InteractiveFly: GeneBrief

Rab2: Biological Overview | References

BIOLOGICAL OVERVIEW

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. This study uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. It was shown that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, the screens uncover an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, (Jiang, 2014; Takáts, 2014) suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms (Fujita, 2017).

Differentiated muscle cells, or myofibers, are highly organized in order to coordinate the roles of specialized subcellular structures involved in contraction. Myofibril bundles of sarcomeres provide the contractile force. The power of contraction, however, requires synchronous sarcomere function under control of the 'excitation-contraction coupling' system that includes two membranous organelles, the sarcoplasmic reticulum (SR) and Transverse (T)-tubules (Al-Qusairi, 2011). The T-tubule membrane network is continuous with the muscle cell plasma membrane, with tubulated membranes that invaginate radially inward in a repeated pattern at each sarcomere. With excitation-contraction coupling, neuromuscular action potentials are transmitted along the muscle T-tubule membrane to the SR junction, or dyad/triad, triggering coordinated SR Ca2+ release and synchronous sarcomere contractions (Al-Qusairi, 2011). Formation of organized T-tubule membranes is thus critical for muscle function (Takeshima, 2015). Mechanisms must also remodel the T-tubule membrane network with ongoing myofiber reorganization in response to muscle use, damage, atrophy and aging. However, the extent and mechanisms of T-tubule remodeling remain largely unknown, in part due to challenges with observing T-tubule membrane network dynamics within intact mammalian myofibers (Fujita, 2017).

The T-tubule network includes both transversal and longitudinal tubular membrane elements that form and mature with myofiber differentiation and growth. In mouse skeletal muscle, mostly longitudinal tubular membranes initially present in embryonic muscle are remodeled postnatally with expansion to predominantly transversal tubular elements. In contrast, both longitudinal and transversal T-tubule elements are maintained in adult mammalian cardiac muscle and in insect muscles. Relatively few molecular factors are known to shape the T-tubule network, and perhaps not surprisingly, all of which so far encode for membrane-associated functions (CAV3, DYSF, BIN1/Amph2, MTM1, DNM2). Mutations in each also are associated with human myopathy and/or cardiomyopathy with T-tubule disorganization, pointing to the critical importance of membrane-mediated mechanisms to maintain the T-tubule membrane network (Fujita, 2017).

Drosophila is a powerful system for insights into the functional requirements for T-tubule formation and remodeling. The BIN1 BAR-domain protein has a conserved function involved in membrane tubulation required for T-tubule formation that was first described for the single Drosophila homolog, Amphiphysin. The amph null mutant flies lack transversal T-tubule element membranes in myofibers at all developmental stages, corresponding with both larval and adult mobility defects. In contrast, the myotubularin (mtm) fly homolog of mammalian MTM1/MTMR2/MTMR1 subfamily of phosphatidylinositol 3-phosphate phosphatases is required only at later stages in development for T-tubule remodeling. While mtm loss of function has no obvious effects on larval muscle T-tubule organization or function, mtm-depleted post-larval stage muscles lack transversal T-tubule membranes with adult mobility defects in eclosion and flight. Together, the amph and mtm mutant conditions that both lack transversal T-tubule elements in post-larval stage muscle yet different early development requirements underscores that distinct mechanisms are involved in T-tubule formation (amph-dependent) versus maintenance/remodeling (amph- and mtm-dependent) (Fujita, 2017).

In Drosophila, a set of larval body wall muscles that persist as viable pupal abdominal muscles, called dorsal internal oblique muscles (IOMs), are essential for adult eclosion. During metamorphosis, changes in IOM cell size and myofibril content have been noted. Previous studies have shown that wildtype IOMs undergo dramatic cortical and membrane remodeling with costamere integrin adhesion complex disassembly and reassembly at discrete pupal stages (Ribeiro, 2011). In contrast, the mtm-depleted IOMs exhibited persistent disassembly or a block in reassembly of integrin costameres along with the loss of transversal T-tubule membranes at late pupal stages, but without any precocious cell death (Ribeiro, 2011). A striking feature in the mtm-depleted IOMs was the accumulation of endosomal-like membranes decorated with integrin and T-tubule markers, Amph and Discs large (Dlg1, a PDZ protein). Altogether, these results suggest that T-tubule membranes may undergo disassembly-reassembly with normal myofiber remodeling, including the delivery of disassembled T-tubule membrane into an endomembrane trafficking pathway. The role for a molecular-cellular program in control of T-tubule remodeling that is at least partially distinct from that involved in initial T-tubule formation raises many questions about possible mechanisms, including the regulation of T-tubule organization and dynamics, the membrane fate(s) and source(s) with disassembly-reassembly, respectively, and the specific membrane trafficking routes and effectors involved. Possible hints may come from studies of other specialized dynamic cell membrane invaginations shown to involve endosomal and Golgi membrane trafficking pathways, such as cellularization of Drosophila syncytial embryos and the tubulated demarcation membrane system in megakaryocyte platelet formation (Fujita, 2017).

Membrane trafficking relies on the large family of Rab GTPases, with over sixty Rabs in humans and thirty in flies. The different Rabs are under distinct spatiotemporal regulation for recruitment, activation and functions at specific membrane compartments or domains. Guanine nucleotide exchange factors (GEFs) convert specific inactive GDP-bound Rabs to an active GTP-bound form. Active Rab-GTP then recruits a range of specific effector proteins to the membrane that mediate key trafficking functions, including cargo selection, vesicle budding, transport, tethering and fusion. Subsequently, GTPase-activating proteins (GAPs) deactivate Rabs by promoting GTP hydrolysis. Many membrane compartments have been defined by well-established localized functions of specific Rabs, for example: ER (Rab1), Golgi (Rab1, Rab6), secretory vesicles (Rab8), early endosomes (Rab5, Rab21), recycling endosomes (Rab11, Rab35), late endosomes (Rab7, Rab9), lysosomes (Rab7) and others. Thus, identifying the specific Rabs required for a cellular process can provide clues to potential underlying membrane trafficking mechanisms involved. However, examples exist of Rabs with multiple known sites of function or yet unknown functions, and conversely, certain cellular processes - like T-tubule remodeling - lack defined roles yet for any Rabs (Fujita, 2017).

This study utilized the advantages of Drosophila IOMs to screen for Rab GTPases and related membrane trafficking functions required for T-tubule remodeling in intact muscle. The results show that the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and reassembled in IOMs during Drosophila metamorphosis. Autophagy, the membrane trafficking process for degradation of cytoplasmic contents by delivery to lysosomes, is upregulated with IOM remodeling where it plays an indispensable role for progression through T-tubule disassembly to reassembly. Genetic analysis of IOM remodeling also reveals an unexpected and broad role for Rab2 in autophagy in flies and mammals. From these data, it is proposed that Rab2 localizes to autophagosomes where it interacts with the HOPS complex, which in turn, mediates tethering and trans-SNARE complex formation with Rab7-marked lysosomes to promote autophagosome-lysosome fusion. Together, these results show that Drosophila IOM remodeling provides an unprecedented in vivo context for discovery and analysis of T-tubule dynamics with relevance to human myopathy, as well as an ideal system due to high membrane flux to study fundamental trafficking pathways (Fujita, 2017).

This study has characterized a wildtype myofiber remodeling program by confocal and electron microscopy in intact muscles in vivo. In Drosophila IOMs during metamorphosis, the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and then reassembled. This process highlights that myofibers harbor distinct programs for initial T-tubule formation versus regulated T-tubule remodeling. This likely includes additional mechanisms for T-tubule membrane disassembly and renovation, features that reflect those seen with mammalian myofiber atrophy and recovery. The Drosophila body wall muscles provide an unprecedented system permitting a combination of powerful visualization and systematic perturbation analysis, including the first genetic screens, of T-tubule dynamics and organization (Fujita, 2017).

Autophagy is upregulated with the onset of IOM remodeling during metamorphosis. Further, disruption of autophagy initiation, autophagosome formation or clearance all induced loss of T-tubules with a block in IOM remodeling at/after T-tubule disassembly. This is the first report of a non-cell death role of autophagy in Drosophila metamorphosis. The role of autophagy in IOMs that persist and redifferentiate during metamorphosis is clearly different from its roles in pupal midgut and salivary gland cells that undergo autophagic forms of cell death. There are multiple speculative direct or indirect role(s) for autophagy specifically in T-tubule membrane remodeling: (1) a direct role in T-tubule membrane recycling, as a means to deliver disassembled T-tubule membrane via autophagosomes to lysosomes or related organelles for intracellular storage, then later redeployed to contribute to T-tubule reassembly; (2) an indirect role in cell renovation, including T-tubule membrane clearance, to permit cell space for redifferentiation; or (3) an indirect role in cell metabolism, to support cell survival and/or the energy cost of redifferentiation with starvation during metamorphosis. Most likely, autophagy serves some combination of these roles in IOM remodeling (Fujita, 2017).

How could autophagy play a direct role in T-tubule remodeling? It was surprising that mCD8:GFP-positive small vesicles accumulated to a similar degree as autophagosome numbers in IOMs when autophagosome-lysosome fusion was blocked. This suggests that mCD8:GFP localizes to autophagosomes during IOM remodeling. It is possible that T-tubule membranes are a source of autophagosomal membrane, at least in part: mCD8:GFP labels the muscle plasma membrane and T-tubules in larval muscle precursor cells of IOMs, and T-tubule disassembly coincides with the upregulation in autophagy early in metamorphosis. Also, disruption of autophagy induction blocked normal progression in disassembly and remodeling of T-tubule-derived mCD8:GFP-marked membranes. In the absence of autophagy initiation, mCD8:GFP-positive stacked membranes were observed, likely retained or partially disassembled T-tubules. It is proposed that T-tubules are remodeled through autophagosomes. It is important to note that T-tubules are not an apparent autophagic cargo, but instead, a possible source of autophagosome membrane. In this scenario, T-tubules are disassembled into autophagosomes and then reassembled from subsequent autolysosome-related structures, both of which successively increased in numbers during wildtype IOM remodeling (Fujita, 2017).

Alternatively or additionally, other roles for autophagy could indirectly impact T-tubule remodeling. Extensive IOM atrophy with nearly complete disassembly of the contractile and excitation-contraction systems by 1d APF is followed by a rapid re-differentiation within hours after 3.5d APF. Autophagy could be required to simply clear away and degrade the old contraction systems in order to make space to rebuild and realign new systems, as well as permit the normal central repositioning of nuclei away from the myofiber cortex. However, the persistent block in early IOM remodeling with autophagy disruption suggests that the remodeling normally proceeds through a progression of interrelated steps rather than independent programs for disassembly and reassembly. Autophagy also has a well-established role in metabolic homeostasis through the recycling of amino acids and turnover of damaged mitochondria in the lysosome. The current data suggest that mitochondria are a major autophagic cargo with IOM remodeling. In conditions that disrupted autophagy initiation (Atg1, Atg18 RNAi), the cytoplasm was abnormally filled with mitochondria in IOMs at 4d APF. Consistent with that, a significant portion of autophagosomes harbored intact mitochondria when autophagosome-lysosome fusion was blocked (Rab2, Rab7 or Stx17 RNAi). This is different from observations in larval muscle, in which mitochondria were notably absent in autophagosomes that accumulated with a block in autophagy. It is possible that mitophagy, a selective form of autophagy for mitochondrial turnover, is upregulated and could play both metabolic and cell renovation roles in IOM remodeling. Interestingly, the autophagy-blocked IOMs remained viable throughout metamorphosis, suggesting that autophagy is not absolutely required for cell survival through the starvation with metamorphosis (Fujita, 2017).

Through a systemic screen of all Drosophila Rab GTPases, an unexpected role was uncovered for Rab2 in autophagy. The striking Rab2 RNAi IOM phenotype was shared with RNAi of other functions known to be specifically required for autophagosome-lysosome fusion. Genetic blockade of autophagosome-lysosome fusion resulted in a dramatic phenotype, with massive accumulations of autophagosomes within IOMs. Previously, autophagosome-lysosome fusion was shown to involve the cooperative functions of Rab7, the HOPS tethering complex, and a trans-SNARE complex between Stx17, SNAP29 and VAMP7/8. Among these tethering and fusion functions, it has been shown that Stx17 (a hairpin SNARE) is recruited to autophagosomal membranes, while Rab7 and VAMP7/8 localize to endolysosomal membranes. Stx17 localizes to autophagosomes as well as to the ER and mitochondria, but the HOPS complex directly associates and colocalizes with Stx17 only at autophagosomes (Jiang, 2014; Takats, 2014). This suggests that Stx17 is not a sole determinant for HOPS complex recruitment (Fujita, 2017).

It is proposed that Rab2 is required for the autophagosomal recruitment of the HOPS complex. Rab2 specifically localized to completed autophagosomes, and Rab2 had an affinity with the HOPS complex, as does Stx17. It is envisioned that upon completion of autophagosome biogenesis/maturation, Rab2 and Stx17 are recruited to the outer autophagosomal membrane. Then, the HOPS complex is subsequently recruited to autophagosomes in a Rab2-depedent manner through coincident interactions with both Stx17 and Rab2 (see Hierarchal analysis of Rab2 and factors involved in autophagosome-lysosome fusion). At the same time, the HOPS complex binds Rab7 on lysosomes. In turn, the HOPS complex tethers autophagosomes and lysosomes to promote trans-SNARE complex formation between Stx17, SNAP29 and Vamp7/8 and ultimately autophagosome-lysosome fusion (Fujita, 2017).

Rab2 role in autophagy discovered in fly muscle relates to a broader autophagy requirement in other cell types and across species. The localization of Rab2 on autophagosomes in Drosophila IOMs was conserved for both Rab2A and Rab2B in mouse embryonic fibroblasts (MEFs). As in flies, the Rab2A/2B double knockout led to a delay or block in autophagy clearance as indicated by accumulation of LC3/Atg8. However, the specific Rab2 loss-of-function phenotypes were not identical. While Rab2 was required for autophagosome-lysosome fusion in fly IOMs, the Rab2A/2B double knockouts in MEFs indicated a requirement at a later step in autophagic clearance. Interestingly, this disparity in autophagy phenotypes across species is also seen with Rab7. In flies and yeast, Rab7/Ypt7 is essential for autophagosome-lyososome/vacuole fusion, while mammalian Rab7 knockdowns more clearly indicate a required role in autolysosome maturation. Other examples indicate that the autophagosome-lysosome fusion machinery is not highly evolutionarily conserved. The Stx17-SNAP29-VAMP7/8 trans-SNARE complex is conserved in Drosophila and mammals, but not in yeast, where no autophagosomal SNARE has been reported so far. Moreover, budding yeast do not encode for Rab2 (Fujita, 2017).

Altogether, it is plausible that Rab2 is required for autophagosome-lysosome fusion efficiency, and Rab2-dependency is variable across different tissues or species. Two possible models could explain the different Rab2 autophagy requirements in flies and mouse cells. First, it is suggested that autophagosomes sequentially fuse with endosomes then lysosomes to become amphisomes and autolysosomes, respectively. If either of the steps requires Rab2A/2B, then intermediates with partially degraded contents could accumulate in double knockout MEFs. Alternatively, an autophagosome may normally fuse with multiple lysosomes to ensure full degradation of its contents. In the absence of Rab2A/2B in MEFs, autophagosomes could still fuse but not with a sufficient number of lysosomes, resulting in an accumulation of partially digested autolysosomes (Fujita, 2017).

Rab2 has been previously associated with transport events at the Golgi apparatus, ER-to-Golgi traffic and secretory granule formation, as well as in a C. elegans endocytic/phagocytic pathway. Gillingham et al. systematically explored Rab effectors in Drosophila cultured cells, and found that Rab2 interacts with the HOPS complex besides known Golgi-resident effectors (Gillingham, 2014). The interaction between Rab2 and HOPS complex is also conserved in mammals, and the unexpected Rab2 localization to autophagosomes was found. Thus, it is likely that Rab2 exerts multiple functions through interaction with different effectors at different places. A possible Rab2 function in the endosome-lysosome system that affects autophagic flux cannot be excluded, although no clear lysosomal defects were detected in Rab2A/B knockout MEFs. Several other factors that localize to autophagosomes or late endosomes-lysosomes, including Atg14, PLEKHM1 and EPG5, have been shown to control autophagosome maturation. It is plausible that Rab2 contributes to autophagosome maturation through both a direct role in the fusion mechanism and an indirect role in endo-lysosome maturation, the same as Rab7 and the HOPS complex (Fujita, 2017).

How Rab2 localizes to autophagosomes remains unclear. Localization of Rab2 on autophagosomes in IOMs did not depend on HOPS complex subunits, Vps39 and Vps41, or on Stx17. Further studies will be needed to determine the identities of the Rab2 guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) that regulate Rab2 GTPase activity in autophagosome-lysosome fusion. A conserved TBC domain protein, OATL1/TBC1D25, is a strong candidate for a Rab2 GAP, given OATL1 localization to autophagosomes and involvement in autophagosome-lysosome fusion. Further, it was reported that OATL1 directly bound to and showed GAP activity for Rab2A (Fujita, 2017).

Autophagy is critical for the maintenance of myofiber homeostasis in mammalian skeletal muscle. It is known that several myopathies are associated with excess accumulation of autophagic structures in muscle. Further, loss of autophagy in mouse skeletal muscle shows anomalies, including abnormal mitochondria, disassembled sarcomeres and disorganized triads, as also seen in aged muscle. It is established that autophagy is down-regulated during the course of aging. This evidence points to a possible significance of autophagy in myofiber remodeling and in T-tubule maintenance. Jumpy/MTMR14 PI3-phosphatase and Dynamin-2 (DNM2) GTPase, two causative genes of human centronuclear myopathy, are required for not only T-tubule maintenance but also proper progression of autophagy. Based on these reports and the current findings, it is speculated that their roles in T-tubule maintenance are mediated, at least in part, through autophagy (Fujita, 2017).

Signaling pathways that regulate atrophy and hypertrophy in Drosophila have been identified, however, the mechanisms and direct mediators of muscle remodeling remain largely unknown. IOM remodeling is a good model to study the mechanisms of muscle remodeling, given that the signaling pathways that control muscle remodeling are conserved between Drosophila and mammals. Advantages of the IOM system are not only its genetic tractability, but also its reproducibility and structure. As a relatively giant single cell along the body wall, IOMs enable tracking of a single cell and its subcellular organization during metamorphosis. The results show that studies in IOMs can provide new insights into the mechanisms of muscle remodeling as well as regulation of fundamental membrane trafficking pathways, such as autophagy and endocytosis (Fujita, 2017).

Rab2 promotes autophagic and endocytic lysosomal degradation

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. This study shows that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. This study demonstrates that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. RAB2A is also required for autophagosome clearance in human breast cancer cells. In conclusion, Rab2 has been identified as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes (Lorincz, 2017).

The two main pathways of lysosomal degradation are endocytosis and autophagy. Double-membrane autophagosomes (generated in the main pathway of autophagy) and endosomes can fuse with each other to generate amphisomes, and mature into degradative endo- and autolysosomes, respectively, by ultimately fusing with lysosomes. One of the main regulators of intracellular trafficking and vesicle fusions are Rab small GTPases. Active, GTP-bound Rab proteins recruit various effectors including tethers and molecular motors, of which Rab7 is the only known direct regulator of both autophagosome-lysosome and endosome-lysosome fusions (Lorincz, 2017).

The tethering complex homotypic fusion and vacuole protein sorting (HOPS) was identified in yeast, and it simultaneously binds two yeast Rab7 (Ypt7) molecules on its opposing ends. In animal cells, Rab7 binds to RILP, ORPL1, FYCO1, and PLEKHM1 to recruit dyneins and HOPS and ensure the fusion of late endosomes and autophagosomes with lysosomes. This way, HOPS could cross-link two Rab7-positive membranes to prompt tethering and fusio. Rab7 is present on lysosomes, autophagosomes, and endosomes, but it is not clear whether another Rab is involved in degradative auto- and endolysosome formation, which also requires transport of hydrolases from the Golgi (Lorincz, 2017).

Rab2 is known to control anterograde and retrograde traffic between the ER and Golgi. A recent biochemical screen identified Rab2 as a direct binding partner of HOPS, and active Rab2 was found to localize to Rab7-positive vacuoles in cultured Drosophila melanogaster cells (Gillingham, 2014). This study proposes an updated model in which Rab7 and Rab2 coordinately promote the HOPS-dependent degradation of autophagosomes and endosomes via fusion of these as well as biosynthetic vesicles with lysosomes (Lorincz, 2017).

Rab2 is highly conserved among higher eukaryotes, including Drosophila melanogaster and humans. The HOPS subunits Vps39 and Vps41 directly bind to Ypt7/Rab7 in yeast, whereas their interaction may be indirect in mammalian cells. No binding was detected between Drosophila Rab7 and Vps39 or Vps41, whereas GTP-locked Rab7 bound to its known effector PLEKHM1 in yeast two-hybrid (Y2H) experiments. Vps39 directly bound Rab2^GTP in both Y2H and recombinant protein pull-down experiments, and Rab2^GTP immunoprecipitated endogenous Vps16A (another HOPS subunit) from fly lysates. Consistently, it has been reported that recombinant mammalian RAB2A pulls down Vps39 but not Vps41 from cell lysates (Kajiho, 2016), and human HOPS subunits did not show Rab7 binding in Y2H experiments (Lorincz, 2017).

To address whether Rab2 functions in autophagy and endocytosis, rab2 was knocked out by imprecise excision of a transposon from the 5' UTR. The resulting rab2^d42 allele carries a 2,047-bp deletion, which removes most of the protein coding sequences of both predicted Rab2 isoforms and eliminates protein expression. Rab2 mutant animals die as L2/L3-stage larvae, and their viability is fully rescued by expression of YFP-Rab2 (Lorincz, 2017).

Larval fat cells are widely used for autophagy analyses because of their massive autophagic potential. Numerous Lysotracker Red (LTR)-positive vesicles appear upon starvation, which represent newly formed autolysosomes with likely increased v-ATPase-mediated acidification in these cells. LTR dot number and size (and signal intensity as a likely consequence) decreased in rab2-null cells compared with controls, which was rescued by expression of YFP-Rab2. RNAi knockdown of Rab2 in GFP-marked fat cell clones also impaired starvation-induced punctate LTR staining compared with surrounding GFP-negative cells (Lorincz, 2017).

A 3xmCherry-Atg8a reporter that labels all autophagic structures via retained fluorescence of mCherry inside autolysosomes revealed increased number and decreased size of such vesicles in both starved rab2 RNAi and mutant fat cells. A dLamp-3xmCherry reporter of late endosomes and lysosomes showed similar changes in rab2 RNAi or mutant fat cells of starved animals. Tandem tagged mCherry-GFP-Atg8a reporters are commonly used to follow autophagic flux, because GFP is quenched in lysosomes, whereas mCherry signal persists. Knockdown of rab2 prevented the quenching of GFP that is seen in starved control fat cells: dots positive for both GFP and mCherry accumulated, raising the possibility that Rab2 promotes autophagosome-lysosome fusion, similar to HOPS. Colocalization of 3xmCherry-Atg8a with the lysosomal hydrolase cathepsin L (CathL) was examined. The overlap of these markers of autophagic and lysosomal structures strongly decreased in rab2 mutant fat cells compared with controls, and rab2 RNAi also impaired endogenous CathL-positive vesicle formation, suggesting that formation of degradative autolysosomes requires Rab2 (Lorincz, 2017).

These phenotypes resembled the autophagosome-lysosome fusion defect of mutants for the autophagosomal SNARE syntaxin 17, HOPS, and Rab7. Accordingly, ultrastructural analysis of starved fat cells revealed accumulation of double-membrane autophagosomes and small dense structures likely representing amphisomes, similar to HOPS mutants. Recently, rab2 RNAi was reported to cause accumulation of autophagosomes in Drosophila muscles and enlarged amphisomes in fat cells (Fujita, 2017). Autophagosome accumulation in our rab2-null mutant fat cells is likely caused by a complete loss-of-function condition (Lorincz, 2017).

Western blots detected increased levels of the selective autophagy cargo p62/Ref2p, along with both free and lipidated autophagosome-associated forms of Atg8a in starved rab2 mutants. Basal autophagic degradation was also impaired in rab2 mutants, based on increased numbers of endogenous Atg8a and p62 dots in well-fed conditions (Lorincz, 2017).

The importance of Rab2 for autophagic degradation was confirmed in human cells. Knockdown of RAB2B had no effect on endogenous LC3 structures in breast cancer cells, whereas RAB2A or combined siRNA treatment caused accumulation of autophagic vesicles. LC3 accumulated within Lamp1-positive structures upon RAB2A knockdown, which likely represent amphisomes unable to mature into autolysosomes in these cells, consistent with the recently reported role of Rab2 homologs for degradation of autophagic cargo in mouse embryonic fibroblasts (Fujita, 2017; Lorincz, 2017 and references therein).

To analyze the possible involvement of Drosophila Rab2 in endosomal degradation, dissected nephrocytes were incubated with fluorescent avidin for 5 min. Trafficking of this endocytic tracer was clearly perturbed in rab2 mutant cells, similar to vps41/lt and rab7 mutants. Loss of HOPS leads to enlargement of late endosomes. Similarly, Rab7 endosomes are enlarged in rab2 mutant nephrocytes compared with control or rescued cells. Importantly, fluorescent avidin was trapped in Rab7 endosomes and failed to reach CathL-positive lysosomes after a 30-min chase in rab2 mutants. LTR staining showed the presence of acidic vacuoles in rab2 mutant nephrocytes, which probably include the enlarged late endosomes in rab2 mutant nephrocytes, based on ultrastructural analysis . Aberrant late endosomes accumulated in mutant cells, which were apparently unable to fuse with neighboring acid phosphatase-positive lysosomes. Of note, the number of acid phosphatase-positive lysosomes also decreased in mutant nephrocytes, suggesting that Rab2 promotes both endosome-lysosome fusion and biosynthetic transport to lysosomes (Lorincz, 2017).

GTP-locked, constitutively active Rab2^GTP redistributes from the Golgi onto Rab7 vacuoles in cultured Drosophila cells (Gillingham, 2014). Similarly, Rab2^GTP colocalized with endogenous Rab7 in starved fat cells, unlike wild-type Rab2. Rab2^GTP appeared as large pronounced rings around LTR-positive autolysosomes in starved fat cells, unlike wild-type Rab2. Similarly, Rab2^GTP formed rings around lysosomes and autophagic structures marked by dLamp-3xmCherry and 3xmCherry-Atg8a, respectively. Of note, small Rab2^GTP dots often closely associated with large Rab2^GTP rings in these experiments, raising the possibility that Rab2 vesicles fuse with autolysosomes. Finally, wild-type Rab2 or Rab2^GTP modestly overlapped with autophagosomes marked by endogenous Atg8a (Lorincz, 2017).

These localization and loss-of-function data pointed to Rab2 as a positive regulator of autolysosome formation. Indeed, fat and midgut cells expressing Rab2^GTP contained enlarged and brighter 3xmCherry-Atg8a autophagic structures and dLamp-3xmCherry lysosomes compared with surrounding control cells, suggesting that Rab2 controls autolysosome size. Increased lysosomal input or a block of degradation can cause enlargement of autolysosomes. Systemic expression of Rab2^GTP did not impair the viability of animals, and Western blots of starved L3 larval lysates revealed no changes in p62 and Atg8a levels, suggesting that autophagic degradation proceeds normally in cells expressing Rab2^GTP. Thus, Rab2^GTP may increase autolysosome size by accelerating fusions with other vesicles. Importantly, expression of GTP-locked, active Rab7 did not increase the size of autophagic structures. Rab7 is required for autophagosome-lysosome fusion, and its knockdown prevents the formation of large, bright 3xmCherry-Atg8a-positive autolysosomes: these cells contain only small, faint autophagosomes. Similarly, only small, faint 3xmCherry-Atg8a dots appeared in Rab2^GTP-expressing fat cells undergoing Rab7 RNAi, indicating that Rab2-dependent fusions also require Rab7 and there is no functional redundancy between them (Lorincz, 2017).

Eye pigment granules are lysosome-related organelles. Changes in lysosomal transport often lead to eye discoloration caused by pigment granule alterations, such as in HOPS mutants. Rab2^GTP expression led to a slight darkening of eyes and appearance of enlarged pigment granules, consistent with the role of Rab2 in promoting lysosomal fusions (Lorincz, 2017).

Several homo- and hetero-typic fusions occur during endosome and autophagosome maturation into degradative lysosomes. Known metazoan factors acting at lysosomal fusions include HOPS and EPG5 tethers and Rab7 together with its effectors. Because biosynthetic transport to lysosomes also requires input from Golgi, the role of Golgi-associated Rab2 in various lysosomal fusions fits well into this picture. Consistently, Rab2 promotes breakdown of phagocytosed apoptotic bodies and lysosome-related acrosome biogenesis (Lorincz, 2017).

Accumulation of unfused autophagosomes and enlarged late endosomes in rab2 mutants resembles the fusion defect of rab7 mutant cells. The decreased function of lysosomes in rab2 mutants is unlikely to account for these fusion defects, because we have shown that autophagosome-lysosome fusion proceeds and gives rise to enlarged, nondegrading autolysosomes in fat cells with perturbed acidification or biosynthetic transport to lysosomes (Lorincz, 2017).

The role of Rab2 in the fusion of lysosomes with other vesicles is also supported by the autolysosomal localization of its active form and by its binding to the Vps39-containing end of HOPS, the tethering complex required for autophagosomal, endosomal, and biosynthetic transport to lysosomes. Consistently, Rab2 recruits HOPS to Rab7-positive vesicles in cultured Drosophila cells (Gillingham, 2014). Expression of Rab2^GTP increases degradative autolysosome and pigment granule size, suggesting that it is rate limiting during these fusion reactions, unlike Rab7. This is supported by low levels of wild-type Rab2 on these organelles, unlike wild-type Rab7 that is abundant on autophagosomes, late endosomes, and lysosomes. Consistent with this, it has been recently shown that expression of RAB2A^GTP also increases Rab7 vesicle size in human cells (Kajiho, 2016). Based on binding of Rab2 to one end of HOPS, an updated model is proposed of lysosomal fusions in animal cells. It is hypothesized that GTP-loaded Rab2 is transported on Golgi-derived carrier vesicles toward Rab7 positive vesicles, and its interaction with Vps39 promotes fusions. Vps41 located on the other end of HOPS may bind Rab7 vesicles via adaptors such as PLEKHM1. These interactions help the tethering and fusion of autophagic, endocytic, and lysosomal vesicles to generate degrading compartments. Lysosomal membranes may contain active Rab2 for only a short period of time, and it likely dissociates upon GTP hydrolysis to limit organelle size. Rab asymmetry is also observed during homotypic vacuole fusion in yeast: GTP-bound Ypt7/Rab7 is necessary on only one of the vesicles, and its nucleotide status is irrelevant on the opposing membrane. Importantly, Rab7 directly interacts with both ends of HOPS in the absence of a Rab2 homolog in yeast. This difference may explain why yeast cells contain one large vacuole instead of the many smaller lysosomes seen in animal cells. Collectively, these data indicate that Rab2 and Rab7 coordinately promote autophagic and endosomal degradation and lysosome function (Lorincz, 2017).

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila

Mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. This study performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (dICA69) as one of the key regulators of morphological differentiation of larval neuromuscular junction (NMJ). Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of dICA69 deforms liposomes in vitro. Full length and ICAC but not the N-BAR domain of dICA69 which induces filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, dICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of dICA69 or its interactor dPICK1 alters synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing dPICK1 or dRab2 levels phenocopies dICA69 mutation. Interestingly, dRab2 regulates not only synaptic iGluR but also dICA69 levels. Thus, these data suggest that: a) dICA69 regulates NMJ organization through a pathway that involves dPICK1 and dRab2, and b) dRab2 genetically functions upstream of dICA69 and regulates NMJ organization and targeting/retention of iGluRs by regulating dICA69 levels (Mallik, 2017).

This study demonstrates that ICA69 regulates NMJ structural organization and synaptic levels of glutamate receptor clusters. The findings suggest a model in which Rab2 functions genetically upstream of ICA69 to regulate its synaptic level, which in turn regulates the Spectrin cytoskeleton and iGluRs at the NMJ (Mallik, 2017).

The requirement of ICA69 for Drosophila NMJ organization is strongly supported by its enrichment in the postsynaptic Spectrin-rich scaffold. Consistent with this idea, ICA69 mutants or animals with downregulated ICA69 levels show reduced arborization and bouton numbers at the NMJ. Several studies have shown that cytoskeletal regulation is a key process for NMJ development. Multiple lines of evidence suggest that ICA69 promotes NMJ growth by regulating the cytoskeletal network surrounding the SSR. First, ICA69 is highly enriched at the NMJ in the same microdomain as Spectrin. Second, ICA69 induces filopodia in cultured cells and relocalizes positive regulators of actin polymerization at the filopodia. Third, mutation in ICA69 significantly reduces α-Spectrin levels. The Actin-Spectrin scaffold at the postsynapse has been implicated in regulation of NMJ organization in postembryonic development in Drosophila. This study reveals a crucial requirement of ICA69 in regulating synaptic α-Spectrin levels and indicates that ICA69 is required for the assembly of Actin-Spectrin scaffolds surrounding the SSR. Whether localization and/or stability of postsynaptic Spectrin-Actin scaffold depends on direct interaction between scaffold components and ICA69 or on some unknown signaling mechanism needs to be further investigated (Mallik, 2017).

For the proper establishment of NMJ connections, neurons as well as muscles require trafficking of various synaptic proteins. Rab GTPases and their regulators are considered to be some of the most important signaling molecules for intracellular trafficking. Interestingly, nearly half of all the Drosophila Rab proteins function specifically in neurons and a few of them localize to the NMJs. ICA69 physically associates with Rab2 and has been suggested as one of its effectors in regulating dense core vesicle maturation in Caenorhabditis elegans. This study found that Rab2 endogenous regulatory sequence-driven Rab2EYFP is detectable in the larval muscles as punctate structures, suggesting its involvement in NMJ organization. This idea is supported by four compelling pieces of evidence. First, ubiquitous or muscle-specific knockdown of Rab2 phenocopies ICA69 mutants. Second, knockdown of Rab2 significantly reduces synaptic α-Spectrin levels. Third, Rab2 directly regulates synaptic ICA69 levels. Fourth, co-expressing an ICA69 transgene and Rab2 RNAi rescues the morphological defects of Rab2 RNAi. Based on these observations, it is suggested that Drosophila Rab2 functions genetically upstream of ICA69. Like Rab2, PICK1 depletion reduced synaptic ICA69 levels and phenocopied the NMJ morphological defects observed in ICA69 mutants or after Rab2 depletion. Moreover, simultaneous knockdown of ICA69 and PICK1 or of ICA69 and Rab2 did not show an additive effect on the NMJ structural defects. These observations support the notion that ICA69, PICK1 and Rab2 might function in the same genetic pathway to regulate NMJ structural organization (Mallik, 2017).

In mammalian neurons, ICA69 is, surprisingly, not enriched at the synapses and negatively regulates AMPA receptor trafficking. Hence, it is expected that ICA69 mutants would have normal, if not more, iGluR clusters at the NMJ. Contrary to this expectation, reducing the ICA69 level resulted in reduced GluRIIA as well as GluRIIB glutamate receptor clusters. A recent study has shown that ICA69 and PICK1 stability is interdependent in Drosophila brain. Thus, it is likely that iGluR clusters at the NMJ are regulated by levels of ICA69 and PICK1 in muscles (Mallik, 2017).

How does ICA69 reduce iGluR levels both in knockdown and overexpression scenarios? It is suggested that the endogenous stoichiometry of ICA69 and PICK1 is crucial for normal synaptic targeting of iGluRs at the Drosophila NMJ. Reducing ICA69 destabilizes the ICA69-PICK1 heteromeric complex thereby reducing PICK1 availability for synaptic targeting of iGluRs. Overexpression of ICA69 forms more of the ICA69-PICK1 inhibitory complexes, which reduces synaptic targeting of iGluRs. Hence, the idea that the endogenous level of ICA69 is crucial for maintaining normal glutamate receptor clusters at the synapses is supported (Mallik, 2017).

The data suggest that ~40% simultaneous reduction of GluRIIA/IIB/III at Drosophila NMJ synapses has no major consequence on larval synaptic physiology. Three possibilities are suggested to explain this. First, the relative levels of GluRIIA and GluRIIB subunits are crucial for determining the efficacy of synaptic transmission at the Drosophila NMJ synapse . The decrease for each of the GluRIIA, -IIB and -III subunits in the ICA69 mutant is almost identical; ~40% compared with controls. This hints towards a homeostatic compensatory mechanism whereby ~60% of the receptor subunits are sufficient to form enough functional receptor complexes, which can maintain normal synaptic strength. Second, the amount of GluRIII is reflective of the sum of GluRIIA and -IIB complexes together, and GluRIII is essential for the localization of GluRIIA and IIB subunits. A 40% decrease in GluRIII staining correlates well with an identical decrease in GluRIIA and -IIB staining. It is plausible that there is essentially negligible change in functional glutamate receptor assembly at ICA69 mutant synapses. Third, ICA69 possibly plays a role in trafficking glutamate receptors to the postsynaptic density and is not rate limiting in the formation of functional glutamate receptor complexes. Thus, ICA69 mutants exhibit normal synaptic physiology without embracing other compensatory mechanisms such as reduced quantal size or increased quantal content (Mallik, 2017).

How might the iGluR levels relate to the NMJ growth? A tight correlation exists between the amount of synaptic glutamate receptors and the NMJ morphology. Downregulation of iGluRs in muscles has been shown to reduce the number of boutons. Similarly, hypomorphic mutants of GluRIII or GluRIIA have reduced bouton numbers. Consistent with this, overexpression of GluRIIA induces arborization and bouton number. Moreover, mutants with altered synaptic iGluR levels also show altered bouton numbers. For instance, neto and filamin (cheerio) mutants show reduced iGluR levels and bouton numbers. One of the possible mechanisms by which glutamate receptors can alter the NMJ morphology is through regulation of synaptic phospho-MAD levels. As the iGluRs (for instance, GluRIID) have also been shown to localize in central neuropil, it remains a possibility that the endogenous pattern of central electrical activity could also play crucial roles in sculpting the NMJ during development (Mallik, 2017).

Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo

Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. This study analyzed the in vivo movement of 16 Rab GTPases within Drosophila larval axons and showed that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, these findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease (White, 2015).

This study has identified a novel role for HTT in the regulation of the axonal transport of a particular sub-set of Rab-containing vesicles under physiological conditions. In vivo observations have led to two major findings; (1) HTT differentially regulates the movement of a specific sub-set of Rab-containing vesicles within axons, and (2) HTT is present on these Rab-containing vesicles during axonal transport. At least two possible mechanisms could exist by which HTT exerts a differential control on Rab motility, (1) by associations with specific Rab-containing vesicles, and/or (2) by regulating the motors on moving Rab-containing vesicles, although these two pathways may not be mutually exclusive. Collectively, these findings provide new insight into the normal physiological role of HTT which, when disrupted, may contribute to disease pathology observed in HD (White, 2015).

Different Rab-containing vesicles, even those within the same sub-cellular compartment, move at varying velocities, suggesting that Rab proteins found in the same compartment are differentially regulated. Indeed, different regulatory mechanisms could exist as Rab proteins control trafficking in both the secretory and endocytic pathways. Some Rab proteins are in distinct sub-sets of neurons suggesting that Rabs have roles in diversely regulated mechanisms, and HTT may function to differentially regulate the motility of these neuronal Rab proteins via Rab protein specific associations. Previously, work found that reduction of HTT perturbed the motility of Rab11-containing vesicles but not Rab5-containing vesicles. Since Rab11 is a marker for recycling endosomes and Rab5 is a marker for early endosomes, it is hypothesized that HTT influences the axonal motility of all recycling endosomes. Contrary to this, however, the current systematic in vivo analysis found that HTT does not influence the motility of all Rab proteins found in recycling endosomes, but rather, HTT only affects the movement of particular Rab proteins located in many different compartments. Reduction of HTT function perturbed the bi-directional motility of Rab19, a recycling endosomal Rab, while no effect was seen in the motility of other recycling endosomal Rab proteins. Additionally, reduction of HTT perturbed the bi-directional motility of Rab3, a Rab protein known to be present in synaptic vesicles. Intriguingly, reduction of HTT perturbed the retrograde movement of Rab7 (present on late endosomes), while the anterograde movement of Rab2 (present in ER-Golgi associated compartments) was stimulated by reduction of HTT. Perhaps this differential regulation that is observed with reduction of HTT may be caused by the existence of different HTT-Rab-containing motor complexes. Either several HTT-Rab-containing vesicle complexes may exist or alternatively more than one Rab could be present with a single HTT-Rab-containing vesicle. Perhaps, during long distance transport within axons, HTT-mediated regulation of specific Rab-containing vesicles is required for particular functions at the synapse. Indeed, similar to many Rab proteins, roles for HTT in endocytosis, intracellular trafficking and membrane recycling have also been proposed (White, 2015).

Specific associations between HTT, Rab proteins and linker proteins could perhaps dictate one potential mechanism by which HTT-mediated differential regulation of Rab-containing vesicle motility occurs. It is thought that associations between HTT and motors are facilitated by HTT associated proteins (HAPs). Pal (2006). showed that HTT can mediate the transport of a Rab5-HAP40-HTT-containing early endosome on actin filaments via associations with myosin, the actin motor. HTT can also interact with myosin via optineurin. Optineurin is a binding partner that links both myosin and HTT to the Golgi network via Rab8 for ER-Golgi trafficking in the secretory pathway. HTT can also associate with Rab8 through FIP-2 for regulated cell polarization and morphogenesis. Since reduction of HTT altered the sub-cellular localization of Rab8 the current observations suggest that HTT can play a role in linking Rab8 to vesicles; enabling associations with MT motors during axonal transport. While the involvement of optineurin or FIP-2 in the association of HTT and Rab8 in the context of axonal movement is still unclear, what is clear is that HTT is likely required for the membrane-bound state of Rab8 during axonal transport under physiological conditions (White, 2015).

It has been proposed that a HTT-Rab11-motor complex likely exists during axonal transport. The motility of Rab11-containing vesicles was perturbed with reduction of HTT. Both kinesin-1 and dynein motors were required for MT motility of Rab11. Additionally, membrane binding of Rab11 was decreased in HD knock-in mice, suggesting that similar to Rab8, HTT is also likely required for the membrane-bound state of Rab 11. The Rab11 effector Rip11 regulates the endocytic recycling pathway by forming a complex with Rab11 and kinesin II. Rip11 is also important for the trafficking of Rab11 from apical recycling endosomes to the apical membrane. Perhaps Rip11 may act as a linker that connects Rab11 and HTT similar to optineurin linking Rab8 and HTT. Rabphilin-3A, a Rab3 effector molecule may link Rab3 vesicles to HTT during axonal transport. Studies have shown that Rab3 and Rabphilin-3A are both transported by fast anterograde transport and associate with synaptic vesicles. Thus, although further study is needed, Rab-associated proteins could aid in linking specific Rab-containing vesicles with HTT during axonal transport (White, 2015).

Alternatively, HTT-mediated differential regulation of Rab protein motility could result due to changes in motor protein regulation. Indeed, previous work postulated HTT as a molecular switch that determines the direction of movement during axonal transport. HTT is phosphorylated by Akt (protein kinase B) (a serine-threonine kinase) at serine 421. Constitutively phosphorylated (S421D) HTT can recruit kinesin-1 to the dynactin complex to facilitate anterograde transport while disruption of phosphorylation at S421 (S421A) prevents kinesin association with HTT and the motor complex, enabling retrograde transport. Perhaps HTT's role as a molecular regulator during axonal transport could result in the HTT-mediated motility changes were observe in this study, since reduction of HTT not only perturbed the bi-directional motility of Rab3 and 19, and the retrograde motility of Rab7, but also stimulated the anterograde motility of Rab2, via specific changes to motility parameters; vesicle velocities, pause duration/frequencies and run lengths. While the functional significance of the differential regulation of Rab motility and the mechanistic steps of how HTT controls motors in the context of the different Rab-containing complexes are still unclear, perhaps particular Rab proteins could also exert a regulatory function during vesicle motility by affecting the phosphorylation state of HTT and changing the direction of vesicle movement. Interestingly, several Rabs have been shown to be effectors of kinases. Rab5 and Rab7 are thought to be effectors of PI3K, which is an upstream activator of Akt. Additionally, it has been shown that HTT can act as a scaffold to transport glycolytic machinery down the axon that is required for vesicular motility. Reduction of HTT could decrease glycolysis disrupting the motility of Rab-containing vesicles. Further experiments will be needed to dissect the mechanistic steps involved in the differential regulation of these different HTT-Rab-containing complexes during axonal transport under physiological conditions (White, 2015).

An intriguing result from this analysis was that reduction of HTT influenced the retrograde transport of Rab7, although Rab7-containing vesicles moved bi-directionally. Previous work has implicated Rab7 in neurotrophin receptor trafficking, particularly in the retrograde transport of TrkB/p75NTR-positive signaling endosomes in motor neurons. Consistent with this, CMT2B Rab7 mutants altered trafficking and signaling of retrograde NGF/TrkA trophic signal. Thus, since HTT and Rab7 co-localize on moving vesicles during axonal transport a HTT-Rab7-containing signaling endosome could exist during axonal transport. Alternatively, since Rab7 is a marker for late endosomal and lysosomal compartments, and HTT and dynein were found to be required for the perinuclear positioning of lysosomes, perhaps a HTT-Rab7-containing lysosome could exist during axonal transport. Work has also shown that Rab7 and LC3 (a marker for autophagosomes) are together during the transport of autophagosomes at growth cones and that the retrograde movement of autophagosomes is required for their maturation. Interestingly Rab7 interacting lysosomal protein (RILP) was shown to control lysosomal transport by recruiting dynein-dynactin to Rab7-containing late endosomes/lysosomes. The FYVE (Fab1-YotB-Vac1p-EEA1) and coiled-coil domain-containing 1 protein (FYCO1) was found to function as an adaptor to link autophagosomes to kinesin via Rab7. Additionally, both HTT and HAP1 were identified as regulators of autophagosome transport in neurons. Thus, the current results are consistent with these observations and suggest that perhaps a HTT-Rab7-authophagosome complex and/or a HTT-Rab7-signaling endosomal complex could exist under physiological conditions(White, 2015).

Surprisingly this analysis also revealed that reduction of HTT stimulated the anterograde velocity of Rab2, although Rab2-containing vesicles moved bi-directionally. Rab2 is known to regulate the anterograde and retrograde trafficking of vesicles between the Golgi, the ER-Golgi intermediate compartment and the ER. Rab2 was also one of the Rab proteins that showed the most neuronal sub-cellular localization behaviors: synaptic enrichment with expression of a CA form and loss of synaptic localization with the dominant negative form , suggesting a role for Rab2 at the synapse. While roles for HTT at synapses have been documented, perhaps HTT may function to regulate the anterograde motility of a Rab2-containing complex, although the functional significance for this complex at the synapse is still unknown (White, 2015).

Rab dysfunction has been implicated in many neuronal diseases. For example, a missense mutation in Rab7 was demonstrated in the myelin and axonal disorder Charcot-Marie Tooth disease Type 2B. Altered expression of Rab1, Rab8, and Rab2 was shown to cause Golgi fragmentation in Parkinson's disease. Expansion of a hexanucleotide repeat in C9ORF72, a Rab-associated GEF, was seen in both Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD), suggesting that this mutant form of the GEF may contribute to the physiology of the disease through Rab dysfunction. Defects in the recycling of Rab7 from lysosomes to early endosomes impaired the transport and degradation of amyloid beta (Aβ) in Alzheimer's disease (AD). Rab6 was shown to modulate the unfolded protein response due to ER stress in AD. Interestingly, defects in Rab11 function were recently observed in HD. Expression of Rab11 was decreased in HD mouse models, and Rab11 activation was impaired by mutant HTTA. Over expression of Rab11 rescued neurodegeneration, dendritic spine loss, synaptic defects and behavioral defects in HD models in both mice and Drosophila. Perhaps defects to HTT-mediated axonal transport of a specific sub-set of Rab-containing vesicles could contribute to neurodegeneration and synaptic defects observed in HD. Thus this work could highlight a potential novel therapeutic pathway for early treatment of HD pathology (White, 2015).

Toward a comprehensive map of the effectors of rab GTPases

The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, a comprehensive set of Drosophila Rabs was used for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, numerous Rab-specific interactions were found with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. Over 25 interactions were confirmed for Rab2, Rab4, Rab5, Rab6, Rab7, Rab9, Rab18, Rab19, Rab30, and Rab39. These include tethering complexes, coiled-coiled proteins, motor linkers, Rab regulators, and several proteins linked to human disease (Gillingham, 2014).

Rab2 is widely conserved in evolution, being present in mammals, plants, and protozoa. It has been lost from a few organisms, including budding yeasts, and its role is not entirely clear. It is found on the Golgi apparatus and has been proposed to have a role in ER-to-Golgi traffic or in the formation of secretory granules. Deletion of Rab2 from C. elegans perturbs secretory granule formation and the maturation of phagosomes, suggesting a role in the endocytic pathway. Some Rab2 effectors have been identified, all of which are known Golgi proteins. These include golgin-45, and ICA-69 a BAR domain protein. Their function is unknown, but C. elegans ICA-69 is also required for the formation of secretory granules. Rab2 has also been reported to bind to the Golgi coiled-coil protein complex GM130/p115, and previous studies found that Drosophila Rab2 binds to further golgins, GMAP, dGCC88, dGCC185, and dGolgin-245 (Sinka, 2008; Gillingham, 2014 and references therein).

Five of these seven known effectors showed highly specific interactions with Rab2 in both sets of conditions, and a sixth, dGolgin-245, was found in one set. The exception is ICA-69 (CG10566), whose mRNA is expressed at the lowest levels in S2 cells, being primarily expressed in the nervous system. In addition, many other proteins bound specifically to the Rab2 column, including those with a role in membrane traffic and others previously uncharacterized (Gillingham, 2014).

Both sets of conditions produced subunits of the HOPS complex that is found on endosomal membranes and acts in membrane tethering and fusion. In yeast, a second complex called CORVET shares four subunits with HOPS, with each complex having additional unique subunits. CORVET binds to yeast Rab5 via a CORVET-specific subunit, Vps8, but nothing has been reported about Rab interactions in metazoans or even whether the subunits form this pair of complexes. Notably, four of the shared subunits were also found with Rab5 using detergent lysis conditions. However, the pattern of additional subunits was not the same for the two Rabs. Vps39 and Vps41 were only found with Rab2, while Vps8 was found only with Rab5. Strikingly, the Rab2 subunit set corresponds to those found in HOPS rather than CORVET, indicating that the two distinct tethering complexes found in yeast are conserved in metazoans and that HOPS interacts with Rab2 while CORVET interacts with Rab5. Of the two HOPS-specific subunits, the largest number of spectra was found with Vps39, and so it seemed a good candidate to interact with GTP-bound Rab2, and this was confirmed by yeast two-hybrid and in vitro binding (Gillingham, 2014).

An interaction between Golgi-localized Rab2 and an endosomal tether may seem surprising, but it provides a possible explanation for the effects of Rab2 deletion on phagocytosis in C. elegans. Notably, although Rab2 was on the Golgi when expressed in S2 cells, when it was expressed as a GTP-locked form (Rab2Q65L), large swollen structures were observed that colocalized with the late endosomal marker Rab7. The Rab2Q65L could recruit Rab2 effectors to these structures, including Vps39, and it was possible to use this to map the interaction to the N-terminal region predicted to form a β-propeller. It is speculated that Rab2 can traffic on carriers from Golgi to endosomal structures, with GTP hydrolysis being required for its release from endosomes (Gillingham, 2014).

The dynein adaptor BicaudalD (BicD) is a known effector for Rab6 but was also in both sets of Rab2 eluates. The interaction with Rab2 was GTP specific, suggesting that Rab2 also contributes to the recruitment of minus-end-directed motors to the Golgi. BicD also showed GTP-dependent binding to Rab30 and to the Rab2 relative Rab39 (Gillingham, 2014).

CG4925 (Golgin104) is the Drosophila ortholog of human C10orf118 with both proteins predicted to be primarily coiled-coil. It bound the GTP form of Rab2 by affinity chromatography and yeast two-hybrid, via the C-terminal 200 residues. Epitope-tagged CG4925 localized to the trans-Golgi in S2 cells and relocalized to the enlarged endosomes induced by overexpression of Rab2Q65L. The mammalian protein is also on the Golgi and binds Rab2 and the Golgi via its C-terminal region. Given that extensive coiled-coils and C-terminal attachment via a G protein are typical for the golgin coiled-coil proteins, it is proposed that this protein is named golgin-104/dGolgin-104 in humans and flies (Gillingham, 2014).

CG9590 is the Drosophila ortholog of FAM114A1/2, two closely related human proteins of unknown function. Screening a panel of Rabs by yeast-two hybrid showed binding to Rab2 and a weaker interaction with Rab14. GFP-tagged CG9590 localized to the Golgi in S2 cells and bound the GTP-form of Rab2 and, more weakly, Rab14 by chromatography. It did not relocalize to the Rab2Q65L-induced swollen endosomes, suggesting that its membrane recruitment is stabilized by additional interactions. GFP-tagged human FAM114A1 and FAM114A2 were also Golgi localized (Gillingham, 2014).

CG32485 is a member of the CRAL-TRIO (or Sec14) family that binds lipids and other hydrophobic molecules. It lacks a clear mammalian ortholog but has close relatives in plants and fungi. A Rab2-GTP-specific interaction was confirmed by affinity chromatography and yeast two-hybrid interactions, and a GFP-tagged form of the protein showed faint but reproducible Golgi staining (Gillingham, 2014).

Investigating every protein enriched on the Rab2 column was beyond the scope of this study, but for those looking at the list of unconfirmed interactions, CG15523 (Drosophila Vps13B), the TBC domain Rab GAP CG5337 (Drosophila TBC1D16), and the BEACH-domain family member Mauve (CG42863) can be highlighted as all binding Rab2 in both data sets and being known components of membrane traffic (Gillingham, 2014).

Search PubMed for articles about Drosophila Rab2

Ailion, M., Hannemann, M., Dalton, S., Pappas, A., Watanabe, S., Hegermann, J., Liu, Q., Han, H. F., Gu, M., Goulding, M. Q., Sasidharan, N., Schuske, K., Hullett, P., Eimer, S. and Jorgensen, E. M. (2014). Two Rab2 interactors regulate dense-core vesicle maturation. Neuron 82(1): 167-180. PubMed ID: 24698274

Aizawa, M. and Fukuda, M. (2015). Small GTPase Rab2B and Its Specific Binding Protein Golgi-associated Rab2B Interactor-like 4 (GARI-L4) Regulate Golgi Morphology. J Biol Chem 290(36): 22250-22261. PubMed ID: 26209634

Al-Qusairi, L. and Laporte, J. (2011). T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases. Skelet Muscle 1(1): 26. PubMed ID: 21797990

Fujita, N., Huang, W., Lin, T.H., Groulx, J.F., Jean, S., Kuchitsu, Y., Koyama-Honda, I., Mizushima, N., Fukuda, M. and Kiger, A.A. (2017). Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy. Elife pii: e23367. PubMed ID: 28063257

Gillingham, A. K., Sinka, R., Torres, I. L., Lilley, K. S. and Munro, S. (2014). Toward a comprehensive map of the effectors of rab GTPases. Dev Cell 31(3): 358-373. PubMed ID: 25453831

Jiang, P., Nishimura, T., Sakamaki, Y., Itakura, E., Hatta, T., Natsume, T. and Mizushima, N. (2014). The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol Biol Cell 25(8): 1327-1337. PubMed ID: 24554770

Lorincz, P., Toth, S., Benko, P., Lakatos, Z., Boda, A., Glatz, G., Zobel, M., Bisi, S., Hegedus, K., Takats, S., Scita, G. and Juhasz, G. (2017). Rab2 promotes autophagic and endocytic lysosomal degradation. J Cell Biol. PubMed ID: 28483915

Mallik, B., Dwivedi, M. K., Mushtaq, Z., Kumari, M., Verma, P. K. and Kumar, V. (2017). Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila. Development 144(11): 2032-2044. PubMed ID: 28455372

Kajiho, H., Kajiho, Y., Frittoli, E., Confalonieri, S., Bertalot, G., Viale, G., Di Fiore, P. P., Oldani, A., Garre, M., Beznoussenko, G. V., Palamidessi, A., Vecchi, M., Chavrier, P., Perez, F. and Scita, G. (2016). RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs. EMBO Rep 17(7): 1061-1080. PubMed ID: 27255086

Ribeiro, I., Yuan, L., Tanentzapf, G., Dowling, J. J. and Kiger, A. (2011). Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance. PLoS Genet 7(2): e1001295. PubMed ID: 21347281

Sato, K., Roboti, P., Mironov, A. A. and Lowe, M. (2015). Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210. Mol Biol Cell 26(3): 537-553. PubMed ID: 25473115

Sinka, R., Gillingham, A. K., Kondylis, V. and Munro, S. (2008). Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins. J Cell Biol 183(4): 607-615. PubMed ID: 19001129

Takats, S., Pircs, K., Nagy, P., Varga, A., Karpati, M., Hegedus, K., Kramer, H., Kovacs, A. L., Sass, M. and Juhasz, G. (2014). Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol Biol Cell 25(8): 1338-1354. PubMed ID: 24554766

Takeshima, H., Hoshijima, M. and Song, L. S. (2015). Ca(2)⁺ microdomains organized by junctophilins. Cell Calcium 58(4): 349-356. PubMed ID: 25659516

White, J. A., Anderson, E., Zimmerman, K., Zheng, K. H., Rouhani, R. and Gunawardena, S. (2015). Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo. Hum Mol Genet 24(25): 7182-7195. PubMed ID: 26450517

date revised: 1 March 2024

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