Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Rab8

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_19

Synonyms

Historical Background

Ras-like Rab GTPases (guanosine triphosphatases) are regulators of membrane trafficking. Identification of two Ras-like GTPases, Sec4p and Ypt1p, which are involved in regulating secretion in yeast, rapidly led to the discovery of additional small GTPases in mammalian cells. Rab8 was among the first group of mammalian Rab GTPases to be identified and is a close functional and sequence homolog of the yeast proteins Ypt2 and Sec4p. A second isoform, Rab8b (b for basophil), was cloned from mast cells in 1996. Rab8b shares 83% sequence identity with MEL/Rab8 (now also termed Rab8a) primarily over amino acids 1–152. The two isoforms display significant overlap in tissue distribution (coexpressed in liver, skeletal muscle, and testis), though Rab8 is much more abundant in lung and kidney, while Rab8b is more prevalent in heart, brain, and spleen. This review presents a synopsis of recent developments on Rab8 function. As detailed in the following sections, Rab8 regulates transport from the trans-Golgi network to the basolateral plasma membrane of epithelia, neuronal dendrites, and the ciliary membrane. Rab8 is required in cellular polarization, cellular signaling and development. Consequently, alterations in Rab8 expression and localization affect numerous cellular events and organ systems.

Rab8 Function

Analyses of Rab8 function across diverse cell types demonstrate that Rab8 plays a pivotal role in exocytic and endocytic pathway interfaces and is particularly important in exocytic events (Fig. 1). Rab8 predominately localizes to budding vesicles at the trans-Golgi network, recycling endosomes, exocytic vesicles, and ruffling plasma membrane domains. From the trans-Golgi network, Rab8 regulates delivery of newly synthesized lysosomal enzymes to endosomes (del Toro et al. 2009), exocytosis of regulated secretory vesicles and melanosomes (Faust et al. 2008; Wandinger-Ness and Deretic 2008; Sun et al. 2010), and plasma membrane export to neuronal dendrites, as well as to the basolateral, apical and ciliary membrane domains of epithelia (Bravo-Cordero et al. 2007; Wandinger-Ness and Deretic 2008). An emerging theme is that Rab8-mediated transport of newly synthesized ciliary membrane proteins, basolateral and apical cargo frequently occurs via recycling endosomes in coordination with Rab10 and Rab11a (Schuck et al. 2007; Cramm-Behrens et al. 2008; Wandinger-Ness and Deretic 2008; Knodler et al. 2010; Ward et al. 2011; Westlake et al. 2011). On the endosomal circuit, Rab8 may cooperate with Arf6, Rab11a or Rab13 to regulate externalization of recycling receptors (transferrin and glutamate), cell adhesion molecules (integrins and E-cadherin), and lipid regulators (ABCA1), often to specialized domains such as membrane protrusions, junctions and postsynaptic membranes (Hattula et al. 2006; Nagabhushana et al. 2010; Rahajeng et al. 2010; Roland et al. 2011).
Rab8, Fig. 1

Rab8 trafficking routes. Rab8 plays an important role in both exocytic and endocytic pathway interfaces and is particularly important in exocytic events. Through interactions with different protein complexes, Rab8 regulates delivery of newly synthesized lysosomal enzymes to endosomes, exocytosis of secretory vesicles, and cargo export basolateral, apical and ciliary membrane domains of epithelial cells. RE recycling endosome, EE/SE early endosome/sorting endosome, LE late endosome

Rab8 function in membrane trafficking is intimately connected to the cytoskeleton. Rab8 is associated with microtubules, actin and intermediate filaments, and expression of mutant Rab8 variants results in significant perturbations of both microtubule and actin networks (Omori et al. 2008; Wandinger-Ness and Deretic 2008). Rab8 has not been shown to bind microtubule-based motors, and the perturbation of microtubules induced by mutant Rab8 may be linked to Rabin8-dependent activation of Rab8 at centrosomes and the dependence of microtubule-based cilia on Rab8 membrane transport (Wandinger-Ness and Deretic 2008; Knodler et al. 2010; Westlake et al. 2011). On the other hand, Rab8-dependent actin filament association depends on direct Rab8 interactions with myosin V(a,b,c) or myosin VI motor proteins, which in turn facilitate directed vesicle trafficking (Wandinger-Ness and Deretic 2008; Roland et al. 2009, 2011). Rab8-myosin VI interactions direct basolateral cargo to the plasma membrane (Wandinger-Ness and Deretic 2008). Rab8/Rab27/myosin Va regulate the final stages of melanosome docking to the plasma membrane via actin filaments (Wandinger-Ness and Deretic 2008). Rab8-Rab11-myosin Vb and Rab8-myosinVb interactions are involved in apical lumen formation and insulin-dependent GLUT4 exocytosis, respectively (Ishikura and Klip 2008; Roland et al. 2011). Rab8 and myosin Vc likely cooperate in the early stages of regulated secretion involving tubule-dependent transport to the cell periphery of exocrine epithelia followed by hand-off in the cell periphery to Rab27a vesicles whose cortical actin anchoring depends on myosin Vc (Jacobs et al. 2009). The three isoforms of myosin V also act as scaffolds for the binding of multiple Rab GTPases cooperating on linked pathways, including Rab8, Rab10, Rab11a, and Rab27 (Roland et al. 2009). For example, Rab8 as well as Rab10 interact via an alternatively spliced exon D in myosin Va and Vb or an exon D-like domain in myosin Vc, while Rab27a interacts via exon F in myosin Va (Roland et al. 2009). Thus, regulated interactions of distinct myosin isoforms with distinct combinations of Rab GTPases may partially explain cell-type-specific regulation of Rab8-dependent pathways (Roland et al. 2009, 2011). However, questions remain as to how Rab8 functions cooperatively with other GTPases and how specific functions are regulated, for example: Is Rab8 bound simultaneously with multiple Rab GTPases to individual myosin motors? Are there hand-offs between Rab8 and other GTPases at discrete locations and how do they occur? How are specific effectors involved in discrete transport steps enriched at Rab8-positive membranes?

As illustrated by the given examples, Rab8 serves as a key regulator in communication between membrane transport circuits, the cytoskeleton and cellular signaling. Due to the critical role that Rab8 plays in multiple cellular pathways, loss of Rab8-protein interactions and regulation can result in numerous human diseases including open angle glaucoma, retinitis pigmentosa, microvillus inclusion disease, Huntington’s disease, and cystic kidney disease.

Rab8 Regulation

As with other Rab family members, Rab8 cycles through membrane-associated, GTP-bound “on” states and cytoplasmic, GDP-bound “off” states. Membrane association depends on isoprenylation by protein geranylgeranyl transferases (primarily REP/GGTase II or GGTase I, minor pathway) [reviewed in (Wandinger-Ness and Deretic 2008)]. Activation of membrane-bound Rab8 through GTP binding may be facilitated by one of several guanine nucleotide exchange factors (GEFs) such as MSS4, Rabin8/Rabin3, Rabin 3-like GRAB, or retinitis pigmentosa GTPase regulator (RPGR). In the activated state, Rab8 is a scaffold for numerous effectors and kinases that cooperatively serve in the temporal and spatial regulation of Rab8 transport. Upon completion of the transport cycle, nucleotide hydrolysis and Rab8 inactivation can be mediated by two known GTPase activating proteins (GAPs), AS160 (Akt substrate)/Tbc1d4 (Tre-2/Bub2/Cdc16) domain containing protein or Tbc1d30/XM_037557, for which multiple isoforms may exist (Wandinger-Ness and Deretic 2008). Interestingly, AS160 also serves as a GAP for Rab10 and Rab13 suggesting that GTPases that function on interrelated pathways may also be coregulated (Ishikura and Klip 2008; Sun et al. 2010). Inactive Rab8-GDP is bound and recycled from membranes to the cytosol by GDP dissociation inhibitor-2 (GDI-2) (Shisheva et al. 1999). Given Rab8 involvement in multiple transport pathways, some of which are highly cell type specific, it remains to be clarified if and how Rab8 function is regulated in a site-specific manner.

Rab8 Effectors, Signaling Integration, and Disease

Spatial and temporal regulation of Rab8 trafficking is partially dependent on signaling receptors, as well as effector and cargo protein interactions, thereby conferring specificity to Rab8-mediated targeting in accordance with cell need and cell type (Wandinger-Ness and Deretic 2008). For example, Rab8 interactions differentially regulate α2B- and β2-adrenergic receptor transport from the trans-Golgi network to the plasma membrane (Dong et al. 2010). Triggered by integrin-mediated adhesive events, Rab8 controls the polarized and regulated exocytosis of matrix metalloprotease MT1-MMP/MMP-14, which is critical for collagen degradation and tumor cell invasion (Bravo-Cordero et al. 2007). Specific cell types use conserved trafficking mechanisms during different stages of polarization. For example, Rab8, Rab11, Rabin8, and the exocyst complex are used with different effectors to traffic apical protein cargo during lumen formation and ciliary protein cargo during ciliogenesis upon reaching a fully polarized, ciliated state (Fig. 2). Loss of cargo, especially signaling proteins, to the proper target membrane results in aberrant signaling and disease. Although an assortment of diverse Rab8a effectors and cargo are known, further study is needed to determine if Rab8a activity is regulated by distinct sets of regulators within specific cellular compartments.
Rab8, Fig. 2

Rab8 exocytic trafficking in ciliated and polarizing epithelial cells. (a) Ciliogenesis and cilial transport is regulated in part by Rab8-mediated vesicle trafficking. Vesicular transport of membranous protein cargo such as polycystin-1 begins with formation of a Golgi-exit complex consisting of Arf4 and Rab11 and recruitment of the Arf4 GAP (ASAP1) and Rab11 effector FIP3. TRAPPII components and Rab11 are required for recruitment and vesicular trafficking of Rabin8. Near the centrosome Rabin8 recruits and activates Rab8, which facilitates transit and docking with BBSome. Vesicle fusion is thought to require Rab8, Rab10, the exocyst complex and Cdc42. Kinesin and dynein motor protein isoforms transport proteins within the primary cilium, though the link between Rab GTPases and motor proteins within the cilium has not been defined. (b) Rab8, Rabin8, exocyst components and Cdc42 also participate in Rab11-mediated vesicle transport (recycling endosome transition to exocytic vesicle) in polarizing epithelial during the formation of tubule lumens. These vesicles interact with myosin Vb to transport to the apical surface via the actin cytoskeleton. Rab11 interacts with Rabin8, which in turn recruits and activates Rab8. Rab8 recruits Tuba, the Cdc42 GEF, which activates Cdc42. Cdc42 and Par6 recruit aPKC, which then recruits Par3-Sec8 and Sec10. Interactions with the exocyst and Rab GTPases promote association with the plasma membrane

Rab8-mediated trafficking is required to maintain cellular polarity and homeostasis, which when altered can lead to tumor formation and malignancy. Membrane type 1-matrix metalloproteinase (MT1-MMP, also known as MMP-14) plays a key role in tumor invasiveness by participating in proteolytic degradation of surrounding tissues through activation of MMP2. MT1-MMP is exocytosed via Rab8, and trafficking to the plasma membrane is enhanced with expression of constitutively active Rab8 mutants, whereas RNAi knockdown of Rab8 prevents MT1-MMP from reaching the plasma membrane and thus prevents collagen degradation (Bravo-Cordero et al. 2007). Of note, MT1-MMP activation of MMP2 and MMP9 from pro- to active form is dependent upon interaction between MSS4 (a Rab8 GEF) and a-integrin chains (Knoblauch et al. 2007). The MSS4 binding sites for α-integrin chains and Rab8 are likely competitive. Decreased expression of MSS4 affects assembly and remodeling of the extracellular matrix. Rab8 expression can be up-regulated in breast cancer malignancies and associated lymph node metastases, and further studies on Rab8 mechanisms in tumorigenesis and malignancy may pave the way for the development of small molecule treatments with Rab8 or associated effectors as therapeutic targets.

Rab8 mediates basolateral trafficking and protein transport to adherens and tight junctions. Rab8 and Rab13 interact with effector MICAL-L2 (Molecule Interacting with CasL-Like 2) via competitive binding and formation of distinct independent Rab8a-MICAL-L2 and Rab13-MICAL-L2 protein complexes. Rab-MICAL-L2 protein interactions dictate trafficking specificity by regulating E-cadherin recycling to adherens junction (further evidenced by Rab8 knockdown causing delay of E-cadherin delivery to adherens junctions in calcium switch assays), and Rab13-MICAL-L2 interactions mediate recycling of occludin to tight junctions (Rahajeng et al. 2010). MICAL-1, MICAL-2, MICAL-3, MICAL-L1, and MICAL-L2 make up a family of large proteins with two to three common domains that mediate vesicular transport and cytoskeleton organization (Rahajeng et al. 2010). Rab8 interacts with at least three MICAL family members. Rab6-dependent recruitment of Rab8 to exocytic vesicles involves a MICAL3 intermediate in Rab8-ELKS interactions (Grigoriev et al. 2011). A related MICAL-like protein, MICAL-L1, regulates Rab8 in endocytic recycling. MICAL-L1 is proposed to serve as a Rab8 effector that serves to stably link Rab8 and EHD1 (Eps15 homology domain 1) on endocytic recycling vesicles (Rahajeng et al. 2010). EHD ATPase scaffolding proteins localize to tubular and vesicular membranes, regulate endocytic trafficking, and coordinate activity with Rab GTPases through interaction with Rab effectors. EHD also mediates GLUT4 recycling. Thus, Rab8 in conjunction with MICAL and EHD proteins form scaffolds that are crucial for integrating basolateral and endocytic pathways.

Over the last 3 years insulin-dependent signaling has been linked with Rab-mediated vesicle trafficking. Insulin-dependent glucose uptake is mediated by regulation of the surface recycling of GLUT4 transporters. AS160/TBC1D4 serves as a Rab GAP and AKT signaling target and is phosphorylated in response to insulin. Upon phosphorylation AS160 GAP activity is likely inhibited to allow activated Rab targets to function, thereby releasing the brakes on vesicle docking and fusion and allowing GLUT4 insertion at the plasma membrane of myoblasts (Randhawa et al. 2008). TBC1D1 is a second GAP that coregulates Rab activation in response to insulin stimulation. Rab8a and Rab14 are targets of the GAP TBC1D1 in skeletal myotubes and AS160, along with Rab13, in myoblasts (Ishikura and Klip 2008; Sun et al. 2010). The Rab8, Rab10, and Rab14 GTPases are required for insulin-induced GLUT4 trafficking, with GTP loading stimulated by insulin in a cell-type-specific manner (Ishikura and Klip 2008; Sun et al. 2010). Rab8 and Rab13 act as a Rab regulatory cascade that is activated sequentially to promote GLUT4 translocation in muscle cells, while Rab10 functions in adipocytes. Downregulation of GLUT4 translocation is thought to be achieved through the interaction of active Rab8a with the myosin Vb motor protein, thereby altering Rab8a localization and negatively impacting GLUT4 translocation (Ishikura and Klip 2008; Sun et al. 2010). This example is illustrative of how signaling and Rab8-regulated membrane trafficking are closely intertwined and modulated through cell-type-specific processes.

Noc2, rabphilin, Rim2 and Slp4/granuphilin are members of the  synaptotagmin-like family and all bind to multiple Rab GTPases, including Rab3a, Rab8a, and Rab27a. Noc2 and rabphilin bind to active, GTP-bound Rab8a suggesting that both may serve as Rab8a effector proteins. Rim2 has been predominately studied in regulated secretion within presynaptic nerve terminals and insulin-secreting cells, where it functions in regulated exocytosis (Yasuda et al. 2010). Rabphilin is expressed in neuronal, neuroendocrine, intestinal goblet cells and kidney podocytes. Slp4 is also expressed in neuroendocrine cell dense core vesicles and additionally localizes to the insulin-containing vesicles of pancreatic beta cells. All the members of the synaptotagmin-like family are implicated in regulated secretion and are of interest due to the fact that synaptotagmin-like proteins also bind plus-end directed myosin motors and thus, may bridge Rab8-regulated vesicle docking and fusion to cytoskeletal translocation.

Further evidence for a link between Rab8 and myosin motors is provided by studies in enterocytes. Rab8a is essential for localization of apical proteins and maintenance of the small intestine (Fig. 1) (Sato et al. 2007). Microvillus inclusion disease of the small intestine is characterized by microvillar atrophy and malabsorption. Rab8 conditional knockout in mice causes microvillus inclusion disease and one case of human disease has been linked to decreased Rab8 mRNA and protein expression in the enterocytes of the small intestine. To date, no further work has been reported on the role of Rab8 in microvillus inclusion disease. However, several reports have linked myosin Vb mutations to microvillus inclusion disease, and myosin Vb mutations cause disruption of epithelial cell polarity, as evidenced by loss of microvilli on the surface of intestinal absorptive cells and microvilli present within intracellular inclusion (Ruemmele et al. 2010). Given that both Rab8a and myosin Vb defects can result in microvillus inclusion disease, it is interesting to speculate that, as is the case in insulin-dependent signaling, the motor protein myosin Vb may interact with Rab8a and direct trafficking to the apical surface, which when perturbed, inhibits surface expression of microvilli. Further roles for Rab8 and apical protein targeting were brought to light through the study of zymogen granules in the exocrine pancreas. Rab8 localizes to zymogen granules and facilitates delivery of digestive enzymes to the apical surface, evidenced by the fact that Rab8 knockdown decreases granule numbers and causes granule proteins to accumulate in the Golgi (Wandinger-Ness and Deretic 2008). The authors speculate that Rab8-zymogen trafficking may depend on a clathrin/AP-1/dynamin association at the Golgi.

At the Golgi, Rab8a interfaces with optineurin (FIP-2) to promote cargo export, which is mediated by clathrin adaptor complex AP-1 (del Toro et al. 2009). Optineurin localizes to the cytosol, Golgi, and recycling endosomes and participates in vesicular trafficking. Optineurin binds active, but not GDP-bound, Rab8, thus suggesting that optineurin serves as a downstream effector. Optineurin interacts with myosin VI, a minus-end directed motor, to link Rab8 positive membranes to the actin cytoskeleton [reviewed in (Wandinger-Ness and Deretic 2008)]. The optineurin E50K mutation causes glaucoma and has been shown to impair endocytic trafficking (as demonstrated by impaired transferrin uptake) and slows the velocity of Rab8-GFP positive vesicles (Nagabhushana et al. 2010). Interestingly, mutant E50K optineurin completely abolishes optineurin-Rab8 interactions at the Golgi (Chi et al. 2010). In mice, the E50K mutation causes massive apoptosis and degeneration of the retina, but not broader neuronal degeneration (Chi et al. 2010). The composite data suggest there may be a conserved mechanism for zymogen and optineurin-mediated trafficking in exocrine cells and neurons.

Optineurin-Rab8 interactions also come into play in Huntington’s disease. Huntington’s disease results from abnormal expansion of a polyglutamine tract in the N-terminus of the huntingtin protein and has defects in lysosome function. Huntingtin regulates post-Golgi trafficking of secreted proteins and interacts with the optineurin-Rab8 complex. Huntingtin links Rab8/optineurin vesicles to microtubules via interactions with via HAP1 (a Trio-like protein with a Rac1 GEF domain), dynactin (p150glued) and dynein [reviewed in (Wandinger-Ness and Deretic 2008)]. Expression of huntingtin mutants or decreased huntingtin expression decreases Rab8 and optineurin localization at the Golgi and inhibits clathrin and optineurin/Rab8-dependent trafficking to lysosomes (del Toro et al. 2009). Loss of huntingtin causes loss of Rab11 in isolated membranes, and Rab11-GDP was shown to interact with huntingtin ~30-fold greater than Rab11-GTP. Rab8 and Rab11 both play a role in polarized outgrowth and are preferentially located in the somatodendritic domain of neurons, and Rab8 functions in neuronal maturation and polarized transport. For example, Rab8 is required for the transport and insertion of (AMPA)-type glutamatergic receptors, which mediate the fast synaptic transmission throughout the nervous system, into the postsynaptic compartment. AMPA receptors are recycled back to the membrane via Rab11 recycling endosomes, suggesting a potential Rab11/Rab8 trafficking pathway similar to those found in neuronal photoreceptor cells. However, the generalizability of the mechanism remains to be studied further.

Rab8a in Cilial Transport

The role of Rab8 in cilial transport and cilial-mediated signaling was suggested approximately 16 years ago (Wandinger-Ness and Deretic 2008), and the body of literature supporting this hypothesis has grown significantly over the last 5 years. The primary cilium is a specialized organelle that sits atop most cell types in the body and serves as an antenna to sample the extracellular space and transmit signals to the cell body. Accordingly, components of several signaling pathways such as hedgehog, WNT and JAK-STAT, which regulate cellular growth, proliferation, differentiation, and polarization, are found within the primary cilium. Cilial structures are evolutionarily conserved but can have cell-type-specific modifications to detect and respond to various stimuli such as mechanical, physical, chemical, or temperature sensation. For example, the modified cilium in photoreceptor cells responds to light, whereas the primary cilia in kidney epithelia are thought to be chemo-mechano sensors. Loss or aberrant localization of membrane-bound and cytoplasmic ciliary proteins at the site of the primary cilia is associated with a group of inherited diseases known as the ciliopathies. Ciliopathies encompass a broad range of genetic mutations and phenotypes. Some genetic mutations cause single organ, adult onset and progressive disease such as autosomal dominant polycystic kidney disease (ADPKD) and retinitis pigmentosa, whereas other genetic mutations affect multiple organs. An example of a multiorgan disorder is Bardet-Biedl Syndrome (BBS), which exhibits phenotypes that include (but are not limited to) obesity, polydactyly, retinopathy, mental impairment, and kidney abnormalities.

Rab8 function aligns strongly with the defined responsibilities of primary cilia, namely, regulation of epithelial differentiation and maintenance, development and organ function through cellular signaling. Expression of GTP-locked Rab8 induces cilial extension, whereas depletion of Rab8 inhibits cilia formation and trafficking, which often results in renal and retinal defects (Nachury et al. 2010). Numerous studies have linked Rab GTPases to the transport of membrane-bound ciliary proteins to the primary cilium; however, the distinct mechanisms are not well defined. Many questions about ciliary trafficking have been brought to the forefront of basic cell biology research, such as: How many ciliary trafficking routes exist? How are they modulated for specific cell types? What are the signaling pathways that drive ciliary transport? What are the mechanisms that switch cellular transport to direct ciliary trafficking of molecules with multiple localization patterns? How do vesicular transport pathways cooperate with other trafficking pathways such as intraflagellar transport (IFT)?

In the context of cilial function, Rab8 trafficking is best characterized in the rhodopsin transport model. Rhodopsin requires several small GTPases, including Rab8, to shuttle from the Golgi to an elaborate primary cilium, known as the rod outer segment, of photoreceptor cells; a Rab8 GDP-locked mutant (Rab8-T22 N) inhibits docking and fusion of rhodopsin-containing exocytic vesicles in transgenic frogs which results in dramatic retinal degeneration (Wandinger-Ness and Deretic 2008). Rab8 in concert with the Rab6, Rab11, and Arf4 GTPases is responsible for rhodopsin transport from the Golgi to the rod outer segment, in concert with regulatory proteins (e.g., ASAP, the Arf4 GAP) and effector proteins (e.g., FIP3) (Mazelova et al. 2009a). Mutations in the extreme C-terminus of rhodopsin impair interactions between rhodopsin and the GTPase trafficking complex, which leads to autosomal dominant retinitis pigmentosa. The altered interaction results in aberrant trafficking of rhodopsin to the rod outer segment, and explains at the molecular level the cause of retinitis pigmentosa. These careful studies were the first to reveal GTPase-mediated vesicular hand-offs in ciliary transport, beginning with interaction of a cargo protein with Rab6 and Rab11 in the Golgi. The cargo recruits and binds Arf4 via a specific targeting sequence to facilitate vesicle budding and Arf4 cooperates with Rab11. ASAP1, the Arf4 GAP, recognizes membrane curvature and is recruited to bind Arf4 to promote GTP hydrolysis and removal of Arf4 from the ciliary-targeted vesicle. FIP3, the Rab/Arf effector also bridges Arf and Rab GTPases via interactions with Rab11 and ASAP1 (Fig. 2a). The described pathway of rhodopsin transport prompted further questions about mechanism conservation between different cell types, the identification of other trafficking molecules that participate in the GTPase vesicle ciliary relay, and the composition of the coat complex of the ciliary transport vesicles.

Further studies in renal epithelial cells provided clues to conservation of Rab-mediated ciliary trafficking. In normal cultured renal cells, Rab8 can be found at the perinuclear Golgi region, whereas in cultured ADPKD cells, Rab8 is mislocalized to disperse vesicles (Wandinger-Ness and Deretic 2008). Polycystin-1, a protein that when mutant causes ADPKD, uses the same GTPase transport mechanism as rhodopsin to traffic to primary cilia of renal epithelial cells (Ward et al. 2011). Thus, the trafficking mechanism consisting of Rab GTPases (Rab6, Rab11, Rab8), Arf4 GTPase, and Arf GAP ASAP1 is conserved between retinal photoreceptor cells and renal epithelial cells (Ward et al. 2011). In contrast, fibrocystin, a protein mutant in patients with autosomal recessive PKD, also utilizes Rab8 to traffic to the primary cilium, but the fibrocystin C-terminal sequence does not bind Rab6 and Rab11 (nor Rab17, Rab23, and IFT20), suggesting that Rab8 may serve as the common GTPase between different vesicular transport mechanisms leading to ciliary delivery (Follit et al. 2010).

Until recently, the mechanism of Rab8 recruitment and activation along the cilial trafficking route was an enigma. For example, in the sequential transport of cargo that traffics with Rab6, then Rab11, and finally with Rab8 vesicles, how is Rab8 recruited to the exocytic vesicle? Identification of the interaction between Rabin8, a Rab8 GEF, and a complex of Bardet-Biedl proteins (known as the BBSome) at the centriole provided insight into the GTPase coordination and localized GTPase activation required for ciliary formation. The BBSome-Rabin8 interaction was identified at the ciliary base within pericentriolar recycling endosomes, offering the first clue for spatial regulation (Wandinger-Ness and Deretic 2008). Here BBSome components assist in recruitment of Rab8 to the pericentriolar recycling endosome for nucleotide exchange and activation, presumably by Rabin8. Further studies revealed that transport protein particle II complex (TRAPPII) components (C3, C9 and C10) and Rab11 are required for vesicular trafficking of Rabin8 to the centrosome, where Rabin8 subsequently recruits and activates Rab8 (Fig. 2a) (Knodler et al. 2010; Westlake et al. 2011). Thus, Rabin8 serves both as a Rab11 effector and as a Rab8 activator and both Rab8 and Rab11 are required for ciliogenesis. Together, the data highlight the involvement of a spatially and temporally regulated GTPase cascade in cilial formation and ciliary membrane protein targeting.

The dissection of a specific Golgi to cilia pathway is revealing key players in a ciliary targeting, but there are numerous examples of Rab8 involvement in cilial targeting that do not seem to follow the above-mentioned GTPase sequence and the unifying mechanisms remain elusive. CEP290 mutations are linked to several inherited cystic diseases including Senior-Löken syndrome, nephronophthisis, Joubert syndrome, Meckel-Grüber syndrome, and BBS. CEP290 protein satellites the base of the primary cilium, interacts with pericentriolar material 1 (PCM-1), and CEP290 knockdown significantly inhibits Rab8 localization to the primary cilium (Kim et al. 2008); however, the mechanism that mediates CEP290/PCM-1 Rab8 recruitment to the primary cilium remains undefined. An alternate Rab8-mediated ciliary trafficking mechanism has been suggested in Caenorhabitis elegans worms in the context of the transmembrane olfactory receptor ODR-10 (Kaplan et al. 2010). Rab8 associates with AP-1 in the clathrin-dependent delivery of ODR-10 to dendritic sensory cilia. Overexpression of GTP-locked Rab8 or mutations in the clathrin heavy chain perturbed ODR-10 trafficking and caused ODR-10 to localize to all plasma membrane compartments. Therefore, AP-1 and Rab8 likely cooperate to direct ODR-10 to the dendritic cilium. The authors suggested that a default secretory pathway directs proteins to all plasma membrane regions, and that this default pathway can be re-routed by AP-1 and Rab8 activation. The level of Rab8 activity may serve as the determining factor in protein destination. If so, then active Rab8 may serve as the switch between a general secretory and cilial-directed transport pathway. The authors’ leading model predicts that AP-1 functions at the trans-Golgi network in a clathrin-dependent manner, where the cargo is packaged into budding vesicles. Upon leaving the Golgi, the vesicle uncoats, fuses with Rab8 positive vesicles, and targets to the cilium. It is interesting to speculate that Rab8 vesicles that traffic ODR-10 from neuronal soma to the dendritic cilium may resemble secretory granules in exocrine cells and contain components of the pericentriolar recycling endosomes described in epithelial cells.

At site of the cilium, Rab8 serves as an evolutionarily conserved regulator of ciliary trafficking in several cell types. In turn, the exocyst complex, first identified in Saccharomyces cerevisiae, also serves as a conserved trafficking regulator and as an effector of several GTPases. The exocyst complex is comprised of several subunits and regulates polarized secretion via docking of intracellular vesicles to the plasma membrane. The exocyst also localizes to the primary cilium of renal cells, and Sec6 and Sec8 exocyst components are overexpressed or diminished in ADPKD cells. The exocyst complexes with small GTPases including RalA, Rho1, and Rab8, and regulates function via interactions with GAPs and other molecules such as aPKCs and the Arp2/3 complex (Hertzog and Chavrier 2011). Rab8a-Sec6/8 interactions are thought to control vesicle docking and fusion with the basolateral plasma membrane, thus linking the exocyst with Rab GTPase trafficking. In photoreceptor cells, Rab8 cooperates with phosphatidylinositol (4,5)-bisphosphate, moesin, Rac1 and actin to tether and fuse vesicles to the base of the modified photoreceptor cell cilium. The Sec6/8 complex likely serves as a Rab8 effector during GTPase-mediated vesicular trafficking, as Sec8 colocalizes with Rab8 at fusion sites of vesicles transporting rhodopsin, and, like Rab8, the exocyst localizes to the primary cilia of renal epithelial cells (Fig. 2a) (Nachury et al. 2010). In the case of retinal cells, the Sec6/8 complex coordinates with syntaxin 3 and SNAP-25, whose interactions are regulated by omega-3 docosahexaenoic acid, to regulate rhodopsin delivery (Mazelova et al. 2009b).

Rab8, Rab11, Rabin8 and exocyst components play dual roles in renal epithelial cells. Once renal cells are polarized, Rab8, Rab11 and Rabin proteins participate in delivery of trans-membrane protein cargo to the primary cilium. However, during polarization and lumen formation, Rab11, Rabin 8 and Rab8 mimic a yeast trafficking pathway and traffic cargo, such as podocalyxin, from the trans-Golgi to the forming pre-apical membrane (Fig. 2b). Rab11 recruits Rabin8, which recruits and activates Rab8. Rab11 (and potentially Rab8) also recruits Sec15a, an exocyst component, which binds Sec10 at the plasma membrane. Rab8 recruitment to the transport vesicle enhances active Cdc42 localization, likely driven by Rab8 activity on Tube, the Cdc42 GEF. Cdc42 and Par6 recruit aPKC, which then recruits Par3-Sec8 and Sec10. Thus, similar Rab8 trafficking mechanisms are utilized throughout the cellular polarization process, but the role that Rab8 plays in the regulation and switch between different target destinations remains to be explored.

One important question in the field of ciliary trafficking is how the intraflagellar transport (IFT) system, which transports non-membranous particles to and within the primary cilium, interfaces with the Rab8 GTPase-mediated vesicular trafficking pathway. The IFT protein Elipsa provided one of the first links to IFT-vesicular cross talk. Elipsa localizes to primary cilia and interacts with Rab8 via the Rab8 effector, Rabaptin5 (Omori et al. 2008). Elipsa also directly interacts with IFT20, which has been shown to localize to the Golgi and within primary cilia, and when knocked down, decreases the amount of polycystin-2 delivered to primary cilia (Follit et al. 2010). Combined, these data suggest that IFT plays a role in the transport of membrane-bound proteins to the primary cilium. Taken together, one can speculate that membrane-bound proteins interact in a complex associated with GTPase-mediated vesicular transport, which in turn interacts via effector proteins, such as Rabaptin5, with IFT components. Of note, IFT molecules have been linked to the formation of the immune synapse in T-lymphocytes (Nachury et al. 2010), and immune synapse formation shares similarities with cilial trafficking, namely, the use of IFT20, IFT57, IFT88, Kif3a motor protein. In contrast, IFT transport was not affected when ODR-10 transport was perturbed by Rab8 and clathrin manipulation (Kaplan et al. 2010). Further studies are necessary to dissect out the overlapping and independent roles of IFT and Rab8-mediated vesicular transport pathways to cilia.

Though advances have been made in the context of Rab8 and cilial trafficking, numerous questions remain about coat complexes used by cilia-destined vesicles in various cell types. Since Rab8, along with FIP-2, facilitates AP-1-mediated cargo export from the Golgi and may be associated with AP-1 in zymogen granule transport, could AP-1 (an Arf4 effector) also serve to generate the exocytic vesicle coat in mammalian epithelial cells? Alternatively, going back to the Rab GTPase-mediated transport in epithelial cells (Fig. 2), note that Arf4 facilitates vesicle budding. Arf1 and Arf4 regulate COPI recruitment and TRAPPII components bind COPI (Angers and Merz 2011). Thus, COPI may serve as the initial vesicle coat for some ciliary targeted vesicles. The coat-like BBSome plays a role in Rab8 recruitment and activation at the pericentrosomal region and the complex shares similarities with the COPI and clathrin coat complexes (Nachury et al. 2010). Therefore, after initial budding, does the vesicle coat morph to resemble components of the BBSome, or can the BBSome itself become the late-stage Rab8-positive vesicle coat prior to fusion with the ciliary membrane? Further questions arise about how Rab8, a potentially central ciliary component, may play a role in recruitment of the motor proteins and tethering components for ciliary transport vesicles. Recently, helical SNARE [Soluble NSF Attachment Protein (SNAP) Receptor] tethering proteins have been incorporated into the rhodopsin trafficking model (Mazelova et al. 2009b). Syntaxin 3 and SNAP-25 regulate rhodopsin delivery and localize to the base of the cilium, where Rab8 recruitment and activation is predicted to occur. SNARE proteins can function as Rab effectors or GEFs and future studies may reveal that SNARE proteins, in addition or as an alternative to the BBSome and Rabin8, may play a role in Rab activation at the primary cilium.

Summary

Vesicular transport can be simplified into three major steps: budding from the donor membrane, translocation of the trafficking vesicle along the cytoskeleton, and docking and fusion with the acceptor membrane compartment (Wandinger-Ness and Deretic 2008). Rab GTPases facilitate this process by serving as molecular scaffolds and interacting with cargo, proteins that promote vesicle budding and coat proteins, and through recruitment of motor protein complexes and tethering molecules. Rab8 is involved in several transport pathways and interfaces with endocytic pathways, regulates exocytosis, and coordinates regulation of the cytoskeleton and trafficking of diverse cargo to multiple subcellular destinations. Rab effectors, motor proteins and specific GEFs, GAPs and kinases mediate regulation of Rab8. Defects in Rab8-mediated trafficking have profound effects on cell morphogenesis, cytoskeletal organization, and cellular polarity. Rab8 serves to coordinate vesicle recycling and delivery of newly synthesized vesicle components to target membranes and functions as a nexus between vesicular endocytic and exocytic pathways. There are a number of unanswered questions in the field of vesicle trafficking: How do cells temporally and spatially regulate the sorting decisions for GTPases? What are the molecular switch mechanisms that cause a shift of shuttling cargo from one target to another? What signals cause Rab8 to associate with different effectors and how are these signals processed? Further investigation of Rab8 in diverse cell types and polarization states is predicted to reveal the unifying and diverse mechanisms of GTPase trafficking and lead to specific signaling targets for small molecule therapies for tumor invasion, cyst formation, and neurologic disease.

Notes

Acknowledgments

We acknowledge fellowships from NCRR INBRE 5P20RR016480 and NIGMS 1K12GM088021 to HW and research support from NIDDK DK50141 to AWN.

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Pathology and Cancer CenterUniversity of New Mexico Health Sciences CenterAlbuquerqueUSA