Rab and Arf Proteins at the Crossroad Between Membrane Transport and Cytoskeleton Dynamics

Rab and Arf proteins at the crossroad between membrane transport and cytoskeleton dynamics

Ingrid Kjos1, Katharina Vestre1, Noemi Antonella Guadagno1, Marita Borg Distefano1, Cinzia Progida1*

1Department of Biosciences, University of Oslo, Norway *Corresponding author: Cinzia Progida, [email protected]

Keywords: small GTPases, Rab, Arf, Vesicular transport, Actin, Microtubule

Abstract The intracellular movement and positioning of organelles and vesicles is mediated by the cytoskeleton and molecular motors. Small GTPases like Rab and Arf proteins are main regulators of intracellular transport by connecting membranes to cytoskeleton motors or adaptors. However, it is becoming clear that interactions between these small GTPases and the cytoskeleton are important not only for the regulation of membrane transport. In this review, we will cover our current understanding of the mechanisms underlying the connection between Rab and Arf GTPases and the cytoskeleton, with special emphasis on the double role of these interactions, not only in membrane trafficking but also in membrane and cytoskeleton remodeling. Furthermore, we will highlight the most recent findings about the fine control mechanisms of crosstalk between different members of Rab, Arf, and Rho families of small GTPases in the regulation of cytoskeleton organization.

Introduction

The cytoplasm of eukaryotic cells contains membrane-enclosed compartments that constantly communicate with each other and with the extracellular environment. For this reason, the cells are provided with a highly regulated transport system that ensures the delivery of proteins and other molecules to their appropriate destinations. This transport system mainly consists of cargo-containing vesicles that bud off from the membrane of a donor compartment and are delivered to their target compartment where they fuse and release their cargo. The transport of vesicles relies on the cytoskeleton, a system of filamentous proteins which is also important for establishing and maintaining cell structure, shape, and motility.

The different fibers constituting the cytoskeleton are microtubules, actin and intermediate filaments. Both microtubules and actin filaments are polarized structures with a minus- and a plus-end. Microtubules are more rigid than actin filaments and function in long-distance transport and positioning of membrane-enclosed organelles [1, 2]. Actin filaments are more flexible and drive short- range transport, or help shaping the cell. Intermediate filaments provide mechanical strength. However, there is also evidence that supports their involvement in intracellular transport [3].

Vesicles and organelles can be connected to cytoskeleton filaments by motor proteins that convert energy from ATP hydrolysis into mechanical work to travel along the cytoskeleton. Specific molecular motors bind to actin filaments or microtubules. Dynein and kinesins are microtubule motor proteins. Normally, microtubules direct their minus-ends towards the microtubule organizing centers in the perinuclear region of the cell, and their plus-ends towards the cell periphery. The kinesin family, except for the kinesin-14 subfamily, performs plus-end-directed movement on microtubules, while dynein and kinesin-14 execute microtubule minus-end-directed transport [4]. The molecular motors binding to actin filaments belong the myosin superfamily [5].

In the cell, there is a continuous crosstalk between the cytoskeleton and membrane trafficking, and a plethora of proteins are involved in the coordination of this interplay. The members of the Ras superfamily of small GTPases are key regulators of these processes. The Ras superfamily consists of more than 150 different members in humans. These small GTPases share structural and biochemical similarities, and function as molecular switches that change conformation by alternating between an active GTP- and an inactive GDP-bound state. The Ras superfamily is divided into five major families, namely Ras, Ran, Rho, Rab, and Arf. The Ras GTPases are known for their role in signaling pathways. The Ran protein facilitates transport of RNA and proteins between the nucleus and cytoplasm. Finally, the members of the Rho family regulate actin cytoskeleton organization and dynamics, and the Rab and Arf families of GTPases are known for their role in regulating intracellular transport [6].

In this review, we will focus on the crosstalk between Rab and Arf proteins and the cytoskeleton. Indeed, although these small GTPases are important for intracellular transport, they are also emerging as regulators of the cytoskeleton, and should therefore be considered coordinators of membrane and cytoskeleton dynamics.

Interaction between membranes and cytoskeleton in vesicular transport

Rab and Arf GTPases Like the other members of the Ras superfamily, Rab and Arf proteins function as molecular switches and preferentially interact with effectors in their GTP-bound state. The cycling between GTP- and GDP-bound forms is tightly regulated by different factors, including guanine nucleotide exchange factors (GEFs), that stimulate the exchange of GDP for GTP, and GTPase activating proteins (GAPs), that promote GTP hydrolysis and thereby control the lifetime of the active small GTPase [7].

The first members of the Rab family were identified in yeast in the 1980s [8, 9], and today more than 60 members have been revealed in humans [10]. Each Rab protein can interact with a variety of

molecular effectors, providing the Rab family with high functional diversity. Indeed, all the intracellular trafficking steps are dependent on different Rabs interacting with specific effectors. For sorting of cargo and vesicle formation, Rabs interact with sorting adaptors as well as phosphoinositide kinases and phosphatases [11-14]. They also interact with motor proteins to mediate vesicle transport towards the acceptor membrane [15-17], while for tethering, docking or fusion the Rabs recruit either tethering factors or SNAREs [18-21].

The Arf family consists of 6 Arf proteins, as well as Sar proteins and more than 20 Arf-like (Arl) proteins [22-24]. Similarly to Rabs, also the members of the Arf family are highly conserved and localize to different intracellular compartments where they bind to specific effectors. In this way, Arf proteins regulate sorting and vesicle formation by recruiting coat components onto membranes. For example, activated Arf1 recruits COP1 coat protein complexes to the Golgi [25, 26]. Various adaptor proteins, such as AP1 and the GGAs, are also recruited by Arf GTPases from the cytosol onto TGN membranes. This induces membrane curvature and the formation of clathrin-coated vesicles [27, 28]. While Arfs 1-5 localize mainly to the Golgi complex and regulate secretory trafficking, Arf6 is present on the plasma membrane and regulates endocytic membrane trafficking [23]. The Arl proteins have various localizations, including the cytosol, nucleus, and mitochondria, and seem to have more diverse roles than the Arf proteins [29]. Indeed, only a fraction of them have been shown to regulate trafficking like the Arf proteins, as for example the TGN-localized Arl1 or the lysosomal Arl8 [30, 31].

Rab and Arf GTPases interact with motor proteins either directly or indirectly through a linker protein (Tables 1-2). By interacting with different motor proteins they link membranes to the cytoskeleton and mediate transport along microtubules or actin filaments to specific locations. The precise coupling of motors and vesicles through Rab or Arf proteins ensures that the cargo vesicle is directed to the right place in the cell (Figure 1).

Microtubule-based membrane transport Earlier reports indicate that Rab proteins can directly bind to dynein for retrograde traffic [32, 33]. However, more recent evidence demonstrates that dynein movement is dependent on dynactin, and thus Rabs rather bind to adaptors that help join the dynein/dynactin complex for retrograde transport [34]. One of the best characterized examples of Rab-mediated endosomal movement toward the microtubule minus-end is Rab7a. By recruiting the dynein-dynactin complex onto late endosomal membranes, Rab7a mediates endosomal transport toward the perinuclear region of the cell. The interaction between Rab7a and dynein is not direct, but occurs through the Rab7a direct interactor RILP [16, 35]. Indeed, expression of a truncated form of RILP leads to dispersion of late endosomes and lysosomes by preventing the recruitment of the dynein/dynactin complex [16, 35]. Interestingly, RILP is also an effector for Rab36, and through this interaction it mediates retrograde transport of melanosomes in melanocytes [36].

The intracellular distribution and the transport direction of an organelle can be regulated by multiple Rabs interacting with different motors. For example, on early endosomes Rab4 recruits dynein LIC-1 to regulate sorting of vesicles [33], and Rab1 recruits the minus-end-directed kinesin motor KIFC1 [37]. In addition, Rab5 mediates early endosome plus-end directed motility by recruiting the kinesin KIF16B through its effector, the phosphatidylinositol-3-OH kinase hVPS34 [38, 39]. Indeed, while expression of dominant-negative mutants or silencing of KIF16B repositions early endosomes to the perinuclear region and accelerates cargo degradation, the overexpression of KIF16B induces repositioning of early endosomes to the cell periphery and thereby inhibits transport to the degradative pathway [39].

Not only can different Rabs work on the same organelle to recruit diverse motor proteins, but a single Rab protein can also interact with several kinesins and dyneins. One of the most striking examples is Rab6 that has been reported to interact with several different motors and adaptor proteins, both plus- and minus-end directed. Rab6 interacts with the dynein adaptor proteins BICD2 and BICD1, but also

with the dynactin subunit p150glued, which possibly promotes Rab6-dependent recruitment of dynactin to the Golgi to mediate microtubule-dependent motility of Golgi-associated vesicles [40]. Moreover, Rab6 mediates exocytic vesicle transport to the cell surface by interacting with members of the kinesin-1 subfamily [41]. Rab6 also binds to kinesin KIF1C and the dynein/dynactin motor complex through Bicaudal-D-related protein 1 (BICDR-1) in the early phase of neuronal differentiation, taking part in regulation of the secretory trafficking [42]. More recently, Rab6 was found to interact directly with both the motor domain and the C-terminus of KIF1C. The binding of the motor domain prevents KIF1C from microtubule association and thereby decreases the delivery of vesicles to the cell surface [43]. The complexity of the network of interactions between Rab6 and different motor proteins might be at least partially explained by the multiple intracellular pathways regulated by this small GTPase at the Golgi. However, it remains poorly understood what determines the specific recruitment of the different motors for the regulation of the individual pathways.

Another example of a Rab interacting with several microtubule motors is Rab11. The Rab11 effector Rab11-FIP3 recruits the dynein light intermediate chain 1 (DLIC-1) to drive membrane trafficking from peripheral sorting endosomes to the centrally located endosomal-recycling compartment (ERC) [44]. Another Rab11-binding protein, Rip11/FIP5, interacts with KIF3A/B to mediate the sorting of internalized receptors to a slow recycling pathway towards perinuclear recycling endosomes [45]. How KIF3A/B, subunits of the plus-end directed motor kinesin-2, can mediate transport to the centre of the cell is unclear. It has been hypothesized that kinesin-2 could mediate the dissociation of endosomes from the actin cytoskeleton at the periphery of the cell; however the molecular mechanisms involved remain to be determined. Finally, Rab11 also associates with KIF13A to generate tubules from recycling endosomes [46].

Other Rabs connecting vesicles to the microtubule cytoskeleton are Rab2, Rab3 and Rab27a. Rab2 directly interacts with the microtubule-binding proteins aPKCι/λ and GAPDH on vesicular tubular clusters where they together form a complex that is important for retrograde transport between the Golgi and ER [47-49]. Rab2 is also able to recruit dynein in a PKCι-dependent manner to control microtubules and motor recruitment to vesicular tubular clusters [49]. In axons, the Rab3 effector DENN/MADD (Rab3-GEP) binds to KIF1Bβ and KIF1A to transport synaptic vesicle precursors towards the cell surface [50], while in cytotoxic T lymphocytes, Rab27a is linked to kinesin-1 to mediate lytic granule secretion [51].

Also members of the Arf family of GTPases can interact with microtubule motors [52]. For example, Arf6 interacts with the kinesin-1 and dynactin/dynein adaptor JNK-interacting protein 3 and 4 (JIP3 and JIP4) to mediate the rapid recycling of endosomes to the plasma membrane [53, 54], while Arl8 binds directly to the kinesin-1 linker SKIP to control the plus-end-directed trafficking of lysosomes [55-57]. BLOC-one-related complex (BORC) recruits Arl8 to lysosomes and together they regulate lysosome positioning and motility through recruitment of two distinct kinesin types: kinesin-1 (KIF5B), and kinesin-3 (KIF1Bβ and KIF1A) that mediates lysosome transport in the perinuclear and peripheral regions of the cell respectively [58, 59].

It has been suggested that the balance between Arf and Rab GTPases contributes to lysosome positioning. Indeed also Rab7a determines lysosome movement as it can either recruit the dynein- dynactin complex to mediate microtubule minus-end mediated transport, as mentioned earlier, or kinesin-1 through the binding to FYCO and protrudin for lysosome movement towards the cell periphery [60-63], (Figure 1).

Actin-based membrane transport In contrast to the long-range transport mediated by microtubule motor proteins, the myosin-mediated tranport along actin filaments normally covers shorter distances. For example, Rab27a works in a complex together with MyRIP and myosin Va to regulate the final steps of exocytosis by mediating the interaction between cortical actin and secretory granules [64, 65].

Class V myosins are efficient processive motors and it is therefore not surprising that they are involved in several intracellular transport pathways. In humans, there are three class V myosin genes encoding myosin Va, myosin Vb and myosin Vc [66]. Myosin Vb can interact with a number of Rab GTPases, such as Rab8, Rab11 and Rab10, and these Rab/myosin V-complexes control different pathways for recycling of proteins to the plasma membrane, and for polarization of epithelial cells [67]. Rab11a directly interacts with the myosin Vb-interacting protein Rab11-FIP2, thereby recruiting myosin Vb to recycling endosomes, and thus it regulates plasma membrane recycling steps [68, 69]. Rab8a also interacts with myosin Vb, but co-localizes to a tubular network which does not contain Rab11a, suggesting that Rab8a and Rab11a define distinct recycling pathways that both use myosin Vb [70]. Additionally, Rab8a interacts with myosin Vc, and the overexpression of GFP-myosin Vc tail perturbs the distribution of Rab8a-positive compartments [70, 71]. More recently, a study looking for Rab-myosin Va interactions by yeast two-hybrid screening found ten additional Rab proteins that interact with myosin Va, namely Rab3b, Rab3c, Rab3d, Rab6a, Rab6a´, Rab6b, Rab11b, Rab14, Rab25 and Rab39b [72]. However, in the same work it was demonstrated that Rab10 and Rab11 appear to be the major determinants for recruitment of myosin Va to intracellular membranes [72].

Class VI myosins are unique in that they walk towards the minus end of the actin filaments [73]. They are involved in membrane trafficking events such as the movement of newly formed vesicles from the plasma membrane towards the early endosomes, or the fusion of secretory vesicles at the plasma membrane through the indirect interaction with Rab8 mediated by optineurin [74-76]. Another myosin motor that is known to regulate intracellular transport is myosin IIA. Myosin IIA interacts with Rab3a in a complex together with the Rab3a effector synaptotagmin-like protein 4a (Slp4-a) [77]. This complex is responsible for the peripheral positioning of lysosomes and is involved in the regulation of lysosome exocytosis and plasma membrane repair.

In addition to transport mechanisms that depend on actin motors, the movement of vesicles along the actin cytoskeleton can also be induced by actin polymerization to drive long-range transport. Similarly to the strategy used by some pathogens such as Listeria and Rickettsia, the constitutively active mutant of Arf6 by recruiting the Arp2/3 complex induces actin assembly that causes the movement of vesicles [78, 79]. Moreover, a new actin-dependent pathway for long-range transport was more recently reported in mouse oocytes [80]. In this pathway, Rab11 forms a ternary complex with the actin nucleator Spir-2 and myosin Va, thus coordinating myosin motor activity and F-actin nucleation [80, 81]. As a consequence, Rab11-positive vesicles organize their own actin tracks facilitating long-range movement [80] (Figure 1). This mechanism is different from the one used by Arf6 and Listeria, and indeed is poorly affected by Arp2/3 complex inhibitors [78, 80].

Cooperation between microtubules and actin cytoskeleton To achieve the transport and delivery of vesicles from a donor membrane all the way to its destination, there is often cooperation between microtubule- and actin-mediated transport. An example is represented by the regulation of melanosome transport and positioning. Melanosomes are cell-type specific lysosome-related organelles that serve as the site for synthesis, storage and transport of melanin pigments [82], and several Rab proteins regulate their transport. Melanosomes move along microtubules in a bi-directional manner mediated by both kinesin and dynein motors. When they reach the cell periphery, they attach to the peripheral actin through myosin motors [83]. While melanosome retrograde movement is mediated by Rab36, the transport towards the cell periphery is mediated by Rab1a that recruits the microtubule plus-end-directed motor kinesin-1 (KIF5B+KLC2) to melanosomes through its effector SKIP (SifA and kinesin-interacting protein/PLEKHM2), [84]. Thus, Rab1a regulates the distribution of melanosomes in concert with Rab36, and a shift in the balance between these two Rabs could be the cause for the altered melanosome distribution seen in skin after sun exposure [85].

At the cell periphery, other Rabs link melanosomes to the actin cytoskeleton through myosin motors. Rab32 and Rab38, which are key components in the biogenesis of melanosomes, interact with myosin Vc [86]. In addition, Rab27a also links melanosomes via melanophilin or MyRIP to myosin Va [87-

89]. MyRIP also bridges Rab27a with myosin VIIa [90, 91], meaning that the same Rab can interact with different myosin motors. The C-terminal conserved regions of melanophilin and MyRIP, which are not essential for myosin Va binding, are also shown to bind directly to actin [91]. The MyRIP- mediated binding to actin could be how Rab27a aids tethering of melanosomes to the actin network also after detachment from myosin.

Moreover, Rab27a works together with Rab7a to regulate subsequent stages of melanosome biogenesis by recruiting different motor proteins. Rab7a, through its effector RILP, recruits dynein to mediate the transport of early and intermediate melanosomes along microtubules. Afterwards, when the melanosomes mature, this complex is replaced by the Rab27a-melanophilin-myosin Va complex, and the melanosomes switch from microtubule- to actin-based transport [83], (Figure 1). The importance of Rab27a and its interactors melanophilin and myosin V in melanosome transport is reflected in their association with Griscelli syndrome, a rare autosomal recessive disorder that results in hypopigmentation of skin and hair that can be caused by dysfunction of any of these proteins [92- 94].

Another well studied intracellular transport route involving cooperation of several Rab proteins together with microtubule and actin motors is the transport of the glucose transporter GLUT4. In unstimulated insulin-sensitive cells, such as fat and muscle cells, GLUT4 is mainly present in intracellular vesicles. Insulin stimulation activates Rab4 on GLUT4 vesicles leading to the binding of Rab4 with the microtubule plus-end-directed motor KIF3 [95]. This promotes the transport of GLUT4-containing vesicles to the plasma membrane and their release, and ensures the proper control of glucose levels in blood and tissues [96]. Rab5 is also associated with GLUT4-containing vesicles, but on the contrary to Rab4, Rab5 interacts with the minus-end-directed motor dynein. Insulin signaling inhibits Rab5 activity and the interaction between dynein and microtubules, and thereby limits the inward movement of GLUT4 [97]. Insulin also stimulates the binding between Rab8a and myosin Va which mobilizes GLUT4 vesicles toward the cell surface regulating their exocytosis [98]. Also Rab10 interacts with myosin Va and is involved in the final steps of insulin-stimulated transport to the cell surface [99]. Thus, the insulin-dependent translocation of the GLUT4 transporter is regulated by the coordinated action of many different Rabs and motor proteins.

Actin cytoskeleton-dependent membrane remodeling Rab and Arf GTPases interact with motor proteins not only to mediate vesicle and organelle motility, but also to control membrane remodeling. Indeed, even though actin polymerization by itself can promote membrane bending for endocytosis as shown for example in yeast [100, 101], molecular motors together with the cytoskeleton also influence membrane remodeling. By exerting force on membranes to induce curvature, they contribute, for example, to clathrin-coated vesicle budding at the plasma membrane [102, 103] and TGN [104, 105].

Members of class I myosins associate with a number of organelles to regulate membrane remodeling. Myosin Iα, for example, mediates not only the movement of lysosomes along actin filaments in cooperation with microtubules, but also the traffic of proteins in multivesicular endosomes (MVEs) by modulating the morphological organization of the early sorting MVEs [106, 107]. Myosin Ib regulates actin assembly and TGN remodeling, thus promoting the formation of post-Golgi carriers [108]. Also other myosins, including myosin18A [109] and myosin IIA, have been implicated in carrier formation at the TGN [110-112]. However, recent evidence showing that myosin 18A lacks motor activity and does not localize to the Golgi apparatus questions the contribution of myosin 18A to TGN carrier formation [113].

Myosin IIA is required for the assembly of basolateral transport vesicles from the TGN of polarized cells [114]. Furthermore, the coiled-coil region of myosin IIA associates with Rab6, suggesting that this motor is tethered to the Golgi by its tail and, by sliding along actin filaments via its head domains, it generates the force necessary for the formation of tubules or vesicles at the TGN [112]. In addition to the previously mentioned kinesins and dynein adaptors, Rab6 also interacts directly with the plus-

end-directed motor kinesin KIF20A (Rabkinesin-6/MKlp2/Rab6-KIFL) [15]. Even though it was shown that KIF20A is required for cytokinesis and is not expressed in interphase cells, the role of KIF20A in Rab6-mediated membrane traffic at the Golgi was not ruled out, as membrane trafficking is required during cell division [115]. Indeed, it has recently been demonstrated that KIF20A anchors Rab6 to the Golgi membranes near microtubule nucleating sites [116]. It has been suggested that Rab6, through recruitment of myosin IIA and KIF20A, couples the actin and microtubule cytoskeleton at the Golgi/TGN membrane. This enables spatial coordination for contractility in the process of carrier fission at the Golgi membrane and the subsequent vesicle movement along microtubules [116], (Figure 2).

Also Arf GTPases contribute to the regulation of actin cytoskeleton-dependent remodelling of membranes. One way to achieve this is by interacting with different lipid-modifying enzymes. Indeed, Arf GTPases can directly modulate local phosphoinositide synthesis, which in turn affects several actin regulatory proteins [117, 118]. Both Arf1 and Arf6 have been shown to recruit kinases that increase the local production of phosphatidylinositol 4,5 bisphosphate (PIP2) [119, 120]. This phospholipid binds directly to various cytoskeletal proteins, including the actin-nucleating factor Wiskott-Aldrich syndrome protein (WASP) [121]. PIP 2 also modulates the conformation of proteins at the interface between the cytoskeleton and the membrane, such as moesin and vinculin [117].

Arf6 coordinates membrane and cytoskeleton remodelling at the plasma membrane where it activates phosphatidylinositol 4-phosphate 5-kinase and triggers a local production of PIP2 that is involved in membrane ruffling formation [120]. Arf1 is involved in Golgi membrane dynamics. Arf1-driven lipid modification recruits spectrin to the Golgi to regulate the structure and function of this organelle [122]. The association of a complex consisting of the actin-binding protein cortactin and of dynamin, a large GTPase that mediates the formation of vesicles, with actin at the Golgi is also dependent on Arf1, and essential for the formation of nascent secretory vesicles [123].

Cytoskeleton regulation by membrane trafficking GTPases

Rab and Arf GTPases as regulators of cytoskeleton organization In addition to regulate different steps of membrane trafficking, Rab and Arf proteins also contribute to the regulation of cytoskeleton assembly by means of different mechanisms. One example is the Spir- 2-myosin Va-Rab11 complex described previously. Another mechanism involves Rab5. Rab5 functions as a signaling protein in actin remodeling triggered by receptor tyrosine kinases (RTKs). Upon activation by RTKs, Rab5 participates in the formation of circular ruffles through RN-tre which acts both as a Rab5 GAP and effector. Indeed, RN-tre interacts with the actin cross-linking protein actinin-4 that is implicated in actin bundling at the plasma membrane, leading to circular ruffles [124]. Rab5 also interacts with the actin-bundling proteins L- and T-plastin, and through these bindings it influences actin dynamics and endocytic activity [125, 126].

GDP-bound Rab27a interacts directly with another actin-bundling protein, coronin 3, and is necessary for endocytosis and recycling of insulin granules in pancreatic β-cells. The insulin secretagogue glucose induces the conversion of GTP-bound Rab27a to GDP-bound Rab27a and thereby the binding to coronin 3. In this way, Rab27a controls endocytosis of insulin granules by modulating actin assembly through coronin 3 [127].

Arf GTPases can also affect cytoskeleton dynamics. In addition to the previously described mechanisms used to remodel membranes by acting on the actin cytoskeleton, Arfs can also influence actin organization by interacting with myosin IIA via their GEFs and GAPs [128]. For example, ASAP1, which functions as an Arf GAP, binds directly to myosin IIA. Knockdown of ASAP1 reduces colocalization of F-actin and myosin IIA, and affects myosin-dependent processes such as maturation of focal adhesions, cell migration and spreading [129]. Also, the Arf GEFs BIG1 and BIG2 interact directly with myosin IIA, and negatively regulate its activity by promoting myosin light chain dephosphorylation [130]. Similarly, a Rab protein, Rab7b, regulates the actin cytoskeleton

assembly and stress fiber formation by direct interaction with myosin IIA. Importantly, this interaction has a double role. Indeed, it is also important for proper Rab7b-endosomal dynamics, as myosin IIA inhibition slows down, enlarges and clusters Rab7b-positive endosomes in the perinuclear region [131].

Rab proteins can also inhibit actin assembly. An example is Rab1. Rab1 directly interacts with WASP homologue associated with actin, membranes, and microtubules (WHAMM), a protein that drives Arp2/3-mediated actin assembly important for membrane tubule elongation. By recruiting WHAMM to dynamic tubulo-vesicular membranes, Rab1 negatively regulates its actin nucleation activity and thereby slows down actin assembly [132].

A few Rabs can regulate cytoskeleton dynamics by association with members of the molecules interacting with CasL (MICAL)-family proteins that are regulators of the actin cytoskeleton. MICALs, by oxidizing actin molecules, lead to actin depolymerization [133]. Rab13 performs many functions on the cytoskeleton through its effector MICAL-like protein 2 (MICAL-L2, also known as JRAB), an interactor of the actin binding proteins actinin-1 and actinin-4, and of filamentous actin that regulates actin cross-linking and stabilization [134, 135]. Rab13 and MICAL-L2 works together to regulate endocytic recycling of occludin, a component of tight junctions, by a mechanism involving the association between MICAL-L2 and the actin cytoskeleton [134]. Indeed, it has been suggested that Rab13, together with MICAL-L2, regulates reorganization of the actin cytoskeleton throughout epithelial junctional development [136]. In muscle cells, insulin stimulation promotes the interaction between Rab13 and MICAL-L2, and they further form a complex together with actinin-4 [137]. Through actinin-4, this complex is connected to GLUT4, and thereby localizes GLUT4 vesicles to the cell periphery to enable fusion with the plasma membrane. Thus, Rab13 and MICAL-L2 work together to coordinate vesicular trafficking and reorganization of the actin cytoskeleton.

Rab35 plays a role in endosome recycling through the effectors MICAL-L1 [138] and Arf6 GAP centaurin β2 (ACAP2) [139]. Rab35 recruits both MICAL-L1 and ACAP2 to recycling endosomes, which again recruits EHD1 to promote fission of tubular endosomes [138, 140, 141]. In phagocytosis, Rab35-dependent recruitment of ACAP2 to inactivate Arf6 promotes the formation of actin-rich protrusions which are necessary for final phagosome closure [142, 143].

Even though MICAL family proteins are regulators of the actin cytoskeleton, it has been suggested that MICAL-1 and -3 link Rab1 to microtubules [144, 145]. Indeed, Rab and Arf GTPases modulate not only actin cytoskeleton remodeling, but also microtubules and intermediate filament dynamics. Members of the Arf family of GTPases can affect microtubule-dependent processes by interacting with proteins that bind to microtubules [52]. The best characterized example is Arl2 that associates with the tubulin folding chaperone cofactor D, a β-tubulin GTPase activating protein, thereby regulating microtubule dynamics [146].

Some Rabs interact with intermediate filaments, either for the regulation of intracellular transport or to modulate filament assembly. However, these types of interactions are much less characterized. Rab7a interacts directly with two types of intermediate filament proteins, peripherin and vimentin, regulating their assembly and function [147, 148]. Peripherin is a neuron-specific intermediate filament protein, and its interaction with Rab7a has a possible role in the Charcot-Marie-Tooth type 2B disease, a peripheral ulcero-mutilating neuropathy. It has been demonstrated that Rab7a mutant proteins associated with this disease bind stronger to peripherin than wild-type Rab7a and lead to an increase in soluble peripherin, suggesting an alteration in neurofilament dynamics [148]. Vimentin is a class III intermediate filament protein. Its assembly and function are regulated by phosphorylation and Rab7a positively affects vimentin phosphorylation, thereby decreasing the amount of filamentous vimentin in the cell [147]. Also Rab9 interacts with vimentin. Alteration of this interaction caused by lipid accumulation in late endosomes in Niemann-Pick type C1 disease leads to late endosome dysfunction and vimentin intermediate filament hypophosphorylation [149]. Thus, the late endosomal Rab7a and Rab9 interact with and affect intermediate filament assembly.

It is therefore becoming clear that Rho proteins are not the only small GTPases able to direct cytoskeleton organization. As many interactions, direct or indirect, between Rab or Arf proteins and the cytoskeleton have been identified (Tables 1-2), we expect that in the future many novel mechanisms will be characterized and will reveal how these small GTPases work in concert with the cytoskeleton not only to mediate intracellular trafficking.

Cross-talk with Rho GTPases Increasing evidence indicates that Arf and Rab GTPases can influence cytoskeleton dynamics by regulating the activity and/or location of Rho proteins (Figure 3). Arfs were the first identified small GTPases able to influence cytoskeleton dynamics through cross-talk with Rho GTPases. For instance, Arf6 is involved in the cross-talk with Rac1 at the plasma membrane. Arf6 is required for Rac1- mediated membrane ruffling [150-152], and its activation triggers cell migration through activation of Rac1 [153, 154]. Several mechanisms by which Arf6 modulates Rac1 activity have been proposed. Arf6 can regulate the transport of Rac1 from endosomes to the plasma membrane [150], and is involved in the trafficking of lipid raft components required for Rac activation and coupling to effector proteins [118]. However, Arf6 affects Rac1 not only through its role in intracellular transport. Pulldown assays have demonstrated a direct interaction between the two GTPases, and that GTP- bound Arf6 interacts preferentially with GDP-bound Rac1[152]. Subsequently, Arf6 can modulate Rac1 activity by recruiting the Rac GEFs Dock180/Elmo complex [154], as well as Kalirin5 and Trio [155]. The ability of Arf6 to induce changes in the local membrane phospholipid composition is also likely to affect Rac activation. Indeed, Arf6-induced PIP2 production can be involved in the recruitment of Rac GEFs as they bind PIP2 via their PH domain [154, 156, 157]. Arf6 also binds directly to POR1, a Rac1-interacting protein that plays a role in membrane ruffling, to mediate cytoskeleton reorganization [158].

Rac1 exerts its effect on the cytoskeleton by activating the WAVE regulatory complex (WRC), which functions as an actin nucleation promoting factor. Although Rac1 is able to individually bind and activate the WRC in vitro, this interaction is of low affinity [159, 160]. Indeed, efficient activation of the WRC also requires an Arf GTPase bound to the membrane. Arf1 binds directly to the WRC, and cooperates with Rac1 to activate the complex and induce actin polymerization [160]. Arf6, on the other hand, does not bind the WRC directly, but it activates the complex by recruiting the Arf1 GEF ARNO to the plasma membrane [161]. By activating Arf1, ARNO enables WRC-dependent actin assembly and membrane ruffling [161]. In addition to activating Rac1, Arf6 downregulates the activity of RhoA, resulting in depletion of stress fibers [162] and promotes the delivery of Cdc42 and its GEF βPIX to the leading edge of migrating cells [163].

Arf1 also recruits Rho GTPases to TGN membranes to regulate actin polymerization during vesicle formation at the Golgi. Recruitment of active Rac1 promotes WAVE and Arp2/3-dependent actin nucleation during the initial stages of carrier formation, thus coordinating the assembly of clathrin coat components and actin polymerization [164]. In addition, Arf1 is involved in Cdc42 crosstalk. By interacting with COPI, Arf1 is essential for the recruitment of Cdc42 to Golgi membranes [165], (Figure 2), and can also recruit the Cdc42 GAP ARHGAP21 (originally reported as ARHGAP10) to downregulate Cdc42 activity [166]. Similarly, Arf1 recruits ARHGAP21 to the plasma membrane to control Cdc42 dynamics during clathrin-independent endocytosis [167]. Finally, Arf1 also modulates the activity of RhoA and RhoC, which in turn affects the phosphorylation of the myosin light chain influencing cancer cell invasiveness [168].

More recently, a similar crosstalk was discovered to occur also between members of the Rab and Rho GTPase families. One of the first evidence is the crosstalk between Rab5 and Rac. Rab5 regulates Rac activation by regulating Rac membrane trafficking leading to actin dynamics required in migrating cells [169]. In addition, the Rab5 effector and RhoD binding partner Rabankyrin-5 coordinates cellular processes regulated by these two small GTPases, including the internalization of platelet- derived growth factor receptor β (PDGFR-β) [170].

A crosstalk between Rab7b and RhoA has also been reported. Active RhoA positively regulates kinases responsible for myosin light chain phosphorylation, thereby stimulating the formation of stress fibers and cell migration. By modulating this pathway, Rab7b is able to influence actin cytoskeleton organization, the formation of stress fibers and cell migration [131]. The Rab27a effector Slp1 (also known as JFC1) interacts with the RhoA-GAP Gem-interacting protein (GMIP). Slp1, by inactivating RhoA, promotes local actin depolymerization and thereby facilitates vesicle movement through the cortical actin toward the exocytic active zone during azurophilic granule exocytosis [171]. Rab7a and Rac1 directly interact, and this interaction has been proposed to regulate late endosomal transport between microtubules and microfilaments in order to control ruffled border formation [172]. By modulating Rac1 activation, Rab7a can also regulate cell migration [173]. Interestingly, there is a two-way crosstalk between Rab7a and Rac1. Indeed, active Rac1 recruits the Rab7a GAP Armus to promote the local inactivation of Rab7a, which in turn contributes to E-cadherin turnover and therefore to the stability of cell-cell contacts [174].

Several other Rabs are known to modulate Rho activity to mediate cytoskeleton remodeling in different cellular processes. For example, cdc42 activity is controlled by Rab8 for polarization and lumen formation in epithelial cells [175], and by Rab14 for the establishment and regulation of cell polarity [176]. Furthermore, Rab35 modulates Cdc42 activity [177], and during phagocytosis it facilitates the transport of Cdc42 and Rac1 to the plasma membrane [178]. In addition, the role of Rab23 in cell migration and invasion involves the modulation of Rac1 activity [179]. However, the molecular mechanisms behind these regulations are not completely elucidated yet.

Concluding remarks

Even though Arf and Rab proteins were first identified as regulators of vesicular transport, it has lately been shown that they can also promote or inhibit cytoskeleton changes at specific cellular locations by binding directly or indirectly to cytoskeleton remodeling proteins. Nevertheless, our knowledge of the mechanisms involved is still quite limited.

We know that the integration of vesicular transport with cytoskeleton assembly can occur through crosstalk of Arfs or Rabs with Rho GTPases. This is well established for several Arfs [152, 162, 166, 180], and it is starting to emerge as a mechanism also for Rab proteins [131, 170, 171]. Arfs and Rabs can indeed regulate the spatial distribution of different Rho family members as well as their activation state by recruitment of Rho GAPs and GEFs (Figure 3). However, other ways of regulation might exist, and this is a future challenge to solve. Understanding the joint regulation between cytoskeleton dynamics and intracellular transport will help to shed light on different cellular processes, including cell division and migration.

During cell division, endosomal transport facilitates microtubule and actin cytoskeleton remodeling in spindle formation, orientation and function, as well as the actomyosin contractile ring dynamics [181- 183]. Some of the best studied Rabs involved in cytoskeleton remodeling during cell division are Rab6, Rab11 and Rab5. Rab6a splice variant Rab6a′, by directly interacting with the dynactin subunit p150Glued, regulates the dynamics of the dynein/dynactin complex at the kinetochores and alteration of Rab6a′ function leads to a block in the metaphase-anaphase transition [184]. Similarly, also Rab11 and Rab5 are involved in mitotic progression. Rab11 contributes to mitotic spindle organization and orientation by regulating the dynein-dependent localization of recycling endosomes, which contain microtubule-nucleating components, at poles [185]. Rab5 has a role in chromosome congression through the regulation of the nuclear envelope disassembly [186] and of the localization of CENP-F, a centrosome associated protein, to kinetochores [187].

In addition to cell division, intracellular transport can regulate other cytoskeleton-dependent processes such as cell migration and phagocytosis [188-191]. This regulation can occur, for example, by the delivery of activated Rho family members to distinct locations of the plasma membrane to drive cell protrusion formation [169, 178, 192, 193]. Nevertheless, several of these processes require a more

complex cooperation between members of the Rho, Rab and Arf families [194]. One example is the involvement of Rab11, Arf6 and Rac1 in the motility of breast cancer cells, where the Rab11 and Arf6 effector Rab11-FIP3 is necessary for both Arf6 plasma membrane targeting to the leading edge and the regulation of polarized Rac1-dependent actin cytoskeleton dynamics [195-197].

In conclusion, the cooperation of different families of small GTPases is required in several fundamental processes, and alterations in this crosstalk can lead to pathologies such as cancer or neurodegenerative diseases. Therefore, it is important for the future to dissect pathways regulated by multiple GTPases and their coordinated spatio-temporal regulation, rather than investigating cytoskeleton and membrane dynamics separately.

Acknowledgments The work in the authors laboratories is funded by grants from the Norwegian Cancer Society (grant 5760850 to C.P.) and the Research Council of Norway (grant 239903 to C.P.).

Figure legends

Figure 1. Rabs, Arfs and the cytoskeleton in intracellular trafficking. Schematic model illustrating Rab and Arf proteins in the transport of organelles along the cytoskeleton tracks. Magnifications of the boxed areas are shown in the respective insets and illustrate examples of Rab and Arf proteins interacting with different molecular motors. (A) The transport of melanosomes is mediated by different Rabs interacting with dynein/dynactin and myosin motors. (B) Lysosome positioning is determined by the balance of Rab7a and Arl8-mediated transport on microtubules. (C) Example of a transport pathway along the actin cytoskeleton. Here, Rab11 is in complex with the actin nucleator Spir-2, which in turn interacts with myosin Va to promote F-actin nucleation and myosin force generation for vesicle transport.

Figure 2. Rabs, Arfs and the cytoskeleton in membrane remodeling at the Golgi Schematic cartoon showing examples of Rab and Arf proteins involved in membrane remodeling at the Golgi for vesicle fission. Arf1 mediates vesicle formation and dynein-mediated motility by recruiting the Cdc42 GAP ARHGAP21 to the Golgi membranes to downregulate Cdc42 activity and modulate actin assembly (upper-left box). Rab6 mediates the fission and transport of vesicles originated from the Golgi by interacting with both the motor protein KIF20A and myosin IIA (upper- right box).

Figure 3. Crosstalk of Arfs or Rabs with Rho GTPases. Rab and Arf proteins regulate the activation/inactivation of Rho GTPases. Rabs and Arfs are able to recruit Rho GEFs or GAPs and hence determine the activity of the Rho GTPases. This in turn modulates the downstream Rho effectors and intracellular events such as the cytoskeleton dynamics.

Table 1. Rab interactors connecting Rab GTPases to the cytoskeleton

Rab Interactor Function Ref GTPase

Rab1 KIFC1 Early endosome minus-end-directed motility. [37, 198]

SKIP SKIP recruits kinesin-1 to regulate anterograde [84] melanosome transport.

WHAMM Inhibition of actin assembly. [132]

MICAL-1 , MICAL- Unknown. [144, 2 and MICAL-3 145]

Rab2 aPKCι/λ and Microtubule and dynein recruitment to vesicular [47-49] GAPDH tubular clusters.

Rab3 Gas8 Unknown (Gas8 is a microtubule-binding protein). [199]

DENN/MADD DENN/MADD functions as a linker between Rab3- [50] positive vesicles and KIF1Bβ/KIF1A.

Myosin IIA Control of lysosome positioning and plasma [77] membrane repair.

Myosin Va Neuronal vesicle transport. [72, 200]

Rab4 LIC-1 Recruitment of LIC-1 (dynein light intermediate [33] chain-1) on early endosomes.

KIF3 KIF3 associates with microtubules and participates in [95] GLUT4 exocytosis.

Rab5 RN-tre RN-tre interacts with both F-actin and actinin-4, an F- [124] actin bundling protein, thus promoting actin remodelling.

Dynein Binding of dynein to microtubules to regulate GLUT4 [97] movement.

L- and T-plastin Regulation of actin dynamics and endocytic activity. [125, 126]

hVPS34 Recruitment of KIF16B for early endosomal transport [14, 39] to the plus end of microtubules.

Rab6 BICDR-1 Recruitment of KIF1C on Rab6 secretory vesicles. [42]

KIF20A Carrier formation and exit along microtubules from [15, Golgi/TGN membranes. 116]

KIF1C Prevent the interaction of KIF1C with microtubules. [43]

DYNLRB1 Unknown. [32]

BICD1, BICD2 and Recruitment of kinesin-1 and dynein-dynactin to [40, 41, p150glued Rab6a-containing vesicles and Golgi membranes. 201]

Myosin IIA Fission of post-Golgi carriers. [112]

Myosin Va Unknown. [72]

Rab7a Vimentin Regulation of vimentin assembly. [147]

Peripherin Regulation of peripherin organization and function. [148]

FYCO1 Promotes plus end-directed microtubule transport. [202]

RILP Recruits dynein-dynactin to late endosomes and [16, 35] lysosomes.

Rac1 Modulate Rac1 activity and cell migration. [172, 173]

Rab7b Myosin IIA Regulation of Rab7b-vesicle dynamics and actin [131] remodelling.

Rab8 Myosin Va Transport of GLUT4 vesicles towards the cell surface. [98]

Myosin Vb and Transport to target membranes in a recycling pathway. [70, myosin Vc Translocation of GLUT4. 203]

Optineurin Rab8 is linked to myosin VI through optineurin/FIP-2, [204, and this interaction is involved in the secretory 205] pathway.

Rab9 Vimentin Regulation of intracellular lipid transport. [149]

Rab10 Myosin Va, myosin Regulation of distinct trafficking pathways, including [99, Vb and myosin Vc GLUT4 translocation to the plasma membrane. 206, 207]

Rab11 Moesin Regulation of Moesin activation during cell migration. [208]

Rab11-FIP2 Recruitment of myosin Vb for plasma membrane [68, recycling. 196,

209]

Rab11-FIP3 Dynein recruitment to mediate transport from [44, peripheral sorting endosomes to centrally located 196, recycling endosomes. Control of Rac1 intracellular 210] localization.

Rab11-FIP5 Recruitment of KIF3B for transport of internalized [45, receptors through the perinuclear recycling 211] endosomes.

Myosin Va and Maintaining a peripheral distribution of Rab11- [72, 81] myosin Vb positive endosomes. Recruitment of the actin nucleators Spir.

KIF13A Generation of recycling endosomal tubules. [46]

Rab13 MICAL-L2/JRAB Endocytic recycling and actin cytoskeletal [134, organization. 136]

Actinin-4 GLUT4 exocytosis. [137]

Rab14 KIF16B Golgi-to-endosome traffic. [212]

Cdc42 Regulation of Cdc42 activation and establishment of [176] cell polarity.

Myosin Va Unknown. [72]

Rab23 KIF17 Ciliary transport. [213]

Rab25 Myosin Va Unknown. [72]

Rab27a Melanophilin Myosin Va recruitment for melanosome transport. [87, 89, 214, 215]

MyRIP Myosin Va and myosin VIIa recruitment for [65, 90, melanosome transport. Myosin Va recruitment to 91] regulate secretion.

Coronin 3 Endocytosis of the insulin secretory membrane [127, modulating actin assembly. 216]

Slp3 Recruitment of kinesin-1 for lytic granule secretion. [51]

Rab29 Dynein and KIF3A Ciliary transport. [217]

Rab32 Myosin Vc Melanosome biogenesis and secretion. [86]

Rab34 RILP Lysosomal positioning. [218]

Rab35 Fascin Actin bundling. [219]

OCRL Remodelling of lipids and F-actin in the last step of [220] cell division.

MICAL-L1 Recruitment of Rab8, Rab13 and Rab36 to recycling [138, endosomes during neurite outgrowth. 221]

Rab36 RILP Retrograde transport of melanosomes on [36] microtubules.

Rab38 Myosin Vc Melanosome biogenesis and secretion. [86]

Rab39b Myosin Va Unknown. [72]

Rab41 Dynactin 6 Golgi organization. [222]

Table 2. Arf interactors connecting Arf GTPases to the cytoskeleton

Arf Interactor Function Ref GTPase

Arf1 WRC Promotion of actin polymerization. [160]

ARHGAP21 (originally Modulate Cdc42 activity that controls actin [166] reported as ARHGAP10) dynamics at the Golgi.

Cortactin and dynamin Actin recruitment to the Golgi for the regulation [223] of post-Golgi transport.

Arf6 JIP3 and JIP4 Association with kinesin-1 and the dynactin [53, 54] complex to mediate endocytic recycling.

Rac1 Actin remodeling and cell migration. [152]

Kalirin5 and Trio Kalirin5 and Trio function as Rac1 GEFs, [155] leading to activation of Rac1.

POR1 POR1 is a Rac1-interacting protein. Membrane [158] ruffling and cytoskeletal remodelling.

Rab11-FIP3 Targets Arf6 to the plasma membrane of the [224, leading edge, regulating polarized Rac1 225] activation and actin dynamics during cell migration.

Arl2 Tubulin folding chaperone Regulation of microtubule dynamics. [146] cofactor D

Arl8 SKIP Kinesin-1 recruitment to mediate microtubule [57] plus-end motility of lysosomes.

BORC Recruitment of kinesin-1 and kinesin-3 to [58, 59] mediate transportof lysosomes.

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