InteractiveFly: GeneBrief

Rab4: Biological Overview | References

Gene name - Rab4

Synonyms -

Cytological map position - 54C9-54C10

Function - signaling

Keywords - GTPase, endosomal recycling at synapses, maintains gap junctions upon elevated insulin in cultured human cells and in flies, wingless signaling, Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo, epithelial morphogenesis of leg discs, brain, CNS

Symbol - Rab4

FlyBase ID: FBgn0016701

Genetic map position - chr2R:17,573,462-17,574,735

NCBI classification - Rab GTPase family 4

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein
Rab4 orthologs: Biolitmine
Recent literature
White, J. A., 2nd, Krzystek, T. J., Hoffmar-Glennon, H., Thant, C., Zimmerman, K., Iacobucci, G., Vail, J., Thurston, L., Rahman, S. and Gunawardena, S. (2020). Excess Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington's disease. Acta Neuropathol Commun 8(1): 97. PubMed ID: 32611447
Huntington's disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. This study shows that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bi-directional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington's Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, these observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD.
Hodgson, J. J., Buchon, N. and Blissard, G. W. (2022). Identification of Cellular Genes Involved in Baculovirus GP64 Trafficking to the Plasma Membrane. J Virol 96(12): e0021522. PubMed ID: 35608346
The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, this study developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, six host genes were validated and examined that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4, Clathrin heavy chain, clathrin adaptor protein genes AP-1-2β and AP-2&my;, and Snap29. Two gene knockdowns (Rab5 and Exo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. A small amount of GP64 is released from cells in exosomes, and tsome portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface.
Li, Y., Wang, W. and Lim, H. Y. (2023). Drosophila transmembrane protein 214 (dTMEM214) regulates midgut glucose uptake and systemic glucose homeostasis. Dev Biol 495: 92-103. PubMed ID: 36657508
The availability of glucose transporter in the small intestine critically determines the capacity for glucose uptake and consequently systemic glucose homeostasis. Hence a better understanding of the physiological regulation of intestinal glucose transporter is pertinent. However, the molecular mechanisms that regulate sodium-glucose linked transporter 1 (SGLT1), the primary glucose transporter in the small intestine, remain incompletely understood. Recently, the Drosophila SLC5A5 (dSLC5A5) has been found to exhibit properties consistent with a dietary glucose transporter in the Drosophila midgut, the equivalence of the mammalian small intestine. Hence, the fly midgut could serve as a suitable model system for the study of the in vivo molecular underpinnings of SGLT1 function. This study reports the identification, through a genetic screen, of Drosophila transmembrane protein 214 (dTMEM214) that acts in the midgut enterocytes to regulate systemic glucose homeostasis and glucose uptake. dTMEM214 resides in the apical membrane and cytoplasm of the midgut enterocytes, and that the proper subcellular distribution of dTMEM214 in the enterocytes is regulated by the Rab4 GTPase. As a corollary, Rab4 loss-of-function phenocopies dTMEM214 loss-of-function in the midgut as shown by a decrease in enterocyte glucose uptake and an alteration in systemic glucose homeostasis. This study further showed that dTMEM214 regulates the apical membrane localization of dSLC5A5 in the enterocytes, thereby revealing dTMEM214 as a molecular regulator of glucose transporter in the midgut (Li, 2023).

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4 GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. This study reports that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network (Dey, 2017).

The apical compartment of a neuronal cell extends into specific processes forming axons and synapses that are maintained by a distributed endosomal system. For example, vesicle recycling at the synapse, orchestrated by various RabGTPases and associated proteins, renews the readily releasable pool of synaptic vesicles sustaining the neurotransmission. Rab4 is thought to play an important role in balancing the vesicle traffic between the recycling and degradation pathways involved in various cellular processes such as metabolism, cell secretion, and antigen processing. Recruitment of Rab4 on sorting intermediate endosomes allows the transfer of vesicular cargoes from early endosomes to recycling endosomes in axons and at synapses. The activity of Rab4 has also been implicated in the progression of growth cone in Xenopus and the maintenance of dendritic spines in rat hippocampal neurons. Furthermore, endosomal abnormalities found in the cholinergic basal forebrain of patients with Alzheimer's disease correlate with elevated levels of Rab4. All these reports suggest that Rab4-dependent vesicle sorting is essential for the development and maintenance of the nervous system (Dey, 2017).

Vesicular cargoes, including the RabGTPases, originate at the trans-Golgi network, and microtubule-dependent motors transport them to different destinations within a cell. Specific association with motors ensures differential and dynamic subcellular localizations of various RabGTPases according to tissue-specific activities and metabolic demands. For instance, the anterograde trafficking of early, sorting, and late endosomes is dependent on the Kinesin-3 motors KIF13, KIF16B, and KIF1A/IBβ, respectively. Similarly, in Drosophila, Rab5-positive early endosomes associate with Khc-73, which is the ortholog of mammalian KIF13, showing a conserved machinery of Kinesin-Rab interaction. The same vesicles also associate with Dynein for their retrograde movement. It is further indicated that multiple RabGTPases can be grouped together and transported to a destination within a cell where they can engage in different functions. Although the long-range transport of certain presynaptic RabGTPases has been studied in cultured neurons, it is still unclear how the bidirectional movement of Rab-associated vesicles could localize them in distant compartments such as the synapse (Dey, 2017).

This study has shown that Rab4-associated vesicles are transported with an anterograde bias, induced by a specific interaction with the tail domain of Kinesin-2α subunit, which results in its moderate enrichment at the synapses. The rate of pre-synaptic inflow of Rab4 is positively correlated with its activation. Interestingly, this study found that a decreased flow of the Rab4-associated vesicles expanded the region occupied by synapses in the neuropil region of the larval ventral ganglion and enhanced larval motility. Altogether, these results indicate that the flow of Rab4-associated vesicles could maintain synaptic homeostasis in a neuronal network (Dey, 2017).

Heterotrimeric Kinesin-2, implicated in the trafficking of apical endosomes and late endosomal vesicles, was initially thought to bind to its cargoes through the accessory subunit KAP3. However, recent studies have reported that motor subunits could independently interact with soluble proteins through the tail domain. This study shows that the Kinesin-2α tail can also bind to a membrane-associated protein, Rab4. A preliminary investigation in the lab suggests that the interaction in not direct. The RabGTPases are known to recruit a variety of different motors to the membrane, directing intracellular trafficking. Although a previous report indicated that Rab4 could bind to Kinesin-2, this study found that such an interaction leads to the anterograde trafficking of Rab4-associated vesicles in the axon. Rab4 associates with early and recycling endosomes carrying a diverse range of proteins. The FRB-FKBP assay indicated that a particular subset of Rab4-only vesicles associates with Kinesin-2. These vesicles predominantly move toward the synapse in axons. Although Kinesin-2 is also found to bind and transport vesicles containing Acetylcholinesterase (AChE) in the same axon, it was observed that Rab4 does not mark them. Similarly, the post-Golgi vesicles carrying N-cadherin and β-catenin, which are known to associate with Kinesin-2, exclude Rab4. Therefore, the Rab4-associated vesicles transported in the axon are likely to contain a unique set of proteins (Dey, 2017).

An association between RabGTPases and putative transport proteins was identified using coimmunoprecipitation, pull-down, and yeast two-hybrid assays. While these assays provide information on the possible interactions, their nature in a cellular or physiological scenario is unknown. Long-range transport of the Rab27/CRMP2 complex by Kinesin-1 and the Rab3/DENN MADD complex by KIF1A/1Bβ are substantiated by genetic perturbations and imaging. The FKBP-FRB-inducible interaction system used in this study identified Kinesin-2α and KIF13A/B as the key motors for nascent Rab4-associated vesicles. The latter was also shown to associate with Rab5-marked early endosomes. The results excluded the possibility that the Rab4 effectors involved in this interaction could bind to Rab5 and Rab11. In vivo analysis of Rab4-associated vesicular traffic further confirmed that heterotrimeric Kinesin-2 plays a dominant role in the trafficking of nascent Rab4 vesicles toward the synapse (Dey, 2017).

Anterograde axonal movement of Rab4 vesicles is mostly mediated by Kinesin-2, which transports nascent Rab4 vesicles, whereas Kinesin-3 transports sorting endosomes. In a recent study, it was shown that combined activity of Kinesin-1 and Kinesin-2 steers AChE-containing vesicles in the axon (Kulkarni, 2016). A simultaneous association between these two motors was suggested to induce longer runs of AChE-containing vesicles. A similar collaboration between Khc-73 and Kinesin-2 could propel longer runs of Rab4 vesicles. Khc-73 has been implicated in neuronal functions. Khc-73 knockdown in lch5 neurons decreased the motility of Rab4-associated vesicles to a limited extent, which suggests that the motor is only engaged in transporting a relatively small fraction of Rab4-associated vesicles in axons. Khc-73 is a processive motor with an in vitro velocity of 1.54 ± 0.46 &mi;m/s, and this study found that Khc-73 RNAi particularly affected the longer runs. Thus, Khc-73 could support the relatively longer runs (3.05 &mi;m) of Rab4 vesicles in axons. It was previously shown that Rab5-associated vesicles bind to KIF13A/B/Khc-73. Hence, it is inferred that sorting endosomes, positive for both Rab4 and Rab5, can engage both Kinesin-2 and KIF13A/Khc-73 through the cognate adaptors of these two Rabs. However, one needs suitably tagged reagents to establish this conjecture through direct observation in vivo (Dey, 2017).

Local synaptic vesicle recycling mediated by RabGTPases is critical for the availability of fusion-ready synaptic vesicles to sustain neurotransmission. The machinery involved in the translocation of RabGTPases from cell bodies to the synapses is poorly understood. Certain RabGTPases such as Rab3 and Rab27 are known to associate with Kinesin-3 and Kinesin-1, respectively, for their anterograde axonal transport. Rab4 is a protein present on the endosomal sorting intermediates, and it allows for the transfer of cargoes from early endosomes to recycling endosomes. As Rab4 is present in both early and recycling endosomal populations, the logistics of Rab4 transport into the presynaptic terminal has been unclear. The current results show that Rab4-associated vesicles are present throughout the neuron and moderately enriched in the synapse, which is a consequence of a small anterograde bias in their transport (Dey, 2017).

The activity of Rab4 is critical for the physiology of the neuron-like extension of the growth cone. The overexpressed levels and activity of Rab4 found in conjunction with neuropathy, such as Alzheimer's disease, suggest that overactivation of the endosomal system could influence disease progression. In Drosophila, Rab4 is expressed in both neuronal and non-neuronal tissues throughout development with the exception of optic lobe neurons of the pupal brain. These studies project the idea that the expression and activity of Rab4 are crucial for the maintenance of neuronal physiology. This study has shown that the synaptic Rab4 is maintained through axonal transport by heterotrimeric Kinesin-2 and Kinesin-3. As the synaptic localization of Rab4 is dependent on its active form, other factors like PI3K are also implicated in this trafficking. The results further suggest that the function and localization of Rab4 at axon termini are critical for the maintenance of synaptic balance at ventral ganglion. Overactivation of Rab4 in cholinergic neurons reduced the synapse-bearing region of the ventral ganglion, although levels of the marker, Brp, remained unchanged. This observation suggests that local Rab4 activity at the axon termini suppresses synapse formation. Consistent with this conjecture, this study found that the synapse-bearing zone at the neuropil expands when Rab4DN is overexpressed. Although the current data are insufficient to explain the underlying mechanism, they indicate that alteration of the trafficking logistics driven by heterotrimeric Kinesin-2 and Kinesin-3 family motors might play an essential role in the development of the defect. Further analysis of larval behavior and neuronal stability in the ventral ganglion with aging would be useful in uncovering the mechanism (Dey, 2017).

Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies

Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the "insulin paradox"). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders (Augustin, 2017).

A number of experimental results demonstrated the impact of long-term IIS manipulations on the nervous system. For example, systemic injections of IGF-1 mimicked some of the effects of exercise in the brain, and genetically reduced IGF-1 signaling in the whole organism reduced inflammation and neuronal loss in a mouse Alzheimer disease model. Likewise, chronic IIS manipulations only in the nervous system can have consequences on the whole organism: attenuated IR substrate/IR substrate 2 signaling in aging brains promotes healthy metabolism and extends the lifespan in mice, and neuron-specific reduction of IIS increases longevity in Drosophila. At the synaptic level, basal IGF-1 activity has recently been shown to regulate ongoing neuronal activity in hippocampal circuits. While infusion of IGF-1 does not appear to have short-term influence on Cx43 levels in various regions of the rat brain, no study so far has examined the effect of chronic IIS manipulations in the aging nervous system on GJs (Augustin, 2017).

This work demonstrated a role for IIS in regulating the trafficking of gap junctional proteins that is conserved over the large evolutionary distance between Drosophila and humans, and between different cell types. Elevated IIS induces the targeting of GJ proteins to lysosomes and degradation, thereby decreasing their cell surface assembly (Augustin, 2017).

Specifically, reduced insulin signaling throughout adulthood leads to Rab4/11-mediated increase in the synaptic targeting of Shak-B-encoded gap junctional components in the Drosophila escape response circuit, resulting in the maintenance of the 'youthful' functional output even in old flies. Previous studies demonstrated a positive effect of reduced insulin signaling on neuronal circuit function. For example, visual acuity is improved in mice with reduced insulin signaling in the visual cortex. In the nematode C. elegans, mutations of the IR gene resulted in improved chemical transmission at the neuromuscular synapse, and delayed decline in the synaptic function with age. The current findings have revealed a novel restorative and adaptive cellular mechanism by which lowered IIS can maintain electrical transmission in a neuronal circuit during aging, and that could potentially be harnessed to prevent decline in neuronal function. A recent report demonstrated a negative effect of neuron-specific IIS reduction on age-specific walking behavior in Drosophila, suggesting that the effect of insulin signaling depends on the type of neuron(s) mediating a specific behavior. For example, physiological roles of different (chemical) neuronal circuits can be preferentially mediated by either evoked or spontaneous transmission. Interestingly, blockade of insulin signaling has opposing effects on these 2 types of transmission, possibly explaining some of the seemingly contradictory experimental data about the role of IIS in the nervous system. Together, these findings indicate that studies of insulin signaling in the nervous system should be circuit- and synapse type-specific, taking into consideration the physiological properties of the neuronal system under study, and precluding simplified generalizations about the effectiveness of specific IIS manipulations across the nervous system (Augustin, 2017).

Drosophila VAMP7 regulates Wingless intracellular trafficking

Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. This study showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, this study found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. It is hypothesized that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling (Gao, 2017).

There are two models describing how the apically secreted Wg encounters basolateral receptors at receiving cells. One suggests that Wg and receptors can be internalized separately, and then, endosome fusion results in Wg and receptor interaction in the receiving cells. Another model proposes that apically secreted Wg undergoes endocytosis and will be transported to the basolateral surface in the producing cells, then spread to the receiving cells for the interaction with receptors. Therefore, Wg is actively endocytosed in both receiving cells and producing cells (Gao, 2017).

This study found that Wg distribution was affected in both receiving and producing cells in vamp7-/- mutant background. Further investigation indicated that Wg double labeled puncta significantly increased, so did the percentage of Rab4 and Wg double staining puncta. Thus, it is suggested that VAMP7 is required for Wg endocytosis in the both receiving cells and producing cells in Drosophila wing disc, and its mutation leads to Wg accumulating in endocytic organelles but not degradation. Rab4 dependent recycling endosomes can recruit proteins from the early endocytic organelles, which may finally lead to increased level of Wg in Rab4 dependent recycling endosomes (Gao, 2017).

Although endocytosis has been demonstrated for Wg transport, there is still debate about whether endocytosis plays a direct role in the Wg signaling. Classically, the early step of endocytosis is thought to contribute positively to signaling, as early endosomes can recruit signaling components, while subsequent vesicle transport may downregulate signaling by sequestrating signaling components in endosomes or degradating them in lysosomes. This study found that the expression of the Wg target gene sens was reduced in vamp7 mutant cells. One possibility is that Rab4 recycling endosomes may recruit Wg from early endosomes. As a previous report found that the expression of activated forms of Rab4 suppressed the ability of Rab5 to enhance activation of Wg pathway, Wg accumulation in Rab4 recycling endosomes may affect Wg signaling. Another possible reason is that vamp7 mutation enhances the level of Wg signaling inhibitors (Gao, 2017).

Dlp is a membrane-associated glypican that can interact with Wg by its core protein on the cell surface, and suppresses Wg target gene sens. However, the functional significance of interaction between Wg and Dlp inside the cell has not been well elucidated. This study showed that Wg might encounter endogenous Dlp in Rab4 dependent recycling endosomes, and vamp7 mutation could improve the levels of Dlp and Wg in Rab4 dependent recycling endosomes. Previous studies proposed that Dlp competes with Wg receptors to interact with Wg, and the signaling activity may be determined by the relative levels of receptor and Dlp. It is suggested that competition between Dlp and receptors might not only occur on the cell surface but may have started from intracellular vesicles. The increased levels of Dlp and Wg in Rab4 dependent recycling endosomes may lead to Sens reduction (Gao, 2017).

In conclusion, this study showed that an endocytic pathway involving VAMP7 regulates Wg and Dlp trafficking. This route adds another layer of spatial regulation in the Wg signaling pathway. Additional work will be needed to determine the functional significance of this route in other Drosophila tissues and whether vamp7 is required for vertebrate Wnt trafficking (Gao, 2017).

A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. The deubiquitinating enzyme UBPY/USP8 has been identified as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, it was hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. This study blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. Inhibition of endosomal trafficking at different steps in vivo was observed to be systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. These results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo (Jacomin, 2016).

Lysosomes are the primary degradative organelles of the cell. They are found in virtually all eukaryotic cells and were initially described in the 1950s by the Nobel laureate Christian de Duve. Their substrates include all kinds of macromolecules delivered either by endocytosis, phagocytosis or autophagy. Lysosomal biogenesis is orchestrated by the transcription factor EB (TFEB) which activates the transcription of ~500 target genes involved in lysosomal biogenesis and autophagy. On the other hand, lysosomal biogenesis also requires the integration of the endosomal and biosynthetic pathways: newly synthesized lysosomal proteins are delivered to lysosomes either directly from the trans-Golgi network to the endosomal system using the mannose-6-phosphate receptor (MPR) or the Vps41/VAMP7 pathway or indirectly via alternative receptors such as LIMP-2. In Drosophila, defects in the biogenesis of lysosomes and lysosomes related organelles such as eye pigment granules result in defective eye pigmentation which has led to the identification of the 'granule group' proteins including Deep-orange, homologue of Vps18p, Carnation, homologue of Vps33A and Light, homologue of Vps41 (Jacomin, 2016).

The endosomal system constitutes a network of progressively maturing vesicles that is required, among other physiological functions, for the degradation of membrane proteins such as receptors and ionic channels. These proteins enter the endosomal system through clathrin or caveolin-coated vesicles and are then delivered to early endosomes. From here, membrane proteins can either be recycled to the plasma membrane or directed for degradation via the multivesicular bodies (MVB) to late endosomes that eventually fuse with lysosomes. Sorting to the MVB requires the ESCRT (Endosomal Sorting Complex Required for Transport) machinery composed of four distinct complexes called ESCRT-0 to -III. Apart from ESCRT machinery, progression along the endosomal pathway requires the activity of Rab GTPases: Rab5 is located to the clathrin coated vesicles and early endosomes and contributes to endocytic internalization and early endosome fusion; Rab4 is located at the early and recycling endosomes, and is involved in the recycling to plasma membrane; Rab7 is involved in the transport from early to late endosomes and is an essential component of the lysosomes biogenesis and maintenance. Rab GTPases notably recruit tethering and docking machinery to bring membranes closer, after which the SNARE proteins complete the fusion process (Jacomin, 2016).

Previous work has that the deubiquitinating enzyme UBPY is required for lysosomal biogenesis in Drosophila (Jacomin, 2015). However, UBPY is mainly known for playing an important role in the sorting of many membrane receptors in Drosophila and mammalian cells. Given the integration of lysosomal biogenesis and the endosomal system, it was hypothesized that the lysosomal defects observed in UBPY mutant cells might be a consequence of UBPY function in the endosomal system and seek to further test the requirement of ongoing endosomal trafficking for lysosomal biogenesis in vivo. This report shows that inhibition of endosomal trafficking at different steps is associated with defects in lysosomal biogenesis and blockade of autophagic degradation indicating that a functional endosomal system is required for lysosome biogenesis in vivo (Jacomin, 2016). UBPY was previously identified as a new deubiquitinating enzyme affecting lysosomal biogenesis in Drosophila. Earlier studies extensively showed the implication of UBPY in the endosomal pathway in both Drosophila and mammalian cultured cell models. It was hypothesized that the autophagy flux blockade and impaired lysosomes formation induced by Ubpy loss-of-function might be related to its function in the endosomal pathway, suggesting that the overall endosomal process is crucial for lysosomal biogenesis. This report investigated this hypothesis by inhibiting endosomal trafficking at different steps: from the plasma membrane to the endo-lysosomal compartment. Using lysosomal markers such as the lysosomal membrane protein LAMP1 and the lysosomal hydrolase Cathepsin L, it was observed that inhibition of endosomal trafficking consistently resulted in severe lysosomal biogenesis defects. Besides, the autophagic process in the cells presenting a defective endosomal trafficking was constitutively impaired, as revealed by the use of the GFP -- and tandem GFP -- mCherry-tagged Atg8a transgenes, and the accumulation of the autophagy substrate Ref(2)P/p62. Altogether, the results show that a functional endosomal pathway is required for lysosomal biogenesis and, as a consequence, for productive autophagy (Jacomin, 2016).

To date, two alternative models for lysosome biogenesis have been proposed. In the maturation model, endosomes are gradually transformed into lysosomes by the addition (delivery of lysosomal enzymes and membrane proteins from the Golgi apparatus) and removal (by recycling vesicles) of molecules. According to this model, lysosomes would not form without endosomal trafficking. A second model, the vesicular transport model, postulates that endosomes, late endosomes, and lysosomes are stable pre-existing compartments that communicate by continuous rounds of fusion and fission. Although studies in cultured cells are numerous and sometimes contradictory, in vivo evidence supporting any of these models are surprisingly scarce. Rab5 is the only known endocytic protein whose inactivation has been shown to impair the biogenesis of the endo-lysosomal system in vivo. The current results thus confirm the crucial role of Rab5 but also extend this property to other components of the endosomal process, actively supporting the maturation model: fully functional lysosomes are not pre-existing compartments, but instead result from the gradual maturation of endosomes to which lysosomal enzymes are delivered (Jacomin, 2016).

Furthermore, it has been shown that the endosomal and autophagy pathways share several components. In particular, the endosomal Rab5 protein has also been proposed to act at an early stage of autophagy since inhibition of Rab5 activity by overexpression of a dominant negative mutant decreases the number of autophagosomes in cultured mammalian cells. This observation does not fit with the current study indicating that autophagosomes accumulate in fat body cells silenced for Rab5. It is possible that the role of Rab5 in autophagy may be unique to mammals and not conserved in Drosophila. Alternatively, differences in the experimental systems (transient overexpression of a dominant negative form of Rab5 in cultured cells versus clonal impairment in a wild-type organ during larval development) may be at stake. A careful comparison of the autophagic phenotype induced by Rab5 inhibition or silencing in the same experimental model should resolve this point. It is worth noting that the scientific literature is quite contradictory on the requirement of endosomal pathway members for autophagy. Autophagosomes and ubiquitinated protein aggregates have been observed in ESCRT mutant cells, indicating a blockade of autophagic degradation after autophagosomes formation in agreement with the current results. In contrast, other studies have shown that perturbations of the endosomal pathway impair autophagosome formation in cultured cells (Jacomin, 2016).

The results demonstrated that genetic impairment of endosomal trafficking induces lysosomal defects in the widely used Drosophila fat body model. It was further shown that endosomal trafficking - because of its requirement for lysosomal biogenesis - is also required for efficient autophagic degradation. Indeed, these last years, the connection between lysosome biogenesis or function and autophagy has been extensively described, and an increasing body of evidence implicates defective autophagy in the ethology of lysosomal storage disorders, a group of approximately 50 rare inherited metabolic disorders that result from defects in lysosomal function. For example, stalled or blocked autophagy has been observed in the lipid storage disorder Niemann-Pick type C1 (NPC1) disease and in the Gaucher disease, the most prevalent lysosomal storage disorder. Moreover, regulation of these two processes is coordinated by the transcription factor EB (TFEB; Mitf in Drosophila) which drives expression of autophagy and lysosomal genes. By suggesting that these disorders can originate from defects in the endosomal system, the results thus open new avenues in the understanding of lysosomal storage diseases and of the numerous pathologies linked to autophagy deficiencies (Jacomin, 2016).

Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo

The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. This study reports that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. It is proposed that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens (Sorvina, 2016).

Following host invasion, cells of the innate immune system detect microbial pathogens via pattern recognition receptors and peptidoglycan-recognition receptors. These receptors can trigger the activation of immune cells by two distinct intracellular cascades; the Toll-interleukin 1 pathway and the immune deficiency-TNFα pathway. This innate immune signalling initiates a sequence of events that leads to the transcription of pro-inflammatory cytokine and antimicrobial peptide genes. Newly synthesised immune-mediators are transported through the endoplasmic reticulum-Golgi network and delivered to endosomes for final processing and packaging into Rab11 vesicles; ready for exocytosis at the plasma membrane. Endosomal trafficking is regulated by a complex set of vesicular machinery including, for example, A-kinase anchoring proteins, and this study has investigated the role of the Drosophila A-kinase anchoring protein, Pkaap, in the control of innate immune secretion (Sorvina, 2016).

Relatively little is known about how A-kinase anchoring proteins regulate immune defence mechanisms, although they have been previously implicated in modulating the transcriptional activation of cytokines in immune cells. For instance, AKAP13 has a role in the nuclear translocation of transcription factor NF-κB, and therefore the TLR2-dependent activation of pro-inflammatory cytokine secretion. In contrast, AKAP95 was required for targeting of type II regulatory subunit of PKA to suppress the early expression of TNFα in lipopolysaccharide-stimulated RAW264.7 macrophages. D-AKAP2-anchored type I regulatory subunit of PKA is involved in prostaglandin E2 potentiation of lipopolysaccharide-induced nitric oxide synthesis downstream of Toll-like receptors and the expression of interleukin 6 and interleukin 10 in alveolar macrophages. Analysis of pkaap depleted larvae showed a normal activation of immune response pathways, resulting in Dorsal nuclear translocation and antimicrobial peptide drosomycin gene expression, suggesting that Pkaap did not have a role in modulating antimicrobial peptide transcriptional regulation. In future studies the effect of pkaapRNAi on phagocytosis and morphology of Rab4/Rab11 endosomes in haemocytes might be determined as the expression of transgenes was driven by fat body- and haemocyte-specific CG-GAL4 driver. Given that D-AKAP2 is involved in the regulation of transferrin receptor recycling via Rab4/Rab11 endosomes and there were no difference in Rab11-GFP fluorescence in control and pkaapRNAi fat body cells, it was postulated that Pkaap might be involved in antimicrobial peptide intracellular traffic (Sorvina, 2016).

In Drosophila, antimicrobial peptide targeting to the plasma membrane is known to involve Rab4 and Rab11 GTPases (Shandala, 2011). GAPs and GEFs control membrane trafficking by modulating Rab protein activities; and the nucleotide-dependent location of Pkaap to endosomes suggested that it could have a role as both a GAP and GEF. Pkaap colocated with wild-type and GTP-bound constitutively active Rab4, but not the GDP-bound dominant negative form of Rab4, which was consistent with it acting as a GAP for Rab4. Pkaap colocated with wild-type and GDP-bound dominant negative form of Rab11, but not GTP-bound constitutively active Rab11, suggesting that it may also be acting as a GEF for Rab11. This was consistent with observations of D-AKAP2 co-immunoprecipitation studies for both Rab4 and Rab11 GTPases in HEK293 cells and that some AKAPs have been shown to act as molecular switches in regulating GTPase activity. For example, AKAP-Lbc binds to the GDP-bound or nucleotide-free forms of RhoA and possesses Rho-selective GEF activity in HeLa and HEK-293 cells. Thus, by regulating the activity of Rab4 and Rab11, Pkaap could modulate two critical steps during the vesicular traffic of cargo along the immune secretory pathway, cargo sorting in recycling endosomes and exocytosis at the plasma membrane (Sorvina, 2016).

The depletion of pkaap had a direct effect on Rab11 vesicle traffic and antimicrobial peptide immune cargo delivery to the plasma membrane and resulted in the accumulation of enlarged Rab11 vesicles at the cellular periphery. The GDP-bound dominant negative form of Rab11 also reduced the number of small Rab11 vesicles in close proximity to the cell surface and resulted in the accumulation of enlarged Rab11 multivesicular endosomes, suggesting that pkaap depletion trapped Rab11 in a GDP-bound form. These enlarged Rab11 compartments with intraluminal vesicles were identified as multivesicular endosomes by definition. To further explore this question, future work may investigate fat body tissues expressing Rab11-GFP with antibodies detecting other multivesicular endosome markers, such as Hrs or Vps16. Constitutively active Rab11 produced a different vesicular phenotype, with enhanced delivery of small Rab11 vesicles to the plasma membrane. The results also revealed low Pkaap levels in fat body cells expressing constitutively active form of Rab11, suggesting a negative feedback mechanism by which the intracellular level of GEF proteins is regulated, but this is yet to be determined. Immune cargo delivery is dependent on Rab11 vesicles and pkaap depletion abrogated the intracellular traffic and delivery of the antimicrobial peptide Drosomycin to the plasma membrane upon bacterial challenge, but did not cause changes in the size of the Rab4/Rab11 endosomes. Concomitantly, pkaap depletion caused Drosomycin to accumulate in Rab4/Rab11 endosomes and this reduction in antimicrobial peptide delivery coupled with reduced secretion into the haemolymph could explain reduced larval viability following bacterial challenge. Due to limitations in the availability of transgenic stocks that could be used to study secreted proteins in live mode, this study was limited to using Drosomycin-GFP and ideally the findings should be confirmed with other immune mediators. It would also be interesting to determine whether treatment of pkaapRNAi transgenic larvae with beta-lactam antibiotics (e.g. tetracycline) can improve survival rates after infection with Micrococcus luteus (Sorvina, 2016).

The structural changes to Rab4/Rab11 endosomes caused by pkaap depletion could affect the sorting and compartmentalisation of immune cargo in recycling endosomes. This would be consistent with the effects that pkaap depletion had on both of the GTPases Rab4 and Rab11 and the morphology of Rab4/Rab11 endosomes. Instead of correct antimicrobial peptide cargo sorting in Rab4/Rab11 recycling endosomes and packaging into Rab11 vesicles, a large amount of Drosomycin appeared to be degraded. For example, after immune challenge, while drosomycin mRNA expression was not reduced in response to pkaap depletion there was less Drosomycin protein, suggesting that a lysosomal degradative pathway may have been evoked in response to failed sorting in Rab4/Rab11 recycling endosomes; therapeutic agents, such as bafilomycin A and chloroquine, could be used in future studies. The specific effects of pkaap depletion on the dynamics and morphology of different endosomes suggested that pkaapRNAi transgenic larvae could have multiple impairments in the antimicrobial peptide trafficking and secretion pathway that contributed to immune dysfunction (Sorvina, 2016).

It is concluded that Pkaap has a critical role in effecting an innate immune response and is important for Drosophila viability. The colocation of Pkaap with Rab11 endosomes suggested that Pkaap might be acting as a regulator of exocytosis and the effect of pkaap depletion on Rab11 vesicle and antimicrobial peptide cargo delivery would support this hypothesis. However, Pkaap also appeared to have an important role in Rab4/Rab11 recycling endosome morphology and function suggesting that it is also involved in cargo sorting and delivery earlier in the secretory pathway. It appears that Pkaap has a dual role in antimicrobial peptide trafficking and exocytosis making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens. In future studies, the role of Pkaap might be investigated in other secretory tissues, such as the salivary glands, to ascertain if a similar molecular mechanism is used to control non-immune secretion (Sorvina, 2016).

RhoGAP68F controls transport of adhesion proteins in Rab4 endosomes to modulate epithelial morphogenesis of Drosophila leg discs

Elongation and invagination of epithelial tissues are fundamental developmental processes that contribute to the morphogenesis of embryonic and adult structures and are dependent on coordinated remodeling of cell-cell contacts. The morphogenesis of Drosophila leg imaginal discs depends on extensive remodeling of cell contacts and thus provides a useful system with which to investigate the underlying mechanisms. The small Rho GTPase regulator RhoGAP68F has been previously implicated in leg morphogenesis. It consists of an N-terminal Sec14 domain and a C-terminal GAP domain. This study examines the molecular function and role of RhoGAP68F in epithelial remodeling. Depletion of RhoGAP68F impaired epithelial remodeling from a pseudostratified to simple, while overexpression of RhoGAP68F caused tears of lateral cell-cell contacts and thus impaired epithelial integrity. The RhoGAP68F protein localized to Rab4 recycling endosomes and formed a complex with the Rab4 protein. The Sec14 domain was sufficient for localizing to Rab4 endosomes, while the activity of the GAP domain was dispensable. RhoGAP68F, in turn, inhibited the scission and movement of Rab4 endosomes involved in transport the adhesion proteins Fasciclin3 and E-cadherin back to cell-cell contacts. Expression of RhoGAP68F was upregulated during prepupal development suggesting that RhoGAP68F decreases the transport of key adhesion proteins to the cell surface during this developmental stage to decrease the strength of adhesive cell-cell contacts and thereby facilitate epithelial remodeling and leg morphogenesis (de Madrid, 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 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).

The Bro1-domain-containing protein Myopic/HDPTP coordinates with Rab4 to regulate cell adhesion and migration

Protein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes that coordinate with protein tyrosine kinases to control protein phosphorylation during various cellular processes. Using genetic analysis in Drosophila non-transmembrane PTPs, this study identified one role that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays in cell adhesion. Depletion of Mop results in aberrant integrin distribution and border cell dissociation during Drosophila oogenesis. Interestingly, Mop phosphatase activity is not required for its role in maintaining border cell cluster integrity. Rab4 GTPase was further identfied as a Mop interactor in a yeast two-hybrid screen. Expression of the Rab4 dominant-negative mutant leads to border cell dissociation and suppression of Mop-induced wing-blade adhesion defects, suggesting a critical role of Rab4 in Mop-mediated signaling. In mammals, it has been shown that Rab4-dependent recycling of integrins is necessary for cell adhesion and migration. This study found that human HDPTP regulates the spatial distribution of Rab4 and integrin trafficking. Depletion of HDPTP resulted in actin reorganization and increased cell motility. Together, these findings suggest an evolutionarily conserved function of HDPTP-Rab4 in the regulation of endocytic trafficking, cell adhesion and migration (Chen, 2012).

Cell adhesion and cell migration are essential for the development and coordinated function of multicellular organisms. Aberrant regulation of these processes often results in the progression of many diseases, including cancer invasion and metastasis. Accumulating evidence has indicated that dynamic and reversible protein tyrosine phosphorylation is essential for the regulation of cell migration and cell adhesion. While many studies have been devoted to the role of protein tyrosine kinases in these processes, the function of protein tyrosine phosphatases (PTPs) in cell adhesion and migration remains unclear (Chen, 2012).

The dynamic change of integrin-mediated focal adhesions plays a critical role in cell adhesion and migration. Many focal adhesion regulators such as focal adhesion kinase (FAK), Src, p130Cas, and paxillin are tyrosine phosphorylated. The tyrosine phosphorylation of these proteins affects focal adhesion dynamics. Phosphorylation of tyrosine 397 in FAK promotes its association with Src, and the activated FAK-Src complex subsequently regulates focal adhesion dynamics by signaling downstream targets. Several PTPs have been implicated in integrin signaling, cell adhesion and motility. One study has shown that SHP-2 phosphatase influences FAK activity. SHP-2 also promotes Src kinase activation by inhibiting Csk. Depletion of PTP-PEST has been found to lead to the hyperphosphorylation of p130Cas, FAK and paxillin, and a marked increase in focal adhesions. Moreover, PTP1B and PTPα, have also been found to regulate Src phosphorylation and integrin-mediated adhesion (Chen, 2012).

In Drosophila, a total of sixteen putative classical PTPs have been identified. Compared to mammalian PTPs, Drosophila PTP family members are relatively simple, most containing only one gene corresponding to each subtype (except for DPTP10D and DPTP4E, which share similar domain structures). Therefore, Drosophila can serve as an excellent model system for the study of the physiological and developmental function of PTPs. While much research has been devoted to the function of receptor PTPs, the role of non-transmembrane PTPs (NT-PTPs) in Drosophila development remains unknown. One of the most well studied Drosophila NT-PTPs is Corkscrew (Csw). Csw is the ortholog of human SHP-2 which has two SH2 domains at the N-terminus and a PTP domain at the C-terminus. Csw functions as a downstream effecter of Sevenless PTK and is essential for the development of the R7 photoreceptor. Phenotypic analysis showed that Csw can also act downstream of many receptor tyrosine kinases, such as the Drosophila epidermal growth factor receptor (DER) and the fibroblast growth factor (Breathless). PTP-ER has been shown to function as a negative regulator downstream of Ras1 and to be involved in RAS1/MAPK-mediated R7 photoreceptor differentiation. PTP61F, the Drosophila ortholog of human PTP1B and TCPTP, has been reported to interact with Dock, an adapter protein required for axon guidance. PTP61F has recent been shown to coordinate with dAbl in regulating actin cytoskeleton organization via reversible tyrosine phosphorylation of Abi and Kette ). Moreover, dPtpmeg, a FERM and PDZ domain-containing NT-PTP, is reported to be involved in the formation of neuronal circuits in the Drosophila brain, though its molecular function in this process is not known (Chen, 2012).

To explore the functional role of Drosophila NT-PTPs in cell adhesion and migration, genetic analyses was performed to identify NT-PTPs that could modulate border cell migration during oogenesis. This study found that Myopic (Mop), the Drosophila homolog of the human His domain phosphotyrosine phosphatase (HDPTP), plays an important role in maintaining border cell cluster integrity. Depletion of Mop altered the normal distribution of integrin receptor. While Mop has recently been reported to regulate EGFR and Toll receptor signaling, its molecular mechanism has remained elusive. This study found that Mop interacts with Rab4 GTPase in controlling integrin distribution and cell adhesion. It was further demonstrated that human HDPTP is essential for the intracellular positioning of Rab4, integrin trafficking, and cell migration. These findings provide some insight into the mechanisms underlying HDPTP in the regulation of cell adhesion and migration (Chen, 2012).

Accumulating evidence has indicated that vesicular trafficking regulates the distribution of plasma membrane content as well as the localization of cytoskeletal proteins during cell adhesion and migration. Drosophila border cells migrate as a cluster of strongly adherent cells during the development of the egg chamber. During this process, JNK signaling and endocytosis-mediated spatial distribution of receptor tyrosine kinases play a critical role, though mechanisms involved in this process have remained elusive. This identified Mop, the Drosophila homolog of human HDPTP, as a regulator of integrin trafficking. Mop is essential for proper integrin localization and for maintaining border cell integrity during oogenesis. It was further demonstrated that Mop and HDPTP interacts with Rab4 GTPase in both Drosophila and mammals. Rab4 has been shown to regulate integrin recycling and cell migration (Roberts, 2001; White, 2007). The current findings indicate that Mop/HDPTP-mediated endocytic trafficking plays an essential role in integrin-mediated cell adhesion and migration. Mop has been predicted as a nontransmembrane-PTP (Andersen, 2005). However, amino acid sequence analysis revealed that Mop displays several differences from conserved PTP motifs within the phosphatase domain. For example, the catalytic essential aspartic acid (D) within motif 8 (WPDXGXP) is replaced by a lysine residue (K). Although the active site cysteine (C) in the catalytic motif 9 (VHCSAGXGR[T/S]G) could be found, the overall signature motif of Mop was much more divergent compared to other PTPs. Moreover, no Mop tyrosine phosphatase activity could be detected using in vitro phosphatase assays. These results suggest that Drosophila Mop may not be enzymatic active. Alternatively, Mop may exhibit weak phosphatase activity which can not be detected using either pNPP or in gel phosphatase assay. A recent study indicated that human PTPN23/HDPTP exhibits relatively low activity that is comparable with the specific activity of PTP1B D181E mutant. This study also found that expression of Mop-C/S mutant, in which the catalytic cysteine in the active site is replaced by serine, or Mop phosphatase domain deletion mutant rescued the Mop-RNAi induced border cell dissociation defects as effectively as the wild-type Mop, indicating that the putative tyrosine phosphatase activity is not essential for maintaining border cell cluster integrity (Chen, 2012).

In addition to having a C-terminus phosphatase domain, Mop has a sequence similar to that of yeast Bro1 at the N-terminus. The Bro1 domain consists of a folded core of about 370 residues and has been found in many proteins, including Bro1,

Functions of Rab4 orthologs in other species

Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles

Sorting endosomes (SEs) are the regulatory hubs for sorting cargo to multiple organelles, including lysosome-related organelles, such as melanosomes in melanocytes. In parallel, melanosome biogenesis is initiated from SEs with the processing and sequential transport of melanocyte-specific proteins toward maturing melanosomes. However, the mechanism of cargo segregation on SEs is largely unknown. Here, RNAi screening in melanocytes revealed that knockdown of Rab4A results in defective melanosome maturation. Rab4A-depletion increases the number of vacuolar endosomes and disturbs the cargo sorting, which in turn lead to the mislocalization of melanosomal proteins to lysosomes, cell surface and exosomes. Rab4A localizes to the SEs and forms an endosomal complex with the adaptor AP-3, the effector rabenosyn-5 and the motor KIF3, which possibly coordinates cargo segregation on SEs. Consistent with this, inactivation of rabenosyn-5, KIF3A or KIF3B phenocopied the defects observed in Rab4A-knockdown melanocytes. Further, rabenosyn-5 was found to associate with rabaptin-5 or Rabip4/4' (isoforms encoded by Rufy1) and differentially regulate cargo sorting from SEs. Thus, Rab4A acts a key regulator of cargo segregation on SEs (Nag, 2018).

Rab4 orchestrates a small GTPase cascade for recruitment of adaptor proteins to early endosomes

Early, sorting endosomes are a major crossroad of membrane traffic, at the intersection of the endocytic and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations is mediated by a number of distinct coat protein complexes, including adaptor protein 1 (AP-1), AP-3, and Golgi-localized, gamma adaptin ear-containing, Arf-binding (GGAs) protein. Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal membrane, but the mechanisms of coat recruitment and assembly at this site remain poorly understood. This paper reports that the endosomal Rab protein Rab4 orchestrates a GTPase cascade that results in the sequential recruitment of the ADP-ribosylation factor (Arf)-like protein Arl1; the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2; and the class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1 and BIG2 activity with brefeldin A results in the loss of AP-1, AP-3, and GGA-3, but not Arl1, from endosomal membranes and the formation of elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal membranes, inhibits the formation of tubular subdomains, and blocks recruitment of BIG1 and BIG2, Arfs, and adaptor protein complexes to the endosome. Together these findings indicate that Arl1 links Rab4-dependent formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that constitute the endosomal sorting machinery (D'Souza, 2014).

Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11

Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, Rab4, Rab5, and Rab11, three regulatory components of the transport machinery, were studied. Following transferrin as cargo molecule and GFP-tagged Rab proteins it was possible to show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. It is proposed that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane (Sonnichsen, 2000).


Search PubMed for articles about Drosophila

Augustin, H., McGourty, K., Allen, M. J., Madem, S. K., Adcott, J., Kerr, F., Wong, C. T., Vincent, A., Godenschwege, T., Boucrot, E. and Partridge, L. (2017). Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies. PLoS Biol 15(9): e2001655. PubMed ID: 28902870

Chen, D. Y., Li, M. Y., Wu, S. Y., Lin, Y. L., Tsai, S. P., Lai, P. L., Lin, Y. T., Kuo, J. C., Meng, T. C. and Chen, G. C. (2012). The Bro1-domain-containing protein Myopic/HDPTP coordinates with Rab4 to regulate cell adhesion and migration. J Cell Sci 125(Pt 20): 4841-4852. PubMed ID: 22825871

de Madrid, B.H., Greenberg, L. and Hatini, V. (2015). RhoGAP68F controls transport of adhesion proteins in Rab4 endosomes to modulate epithelial morphogenesis of Drosophila leg discs. Dev Biol 399(2):283-95. PubMedID: 25617722

Dey, S., Banker, G. and Ray, K. (2017). Anterograde transport of Rab4-associated vesicles regulates synapse organization in Drosophila. Cell Rep 18(10): 2452-2463. PubMed ID: 28273459

D'Souza, R. S., Semus, R., Billings, E. A., Meyer, C. B., Conger, K. and Casanova, J. E. (2014). Rab4 orchestrates a small GTPase cascade for recruitment of adaptor proteins to early endosomes. Curr Biol 24(11): 1187-1198. PubMed ID: 24835460

Gao, H., He, F., Lin, X. and Wu, Y. (2017). Drosophila VAMP7 regulates Wingless intracellular trafficking. PLoS One 12(10): e0186938. PubMed ID: 29065163

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: 358-373. PubMed ID: 25453831

Jacomin, A. C., Bescond, A., Soleilhac, E., Gallet, B., Schoehn, G., Fauvarque, M. O. and Taillebourg, E. (2015). The deubiquitinating enzyme UBPY is required for lysosomal biogenesis and productive autophagy in Drosophila. PLoS One 10(11): e0143078. PubMed ID: 26571504

Jacomin, A. C., Fauvarque, M. O. and Taillebourg, E. (2016). A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila. BMC Cell Biol 17: 36. PubMed ID: 27852225

Nag, S., Rani, S., Mahanty, S., Bissig, C., Arora, P., Azevedo, C., Saiardi, A., van der Sluijs, P., Delevoye, C., van Niel, G., Raposo, G. and Setty, S. R. G. (2018). Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles. J Cell Sci 131(18). PubMed ID: 30154210

Shandala, T., Woodcock, J. M., Ng, Y., Biggs, L., Skoulakis, E. M., Brooks, D. A. and Lopez, A. F. (2011). Drosophila 14-3-3epsilon has a crucial role in anti-microbial peptide secretion and innate immunity. J Cell Sci 124(Pt 13): 2165-2174. PubMed ID: 21670199

Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J. and Zerial, M. (2000). Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J Cell Biol 149(4): 901-914. PubMed ID: 10811830

Sorvina, A., Shandala, T. and Brooks, D. A. (2016). Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo. Biol Open 5(6):678-88. PubMed ID: 27190105

Biological Overview

date revised: 22 May 2023

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