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Regulating the actin cytoskeleton during vesicular transport Mark Stamnes Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)mediated transport at the Golgi apparatus and on clathrinmediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transportvesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation. Addresses Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA; e-mail: [email protected] Current Opinion in Cell Biology 2002, 14:428–433 0955-0674/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations ARF ADP-ribosylation factor COPI coat protomer I Hip huntington-interacting protein GAP GTPase-activating protein mAbp1 mammalian actin-binding protein 1 PI phosphatidylinositol PIP2 PI 4,5-bisphosphate SH3 Src homology 3 VSVG vesicular stomatitis virus G protein WASP Wiskott–Aldrich syndrome protein

Introduction As the molecular bases for transport-vesicle formation and membrane fusion events become increasingly clear, additional attention is being paid to the regulatory and targeting mechanisms in the secretory pathway. Recently, progress has been made in identifying specific actinbinding proteins and actin signaling molecules that appear to function in the secretory pathway. Importantly, these discoveries are revealing which transport steps in the cell are sensitive to actin regulation and are beginning to suggest roles for the actin cytoskeleton in protein transport. The most progress toward elucidating the mechanisms for regulating the actin cytoskeleton in the secretory pathway comes from studies on the Golgi apparatus and on endocytosis. Progress in these areas is revealing interesting parallels and distinctions in the regulation of actin at these two steps.

Some of these similarities and differences will be highlighted within this review.

The actin cytoskeleton in the early secretory pathway A first line of evidence linking the actin cytoskeleton to protein trafficking in the early secretory pathway comes from studies examining defects in protein transport upon treating cells with actin toxins. Anterograde vesicular stomatitis virus G protein (VSVG) transport through the Golgi was found to be inhibited when cells were treated with cytochalasin B [1]. In a separate study, latrunculin B and botulinum C2 were found to block Golgi-to-ER retrograde transport [2]. Treating cells with cytochalasin D also affects Golgi morphology and positioning, indicating a role for actin in Golgi function [3]. These studies with actin toxins are all consistent with a role for actin in the early secretory pathway. A second line of evidence linking the actin cytoskeleton to the function of the early secretory pathway is the identification of actin-binding and actin regulatory proteins that localize to the Golgi membranes or to Golgi-derived vesicles. A rather long list of actin-binding proteins has been found associated with the Golgi, through both wholecell localization studies and the characterization of isolated membranes or vesicles. Among these proteins are actin itself [4–7], spectrin [4,6,8–13], ankyrin [9,14,15], centractin [5,10], tropomyosin [5], drebrin [6] and mammalian actinbinding protein 1 (mAbp1) [16•]. Owing to space limitations, actin-based myosin motors will not be discussed in detail in this review. However, a large body of literature implicates the actin motor protein myosin, particularly myosin V, in Golgi transport [17]. Actin may play multiple roles in transport, since different classes of Golgi-derived vesicles bind to distinct sets of actin-binding proteins [5]. Besides the actin-binding proteins, proteins involved in Rho-family-mediated actin signaling, such as Cdc42 [16•,18,19], IQGAP [19] and ARAP1 [20•] also localize to the Golgi apparatus (see below). Strong evidence that actin and actin signaling are important for Golgi function comes from recent studies showing that the binding of several actin cytoskeleton-related proteins to the Golgi is regulated by components of coat protomer I (COPI) transport vesicles, which mediate transport within and from the Golgi apparatus. Activation of ADP-ribosylation factor 1 (ARF1), the GTP-binding protein that regulates COPI vesicle coat assembly, causes a dramatic increase in actin and spectrin levels on Golgi membranes [4,6,16•]. ARF regulates spectrin levels on the Golgi through a mechanism involving phosphatidylinositol (PI) metabolism [4,21]. Spectrin is important for Golgi function, since

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Figure 1

Vesicle translocation and targeting

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Mechanisms for activating actin assembly during vesicle formation at the Golgi apparatus and at the plasma membrane. At the Golgi apparatus, activated ARF can stimulate PIP2 production by activating PI4-kinase [4,21]. Increased PIP2 levels recruits spectrin and actin to the Golgi. In addition, Cdc42 is recruited to the Golgi via a binding interaction with coatomer [16•,23•]. Cdc42/coatomer leads to the assembly of a specific actin pool containing mAbp1. Spectrin, Cdc42, WASP and mAbp1 are all implicated in protein transport in the early

secretory pathway. During endocytosis from the plasma membrane, mAbp1, together with other SH3-domain-containing proteins such as syndapins, amphiphysins, cortactin and intersectin, can bind dynamin [29,31]. Note that in yeast, Abp1 can activate Arp2/3-mediated actin polymerization directly and thus might bypass dynamin. Besides interacting with dynamin, intersectin may increase the levels of Cdc42–GTP by binding to and inhibiting a Cdc42/Rac-specific GAP. AP2, clathrin adaptor protein 2.

disrupting its binding with an anti-spectrin antibody or with truncated forms of recombinant spectrin inhibits VSVG transport at the Golgi [4]. The PI-regulated actin/spectrin cytoskeleton, together with Golgi-localized isoforms of ankyrin, have been proposed to mediate interactions with molecular motors and to organize vesicles and cargo proteins during Golgi transport ([22]; see the review by De Matteis et al., this issue [pp 000–000]) .

transport) might indirectly disrupt the function of other pathways (i.e. ER-to-Golgi transport), further characterization will be required to identify the trafficking steps directly influenced by Cdc42.

Cdc42 function during vesicle formation at the Golgi

Cdc42 also regulates the orientation of the Golgi apparatus in the establishment of cell polarity, through a mechanism involving dynein and dynactin function [27]. It will be interesting to characterize whether there are connections between Cdc42-mediated regulation of Golgi polarity and its regulation of protein transport.

While ARF/PI-sensitive regulation of the actin/spectrin cytoskeleton appears to be independent of the vesicle coat protein, recent studies show that ARF1 regulates the actin cytoskeleton on the Golgi also, through a second mechanism involving the Rho-family GTP-binding protein Cdc42 and vesicle coats. When Cdc42 function is disrupted using inhibitory or constitutively activated mutants, several transport defects have been uncovered at the Golgi apparatus. These include ER-to-Golgi transport of VSVG [23•], and retrograde transport of Shiga toxin from the Golgi to the ER [24•]. There are also selective defects in post-Golgi protein sorting and transport. Specifically, expression of Cdc42 mutants in MDCK cells was found to cause defects in the basolateral transport of VSVG and the endogenous marker protein gp58 [25]. Interestingly, a second study showed that mutant Cdc42 inhibited the basolateral transport of low-density lipoprotein receptor from the Golgi and also facilitates apical transport of neurotrophin receptor [26•]. These studies indicate an important role for Cdc42 signaling during protein transport at the Golgi apparatus. Since a direct defect in one pathway (i.e. Golgi-to-ER

The localization of Cdc42 is consistent with a role in regulating Golgi function, since both Cdc42 and its effector, IQGAP, are enriched on the Golgi membranes in several cell types [18,19]. Interestingly, the localization of Cdc42 to the Golgi is dependent on ARF activation, and it is reduced by expression of inhibitory ARF mutants [18]. The sensitivity to ARF indicates a link between the regulation of vesicle formation and Cdc42 function. This notion was greatly strengthened by the finding that Cdc42 is recruited to the Golgi membranes through a binding interaction with the COPI vesicle coat protein coatomer [16•,23•]. When recruited to the Golgi via coatomer, activated Cdc42 stimulates actin polymerization on the membrane [16•]. Actin polymerization is blocked in the presence of the Arp2/3-binding domain of Wiscott–Aldrich syndrome protein (WASP), a known inhibitor of Arp2/3 activation [16•]. Furthermore, constitutively active Cdc42 causes the recruitment of GFP-bound neuronal (N)-WASP to the Golgi [24•]. Microinjecting N-WASP into cells was found to affect Golgi-to-ER retrograde transport of the Golgi

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enzyme galactosyl transferase [24•]. Together, these studies indicate that coatomer/Cdc42 regulate actin polymerization and protein transport through a WASP/Arp2/3-dependent mechanism. Activation of this pathway leads to the recruitment of a distinct set of actin-binding proteins to the Golgi. One actin-binding protein — mAbp1 — is recruited to the Golgi through the function of the coatomer–Cdc42 complex [16•]. mAbp1 is the mammalian orthologue of the yeast actin-binding protein 1 (Abp1). Both Abp1 and mAbp1 have been implicated in protein transport during endocytosis (see below). mAbp1 localizes both to the Golgi region and to the plasma membrane, consistent with its function at both sites in the cell [16•]. Ectopic expression and mislocalization of mAbp1 disrupts ER-to-Golgi transport of VSVG [16•]. Thus, Cdc42, WASP and mAbp1 — three separate components of the coatomer-dependent actin regulatory mechanism (Figure 1) — each can be linked to either anterograde or retrograde protein transport between the ER and the Golgi in whole-cell transport assays [16•,24•]. An important clue regarding Cdc42 function at the Golgi is the finding that the Cdc42–coatomer complex is disrupted in the presence of the carboxy-terminal domain of p23, which binds to coatomer [16•,23•]. p23 is a member of the p24 family of putative cargo receptor proteins that are abundant integral membrane proteins on both COPI and COPII classes of coated transport vesicles [28]. This finding is important because it indicates that regulation of the actin cytoskeleton may be not only sensitive to vesicle coat assembly but also to the presence or absence of cargo proteins within the vesicles. While the connections between vesicle assembly, cargo packaging and actin regulation indicate a role for actin in vesicle release and/or targeting (see Conclusions, below), many questions remain with regard to the role of actin in the early secretory pathway. Indeed, the recent characterization of a novel signaling protein, ARAP1, indicates that there will be additional levels of actin regulation [20•]. ARAP1 contains pleckstrin homology (PH), ARF GAP (GTPase-activating protein) and Rho/Cdc42 GAP domains. On the basis of this domain structure, it could provide links between ARF, PI and Cdc42-based actin regulation. Like mAbp1, ARAP1 localizes both to the Golgi and to the plasma membrane [20•]. Exploring these connections between actin regulation at the Golgi and actin regulation during endocytosis at the plasma membrane may offer insight more generally into the function of the actin cytoskeleton during vesicular transport.

Actin and Cdc42 in the endocytic pathway Numerous recent studies have been characterizing the role of actin and actin regulatory mechanisms in endocytosis. Because of space constraints and the existence of excellent recent reviews on actin and endocytosis [29–31], I will not provide a broad overview of actin’s involvement in endocytosis in this section. Instead, I will focus on aspects that

highlight some of the similarities and distinctions between actin regulation at the Golgi apparatus and actin regulation at the plasma membrane during endocytosis. Key similarities include the regulation of actin by ARF [32–34] and the involvement of mAbp1, Cdc42 signaling and Arp2/3-mediated actin polymerization (Figure 1). Mammalian Abp1 and other actin-binding proteins function during endocytosis

A role for Abp1 in endocytosis has been found both in yeast and in mammalian cells. Surprisingly, the existing data indicate that mAbp1 might act through distinct mechanisms at different sites in the cell in different organisms. mAbp1, together with several other Src homology 3 (SH3)-domain-containing proteins that localize to clathrincoated pits, is important for the recruitment of dynamin to the site of a forming vesicle [29,31,35•]. Dynamin is a GTP-binding protein involved in the scission of nascent clathrin-coated vesicles from the plasma membrane. In yeast, Abp1 can catalyze actin polymerization by activating the Arp2/3 complex directly [36•]; but a similar property has not been shown for mAbp1, which lacks acidic residues important for Arp2/3 binding. However, recent work shows that dynamin regulates the polymerization of actin comet tails [37•,38•]. Thus, during endocytosis Abp1-related proteins could activate actin polymerization either by directly interacting with Arp2/3 or through the recruitment of dynamin. The polymerization of actin comet tails may be involved in the scission or translocation of vesicles [39]; it might also play a role in regulated exocytosis [40]. In yeast, Abp1 interacts genetically with a second actin-binding protein, End4/Sla2, which is required for endocytosis [41]. End4 is homologous with the mammalian proteins huntingtoninteracting protein, Hip1, and Hip1R [42,43]. Studies in mammalian cells have implicated these proteins in endocytosis [43,44•,45–49]. Hip1 and Hip1R localize to clathrin-coated pits via the epsin amino-terminal homology (ENTH) domain that binds to PI 4,5-bisphosphate (PIP2) [50•], and through interactions with both clathrin heavy chain and the AP2 clathrin adaptor [45,49]. Recent data indicate that this protein plays a role distinct from mAbp1. Studies suggest that Hip1 plays a role in coat assembly [44•,47,49] and in localizing clathrin-coated pits along actin filaments at the plasma membrane [46]. The emerging data indicate that actin-binding proteins play multiple important roles in the organization and function of the secretory pathway. Cdc42 function during clathrin-mediated endocytosis

Rho-family GTP-binding proteins regulate actin at the cell surface and regulate endocytosis, and both Cdc42 and Rac have been implicated in endocytosis [29,51•]. Unlike for coatomer, a direct interaction between clathrin-coat proteins and Rho-family members has not been observed. However, the protein intersectin, which localizes to clathrin-coated pits via an eps15 homology (EH) domain, provides a link between clathrin-coat assembly and Cdc42 signaling [29]. Intersectin was shown recently to interact with and inhibit a

Regulating the actin cytoskeleton during vesicular transport Stamnes

Cdc42 GAP protein, CdGAP [52•]. Interestingly, a brain splice variant of intersectin, intersectin-l, contains a Dbl homology domain that acts as a nucleotide exchange factor for the activation of Cdc42 [53•]. Intersectin also contains an SH3 domain which — like mAbp1 — can recruit dynamin to the sites of vesicle assembly [29].

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Conclusions There appear to be distinct differences in the utilization of actin-binding and actin regulatory proteins during transport in the early secretory pathway and in endocytosis. For example, mAbp1 recruitment at the Golgi membranes appears to be in response to Cdc42/WASP/Arp2/3-mediated actin polymerization, whereas at the plasma membrane it may activate actin polymerization. Nevertheless, there are also similarities in these processes which at this stage may be more informative. The most important similarity is that in both cases, regulation of the actin cytoskeleton involves direct interactions between vesicle-coat components and actin regulatory proteins. It seems that regulation of the actin cytoskeleton is closely linked to protein-transport processes involving transport vesicles. Thus, an important role for actin in protein transport is likely to be in the scission of vesicles and the translocation of vesicles from a donor organelle to an acceptor organelle. An emerging question is, why are there so many proteins and such complex mechanisms for regulating actin at the site of vesicle formation? If the scission and targeting of vesicles involve protein-catalyzed reactions, then it is very important that the temporal regulation of both vesicle formation and targeting be coordinated. If the scission and targeting machinery functions before the completion of vesicle formation, transport will be inefficient, as partially completed or partially filled vesicles will be translocated. Similarly, if scission and translocation are delayed, transport will be inefficient, as completed vesicles would accumulate at the donor organelle. Thus, the ability of cargo and vesicle coat proteins to interact with complex actin regulatory mechanisms involved in targeting and scission might help explain how such precise temporal coordination could be accomplished. The recent elucidation of these proteins and mechanisms involved in regulating actin during the endocytic and exocytic functions of the secretory pathway is sure to catalyze numerous future studies. It seems likely that a clearer picture of the role of the actin cytoskeleton in protein transport will soon emerge from this work.

Acknowledgements I thank J Ahluwalia for comments on the manuscript. This work is supported through a grant from the American Cancer Society.

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51. Garrett WS, Chen LM, Kroschewski R, Ebersold M, Turley S, • Trombetta S, Galan JE, Mellman I: Developmental control of endocytosis in dendritic cells by Cdc42. Cell 2000, 102:325-334. Using toxins and the introduction of mutant Cdc24. the authors show a requirement for Cdc42 during endocytosis in dendritic cells. They propose that developmental changes in endocytosis occur through regulating Cdc42 function. 52. Jenna S, Hussain NK, Danek EI, Triki I, Wasiak S, McPherson PS, • Lamarche-Vane N: The activity of the GTPase-activating protein CdGAP is regulated by the endocytic protein intersectin. J Biol Chem 2002, 277:6366-6373. This paper describes the characterization of a Cdc42/Rac-specific GTPase-activating protein (GAP) protein CdGAP, which binds to the SH3

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domain of intersectin. Binding of intersectin inhibited the GAP activity of CdGAP, indicating that it could act to increase the levels of activated Cdc42 or Rac. 53. Hussain NK, Jenna S, Glogauer M, Quinn CC, Wasiak S, Guipponi M, • Antonarakis SE, Kay BK, Stossel TP, Lamarche-Vane N, McPherson PS: Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat Cell Biol 2001, 3:927-932. This paper characterizes a brain-specific splice variant of intersectin that contains additional PH domains and a Dbl homology domain. The authors show that this isoform can catalyze nucleotide exchange on Cdc42.