization and fission thanks to a combination of evanescent field and a pH-sensitive fluorescent cargo, the transferrin receptor-phluorin. Since the epifluorescence of the tag is almost completely quenched upon transition of pH to the acidic environment of vesicles, it allows discrimination between invagination and scission. By means of this technique it is shown that clathrin-coated pits support multiple rounds of vesicle creation, that they elongate inside the cell before scission, and that the peak of cortactin recruitment coincides with scission.
6.
Yarar D, Waterman-Storer CM, Schmid SL: A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. Mol Biol Cell 2005, 16:964-975. One of the main achievements of this paper consists in the demonstration that actin cytoskeleton dynamics are required during all the steps of clathrin-coated pit formation, invagination and scission and for lateral motility. 7.
Ehrlich M, Boll W, Van Oijen A, Hariharan R, Chandran K, Nibert ML, Kirchhausen T: Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 2004, 118:591-605.
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Zoncu R, Perera RM, Sebastian R, Nakatsu F, Chen H, Balla T, Ayala G, Toomre D, De Camilli PV: Loss of endocytic clathrincoated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci USA 2007, 104:3793-3798.
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Daly RJ: Cortactin signalling and dynamic actin networks. Biochem J 2004, 382:13-25.
10. Takenawa T, Miki H: WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement. J Cell Sci 2001, 114:1801-1809. 11. Merrifield CJ, Qualmann B, Kessels MM, Almers W: Neural Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts. Eur J Cell Biol 2004, 83:13-18. 12. Roux A, Uyhazi K, Frost A, De Camilli P: GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 2006, 441:528-531. This study provides relevant insights into the mechanism of dynamin function. The authors show that dynamin has a GTP-dependent twisting activity on membrane tubules that is not sufficient to cause membrane scission unless tension at the membrane tubules is applied. www.sciencedirect.com
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13. McPherson PS: The endocytic machinery at an interface with the actin cytoskeleton: a dynamic, hip intersection. Trends Cell Biol 2002, 12:312-315. 14. Le Clainche C, Pauly BS, Zhang CX, Engqvist-Goldstein AE, Cunningham K, Drubin DG: A Hip1R–cortactin complex negatively regulates actin assembly associated with endocytosis. EMBO J 2007, 26:1199-1210. This important study identifies the functional meaning of the interaction between a clathrin-binding protein, HIP1R, and an actin–dynamin-binding protein, cortactin. HIP1R and cortactin form a complex that caps the actin filaments’ barbed ends preventing filaments’ elongation. Moreover HIP1R inhibits N-WASP activation as well as the association of cortactin with dynamin. A nice discussion on the role of actin in vesicles invagination is also provided. 15. Itoh T, Erdmann KS, Roux A, Habermann B, Werner H, De Camilli P: Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev Cell 2005, 9:791-804. This outstanding paper provides a number of important information: (i) the FCH domain is part of a larger BAR domain-related module (F-BAR), (ii) F-BAR domains bind to phosphatidylserine and phosphoinositides and induce potent bilayer deformation in vitro and membrane tubulation in vivo, (iii) membrane tubulation is counteracted by dynamin and enhanced by latrunculin B-mediated disruption of the actin cytoskeleton. Thus, the function of actin could be to antagonize membrane deformation by stabilizing a scaffold beneath the plasma membrane and/or to participate in vesicle pinching off. 16. Tsujita K, Suetsugu S, Sasaki N, Furutani M, Oikawa T, Takenawa T: Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J Cell Biol 2006, 172:269-279. This paper shows that the F-BAR containing protein FBP17 recruits N-WASP at the plasma membrane where it activates actin polymerization. Moreover it supports the notion that dynamin-dependent vesicle fission is coordinated with actin polymerization. 17. Dawson JC, Legg JA, Machesky LM: Bar domain proteins: a role in tubulation, scission and actin assembly in clathrinmediated endocytosis. Trends Cell Biol 2006, 16:493-498.
25. Legg JA, Bompard G, Dawson J, Morris HL, Andrew N, Cooper L, Johnston SA, Tramountanis G, Machesky LM: N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts. Mol Biol Cell 2007, 18:678-687. 26. Pelkmans L, Puntener D, Helenius A: Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 2002, 296:535-539. 27. Sokac AM, Co C, Taunton J, Bement W: Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs. Nat Cell Biol 2003, 5:727-732. 28. Yu HY, Bement WM: Control of local actin assembly by membrane fusion-dependent compartment mixing. Nat Cell Biol 2007, 9:149-159. This study nicely identifies the signaling pathway underlying the compartment mixing-dependent actin assembly during exocytosis. 29. Malacombe M, Ceridono M, Calco V, Chasserot-Golaz S, McPherson PS, Bader MF, Gasman S: Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42. EMBO J 2006, 25:3494-3503. 30. Alberts P, Rudge R, Irinopoulou T, Danglot L, Gauthier-Rouviere C, Galli T: Cdc42 and actin control polarized expression of TIVAMP vesicles to neuronal growth cones and their fusion with the plasma membrane. Mol Biol Cell 2006, 17:1194-1203. 31. Lipschutz JH, Mostov KE: Exocytosis: the many masters of the exocyst. Curr Biol 2002, 12:R212-R214. 32. Zuo X, Zhang J, Zhang Y, Hsu SC, Zhou D, Guo W: Exo70 interacts with the Arp2/3 complex and regulates cell migration. Nat Cell Biol 2006, 8:1383-1388. 33. Sokac AM, Bement WM: Kiss-and-coat and compartment mixing: coupling exocytosis to signal generation and local actin assembly. Mol Biol Cell 2006, 17:1495-1502. 34. Smythe E, Ayscough KR: Actin regulation in endocytosis. J Cell Sci 2006, 119:4589-4598. 35. Zerial M, McBride H: Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2001, 2:107-117.
18. Takenawa T, Suetsugu S: The WASP–WAVE protein network: connecting the membrane to the cytoskeleton. Nat Rev Mol Cell Biol 2007, 8:37-48.
36. Polo S, Di Fiore PP: Endocytosis conducts the cell signaling orchestra. Cell 2006, 124:897-900.
19. Stossel TP, Fenteany G, Hartwig JH: Cell surface actin remodeling. J Cell Sci 2006, 119:3261-3264.
37. Ridley AJ: Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 2006, 16:522-529.
20. Bershadsky A: Magic touch: how does cell–cell adhesion trigger actin assembly? Trends Cell Biol 2004, 14:589-593. 21. Benesch S, Polo S, Lai FP, Anderson KI, Stradal TE, Wehland J, Rottner K: N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits. J Cell Sci 2005, 118:3103-3115. 22. Innocenti M, Gerboth S, Rottner K, Lai FP, Hertzog M, Stradal TE, Frittoli E, Didry D, Polo S, Disanza A et al.: Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nat Cell Biol 2005, 7:969-976. This study demonstrates the ability of the protein Abi1 to regulate the activity of two distinct actin-assembly machineries. By entering into a complex with WAVE, Abi1 stimulates Rac-dependent membrane protrusions. Conversely, binding of Abi1 to N-WASP results in the regulation of receptor cell surface distribution and internalization. This work provides evidence for a role of N-WASP in clathrin-coated vesicle endocytosis and movement. 23. Suetsugu S, Yamazaki D, Kurisu S, Takenawa T: Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration. Dev Cell 2003, 5:595-609. 24. Orth JD, Krueger EW, Weller SG, McNiven MA: A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization. Cancer Res 2006, 66:3603-3610. The function and the biological implications of the formation of dorsal/ circular ruffles are poorly understood. This work demonstrates how these actin waves act as endocytic structures that can specifically sequester and internalize up to 50% of receptor tyrosine kinases such as the EGF receptor. www.sciencedirect.com
38. D’Souza-Schorey C, Chavrier P: ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 2006, 7:347-358. 39. Fernandez-Borja M, Janssen L, Verwoerd D, Hordijk P, Neefjes J: RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1. J Cell Sci 2005, 118:2661-2670. 40. Wallar BJ, Deward AD, Resau JH, Alberts AS: RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking. Exp Cell Res 2007, 313:560-571. 41. Gasman S, Kalaidzidis Y, Zerial M: RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase. Nat Cell Biol 2003, 5:195-204. 42. Yamana N, Arakawa Y, Nishino T, Kurokawa K, Tanji M, Itoh RE, Monypenny J, Ishizaki T, Bito H, Nozaki K et al.: The Rho–mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src. Mol Cell Biol 2006, 26:6844-6858. 43. Proux-Gillardeaux V, Gavard J, Irinopoulou T, Mege RM, Galli T: Tetanus neurotoxin-mediated cleavage of cellubrevin impairs epithelial cell migration and integrin-dependent cell adhesion. Proc Natl Acad Sci USA 2005, 102:6362-6367. This study shows the localization of the SNARE protein cellubrevin on vesicles trafficking in the lamellipodia and at the distal tip of focal contacts. Consistently, cellubrevin is required for cell adhesion and migration. 44. Pellinen T, Ivaska J: Integrin traffic. J Cell Sci 2006, 119:3723-3731. Current Opinion in Cell Biology 2007, 19:453–458
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45. Jones MC, Caswell PT, Norman JC: Endocytic recycling pathways: emerging regulators of cell migration. Curr Opin Cell Biol 2006, 18:549-557. 46. del Pozo MA, Balasubramanian N, Alderson NB, Kiosses WB, Grande-Garcia A, Anderson RG, Schwartz MA: Phosphocaveolin-1 mediates integrin-regulated membrane domain internalization. Nat Cell Biol 2005, 7:901-908. 47. Ning Y, Buranda T, Hudson LG: Activated epidermal growth factor receptor induces integrin {alpha}2 internalization via caveolae/raft-dependent endocytic pathway. J Biol Chem 2007, 282:6380-6387. 48. Pellinen T, Arjonen A, Vuoriluoto K, Kallio K, Fransen JA, Ivaska J: Small GTPase Rab21 regulates cell adhesion and controls endosomal traffic of beta1-integrins. J Cell Biol 2006, 173:767-780.
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This work provides, for the first time, evidence for a direct binding between Rab proteins and integrin receptors. 49. Zhang H, Berg JS, Li Z, Wang Y, Lang P, Sousa AD, Bhaskar A, Cheney RE, Stromblad S: Myosin-X provides a motor-based link between integrins and the cytoskeleton. Nat Cell Biol 2004, 6:523-531. 50. Powelka AM, Sun J, Li J, Gao M, Shaw LM, Sonnenberg A, Hsu VW: Stimulation-dependent recycling of integrin beta1 regulated by ARF6 and Rab11. Traffic 2004, 5:20-36. 51. Prigozhina NL, Waterman-Storer CM: Decreased polarity and increased random motility in PtK1 epithelial cells correlate with inhibition of endosomal recycling. J Cell Sci 2006, 119:3571-3582.
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Current Opinion in Cell Biology 2007, 19:453–458 This review comes from a themed issue on Membranes and Orgaelles Edited by Peter McPherson and Thierry Galli Available online 5th July 2007 0955-0674/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
Actin polymerization in endocytosis
In mammalian cells, the requirement for actin polymerization in endocytosis has been addressed in particular in clathrin-mediated internalization. Relevant insights into the kinetics of actin remodeling, recruitment of endocytic proteins, and membrane invagination came from studies based on live cell imaging coupled to the use of epifluorescence and pH-sensitive cargoes. These revealed that actin polymerization at endocytic sites is an early event, already occurring during invagination of clathrin-coated pits [4]. Moreover, F-actin dynamics are needed at multiple stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and vesicle scission [5,6], suggesting that actin may play several distinct, and probably coordinated, functions in endocytosis. Vesicles scission depends on the activity of the large GTPase dynamin that is recruited early during clathrin-coated pit formation [4] and accumulates with a strong burst just before vesicles pinching off [7,8]. Concomitant with scission is the recruitment of cortactin [5], a protein that binds to dynamin and to F-actin [9] and activates the Arp2/3 complex, which in turn is responsible for nucleation of actin polymerization [10]. The timing of cortactin recruitment coincides with that of Arp2/3-dependent actin polymerization [5,11], suggesting that cortactin may link actin rearrangements with dynamindependent vesicle scission.
DOI 10.1016/j.ceb.2007.04.017
Introduction The cortical actin cytoskeleton undergoes tightly controlled remodeling concomitant with membrane budding or fusion events occurring at the cell surface. The complex molecular machinery involved is starting to be elucidated. By means of imaging technologies, actin dynamics [1] and vesicle generation and movement [2] can be followed in living cells, thereby providing relevant advances in the understanding of the underlying mechanisms. The involvement of the actin cytoskeleton in vesicular trafficking is not restricted to the plasma membrane. For instance, actin is also required during budding, fission, and transport of Golgi carriers. Since actin contribution to Golgi architecture and function has been recently reviewed [3], it will not be discussed here. I will focus instead on selected achievements over the last 2 years, which highlight the role of actin in endocytosis and exocytosis, and how these trafficking events participate in cell locomotion. www.sciencedirect.com
Recent findings show that the constricting activity of dynamin on membrane tubules results in scission only if tension is applied [12]. Thus an appealing hypothesis is that actin polymerization could promote membrane invagination and provide tension at the vesicle neck cooperating with dynamin in vesicle budding [12]. A number of other endocytic proteins including intersectin-1 and HIPs have been shown to interact directly or indirectly with cortical actin, adding complexity to the picture (reviewed in [13]). Notably, HIP1R, which stimulates the assembly of clathrin coats on the forming vesicles, binds to cortactin preventing its association with dynamin [14]. The HIP1R–cortactin complex works as an actincapping protein; it inhibits actin assembly by blocking actin filament- barbed end elongation [14]. Since HIP1R localizes to the surface of the clathrin-coated vesicles while dynamin is at the neck, it is tempting to suggest a spatial separation of the capping and nucleating activity associated with cortactin. Furthermore, the HIP1R–cortactin dependent local capping activity could limit the growth of the actin network to the neck. This would prevent the Current Opinion in Cell Biology 2007, 19:453–458
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network from surrounding the vesicles and from causing their inward movement into the cytoplasm [14]. An additional link between membrane invagination and actin polymerization has been brought by findings on the activity of membrane-deforming proteins, containing BAR and F-BAR domains [15,16]. The superfamily of BAR proteins shares common features: they participate in membrane bending during endocytosis, they bind to dynamin and to synaptojanin, and some of them also bind to the Arp2/3 activator N-WASP. According to a current model, BAR proteins sense membrane curvature and associate with the invaginating pit further bending it (reviewed in [17]). The ability of F-BAR proteins to induce membrane tubulation in living cells is counteracted by dynamin overexpression and enhanced by disruption of the actin cytoskeleton with latrunculin [15,16]. These observations suggest that the actin cytoskeleton might control the extent of membrane tubulation by regulating rigidity at the plasma membrane, which in turn would allow the constricting activity of dynamin, (as proposed by Roux et al. [12]) finally resulting in vesicle scission. Thus, F-BAR proteins appear to promote membrane invagination coupled to concomitant actin polymerization and dynamin recruitment. An interesting question is whether the requirement for actin remodeling also extends to other modalities of internalization, besides clathrin-mediated endocytosis. Indeed this seems to be the case, as shown by studies of N-WASP and WAVE proteins. In vivo, actin filaments are generated with two distinct modalities. One depends on the activity of formins, F-actin nucleating factors that, via a ‘processive-capping’ mechanism, promote the formation of linear F-actin arrays. The second is based on the N-WASP–Arp2/3 or WAVE–Arp2/3 complex that generate new filaments at the membrane, in a sitedirected fashion, by branching off existing filaments [18–20]. Notably, N-WASP participates in clathrin-dependent endocytosis, and its functional ablation results in the accumulation of receptors at the plasma membrane [21,22]. Conversely, the family of WAVE proteins appears to connect actin polymerization to some forms of clathrin-independent internalization. WAVE1 is required for the formation of dorsal or circular ruffles [23], which are sites of rapid receptor endocytosis [24], while WAVE2 has been localized to macropinosomes and its depletion affects this endocytic route [22,23]. A recent work proposes the involvement of N-WASP in the formation of circular ruffles [25]; whether this would implicate a function for this protein also in dorsal ruffle-dependent endocytosis is still not known. Interestingly, circular ruffles couple internalization of significant amount of activated receptors with extensive actin remodeling at the dorsal cell surface [24], further supporting the notion that the two mechanisms are strictly connected. Current Opinion in Cell Biology 2007, 19:453–458
In another setting, extensive actin remodeling has also been found in caveolae-mediated endocytosis [26]. Thus, the requirement for actin dynamics seems to be a common motif in most of the known internalization processes. Actin polymerization in exocytosis
Local actin assembly at the plasma membrane plays a role during exocytosis as well. Exocytosis controls cell surface expansion and protein secretion by fusing secretory vesicles with the plasma membrane. Live cell imaging studies have shown that F-actin is rapidly assembled around the secretory vesicle upon fusion, suggesting that dynamic F-actin coats might drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments [27]. Thus, the function of actin would be to stabilize the secretory compartment during docking with the plasma membrane and also to drive compensatory endocytosis. Local actin assembly appears to be triggered by compartment mixing between secretory vesicles and the plasma membrane [28]. This is achieved at least in part by incorporation of diacylglycerol from the plasma membrane to the secretory granule and recruitment of protein kinase Cb that activates the GTPase Cdc42 [28]. In turn, Cdc42 stimulates actin coat polymerization via its downstream effectors Toca1 and N-WASP [27,28,29]. Remarkably, Cdc42 coordinates actin remodeling and exocytosis also during neurite outgrowth as shown by the ability of its dominantactive mutant to promote exocytosis of TI-VAMP-containing vesicles in an actin-dependent manner at the growth cone [30]. An important molecular actor of exocytosis is the exocyst, an octameric protein complex conserved through evolution and essential for tethering secretory vesicles to specific domains of the plasma membrane [31]. Recently, one of the exocyst components, Exo70, has been found to directly bind to the Arp2/3 complex. Functional ablation of Exo70 prevents Cdc42-dependent or Rac-dependent actin cytoskeleton remodeling, recruitment of Arp2/3 at the leading edge of lamellipodia, and membrane protrusion, thereby affecting the migratory ability of the cell [32]. In conclusion, both membrane budding and fusion events require the interaction of actin polymerizing factors with the endocytic and exocytic machinery. These interactions appear to regulate actin polymerization at sites of active membrane dynamics. In turn, the remodeling of the actin cytoskeleton might participate in the execution of trafficking events in several ways, which also probably depend on the nature of the event, that include pushing of membrane invaginations, propelling of vesicles, creation of platforms for protein–protein interactions, or modulation of membrane plasticity (see also the following www.sciencedirect.com
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reviews addressing the role of actin in exocytosis and endocytosis [33,34]). Spatial and temporal control of actin polymerization in membrane trafficking
The Rho, Rab, and ARF families of small GTPases play a key role in determining the site and the duration of actin remodeling and membrane trafficking. Their localization at distinct membrane sites is tightly controlled via several mechanisms including post-translational modification by prenylation, association with GDIs (guanine nucleotide dissociation inhibitors), and phosphorylation. In an active GTP-bound form, these GTPases bind to downstream effectors, thus creating membrane-associated signaling platforms where membrane remodeling may occur and actin polymerization is stimulated ([35–37]; advances on the role of ARF proteins are extensively covered in a recent review [38]). An example of GTPase-driven formation of actin coats on vesicles is provided by studies on Rho proteins. Activated RhoB, by recruiting the Diaphanous-related formins mDia1 and mDia2, promotes the polymerization of actin around endosomes and the association of the latter with subcortical actin cables [39,40]. The prevailing model is
that RhoB binding to self-inhibited mDia relieves the inhibition resulting in the nucleation and processive elongation of nonbranched actin filaments at the site of activation. This in turn might generate the forces to propel vesicles in short-range movements. Indeed, treatment with actin depolymerizing or stabilizing drugs blocks the movement of endocytic vesicles, further supporting the requirement for actin dynamics in endosomes motility [40]. In addition, active RhoA has been found in association with PDGF-induced pinosomes, while they traffic through the cell, suggesting that RhoA stimulates actin polymerization thereby enabling pinosomes propulsion into the cytosol [1]. These data add to the previously identified function of another Rho family member, RhoD, which was found to regulate motility of early endosomes along actin fibers via its effector, hDia2C [41]. The coordination between actin cytoskeletal remodeling and membrane trafficking appears to be critical in particular for cell motility. In order to migrate, cells generate oriented lamellipodial extensions. Lamellipodia are formed by coupling strong actin polymerization, promoted by Rac, Cdc42, and Rho [1,42], and membrane protrusion, supported by vesicles delivery and recycling. While
Figure 1
A model for Rab GTPases function in integrin traffic. The migrating cell is extending lamellipodia where actin is polymerized as a meshwork of branched filaments. Integrin-containing structures (focal complexes) are formed at the base of the extending edge of lamellipodia and matured into focal adhesion. Integrins may internalize via Rab21-positive endosome and Rab5-positive endosome and recycle via the Rab4-recycling pathway or the Rab11-recycling pathway. www.sciencedirect.com
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a number of mechanistic studies have extensively addressed the role of actin dynamics in cell migration, the relevance of the exo–endocytic machinery and its link with the cytoskeleton is only starting to be appreciated ([43] and reviewed in [31,44,45]). The exo–endocytic processes have a dual function in cell locomotion: they provide new membranes for extension, and they also control cell spreading and adhesion by regulating the turnover of adhesive molecules at the plasma membrane. As the cell moves forward, adhesive structures, mainly the matrix-adhesive receptors integrins, are internalized and recycled to cell protrusive regions. Integrin internalization may occur, depending on the stimulus and on the type of receptor, via lipid rafts/caveolae [46,47]. Since caveolar trafficking requires actin cytoskeleton dynamics [26], an interesting question is whether actin remodeling also participates in integrin endocytosis. Furthermore, Rab21 and Rab5 have been recently found to bind to b1-integrins and to control their endosomal trafficking [48]. Notably, the motility of Rab21 vesicles close to the plasma membrane requires the actin cytoskeleton, and Rab21 mutants with impaired GTP binding or membrane localization induce the formation of exaggerated adhesion sites [48]. This is in agreement with the recent finding that integrins, in newly forming protrusions, travel on actin cables associated with the actin-based motor protein Myosin X [49]. Finally, integrins recycle via two routes: a Rab4-dependent early endosomes route, named ‘short loop,’ and a second path, which takes place from the perinuclear recycling Rab11-positive compartment, called ‘long loop’ [45]. The ‘long loop’ is regulated by the activity of both ARF6 and Rab11 and it also requires actin rearrangements [50]. Notably, Rab11-dependent endosomal recycling appears to be necessary in particular for polarized cell migration since disruption of its activity results in disorganized motility [51] (Figure 1).
Conclusions A growing body of evidence points to a requirement for actin dynamics in the majority of the membrane trafficking events investigated so far. In particular, future developments will certainly provide a high-definition mechanistic picture of how polarized exo–endocytic traffic of membranes and of adhesive cues is coupled to the actin cytoskeleton. This will be extremely relevant not only for physiology but also for a better understanding of those pathological states characterized by alterations of the normal migratory phenotypes, the first and foremost tumor cell invasion.
Acknowledgements Work in the author’s lab is supported by grants from Associazione Italiana per la Ricerca sul Cancro, the European Community (VI Framework), and the Association for International Cancer Research. The author thanks Giorgio Scita and Guido Serini for critically reading the manuscript. Current Opinion in Cell Biology 2007, 19:453–458
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