Rab11: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Rab11

Synonyms -

Cytological map position - 93B12--13

Function - signaling

Keywords - oogenesis, cellularization, eye development, vesicle transport, golgi apparatus

Symbol - Rab11

FlyBase ID: FBgn0015790

Genetic map position - 3R

Classification - GTP-binding protein domain

Cellular location - cytoplasmic

NCBI links: Gene | HomoloGene

Recent literature
Ji, H. H., Zhang, H. M., Shen, M., Yao, L. L. and Li, X. D. (2015). The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11. Biochem J [Epub ahead of print]. PubMed ID: 25940004
Myosin-5 (DmM5; Didum) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. This study overexpressed DmM5 in Sf9 cells and investigated its regulation using purified proteins. The actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid and dRab11 respectively. GTP-bound dRab11, but not Lightoid, significantly activates DmM5 actin-activated ATPase activity. Moreover, Gln1689 in the GTD was identified as the critical residue for the interaction with dRab11. Based on those results, it is proposed that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx.

Mavor, L. M., Miao, H., Zuo, Z., Holly, R. M., Xie, Y., Loerke, D. and Blankenship, J. T. (2016). Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster. Development [Epub ahead of print]. PubMed ID: 26839362
One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminates in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events have remained unclear. This study shows that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. CRISPR/Cas9 technology was used to N-terminally tag Rab8, which is then used to address both endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, while GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through exocyst tethering complex interactions.

Woichansky, I., Beretta, C. A., Berns, N. and Riechmann, V. (2016). Three mechanisms control E-cadherin localization to the zonula adherens. Nat Commun 7: 10834. PubMed ID: 26960923
E-cadherin localization to the zonula adherens is fundamental for epithelial differentiation but the mechanisms controlling localization are unclear. Using the Drosophila follicular epithelium, E-cadherin transport was genetically dissected in an in vivo model. Three mechanisms were distinguished mediating E-cadherin accumulation at the zonula adherens. Two membrane trafficking pathways deliver newly synthesized E-cadherin to the plasma membrane. One is Rab11 dependent and targets E-cadherin directly to the zonula adherens, while the other transports E-cadherin to the lateral membrane. Lateral E-cadherin reaches the zonula adherens by endocytosis and targeted recycling. This pathway is dependent on RabX1, which provides a functional link between early and recycling endosomes. Moreover, lateral E-cadherin is transported to the zonula adherens by an apically directed flow within the plasma membrane. Differential activation of these pathways could facilitate cell shape changes during morphogenesis, while their misregulation compromises cell adhesion and tissue architecture in differentiated epithelia.

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 [Epub ahead of print]. PubMed ID: 27190105
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.
Catrina, I. E., Bayer, L. V., Yanez, G., McLaughlin, J. M., Malaczek, K., Bagaeva, E., Marras, S. A. and Bratu, D. P. (2016). The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11. RNA Biol: 1-16. PubMed ID: 27654348
To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. This study reports for the first time the expression patterns during oogenesis for the Arf GTPase activating protein Drongo, the fruit fly homolog of AGFG1. Temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. This study shows a first link between the recycling endosome pathway and Drongo and finds that proper Drongo localization at the oocyte's cortex during mid-oogenesis requires functional Rab11.
Choubey, P. K. and Roy, J. K. (2016). Rab11, a vesicular trafficking protein, affects endoreplication through Ras-mediated pathway in Drosophila melanogaster. Cell Tissue Res [Epub ahead of print]. PubMed ID: 27677270
Rab11, a small monomeric GTPase associated with recycling endosomes, is a key molecule in the regulation of vesicular trafficking and is involved in the development and differentiation of many Drosophila tissues through interaction with diverse signaling pathways. This study reportsthat Rab11 affects endoreplication through a Ras-mediated pathway. Suppression of Rab11 activity in salivary glands, an endoreplicating tissue, leads to reduction in size of salivary glands with cells having a small nucleus. Endoreplication-regulating proteins, CycE, E2f1 and Gem, are also down-regulated in Rab11 knocked-down salivary glands suggesting that Rab11 has a role in the process of endoreplication, possibly indirectly through other pathways that regulate cell cycle progression. Ras signaling plays an important role in cell cycle progression through G/S phase transition. Ectopic expression of activated Ras in salivary glands of Rab11 down-regulated individuals rescues the small-sized glands to intermediate size. Furthermore, altered localization of Ras was observed in Rab11 down-regulated salivary glands. It is likely that the low level of endoreplication in the Rab11 down-regulated condition is Ras-mediated.
Colombie, N., Choesmel-Cadamuro, V., Series, J., Emery, G., Wang, X. and Ramel, D. (2017). Non-autonomous role of Cdc42 in cell-cell communication during collective migration. Dev Biol [Epub ahead of print]. PubMed ID: 28143705
Collective cell migration is involved in numerous processes both physiological, such as embryonic development, and pathological such as metastasis. Compared to single cell migration, collective motion requires cell behaviour coordination through an as-yet poorly understood but critical cell-cell communication mechanism. Using Drosophila border cell migration, this study shows that the small Rho GTPase Cdc42 regulates cell-cell communication. Indeed, Cdc42 controls protrusion formation in a cell non-autonomous manner. Moreover, the endocytic small GTPase Rab11 was found to control Cdc42 localisation to the periphery of migrating border cell clusters. Accordingly, over-expression of Cdc42 in border cells rescues the loss of Rab11 function. Thus, this study positions Cdc42 as a new key player in cell-cell communication, acting downstream of Rab11.
Fu, Y., Zhu, J. Y., Zhang, F., Richman, A., Zhao, Z. and Han, Z. (2017). Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes. Cell Tissue Res [Epub ahead of print]. PubMed ID: 28180992
The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. This study analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. These results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease.
Zhang, Y. V., Ormerod, K. G. and Littleton, J. T. (2017). Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity. eNeuro 4(2). PubMed ID: 28303263
Maintenance of neural circuit activity requires appropriate regulation of excitatory and inhibitory synaptic transmission. Recently, glia have emerged as key partners in the modulation of neuronal excitability; however, the mechanisms by which glia regulate neuronal signaling are still being elucidated. This study describes an analysis of how Ca2+ signals within Drosophila astrocyte-like glia regulate excitability in the nervous system. Drosophila astrocytes exhibit robust Ca2+ oscillatory activity manifested by fast, recurrent microdomain Ca2+ fluctuations within processes that infiltrate the synaptic neuropil. Unlike the enhanced neuronal activity and behavioral seizures that were previously observed during manipulations that trigger Ca2+ influx into Drosophila cortex glia, this study found that acute induction of astrocyte Ca2+ influx leads to a rapid onset of behavioral paralysis and a suppression of neuronal activity. It was observed that Ca2+ influx triggers rapid endocytosis of the GABA transporter (GAT) from astrocyte plasma membranes, suggesting that increased synaptic GABA levels contribute to the neuronal silencing and paralysis. Rab11 was identfied as a novel regulator of GAT trafficking that is required for this form of activity regulation. Suppression of Rab11 function strongly offsets the reduction of neuronal activity caused by acute astrocyte Ca2+ influx, likely by inhibiting GAT endocytosis. These data provide new insights into astrocyte Ca2+ signaling and indicate that distinct glial subtypes in the Drosophila brain can mediate opposing effects on neuronal excitability.
Choubey, P. K. and Roy, J. K. (2017). Rab11 is required for tubulogenesis of Malpighian tubules in Drosophila melanogaster. Genesis [Epub ahead of print]. PubMed ID: 28653473
Intracellular vesicular trafficking is one of the important tools in maintaining polarity, adhesion, and shape of epithelial cells. Rab11, a subfamily of the Ypt/Rab gene family of ubiquitously expressed GTPases and a molecular marker of recycling endosomes, transports different components of plasma membrane. This study reports that Rab11 affects tubulogenesis of Malpighian tubules (MTs). MTs are simple polarized epithelial tubular structures, considered as functional analogue of human Kidney. Rab11 has pleiotropic effects on MTs development as down-regulation of Rab11 in principal cells of MTs from embryonic stages of development, results in reduced endoreplication, clustering of cells, disorganized cytoskeleton and disruption of polarity leading to shortening of MTs in third instar larvae. Rab11 is also required for proper localization of different transporters in PCs, essential for physiological activity of MTs. Collectively these data suggest that Rab11 plays a key role in the process of tubulogenesis of MTs in Drosophila.
Del Signore, S. J., Biber, S. A., Lehmann, K. S., Heimler, S. R., Rosenfeld, B. H., Eskin, T. L., Sweeney, S. T. and Rodal, A. A. (2017). dOCRL maintains immune cell quiescence by regulating endosomal traffic. PLoS Genet 13(10): e1007052. PubMed ID: 29028801
Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. This study shows that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that identified in docrl mutant hemocytes, the cause of innate immune cell activation was pinpointed to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spatzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.
Kramer, R., Rode, S. and Rumpf, S. (2019). Rab11 is required for neurite pruning and developmental membrane protein degradation in Drosophila sensory neurons. Dev Biol. PubMed ID: 30871987
Neurons, with their distinct neurites, require elaborate membrane trafficking pathways and regulation to uphold neurite identity and to be able to respond to neuronal or developmental stimuli. In a survey of trafficking regulators required for developmental dendrite pruning in Drosophila sensory neurons, the small GTPase Rab11, a regulator of recycling endosomes, was identified. Dendrite pruning requires the developmentally regulated degradation of the cell adhesion molecule Neuroglian, and loss of Rab11 causes defects in the developmental degradation of Neuroglian and another target, the ion channel Ppk26. Rab11 often links vesicles to molecular motors, and this study finds that loss of the microtubule motor dynein also leads to defective Neuroglian and Ppk26 degradation. Loss of Rab11 also leads to defects in larval dendrite elaboration, and Neuroglian and Ppk26 localization is already altered at this stage. These data highlight the importance of membrane protein recycling during development.
Kramer, R., Rode, S. and Rumpf, S. (2019). Rab11 is required for neurite pruning and developmental membrane protein degradation in Drosophila sensory neurons. Dev Biol 451(1): 68-78. PubMed ID: 30871987
Neurons, with their distinct neurites, require elaborate membrane trafficking pathways and regulation to uphold neurite identity and to be able to respond to neuronal or developmental stimuli. In a survey of trafficking regulators required for developmental dendrite pruning in Drosophila sensory neurons, the small GTPase Rab11 was identified as a regulator of recycling endosomes. Dendrite pruning requires the developmentally regulated degradation of the cell adhesion molecule Neuroglian, and loss of Rab11 causes defects in the developmental degradation of Neuroglian and another target, the ion channel Ppk26. Rab11 often links vesicles to molecular motors, and this study finds that loss of the microtubule motor dynein also leads to defective Neuroglian and Ppk26 degradation. Loss of Rab11 also leads to defects in larval dendrite elaboration, and Neuroglian and Ppk26 localization is already altered at this stage. These data highlight the importance of membrane protein recycling during development.
Nie, Y., Yu, S., Li, Q., Nirala, N. K., Amcheslavsky, A., Edwards, Y. J. K., Shum, P. W., Jiang, Z., Wang, W., Zhang, B., Gao, N. and Ip, Y. T. (2019). Oncogenic pathways and loss of the Rab11 GTPase synergize to alter metabolism in Drosophila. Genetics. PubMed ID: 31213502
Colorectal cancer is a complex disease driven by well-established mutations such as APC and other yet to be identified pathways. The GTPase Rab11 regulates endosomal protein trafficking and previous work has shown that loss of Rab11 caused intestinal inflammation and hyperplasia in mice and flies. To test the idea that loss of Rab11 may promote cancer progression, archival human patient tissues were analyzed and 51 out of 70 colon cancer tissues had lower Rab11 protein staining. By using the Drosophila midgut model, this study has found that loss of Rab11 can lead to three changes that may relate to cancer progression. First is the disruption of enterocyte polarity based on staining of the FERM domain protein Coracle. Second is an increased proliferation due to an increased expression of the JAK-STAT pathway ligand Upd3. Third is an increased expression of ImpL2, which is an IGFBP7 homolog and can suppress metabolism. Furthermore, loss of Rab11 can act synergistically with the oncoprotein Ras(V12) to regulate these cancer related phenotypes.
Otsuka, Y., Satoh, T., Nakayama, N., Inaba, R., Yamashita, H. and Satoh, A. K. (2019). Parcas is the predominant Rab11GEF for rhodopsin transport in Drosophila photoreceptors. J Cell Sci. PubMed ID: 31296556
Rab11 is essential for polarized post-Golgi vesicle trafficking to photosensitive membrane rhabdomeres in Drosophila photoreceptors. This study found that Parcas (Pcs), recently shown to have guanine-nucleotide-exchange (GEF) activity toward Rab11, co-localizes with Rab11 on the trans-side of Golgi units and post-Golgi vesicles at the base of the rhabdomeres in pupal photoreceptors. Pcs fused with the EM-tag APEX2 localizes on 150-300 nm vesicles at the trans-side of Golgi units, which are presumably fly recycling endosomes. Loss of Pcs impairs Rab11 localization on the trans-side of Golgi units and induces the cytoplasmic accumulation of post-Golgi vesicles bearing rhabdomere proteins, as observed in Rab11-deficiency. In contrast, loss of the specific subunits of TRAPPII, another known Rab11-GEF, does not cause any defects on the eye development nor the transport of rhabdomere proteins, however, simultaneous loss of TRAPPII and Pcs shows severe defects on eye development. These results indicated that both TRAPPII and Pcs are required for eye development, but Pcs functions as the predominant Rab11-GEF for post-Golgi transport to photosensitive membrane rhabdomeres.
Martin-Pena, A. and Ferrus, A. (2019). CCB is involved in actin-based axonal transport of selected synaptic proteins. J Neurosci. PubMed ID: 31754011
Synapse formation, maturation, and turnover require a finely regulated transport system that delivers selected cargos to specific synapses. However, the supporting mechanisms of this process are not fully understood. The present study unravels a new molecular system for vesicle-based axonal transport of proteins in male and female flies (Drosophila melanogaster). This study identifies the gene CG14579 as the transcription unit corresponding to the regulatory mutations known as central complex broad, ccb. These mutations were previously isolated for their morphological phenotype in R-neurons of the ellipsoid body, a component of the central complex. Mutant axons from R-neurons fail to cross the midline, which is indicative of an aberrant composition of the growth cone. However, the molecular mechanism remained to be deciphered. This study shows that CCB is involved in axonal trafficking of FasII and Synaptobrevin, but not Syntaxin. These results suggest that axonal transport of certain proteins is required for the correct pathfinding of R-neurons. The molecular network supporting the CCB system was further investigated, and CCB was found to co-localize and co-immunoprecipitate with Rab11. Epistasis studies indicated that Rab11 is positioned downstream of CCB within this axonal transport system. Interestingly, ccb also interacts with Actin and the Actin nucleator Spire. The data revealed that this interaction plays a key role in the development of axonal connections within the ellipsoid body. It is proposed that the CCB/Rab11/SPIRE system regulates axonal trafficking of synaptic proteins required for proper connectivity and synaptic function.
Choubey, P. K., Nandy, N., Pandey, A. and Roy, J. K. (2020). Rab11 plays a key role in stellate cell differentiation via non-canonical Notch pathway in Malpighian tubules of Drosophila melanogaster. Dev Biol. PubMed ID: 31911183
This study reports a novel role of Rab11 in the differentiation of stellate cells via the non-canonical Notch pathway in Malpighian tubules. During Malpighian tubule development caudal visceral mesodermal cells intercalate into the epithelial tubule of ectodermal origin consisting of principal cells, undergo mesenchymal to epithelial transition and differentiate into star shaped stellate cells in adult Malpighian tubule. Two transcription factors, Teashirt and Cut (antagonistic to each other) are known to be expressed in stellate cells and principal cells, respectively, from early stages of development and serve as markers for these cells. Inhibition of Rab11 function or over-expression of activated Notch in stellate cells resulted in the expression of Cut that leads to down-regulation of Teashirt or vice-versa that leads to hampered differentiation of stellate cells. The stellate cells do not transform to star/bar shaped and remain in mesenchymal state in adult Malpighian tubule. Over-expression of Deltex, which plays important role in non-canonical Notch signaling pathway, shows similar phenotype of stellate cells as seen in individuals with down-regulated Rab11, while down-regulation of Deltex in genetic background of Rab11(RNAi) rescues Teashirt expression and shape of stellate cells. These experiments suggest that an inhibition or reduction of Rab11 function in stellate cells results in the faulty recycling of Notch receptors to plasma membrane as they accumulate in early and late endosomes, leading to Deltex mediated non-canonical Notch activation.
Schwartz, R., Guichard, A., Franc, N. C., Roy, S. and Bier, E. (2020). A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways. iScience 23(2): 100865. PubMed ID: 32058973
Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. This study created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border and disruption of intercellular junctions. Toxin-dependent re-distribution of Rab11 to enterocytes' apical surface occurs, and suppression was observed of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). This study also reports that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.

In developing Drosophila photoreceptors, rhodopsin is trafficked to the rhabdomere, a specialized domain within the apical membrane surface. Rab11, a small GTPase implicated in membrane traffic, immunolocalizes to the trans-Golgi network, cytoplasmic vesicles and tubules, and the base of rhabdomeres. One hour after release from the endoplasmic reticulum, rhodopsin colocalizes with Rab11 in vesicles at the base of the rhabdomere. When Rab11 activity is reduced by three different genetic procedures, rhabdomere morphogenesis is inhibited and rhodopsin-bearing vesicles proliferate within the cytosol. Rab11 activity is also essential for development of multivesicular body (MVB) endosomal compartments; this is probably a secondary consequence of impaired rhabdomere development. Furthermore, Rab11 is required for transport of TRP, another rhabdomeric protein, and for development of specialized membrane structures within Garland cells. These results establish a role for Rab11 in the post-Golgi transport of rhodopsin and of other proteins to the rhabdomeric membranes of photoreceptors, and in analogous transport processes in other cells (Satoh, 2005).

Rab11 also functions during oogenesis and during cellularization of Drosophila embryos. The Nuclear fallout and Rab11 function in membrane trafficking and actin remodeling during the initial stages of furrow formation during cellularization. Membrane addition is mediated via endosomal-mediated membrane delivery to the site of furrow formation. Thus Rab11 regulates endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization (Riggs, 2003; Pelissier, 2003). Rab11 is required in endocytic recycling and in the organization of posterior membrane compartments during oogenesis. Rab11 is also required in the organization of microtubule plus ends and osk mRNA localization and translation at the posterior pole (Jankovics, 2001). It is proposed that microtubule plus ends and, possibly, translation factors for osk mRNA are anchored to posterior membrane compartments that are defined by Rab11-mediated trafficking and reinforced by Rab11-Osk interactions (Dollar, 2002).

During photoreceptor terminal differentiation, massive biosynthetic membrane traffic delivers rhodopsin and other phototransduction proteins to an apical plasma membrane subdomain to form photosensory organelles, invertebrate rhabdomeres and vertebrate outer segments. The proper targeting of rhodopsin to this domain is crucial for normal development and, if impaired, leads to retinal degeneration. Rab proteins and their effectors are known to control membrane traffic and maintain distinct organelle identities (Deneka, 2003). Indeed, observations in Xenopus (Deretic, 1995; Moritz, 2001) and Drosophila (Satoh, 1997; Shetty, 1998) support a role for particular Rab proteins in rhodopsin transport to the photosensitive organelles (Satoh, 2005 and references therein).

Surprisingly, these studies on rhodopsin trafficking identified Rab11 as active in exocytosis. Rab11 is thought to regulate endosomal/plasma membrane interactions by controlling membrane traffic through recycling endosomes. These endosomes receive endocytosed plasma membrane and either return it to the cell surface or direct it to degradative pathways. Rab11 localizes to the pericentriolar recycling endosome, the trans-Golgi network (TGN), and post-Golgi vesicles (Chen, 1998; Deretic, 1997; Ullrich, 1996). Dominant-negative Rab11a inhibits apical recycling and basolateral-to-apical transcytosis in polarized MDCK cells (Wang, 2000), blocks stimulus-induced recruitment of endosome-sequestered H+-K+ ATPase-rich membrane to the apical membrane of acid-secreting parietal cells (Duman, 1999), and inhibits exosome release in human leukemic K562 cells (Savina, 2002; Satoh, 2005 and references therein).

In addition to these extensive reports linking Rab11 activity to endocytic pathways, other reports suggest a role for Rab11 in biosynthetic exocytic membrane traffic. In PC12 cells, Rab11 was detected in association with TGN and TGN-derived secretory vesicles (Urbe, 1993). In baby hamster kidney cells, overexpression of dominant-negative Rab11S25N decreased delivery of the basolaterally targeted vesicular stomatitis virus (VSV) G protein to the cell surface (Chen, 1998), and expression of wild-type Rab11a accelerated delivery of new protease activated receptors to kidney epithelial cell surfaces following trypsin exposure (Roosterman, 2003). Of particular interest to this study, the movement of rhodopsin to the apical surface may also be dependent on Rab11. Rhodopsin has been detected in Rab11-positive post-Golgi vesicles of Xenopus retina cell-free extracts (Deretic, 1997). Immature rhodopsin, which is indicative of defective rhodopsin transport, accumulated in Drosophila photoreceptors that expressed dominant-negative Rab11N124I (Satoh, 1998; Satoh, 2005 and references therein).

The movement of rhodopsin and other rhabdomeric membrane proteins has been characterized in the developing Drosophila photoreceptor. This experimental system allows the role of Rab11 in this process to be defined. Vigorous light-dependent endocytosis competes with exocytosis from the outset of rhabdomere morphogenesis. Independent of a requirement in endosomal recycling, Rab11 activity is essential for the initial exocytic rhodopsin delivery to the growing rhabdomere. Loss of Rab11 activity disrupts endocytic pathways, but this is likely to be a secondary consequence of attenuated exocytic delivery. Thus, these results demonstrate Rab11 promotes the trans-Golgi to rhabdomere membrane traffic responsible for elaboration of the sensory membranes of these cells (Satoh, 2005).

Drosophila rhabdomere differentiation begins in mid-pupal life with the establishment of a morphologically and molecularly distinct apical plasma membrane subdomain, which is then amplified and specialized for phototransduction by targeted membrane delivery (Karagiosis, 2004; Longley, 1995). Between ~50% and 60% of pupal development (% pd), nascent rhabdomeres begin to load with TRP, a light-activated Ca2+ channel that serves phototransduction. TRP is first immunodetectable in photoreceptor cytoplasm beginning at about 45% pd and accumulates in large cytoplasmic vesicles by 50% pd. Expression of the major rhodopsin (Rh1), the photosensory protein of photoreceptors R1-6, initiates later, at about 70% pd. Rh1 is first detected as faint diffuse signals and small puncta spread throughout the cytoplasm. In animals raised in standard 12/12 hour light/dark conditions, Rh1 concentrates in large (>200 nm) cytoplasmic vesicles, 'Rh1-containing large vesicles' (RLVs), prior to its appearance in the rhabdomere. During the stage when Rh1 and TRP synthesis overlap, the proteins colocalize in RLVs, suggesting the same vesicle accommodates both rhabdomere proteins. One or more small dots of F-actin decorate each RLV, resembling the actin patches associated with vesicles that mediate transport in yeast and other systems. In both fixed and living preparations, RLVs appear tethered via actin patches to the rhabdomere terminal web (RTW), a specialization of the cortical actin cytoskeleton (Satoh, 2005).

Immunogold electron microscopy using anti-Rh1 identified RLVs as multivesicular bodies (MVBs). The delicate detail of MVBs is poorly preserved by fixation protocols that retain Rh1 antigenicity. However, MVBs are the only organelle observed in the EM with the size, shape and distribution characteristic of RLVs observed in confocal immunofluorescence studies. RLVs label with GFP-tagged Rab7, an endosomal protein that marks the limiting membrane of MVBs in Drosophila. RLVs also immunostain for Hrs, the endosome-associated Hepatocyte growth factor-regulated tyrosine kinase substrate associated with MVBs in Drosophila and other systems. These observations identify RLVs as MVBs (Satoh, 2005).

The early detection of rhodopsin and TRP within MVBs was not anticipated because MVBs are generally considered to be late endosomal compartments, delivering cargo retrieved from the plasma membrane to the lysosomes for degradation. Thus, the presence of Rh1 in an endocytic degradative organelle during the time the cell is increasing its sensory membrane is noteworthy. However, light-dependent endocytosis of Rh1 is well documented in adult photoreceptors, and it is possible that the Rh1 found in RLVs has already been retrieved from the developing rhabdomere (Satoh, 2005).

To investigate this possibility, Rh1 transport was observed in dark-reared flies. Rh1 begins to accumulate in the rhabdomere just after Rh1 expression starts at 70% pd. There are few RLVs in all stages in dark-reared flies. RLVs form within 30 minutes of light exposure, and disappear within 13 hours of return to dark. These observations suggest that in light-reared flies, Rh1 is first transported to the rhabdomere, but light-induced internalization quickly transports Rh1 into RLVs. Thus, even during the developmental period in which the photoreceptor cell is increasing rhabdomeric volume and Rh1 content, vigorous endocytosis can exceed the rate of biosynthetic delivery of Rh1 (Satoh, 2005).

To position Rab11 in the sensory membrane transport pathway, flies expressing the Golgi marker, CFP-galactosyl-transferase and Rab11 were immunolocalized in developing photoreceptors. Rab11 is present on small vesicles (<200 nm in diameter) scattered throughout the cytoplasm. Many of these appear associated with Golgi structures; others are located at the base of developing rhabdomere. Rab11 is not associated with RLVs. To further characterize the Golgi association of Rab11, developing photoreceptors were immunostained for Rab11 and the cis-Golgi marker, Rab1. Rab1 localizes to the convex side of the Golgi, while Rab11 localizes to the opposite, concave, side. Triple staining of CFP-labeled Golgi directly shows Rab1 and Rab11 localize to opposite sides of the Golgi. Therefore, Rab11 must localize to the trans-Golgi surface (Satoh, 2005).

To investigate the possibility that Rab11 is associated with maturing Rh1, blue light was used to trigger synchronized release of Rh1 accumulated in the ER because of lack of the correct chromophore isomer. Rh1 and Rab11 were then immunolocalized in pupal eyes 0, 40, 60, 90 and 180 minutes after blue-light irradiation. Prior to blue light (0 minutes), Rh1 was distributed throughout photoreceptor cytoplasm, colocalizing with an ER marker. At 40 and 60 minutes, most Rh1 colocalized with the Golgi. By 40 minutes, some Rh1 was concentrated in Rab11 immunopositive vesicles, some of these at the rhabdomere base. At both 60 and 90 minutes, Rh1 and Rab11 show extensive colocalization in vesicles at the rhabdomere base. By 90 minutes, there are few Rh1 positive cytoplasmic vesicles, and by 180 minutes most Rh1 is found in the rhabdomeres (Satoh, 2005).

These immunofluorescence results suggest Rh1, upon exit from the ER, associates first with the Golgi, then within Rab11-positive vesicles, before being deposited in the rhabdomere. Thus, the Rab11 localization is consistent with a role in TGN--->rhabdomere transport. Transport visualized in these studies is completed by 180 minutes, in good agreement with previous immunoblot data showing intermediate Rh1 is completely processed into mature 35K Rh1 within 180 minutes (Satoh, 1997). RLVs are not prominent at any time during ER--->rhabdomere transport, consistent with the proposal that RLVs do not participate in biosynthetic traffic (Satoh, 2005).

Thus Rab11 is essential for trafficking two membrane proteins, TRP and Rh1, to the Drosophila photosensory organelle, the rhabdomere. When Rab11 activity is reduced in developing photoreceptors, Golgi-derived TRP- and Rh1-bearing vesicles accumulate in photoreceptor cytoplasm instead of exocytosing to expand the growing rhabdomere. Thus Rab11 activity supports a distinct plasma membrane compartment, the apical plasma membrane subdomain specialized for phototransduction (Satoh, 2005).

Rab11 has been implicated in control of membrane traffic through the pericentriolar recycling endosome. In cultured baby hamster kidney (BHK) cells, return of internalized transferrin receptor to the cell surface is inhibited by dominant-negative Rab11 expression (Ullrich, 1996). During cellularization of Drosophila embryos, apical membrane redeployed to the growing basolateral surface transits a Rab11-dependent recycling endosome (Pelissier, 2003). Rab11 has also been implicated in trans-Golgi to plasma membrane transport. In non-polarized BHK cells in culture, expression of dominant-negative Rab11S25N inhibits transport of a basolateral marker protein marker, vesicular stomatitis virus G protein, but has no impact on delivery of an apical marker protein, influenza hemagglutinin (Chen, 1998). Recent observation that recycling endosomes can serve as an intermediate during transport from the Golgi to MDCK cell plasma membranes raises the possibility that biosynthetic traffic transits a recycling endosome and the site of Rab11 action is at the recycling endosome. However, no pericentriolar endosome has been observed in Drosophila photoreceptors, and Rh1 moves directly from the trans-Golgi to the rhabdomere when released into the biosynthetic pathway by blue light. Thus, there is no evidence for Rh1 moving through an intermediate compartment when en route to the rhabdomere (Satoh, 2005).

Rh1-bearing post-Golgi vesicles and recycling endosome-derived vesicles may both traffick to the cell surface because they share common Rab11 effectors. Rab11 interacts with unconventional class V myosins and expression of dominant-negative Myo-Vb inhibits delivery from early endosomes to the cell surface (Lapierre, 2001). An extensive F-actin terminal web, the RTW, extends from the rhabdomere base into photoreceptor cell cytoplasm (Chang, 2000) and disruption of the photoreceptor actin cytoskeleton inhibits the vesicular traffic that builds crab rhabdomeres. Rab11, together with a Myo-V effector, may promote post-Golgi vesicle motility along the actin RTW to focus delivery to the rhabdomere (Satoh, 2005).

Loss of Rab11 activity also disrupts normal photoreceptor MVB morphology. MVBs are often identified as late endosomal compartments, delivering cargo destined for lysosomal degradation. However, several recent studies show MVBs can be also exocytic carriers, delivering endosomal contents to the cell surface. Examples include the secretory lysosomes of immune system cells, melanosomes of pigment cells, exosomes of maturing red blood cells and secreted vesicles mediating cell-cell signaling. The accumulation of newly synthesized MHCII receptors within MVBs of unstimulated dendritic cells, and the stimulus-induced reorganization of MVBs and appearance of MHCII receptors at the plasma membrane, has led to consideration of MVBs as an exocytic compartment. Autoradiography of crayfish eyes following 3H-leucine injection has shown newly synthesized protein first in the cytoplasm, then in MVBs and then in rhabdomere rhabdomeres, prompting the conjecture that MVBs are a post-Golgi organelle of biosynthetic traffic (Satoh, 2005).

The work reported in this study discounts the possibility that MVBs are exocytic vesicles in Drosophila photoreceptors. It was shown that appearance of Rh1 in the MVBs is dependent on light treatment. Previously, light was shown to trigger endocytosis of rhabdomeric membrane, so this light dependency suggests MVBs originate from an endocytic process. Depletion of Rab5 activity, which is known to regulate the fusion between endocytic vesicles and early endosomes, also eliminates Rh1 and TRP containing MVBs, without affecting Rh1 and TRP transport to the rhabdomere. Thus, all the results support the view that MVBs are endocytic vesicles. The early and rapid appearance of Rh1 and TRP in these vesicles is remarkable, showing that the machinery of light-dependent receptor internalization is fully operational at the outset of morphogenesis. Vigorous light-dependent endocytosis competes with exocytosis from the outset of rhabdomere morphogenesis, internalizing rhodopsin and TRP from the growing sensory membrane even as exocytosis expands it (Satoh, 2005).

Rab5 loss-of-function analysis also supports the view that Rab11 acts before Rab5. In Rab5, Rab11 double mutants, photoreceptors retain the Rab11 phenotype. These results are consistent with a role of Rab11 in the exocytic process, but not with an exclusive role in endocytic recycling. Yet, Rab11 activity is required for accumulation of MVBs. It is proposed that this is an indirect effect of the Rab11 requirement in the exocytic pathway. Rab11 inhibition 'starves' the rhabdomere, the target of Rab11-mediated transport, of required proteins, which in turn slows the rate of endocytosis and eliminates endocytosis-dependent MVBs. In support of this view, Rab11 activity was shown to be required for the presence of labyrinthine channels on Garland cells, membrane specializations that promote vigorous endocytosis. Rab11 loss plausibly depletes membrane components that sustain vigorous endocytosis (Satoh, 2005).

Drosophila and vertebrate photoreceptors share fundamental cellular and molecular mechanisms and Rab family members and their functions are strongly conserved across eukaryotes. Rab11 has been identified in rhodopsin-containing post-Golgi vesicles formed within a vertebrate retina cell-free system (Deretic, 1997), raising the likelihood vertebrate photoreceptors also contain a Rab11-dependent vesicular compartment essential for rhodopsin transport and outer segment development. Failure to traffic Rh1 in Drosophila leads to retinal degeneration, and similar mechanisms are implicated in rhodopsin mutations and other mutations causing the human disease retinitis pigmentosa. The involvement of Rab11 in the post-Golgi processes provides an entry point to discover the cellular components and pathways responsible for elaborating the specialized photosensitive membranes. These events are likely to be key regulators of normal cellular development and the triggering events of retinal disease (Satoh, 2005).

Rab11 is required for membrane trafficking and actomyosin ring constriction in meiotic cytokinesis of Drosophila males

Rab11 is a small GTPase that regulates several aspects of vesicular trafficking. This study shows that Rab11 accumulates at the cleavage furrow of Drosophila spermatocytes and that it is essential for cytokinesis. Mutant spermatocytes form regular actomyosin rings, but these rings fail to constrict to completion, leading to cytokinesis failures. rab11 spermatocytes also exhibit an abnormal accumulation of Golgi-derived vesicles at the telophase equator, suggesting a defect in membrane-vesicle fusion. These cytokinesis phenotypes are identical to those elicited by mutations in giotto (gio) and four wheel drive (fwd) that encode a phosphatidylinositol transfer protein and a phosphatidylinositol 4-kinase, respectively. Double mutant analysis and immunostaining for Gio and Rab11 indicated that gio, fwd, and rab11 function in the same cytokinetic pathway, with Gio and Fwd acting upstream of Rab11. It is proposed that Gio and Fwd mediate Rab11 recruitment at the cleavage furrow and that Rab11 facilitates targeted membrane delivery to the advancing furrow (Giansanti, 2007; full text of article).

The Gio PITP is enriched at the furrow membrane and that it is required for Drosophila cytokinesis (Giansanti, 2006). This study shows that the furrow membrane is also enriched in Rab11 and that Rab11 localization at the equatorial membrane requires the wild-type activity of both giotto (gio) and four wheel drive (fwd). In addition, the wild-type functions of gio, fwd, and rab11 are all required for membrane-vesicle fusion during cytokinesis, because mutations in these genes result in an abnormal accumulation of Golgi-derived vesicles at the equator of telophase cells (Giansanti, 2006). Finally, the results strongly suggest that gio, fwd, and rab11 function in the same cytokinesis pathway. These observations suggest a model for the mechanisms underlying membrane addition to the cleavage furrow during spermatocyte cytokinesis. It is proposed that Gio mediates transfer of PtdIns monomers to the furrow membrane, causing a local enrichment in PtdIns molecules. The association of Gio with this membrane domain may facilitate recruitment of the PtdIns-4-kinase encoded by fwd, which would mediate phosphorylation of PtdIns to PtdIns(4)P, allowing their further phosphorylation to PtdIns(4,5)P2. Fwd may also mediate Rab11 recruitment at the cleavage furrow, allowing targeted Rab11-dependent vesicle fusion events necessary for completion of cytokinesis. It is realized that this is a rather speculative model. Its major drawback is that the subcellular localization and the molecular interactions of the Drosophila Fwd protein are currently unknown. However, studies in S. pombe have shown that one of the PtdIns-4-kinases present in this organism interacts with Cdc4p, a contractile ring protein essential for cytokinesis. This finding indicates that, at least in fission yeast, one of the PtdIns-4-kinases is associated with the cleavage furrow. In addition, a recent study has shown that one of the mammalian PtdIns-4-kinases interacts physically with Rab11 and is required for Rab11 localization in the Golgi complex. The same study has also shown that recruitment of this kinase to the Golgi does not require Rab11 (de Graaf, 2004). These results are consistent with the current findings, and they lead to the belief that Gio, Fwd, and Rab11 are all enriched at cleavage furrow, where they work in concert to ensure proper vesicle docking and fusion (Giansanti, 2007).

Mutations in rab11 cause frequent failures in meiotic cytokinesis of males without affecting cytokinesis of larval brain neuroblasts. The mutations analyzed are obviously hypomorphic since they cause lethality at the larval and pupal stages, whereas rab11 null alleles result in embryonic lethality. Thus, it is possible that the rab11 mutants analyzed retain a residual Rab11 activity that is sufficient for neuroblast cytokinesis but not meiotic cytokinesis. Alternatively, Rab11 may not be required for mitotic cytokinesis. A strong support for a specific involvement of Rab11 in meiotic cytokinesis comes from recent RNAi screens that have shown that Rab11 has little or no role in S2 cell cytokinesis (Giansanti, 2007).

Previous studies have shown that null mutations in fwd and fws disrupt spermatocyte cytokinesis but that they have no observable effects on larval neuroblast mitosis (Brill, 2000; Farkas, 2003; Giansanti, 2004). Thus, at least three proteins involved in membrane traffic, Rab11, Cog5 (encoded by four way stop or fws), and a PtdIns-4-kinase, seem to be specifically required for meiotic cytokinesis. This specificity is unlikely to depend on the peculiar features of the final steps of spermatocyte cytokinesis. In male meiotic cells, the cytoplasmic bridges generated by ring constriction are not severed by a canonical abscission process, as occurs in larval neuroblasts; they instead persist and are stabilized by the formation of a specialized structure called ring canal. Mutations in rab11, fws and fwd inhibit ring constriction and furrow ingression during early telophase and block cytokinesis well before the formation of a cytoplasmic bridge. These observations rule out the possibility that the spermatocyte-specific effects of these mutations reflect problems in the final step of cytokinesis when ring canals are assembled (Giansanti, 2007).

The specific role of Rab11, Cog5, and Fwd in spermatocyte cytokinesis may reflect a specifically high requirement for formation of new membrane at the advancing cleavage furrow. To fulfill this requirement, male meiotic cells may exploit all the extant pathways for membrane addition. These pathways would be redundant in mitotic cell where the requirements for membrane expansion at the advancing furrow are relatively low. Alternatively, the specific requirement of membrane trafficking functions for spermatocyte cytokinesis may reflect the organization of membrane stores within these cells. Spermatocytes contain a large ER that includes astral and parafusorial membranes, and they do not possess a detectable pericentriolar RE. Larval neuroblasts do exhibit a spindle envelope, but, in contrast to spermatocytes, they are devoid of astral membranes and possess pericentriolar REs (Giansanti, 2006). Thus, formation of new membrane during spermatocyte cytokinesis might utilize membrane trafficking activities that are at least in part distinct from those used by mitotic cells, depending on the organization of membrane stores within the two cell types (Giansanti, 2007).

Whatever the reason for their specific sensitivity to mutations that disrupt membrane-related functions, Drosophila spermatocytes are emerging as an extremely useful model system for studying membrane traffic during animal cell cytokinesis. There is indeed growing evidence that the analysis of mutations that disrupt spermatocyte cytokinesis can reveal membrane-trafficking genes that play redundant cytokinetic roles in other animal cell systems (Giansanti, 2007).

Rab11 is required for epithelial cell viability, terminal differentiation, and suppression of tumor-like growth in the Drosophila egg chamber

The Drosophila egg chamber provides an excellent system in which to study the specification and differentiation of epithelial cell fates because all of the steps, starting with the division of the corresponding stem cells, called follicle stem cells, have been well described and occur many times over in a single ovary. This study investigated the role of the small Rab11 GTPase in follicle stem cells (FSCs) and in their differentiating daughters, which include main body epithelial cells, stalk cells and polar cells (see em>rab11-null FSCs give rise to at least two types of cells.... for an illustration of gene expression in ovarian development). This study shows that rab11-null FSCs maintain their ability to self renew, even though previous studies have shown that FSC self renewal is dependent on maintenance of E-cadherin-based intercellular junctions, which in many cell types, including Drosophila germline stem cells, requires Rab11. rab11-null FSCs give rise to normal numbers of cells that enter polar, stalk, and epithelial cell differentiation pathways, but none of the cells complete their differentiation programs, and the epithelial cells undergo premature programmed cell death. This study also showed, through the induction of rab11-null clones at later points in the differentiation program, that Rab11 suppresses tumor-like growth of epithelial cells. Thus, rab11-null epithelial cells arrest differentiation early, assume an aberrant cell morphology, delaminate from the epithelium, and invade the neighboring germline cyst. These phenotypes are associated with defects in E-cadherin localization and a general loss of cell polarity. While previous studies have revealed tumor suppressor or tumor suppressor-like activity for regulators of endocytosis, this study is the first to identify such activity for regulators of endocytic recycling. These studies also support the recently emerging view that distinct mechanisms regulate junction stability and plasticity in different tissues (Xu, 2011).

The invasive behavior of the rab11-null cells is distinct from that described for mutations in characterized Drosophila tumor suppressor genes (tsgs), which include the septate junction organizers, discs large, scribble, and lethal giant larvae, and two regulators of endocytosis, avalanche, and rab5. Thus while previously characterized tsg mutant cells invade surrounding tissues as large multi-layered sheets that remain attached to the epithelium, the rab11-null cells were often completely detached from the epithelium and were in groups containing as few as two cells or as many as 50 or more. In this regard, the invasive behavior of rab11-null cells more closely parallels the behavior of metastatic tumor cells of higher animals. Nevertheless, it is emphasized that there is no direct evidence that rab11-null cells actively migrate, and in fact the possibility cannot be ruled out that their 'invasion' of the germline cysts occurs in a passive fashion, e.g., by their inability to maintain adhesive contacts with neighboring wildtype epithelium cells and subsequent exclusion from the epithelium (Xu, 2011).

In contrast to bona fide tumor cells, the vast majority of rab11-null epithelial cells stopped dividing on schedule, i.e., at stage 6 of oogenesis. A few exceptional cells divided during s7, but none divided after that. It is noteworthy that all of the exceptional (late dividing) cells delaminated from basal side of the epithelium, thus perhaps precluding them from receiving Delta from the germline, which is known to promote a switch from a mitotic cell cycle to an endocycle at s7. The overwhelming majority of rab11-null cells delaminated from the apical side of the epithelium and presumably, then, received Delta, accounting for their mitotic arrest. Drosophila's previously characterized tsgs also have no or only subtle roles in suppressing follicle cell over-proliferation. Indeed, the evidence that these genes suppress over-proliferation stem entirely from analyses of larval tissues, most notably imaginal discs. Whether suppression of over-proliferation in larval tissues is fundamentally different, or simply easier to demonstrate, than suppression of over-proliferation in adult follicle epithelial cells is unclear. To date, it has not been possible to recover rab11-null clones in imaginal discs and other larval tissues, reflecting a unique role for Rab11 in the survival of such cells. In light of these data, it is proposed that Rab11 protein be considered as tumor suppressor-like protein (Xu, 2011).

Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. This study shows that Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. It is proposed that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport (Xiong, 2012).

This study shows that Crag is a novel GEF for Rab11 and that it is required for the post-Golgi transport of Rh1 to the rhabdomeres during light activation. This regulated transport of Rh1, which is independent of Rh1 transport during the development of the photoreceptors, replenishes the loss of Rh1 induced by light stimulation. Loss of Crag leads to accumulation of secretory vesicles in the cytosol of photoreceptor cells, and eventually leads to a light- and age-dependent photoreceptor degeneration (Xiong, 2012).

During development of photoreceptors, Rh1 and other phototransduction proteins are synthesized in the endoplasmic reticulum and transported to the rhabdomeres to build functional photoreceptors. Some molecular players, including Rab11 and exit protein of rhodopsin and TRP (XPORT), have been shown to play a role in this process. Upon light activation Rh1 is converted to metaRh. MetaRh is then converted back into Rh1 on rhabdomere membranes via absorption of another photon, allowing the maintenance of Rh1 levels in the rhabdomere. In wild-type photoreceptors, a portion of metaRh is phosphorylated and endocytosed, and it has been proposed that internalization of metaRh promotes the clearance of dysfunctional proteins and serves as a proofreading mechanism. Internalized Rh1 is then degraded through an endosomal/lysosomal pathway. Obviously, the gradual loss of Rh1 in wild-type photoreceptors leads to the necessity to constitutively synthesize Rh1 and replenish the rhabdomeric pool. This is nicely illustrated with the loss of retinol dehydrogenase (RDH), which is required for the regeneration of the chromophore of Rh1. Loss of RDH leads to progressive reduction in rhabdomere size and light-dependent photoreceptor degeneration (Xiong, 2012).

The data show that Crag is required to maintain homeostasis of Rh1 upon light stimulation. Loss of Crag leads to Rh1 accumulation in the cytosol and, eventually, retinal degeneration in the presence of light. Mutations in genes that affect metaRh1 turnover, such as Calmodulin and arrestin 2, lead to prolonged deactivation time of the photoresponse. Since both ERGs and single-cell recordings of Crag mutant photoreceptors are normal, it is unlikely that Crag is involved in the recycling of metaRh1 to Rh1. To test whether Crag is required for transport of newly synthesized Rh1 in adult photoreceptors, flies exposed to blue light to trigger massive endocytosis and degradation of Rh1, and then the new synthesis and transport of Rh1 back to the rhabdomeres was measured over time. Crag is not required for the synthesis of Rh1. However, in Crag mutants, the newly synthesized Rh1 accumulates in the cytosol. It is proposed that Crag is required for the delivery of newly synthesized Rh1 to the rhabdomeres and that loss of Crag leads to a gradual reduction in the size of rhabdomeres and to degeneration of the photoreceptor cells. Indeed, the time course and morphological features of degeneration associated with loss of Crag are very similar to the phenotypes observed in RDH mutants, further supporting that Crag is involved in the Rh1 synthesis/delivery pathway (Xiong, 2012).

Rab11 has been implicated in various intracellular membrane trafficking processes. Its diverse functions in different membrane compartments are mediated through its downstream effectors in a context-specific manner; many of these functions have been identified in previous studies. However, GEFs for Rab11 in any context have not yet been identified. In vivo and in vitro data provide compelling evidence that Crag is a GEF for Rab11. First, in Drosophila S2 cells, Crag colocalizes and physically interacts with Rab11. Second, Crag preferably binds to the GDP-bound form of Rab11, and the DENN domains are required for binding. Third, Crag is required for the proper localization of Rab11 in photoreceptors upon light stimulation. Fourth, loss of Crag or Rab11 leads to a similar light-induced photoreceptor degeneration. Fifth, expression of Rab11-CA partially rescues the degeneration caused by Crag mutations. Finally, an in vitro GEF assay shows that Crag facilitates the release of GDP from Rab11. It has been previously established that Rab11 is essential for photoreceptor cell development and Rh1 transport during pupal stages. However both rhabdomere morphology and Rh1 localization are normal in Crag clones in newly eclosed flies. Similarly, initial deposition of TRP is also not affected by Crag mutations, in agreement with previous findings that Rh1 and TRP are co-transported to the rhabdomeres during their development. Interestingly, cytosolic localization of TRP is not observed in Crag mutant photoreceptor cells exposed to light, suggesting that during light stimulation, Rh1 and TRP dynamics are distinct. Indeed, internalization of TRP upon light stimulation has not been reported in previous studies. The current data therefore indicate that other GEFs must exist for Rab11 during photoreceptor development, and that Crag is specifically required for Rab11 GDP/GTP exchange during light activation in adult flies. In addition, Crag may function as a GEF for Rab10 in other processes and cells, such as polarized deposition of basement membrane proteins in follicle cells (Xiong, 2012).

The biochemical assay shows that the kinetics of Crag GEF activity is slow when compared to the GEF activity of other DENN-domain-containing proteins such as the Rab35 GEF. Crag exhibits GEF activity against Rab10 with much faster kinetics than against Rab11, indicating that the slow kinetics may be due to properties of Rab11. This is further supported by the slow kinetics of EDTA that triggers GDP release of Rab11. It's possible that the GDP/GTP exchange of Rab11 requires other co-factors besides its GEF, as, for example, documented for Rab6 (Xiong, 2012).

CaM is a ubiquitously expressed calcium sensor. In the Drosophila photoreceptor cells, photoactivation leads to influx of Ca2+ and activation of CaM. It has been shown that CaM is required for the termination of the photoresponse in several steps, including TRP inactivation and conformational change of metaRh. Crag contains a CaM binding site and interacts with CaM in a calcium-dependent manner. In an in vitro GEF assay, the presence of CaM and Ca2+ indeed enhances the GEF activity of Crag. Hence, it is possible that a light-induced increase of intracellular Ca2+ level enhances Crag activity via CaM binding. The activation of Crag/Rab11 then may serve to replenish rhabdomeric Rh1, whose loss is also induced by light stimulation (Xiong, 2012).

In vertebrate rod cells, polarized transport of Rh is mediated by post-Golgi vesicles that bud from the TGN and fuse with the base of the outer segment. Rab11 has been detected on rhodopsin-bearing post-Golgi vesicles in photoreceptors; however, it has not yet been shown that Rab11 is required for Rh trafficking. DENND4 proteins are highly similar to Crag. This study showed that expression of the UAS–human DENND4A construct not only rescues the lethality but also rescues the light-induced photoreceptor degeneration caused by loss of Crag, showing that the molecular function of DENND4A is also conserved. Moreover, three different subtypes of Usher syndrome, an inherited condition characterized by hearing loss and progressive vision loss, have been mapped to the vicinity of the DENND4A locus at 15q22.31. Hence, DENND4A may also function through Rab11 in human photoreceptors, and loss of DENND4A may lead to photoreceptor degeneration (Xiong, 2012).

The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases

Originally identified in yeast, transport protein particle (TRAPP) complexes are Rab GTPase exchange factors that share a core set of subunits. TRAPPs were initially found to act on Ypt1, the yeast orthologue of Rab1, but recent studies have found that yeast TRAPPII can also activate the Rab11 orthologues Ypt31/32. Mammals have two TRAPP complexes, but their role is less clear, and they contain subunits that are not found in the yeast complexes but are essential for cell growth. To investigate TRAPP function in metazoans, this study shows that Drosophila melanogaster have two TRAPP complexes similar to those in mammals and that both activate Rab1, whereas one, TRAPPII, also activates Rab11. TRAPPII is not essential but becomes so in the absence of the gene parcas that encodes the Drosophila orthologue of the SH3BP5 family of Rab11 guanine nucleotide exchange factors (GEFs). Thus, in metazoans, Rab1 activation requires TRAPP subunits not found in yeast, and Rab11 activation is shared by TRAPPII and an unrelated GEF that is metazoan specific (Riedel, 2017).

Rab GTPases control many aspects of subcellular organization. They are typically active on only one particular organelle or vesicle class and so direct the subcellular localization of a wide range of proteins, including membrane traffic machinery, molecular motors, and regulators of phosphoinositide levels or the activity of other GTPases. This role in spatial organization of the cell requires specific guanine nucleotide exchange factors (GEFs) to activate each Rab in only the correct location. GEFs for several Rabs have been identified, and among the best studied are the transport protein particle (TRAPP) complexes. The first TRAPP subunit was identified in yeast in a screen for mutations that interact with a mutation in a SNARE protein, and the corresponding protein was found to be part of a large protein complex that was termed TRAPP. Subsequent work reported the existence of three different TRAPP complexes in yeast. All three share a heptameric core of six proteins (Bet3 being present twice), with TRAPPI having no further subunits, TRAPPII having four additional subunits called Tca17, Trs65, Trs120, and Trs130, and TRAPPIII having one additional subunit, Trs85. The shared TRAPP subunits are essential for membrane traffic through the Golgi apparatus, and consistent with this, TRAPPI was found to act as a GEF for Ypt1 (yeast Rab1), a GTPase essential for ER to Golgi and intra-Golgi traffic. TRAPPIII was initially reported to have a more specific role in activating Ypt1 during autophagy, but recent work suggests that TRAPPI may not exist in vivo and that TRAPPIII is responsible for the majority of Rab1 exchange activity in both secretion and autophagy. In contrast, TRAPPII was proposed to act later in the Golgi as a GEF for the closely related GTPases Ypt31 and Ypt32, yeast orthologues of Rab11. This conclusion was initially questioned, but recent biochemical studies have shown both Rab1 and Rab11 GEF activity for TRAPPII from filamentous fungi and budding yeasts (Riedel, 2017 and references therein).

The shared core TRAPP subunits that are sufficient to act on Ypt1/Rab1 are very highly conserved in evolution and appear to be a universal feature of eukaryotic cells. Mammals have orthologues of all of the yeast TRAPP subunits, including those specific to TRAPPII and TRAPPIII. In addition, coprecipitation experiments have identified two further TRAPP subunits that are not present in yeast. Examination of the proteins associated with each mammalian TRAPP subunit revealed that they form two complexes related to yeast TRAPPII and TRAPPIII, with there being no evidence that mammals have a complex equivalent to TRAPPI, i.e., just the core subunits. Mammalian TRAPPII contains seven core subunits and orthologues of Trs120 (TRAPPC9) and Trs130 (TRAPPC10). Mammalian TRAPPIII contains the same seven core subunits and an orthologue of Trs85 (TRAPPC8) plus three further subunits: TRAPPC13 (an orthologue of yeast Trs65) and the two subunits not found in yeast (TRAPPC11 and TRAPPC12) (Riedel, 2017).

The precise roles of TRAPPII and TRAPPIII in mammals are not fully resolved. When assembled in vitro, the core subunits of the mammalian TRAPP complexes have exchange activity on Rab1, and mammalian TRAPPII has been reported to have the same activity when immunoisolated from cells but to have no activity on Rab11. Moreover, there are also some striking differences to the yeast system. The most obvious is the existence of the two additional subunits in TRAPPIII, TRAPPC11 and TRAPPC12, and these seem unlikely to have minor roles as at least TRAPPC11 is essential for secretion and cell viability. In contrast, the TRAPPII subunits Trs120 and Trs130 are both essential for growth in yeast, and yet in mammals they do not appear to be required for cell viability even though Rab11 is an essential protein. Indeed, loss-of-function mutations in human TRAPPC9 are not lethal but cause mental retardation (Riedel, 2017).

The TRAPP complex subunits found in humans are well conserved across metazoans, and so this study used the tractable genetic system of Drosophila melanogaster to investigate TRAPP in metazoans. Drosophila was found to contain two TRAPP complexes that have the same composition as the human complexes, and this study combined genetics and expression of recombinant TRAPP complexes to investigate their function in vivo and their activity in vitro (Riedel, 2017).

Although recent work has shown that humans and other metazoans have two TRAPP complexes, these are not identical to their yeast counterparts. This study has provided functional evidence from Drosophila to show what is shared between metazoans and yeast and also to resolve some of the apparent paradoxes that have emerged from comparing the yeast and mammalian systems. As in yeast, both of the Drosophila TRAPP complexes have GEF activity on Rab1, whereas TRAPPII also acts as a GEF on Rab11. In the only previous study in which mammalian TRAPP complexes were tested on Rab11, TRAPPCII was immunoisolated from cells using antiserum to TRAPPC9, and activity was found on Rab1 but not Rab11. However, the subunit composition of the isolated complex was not determined, and it is also possible that the antibody or attached beads inhibited access to the Rab11 substrate. A recent study has suggested that mammalian TRAPPII can act on both Rab1 and Rab18 (Rab11 was not tested), although for reasons that are not clear, this study was unable to detect this Rab18 activity within the Drosophila complex (Riedel, 2017).

Beyond these shared properties, there are some clear differences between the yeast and metazoan TRAPP complexes. First, TRAPPIII contains two additional subunits, TRAPPC11 and TRAPPC12, that are absent from yeast. These two subunits are unlikely to have a metazoan-specific role as they are very widely conserved throughout eukaryotic phyla, including even filamentous fungi, and instead appear to have been lost in the budding yeast lineage. TRAPPC11 is distantly related to TRAPPC10, and so it seems likely that in early eukaryotic evolution, there was an ur-TRAPP complex that had two additional subunits, and then both duplicated to make on the one hand TRAPPC8 and TRAPPC9 and on the other TRAPPC10 and TRAPPC11, and hence two TRAPP complexes. TRAPPC11 is essential for the growth of both Drosophila and mammalian cultured cells, and therefore yeast must have found a means to bypass this requirement. Given that the core of shared subunits is sufficient for Rab1 GEF activity, the role of TRAPPC11 seems most likely to be to direct the TRAPPIII Rab1 GEF activity to the correct location. It is possible that yeast have evolved an alternative mechanism to direct TRAPPIII to Golgi membranes via the core subunits. However, it is also possible that TRAPPC11 allows the complex to have an additional activity that budding yeast no longer requires. The finding that in some tissues, TRAPPIII is found on the trans-Golgi as well as the cis-Golgi provides some support for this possibility as Rab1 is widely believed to function only on the cis-Golgi, and consistent with this, this study could only detect YFP-Rab1 on the cis-Golgi in these tissues. In contrast, Drosophila Rab11 is known to be present on the TGN as well as on recycling endosomes (Riedel, 2017).

This work has focused on TRAPPC11 as the TRAPPIII subunit that is essential but absent from yeast. The TRAPPIII subunit that is shared with yeast, TRAPPC8, has been reported to be essential in Drosophila and in mammalian cultured cells, although its yeast orthologue Trs85 is not essential, perhaps for the same reasons suggested in the previous pargaraph as to how yeast survive without TRAPPC11. Metazoans have two further TRAPPIII subunits: TRAPPC13, a distant orthologue of the yeast protein Trs65 that has been suggested to contribute to TRAPP complex structure, and TRAPPC12, a protein that is absent from budding yeasts but present in a diverse range of eukaryotic phyla, suggesting it had been lost during budding yeast evolution. The role of TRAPPC12 is unclear, and it is unrelated to other TRAPP subunits and has even been suggested to have additional roles outside of the TRAPP complex. TRAPPC12 has also been reported to bind to the Sec13/31 component of the COPII coat. However, although this study found this interaction when tagged TRAPPC12 was overexpressed, Sec13/31 was not recovered when TRAPPIII was precipitated with other subunits. This suggests that if this interaction is physiological, then it may represent a distinct function of TRAPPC12 or possibly an intermediate in the assembly of TRAPPIII (Riedel, 2017).

A second striking difference between the yeast and metazoan TRAPP complexes is that although TRAPPII can act on Rab11 in both classes of organism, in metazoans, it shares the role of activating Rab11 with the unrelated SH3BP5 protein family. There is clearly considerable redundancy between the two, as mutations in the SH3BP5 orthologues in Drosophila and C. elegans are viable as are mutations in TRAPPII in Drosophila and mammalian cultured cells. Loss of one or the other Rab11 GEF does have consequences, with loss of TRAPPII from flies causing male infertility and loss of Parcas causing defects in several developmental processes, some of which have been linked to signaling by nonreceptor tyrosine kinases. In C. elegans, loss of the two SH3BP5 orthologues causes defects in embryonic cytokinesis. Thus, it seems likely that each of the two types of Rab11 GEF can provide much of the necessary activation of Rab11, but each also has unique specialized roles in particular cell types. Revealing this overlap in function between the two proteins should greatly assist dissecting the function of each class of GEF. It also seems likely that TRAPPII can contribute to the activation of Rab1 in vivo as both the yeast and Drosophila complexes have activity on Rab1, at least in vitro. This could provide a possible explanation for the observation that at least in S2 cells, the major pool of TRAPPII is found on the cis-Golgi. Indeed, a study of mammalian TRAPPC10 found an epitope-tagged version of the protein to be present on the early Golgi. However, it is clear that the ability of TRAPPII to activate both Rab1 and Rab11 raises a conundrum because there is a growing consensus that Rab GEFs define the location of active Rabs within the cell. However, in this case, the two Rabs are widely believed to act on different compartments and recruit very different effector. It may be that in this case, there are additional mechanisms to restrict the recruitment of the two Rabs to a particular location before activation by TRAPP, such as a GDI displacement factor. Thus, it seems likely that further investigation of the action of the TRAPP complexes will reveal new fundamental principles of how membrane traffic is organized in cells, and this study will hopefully facilitate the pursuit of such studies in metazoan systems (Riedel, 2017).

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

Various bacterial toxins circumvent host defenses through overproduction of cAMP. A previous study has shown that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. This study provides further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies (Guichard, 2017).

Previous studies established that two cAMP toxins, EF from Bacillus anthracis and Ctx from Vibrio cholerae, block Rab11-mediated endocytic recycling of cargo such as signaling ligands and adhesion proteins, ultimately leading to inhibition of Notch signaling and loss of barrier integrity. However, the precise mechanisms by which cAMP overproduction interfered with Rab11-dependent trafficking remained to be explored. This study has examined how cAMP effector pathways converge on discrete nodes of the trafficking process subsequent to the GTP loading step to efficiently interrupt endocytic recycling (Guichard, 2017).

As is typical of small GTPases, Rab11 cycles between active (GTP-bound) and inactive (GDP-bound) conformations, the former permitting interaction with effector proteins to carry out downstream functions. Two types of regulators, activating GEFs and inactivating GAPs provide control for this essential cycle. In the particular case of Rab11, Crag (the Drosophila homolog of human DENND4A) is the only known Rab11-dedicated GEF. Similarly, only one Rab11-specific GAP has been identified: EVI5. Neither of these regulators contains an identified cAMP-binding domain that could provide a direct link between cAMP and upstream regulation of Rab11. Consistent with this inference, it was found that EF acted on Rab11 at a step subsequent to GTP loading. Indeed, transport of vesicles carrying the constitutively activated mutant Rab11*YFP were blocked by EF, while total endogenous levels of Rab11-GTP did not appear to be greatly altered (Guichard, 2017).

Association between Rab11 and its effectors Rip11 and Sec15 was abrogated by EF in several settings, including Drosophila salivary glands and human cells. The Rab11 effector Rip11 is an attractive candidate for mediating some of EF effects, as it contains a verified PKA phosphorylation site located in the central portion of the protein. Indeed, PKA-dependent phosphorylation of Rip11 is required for cAMP-potentiated insulin secretion in pancreatic β-cells. In addition, Ser/Thr phosphorylation is responsible for Rip11 transition from the insoluble to cytosolic fraction in intestinal CACO-2 cells. Although it was not determined whether the latter modification was specifically PKA-dependent, this study proposed a model in which phosphorylation of Rip11 is essential for cycling to a free state following interaction with Rab11 and specific membrane compartments prior to its re-associating with Rab11. The data show that the association between Rab11 and Rip11 can be disrupted by EF in Drosophila and mammalian endothelial or embryonic kidney cells. It is possible that unrelenting phosphorylation of Rip11 by PKA may cause the premature dissociation of Rab11 and its effectors, potentially leading to a failure to reach the AJs. While this PKA-dependent phosphorylation of Rip11 has been demonstrated in human pancreatic cells, it is not known whether it occurs in Drosophila. As dRip11 contains 19 candidate PKA phosphorylation sites, further investigation will be necessary to determine whether phosphorylation of one or more of these sites occurs and promotes the dissociation between dRip11 and Rab11. Intriguingly, Drosophila Sec15 also harbors several putative PKA phosphorylation sites, although such predicted sites are missing in its human counterpart. Importantly, this study found that artificial stimulation of Rap1 also causes a loss in Rab11*/Rip11 co-localization resulting in correlated but separated staining foci of these two proteins, suggesting that the later acting Epac/Rap1 pathway may feedback on this process (Guichard, 2017).

The second branch of the cAMP pathway mediated by the cAMP-regulated GEF Epac and its partner Rap1 contributes significantly to the effect of EF in flies, and surprisingly appears to play the predominant role in mammalian systems examined in this study. In flies, activated Rap1 (Rap1*) causes a wing phenotype more similar to that of EF and Rab11DN than that of PKA*. It has been reported that Rap1* reduces the levels of Rab11 and prevents formation of Sec15 punctae. The present study found that blocking expression of Epac significantly reduces the intensity of the EF phenotype. In addition, Rap1* alters the distribution of Rab11* and inhibits Rab11*/Rip11 co-localization. It is hypothesized that the final exocyst- and SNARE-dependent fusion event with the apical plasma membrane is subjected to inhibition by exuberant Rap1* activity, leading to accumulation of non-functional Rab11* just beneath the plasma membrane. Consistent with this hypothesis, Rap1 has been implicated by many studies in regulating of both cadherin and integrin-mediated cell-cell adhesion. Further indicating a functional connection between Rap1 signaling and Rab11-dependent trafficking, Rap1 and Rab11 over-expressed in human cells co-localize in a recent study. Additional experiments will be required to elucidate the molecular interactions connecting the activities of these two GTPases. The small GTPase RalA is a possible candidate for mediating the activity of Rap1, through activation of the Rap1 effector Rgl1, a positive regulator (GEF) of RalA. Because lowering the dose of Rgl1, or expressing a dominant-negative form of RalA, can suppress Rap1*-induced phenotypes in Drosophila, it has been proposed that RalA may act downstream of Rap1. Also, RalA is known to directly bind to exocyst components Sec5 and Exo84 and plays a central role in regulating exocyst-mediated processes in several settings, including the release of Von-Willebrand Factor from endothelial cells, or insulin secretion in pancreatic β-cells. In addition, a recent study identified Arf6 as a key component acting downstream of RalA, mediating its effect on exocyst-dependent delivery of raft micro-domains to the plasma membrane. Thus, RalA over-activationmay contribute to mediating the effect of cAMP toxins on exocyst inhibition downstream of Rap1, although this hypothesis needs to be tested in future experiments (Guichard, 2017).

Previous work showed that EF caused a drastic reduction in total Rab11 levels in wing epithelial cells. This study found that this effect is also evident in HBMECs treated with ET, but is dependent on cell context, since inhibition of Rab11 function can be uncoupled from reduction in total Rab11 levels in Drosophila salivary glands. This reduction in Rab11 levels is unlikely to derive from transcriptional inhibition, as infection of HBMECs with Bacillis a Sterne did not result in any change in levels of Rab11 transcripts. Similarly, in Drosophila wings, where EF also triggers great reduction in Rab11 protein levels, mRNA transcript levels again were not greatly affected (Valentino Gantz, personal communication to Guichard, 2017). In HBMECs, where Rab11 levels are reduced by ET treatment, it was observed that total levels of cadherins were also severely reduced in ET-treated cells. Although the precise mechanism responsible for the loss of these proteins following ET treatment remains to be explored, it is worth noting that degradation of VE-cadherins has been observed following silencing of Rab11 in human endothelial cells, in which Rab11 is important for stabilizing cadherins at the AJs. Thus, it is possible that following EF intoxication, Rab11 and cadherins are routed to the lysosomal pathway and degraded, further impairing endocytic recycling and junctional integrity. Such an attractive hypothesis could explain the catastrophic loss of cadherins observed in ET-treated cells (Guichard, 2017).

Numerous studies have demonstrated the positive role of physiological induction of cAMP in junction establishment and stabilization, through stimulation of both PKA and Epac. It may therefore seem counterintuitive that cAMP produced by EF or other toxins may exert an opposing effect and jeopardize junctional integrity. In principle, high versus low concentrations, sustained versus transient production, and perinuclear vs cortical subcellular distribution of toxin-delivered cAMP could elicit such opposite outcomes. In the particular case of Rab11-dependent trafficking, low physiological levels of cAMP may exert their positive effects by promoting the release of Rip11 from Rab11, as necessary to allow the final fusion event between recycling endosomes and the plasma membrane. In contrast, pathologically elevated cAMP concentrations may cause premature dissociation of the Rab11-Rip11 complex and permanently block that cycle. Similarly, uncontrolled stimulation of Rap1 by Epac could also have a negative impact on junctional transport: titration of critical partners, failure to return to complete the necessary GTP/GDP cycle, or negative feedback interference with other important steps, could explain the occurrence of this apparent paradox. Another molecule potentially at play during the response to cAMP is the small GTPase RhoA. RhoA can be phosphorylated by PKA, which inhibits its activation and prevents increased endothelial permeability during inflammation, the potential interplay between RhoA and the exocyst downstream of cAMP signaling in EF-intoxicated cells also merits further examination (Guichard, 2017).

The small GTPase Arf6 initiates retrieval of membrane proteins from cell junctions in a wide variety of cells types. Arf6, a member of the ADP-ribosylation factor subfamily, is located at the plasma membrane and some endosomal compartments, and is involved in endocytosis from the plasma membrane, vesicular recycling, and exocytosis. Importantly, Arf6 plays a role during sepsis to mediate acute VEGF-induced vascular permeability. Whether linchpin regulators of opposing vesicular trafficking pathways such as Arf6 and Rab11 interact had not yet been extensively explored. This study presents evidence that these trafficking systems do in fact engage in cross-inhibitory interactions. Consistent with the published role of Arf6 in promoting VE-cadherin endocytosis, the activated form of Arf6 (Arf6*) caused phenotypes similar to those of EF. These findings suggest that the activity of Arf6 negatively feeds back on vesicular transport to the plasma membrane by inhibiting Rab11 function. Previous studies showed that Arf6 physically interacts with the exocyst component Sec10, defining a possible avenue for the observed effects of Arf6 on Rab11 levels and distribution. Given the negative regulation of Rab11 by Arf6 in flies and its known role in compromising barrier function in the mammalian vasculature during sepsis, this study tested whether inhibitors of this pathway might antagonize the effects of EF. In human endothelial cells, it was indeed found that treatment with Slit2, a secreted peptide indirectly blocking Arf6 function, could reverse the effects of EF, restoring junctional integrity. Similarly, pharmacological inhibition of Arf6 by SecinH3, a compound that inhibits the ArfGEF ARNO, potently blocked EF-induced edema in a mouse footpad assay (Guichard, 2017).

An emerging lesson from the current and prior studies is that blocking multiple steps of branching pathways that converge on critical nodes in endocytic recycling may allow pathogens to weaken host protective mechanisms that rely on junctional integrity. For example, LF, the other toxic factor secreted by B.a, blocked exocyst-mediated vesicular docking downstream of Rab11 via inhibition of MAPK signaling. It will be interesting to explore how the various effects of EF and LF are integrated to achieve an efficient inhibition of junctional delivery, and if any compound identified in this study can also block some of the downstream effects of LF. Altogether, this study suggests that a broad range of barrier disruptive diseases ranging from cAMP related toxemia to inflammatory autoimmune diseases that involve positive feedback loops between immune activation and barrier disruption, could potentially be treated with compounds that inhibit Arf6 or Epac/Rap1, or by yet undiscovered compounds that may boost Rab11 activity (Guichard, 2017).

A postsynaptic PI3K-cII dependent signaling controller for presynaptic homeostatic plasticity

Presynaptic homeostatic plasticity stabilizes information transfer at synaptic connections in organisms ranging from insect to human. By analogy with principles of engineering and control theory, the molecular implementation of PHP is thought to require postsynaptic signaling modules that encode homeostatic sensors, a set point, and a controller that regulates transsynaptic negative feedback. The molecular basis for these postsynaptic, homeostatic signaling elements remains unknown. In this study, an electrophysiology-based screen of the Drosophila kinome and phosphatome defines a postsynaptic signaling platform that includes a required function for PI3K-cII, PI3K-cIII and the small GTPase Rab11 during the rapid and sustained expression of PHP. Evidence is presented that PI3K-cII localizes to Golgi-derived, clathrin-positive vesicles and is necessary to generate an endosomal pool of PI(3)P that recruits Rab11 to recycling endosomal membranes. A morphologically distinct subdivision of this platform concentrates postsynaptically where it is proposed to functions as a homeostatic controller for retrograde, trans-synaptic signaling (Hauswirth, 2018).

Homeostatic signaling systems stabilize the functional properties of individual neurons and neural circuits through life. Despite widespread documentation of neuronal homeostatic signaling, many fundamental questions remain unanswered. For example, given the potent action of homeostatic signaling systems, how can neural circuitry be modified during neural development, learning, and memory? Although seemingly contradictory, the homeostatic signaling systems that stabilize neural function throughout life may actually enable learning-related plasticity by creating a stable, predictable background upon which learning-related plasticity is layered. Therefore, defining the underlying molecular mechanisms of homeostatic plasticity may not only be informative about the mechanisms of neurological disease, these advances may be informative regarding how complex neural circuitry is able to accomplish an incredible diversity of behaviorally relevant tasks and, yet, retain the capacity for life-long, learning-related plasticity (Hauswirth, 2018).

Neuronal homeostatic plasticity encompasses a range of compensatory signaling that can be sub-categorized based upon the cellular processes that are controlled, including ion channel gene expression, neuronal firing rate, postsynaptic neurotransmitter receptor abundance and presynaptic vesicle release. Presynaptic homeostatic potentiation (PHP) is an evolutionarily conserved form of neuronal homeostatic control that is expressed at the insect, rodent and human neuromuscular junctions (NMJ) and has been documented at mammalian central synapses. PHP is initiated by the pharmacological inhibition of postsynaptic neurotransmitter receptors. The homeostatic enhancement of presynaptic vesicle release can be detected in a time frame of seconds to minutes, at both the insect and mouse NMJ. This implies the existence of postsynaptic signaling systems that can rapidly detect the disruption of neurotransmitter receptor function and convert this into retrograde, trans-synaptic signals that accurately adjust presynaptic neurotransmitter release. Notably, the rapid induction of PHP is transcription and translation independent, and does not include a change in nerve terminal growth or active zone number (Hauswirth, 2018).

There has been considerable progress identifying presynaptic effector molecules responsible for the expression of PHP. There has also been progress identifying postsynaptic signaling molecules that control synaptic growth at the Drosophila NMJ as well as the long-term, translation-dependent maintenance of PHP. However, to date, nothing is known about the postsynaptic signaling systems that initiate and control the rapid induction and expression of PHP (Hauswirth, 2018).

This paper reports the completion of an unbiased, forward genetic screen of the Drosophila kinome and phosphatome, and the identification of a postsynaptic signaling system for the rapid expression of PHP that is based on the activity of postsynaptic Phosphoinoside-3-Kinase (PI3K) signaling. There are three classes of PI3-Kinases, all of which phosphorylate the 3 position of phosphatidylinositol (PtdsIns). Class I PI3K catalyzes the conversion of PI(4,5)P2 to PI(3,4,5)P3 (PIP3) at the plasma membrane, enabling Akt-dependent control of cell growth and proliferation, and participating in the mechanisms of long-term potentiation. Class II and III PI3Ks (PI3K-cII and PI3K-cIII, respectively) both catalyze the conversion of PI to PI(3)P, which is a major constituent of endosomal membranes. PI(3)P itself may be a signaling molecule with switch like properties, functioning in the endosomal system as a signaling integrator. The majority of PI(3)P is synthesized by PI3K-cIII, which is involved in diverse cellular processes. By contrast, the cellular functions of PI3K-cII remain less well defined. PI3K-cII has been linked to the release of catecholamines, immune mediators, insulin, surface expression and recycling of integrins, and GLUT4 translocation to the plasma membrane, a mediator of metabolic homeostasis in muscle cells. This study demonstrates that Class II and Class III PI3K-dependent signaling are necessary for the rapid expression of PHP, controlling signaling from Rab11-dependent, recycling endosomes. By doing so, this study defines a postsynaptic signaling platform for the rapid expression of PHP and defines a novel action of PI3K-cII during neuronal homeostatic plasticity. This is the first established postsynaptic function for PI3K-cII at a synapse in any organism (Hauswirth, 2018).

Recently, it has become clear that the endosomal system has a profound influence on intracellular signaling and neural development. There is evidence that early and recycling endosomes can serve as sites of signaling intersection and may serve as signaling integrators and processors. Furthermore, protein sorting within recycling endosomes, and novel routes of protein delivery to the plasma membrane, may specify the concentration of key signaling molecules at the cell surface. The essential role of endosomal protein trafficking is underscored by links to synapse development and neurodegeneration. Yet, connections to homeostatic plasticity remain to be established. Based upon the data presented in this study and building upon prior work on endosomal signaling in other systems, it is speculated that postsynatpic PI3K-cII and Rab11-dependent recycling endosomes serve as as a postsynaptic 'homeostatic controller' that is essential for the specificity of retrograde, transsynaptic signaling (Hauswirth, 2018).

The Drosophila kinome and phosphatome were screened for genes that control the rapid expression of PHP. This screen identified three components of a conserved, postsynaptic lipid signaling pathway that is essential for the robust expression of PHP including: (1) class II PI3K, (2) class III PI3K (Vps34) and a gene encoding the Drosophila orthologue of PI4K (not examined in detail in this study). Pi3K68D was shown to be is essential, postsynaptically for PHP. Pi3K68D resides on a Clathrin-positive membrane compartment that is positioned directly adjacent to Golgi membranes, throughout muscle and concentrated at the postsynaptic side of the synapse. Pi3K68D is necessary for the maintenance of postsynaptic PI(3)P levels and the recruitment of Rab11 to intracellular membranes, likely PI(3)P-positive recycling endosomes. Postsynaptic Rab11 and Vps34 knockdown block PHP in an unusual, calcium-dependent manner that phenocopies Pi3K68D. Thus, this study has identified a postsynaptic signaling platform, centered upon the formation of PI(3)P and Rab11-positive recycling endosomes, that is essential for PHP (Hauswirth, 2018).

First it is considered whether postsynaptic Pi3K68D, Vps34 and Rab11 might alter PHP through modulation of postsynaptic glutamate receptor abundance. There is no consistent change in mEPSP amplitude in Pi3K68D mutants or following muscle-specific knockdown of Rab11 or Vps34 that could account for altered PHP. Therefore, functionally, there is no evidence for a change in glutamate receptor abundance at the postsynaptic membrane that could drive the phenotypic effects that were observe. Anatomically, data is presented examining GluR staining levels. In the Pi3K68D mutants, no change was found in GluRIIA levels. GluRIIA subunit containing receptors are the primary mediator of PhTx-dependent PHP. This study also reports a very modest (16%), though statistically significant, increase in GluRIIB levels. Based on these combined data, it seems unlikely that a change in GluR trafficking is a causal event leading to altered expression of PHP. It is noted that previous work showed limited GluRIIA receptor mobility within the PSD at the Drosophila NMJ. Thus, it is speculated that the function of Pi3K68D, Vps34 and Rab11 during PHP is not directly linked to postsynaptic GluR trafficking (Hauswirth, 2018).

Any model to explain the role of PI3K, Vps34 and Rab11-dependent endosomal signaling during homeostatic plasticity must account for the phenotypic observation that PHP is only blocked at low extracellular concentrations. More specifically, in animals deficient for Pi3K68D, Rab11 or Vps34, PHP is fully expressed at elevated calcium, following PhTX application or in the GluRIIA mutant. However, PHP completely fails when extracellular calcium is acutely decreased (following induction) below 0.7 mM [Ca2+]e. Clearly, the PHP induction mechanisms remain fully intact. Instead, the presynaptic expression of PHP has been rendered calcium-dependent. It is important to note that PHP can be fully induced in the absence of extracellular calcium, so the concentration of calcium itself is not the defect. In addition, this study documents trans-heterozygous interactions of Pi3K68D with presynaptic rim and dmp, arguing for the loss of trans-synaptic signaling and a specific function of Pi3K68D in the mechanisms of PHP. In very general terms, it is concluded that PI3K and Rab11-dependent endosomal signaling platform is necessary to enable the normal expression of PHP. Ultimately, some form of retrograde signaling must be defective due to either: 1) the absence of a retrograde signal that should have normally participated in PHP or 2) the presence of an aberrant or inappropriate signal that dominantly obstructs normal PHP expression. Both of these ideas in greater depth (Hauswirth, 2018).

First, the possibility is considered that the absence of postsynaptic PI3K and Rab11 signaling could alter the molecular composition or development of the presynaptic terminal due to the persistent absence of a retrograde signal that controls generalized synapse development or growth. Several observations demonstrate that impaired PHP is not a secondary consequence of a general defect in synapse development. Three independent postsynaptic manipulations are reported (postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34) that have no effect on presynaptic release at any [Ca2+]e, yet block PHP at low [Ca2+]e. In addition, no obvious defect was found in anatomical synapse development (Hauswirth, 2018).

Next, the possibility is considered that postsynaptic PI3K and Rab11 signaling eliminate a retrograde signal that is specific for PHP. It was recently demonstrated that Semaphorin2b (Sema2b) and PlexinB (PlexB) define a retrograde signal at the Drosophila NMJ that is necessary for PHP. However, both Sema2b and PlexB are essential for the rapid induction of PHP, inclusive of experiments at low and elevated extracellular calcium. Further, acute application of recombinant Sema2b is sufficient to fully induce PHP. Since the induction of PHP remains fully intact in the Pi3K68D mutant, and since PHP is rendered calcium sensitive, it suggests that altered Sema2b secretion is not the cause of impaired PHP in the Pi3K68D mutant. Nevertheless, this possibility will be directly tested in the future (Hauswirth, 2018).

Next, the possibility is considered that the loss of PI3K and Rab11 signaling causes aberrant or inappropriate retrograde signaling, thereby impairing the expression of PHP. This is a plausible scenario because the induction of presynaptic homeostatic plasticity suffers from a common problem inherent to many intra-cellular signaling systems: two incompatible outcomes (1. presynaptic homeostatic potentiation and 2. presynaptic homeostatic depression -- PHD) are produced from a common input, and it remains unclear how signaling specificity is achieved. The topic of signaling specificity has been studied in several systems. One system, budding yeast, is a good example. Different pheromone concentrations can induce several distinct behaviors in budding yeast despite having a common input (pheromone concentration) and underlying signaling systems. Signaling specificity degrades in the background of mutations that affect Map Kinase scaffolding proteins. In a similar fashion, presynaptic homeostatic plasticity is induced by a change in mEPSP amplitude. A decrease in mEPSP amplitude causes the induction of PHP, whereas an increase in mEPSP amplitude causes the induction of presynaptic homeostatic depression (PHD). If a common sensor is employed to detect deviations in average mEPSP amplitude, how is this converted into the specific induction of either PHP or PHD? It has been shown that PHD and PHP can be sequentially induced. But, it remains unknown what would happen if the mechanisms of PHP and PHD were simultaneously induced. Under normal conditions this would never occur because mEPSP amplitudes cannot be simultaneously increased and decreased. But, if signaling specificity were degraded in animals lacking postsynaptic PI3K or Rab11, then the expression of PHP and PHD might coincide and create a mechanistic clash within the presynaptic terminal (Hauswirth, 2018).

Signaling and recycling endosomes are, in many respects, ideally suited to achieve signaling specificity during homeostatic plasticity. Signaling specificity can be achieved by mechanisms including sub-cellular compartmentalization of pathways, physically separating signaling elements with protein scaffolds, or through mechanisms of cross-pathway inhibition. Well-established mechanisms of protein sorting within recycling endosomes could physically compartmentalize signaling underlying PHP versus PHD. Alternatively, recycling endosomes can serve as a focal point for signal digitization, integration, and, perhaps, cross-pathway inhibition. Thus, it is proposed that the loss of postsynaptic PI3K and Rab11 compromises the function of the postsynaptic endosomal platform that this study has identified, thereby degrading homeostatic signaling specificity. As such, this platform could be considered a 'homeostatic controller' that converts homeostatic error signaling into specific, homeostatic, retrograde signaling for either PHP or PHD (Hauswirth, 2018).

Other models are considered, but are not favored. It remains formally possible that the calcium-sensitivity of PHP expression could be explained by a partially functioning PHP signaling system. This seems unlikely given that the same phenotype is observed in four independent genetic manipulations including a null mutation in Pi3K68D, postsynaptic expression of kinase dead Pi3K68D, postsynaptic knockdown of Rab11, and postsynaptic knockdown of Vps34. Furthermore, prior experiments examining hypomorphic and trans-heterozygous genetic interactions among essential PHP genes suggest that PHP is either diminished across the entire calcium spectrum or fully functional. So, there is no evidence that partial disruption of PHP could account for calcium-sensitive expression of PHP. Finally, the experiments argue against the possibility that compensatory changes in Vps34 expression partially rescue the Pi3K68D mutant phenotype (Hauswirth, 2018).

Another common signaling module that emerged from the genetic screen is considered. Both CamKII and CamKK were identified as potential hits. The identification of CamKII is supported by prior work showing the expression of dominant negative CamKII transgenes disrupt the long-term maintenance of PHP in the GluRIIA mutant background (Haghighi, 2003). It has been assumed that postsynaptic calcium is used to detect the PhTX or GluRIIA-dependent perturbation. But, the logic remains unclear. PHP is induced by diminished GluR function and, therefore, diminished postsynaptic calcium influx. This should diminish activation of CamKII and yet, loss of CamKII blocks PHP. An interesting alternative model is that calcium and calmodulin-dependent kinase activity facilitate the function of the postsynaptic endosomal membrane system. Both calcium and calmodulin are necessary for endosomal membrane fusion. In this manner, the action of CamKK and CamKII would be entirely consistent with the identification of Class II/III PI3K and Rab11 as homeostatic plasticity genes (Hauswirth, 2018).

This study has uncovered novel postsynaptic mechanisms that drive homeostatic plasticity. Eventually, continued progress in this direction may make it possible to not only reveal how stable neural function is achieved throughout life, but to uncover new rules that are essential for the processing of information throughout the nervous system. In particular, PHP has a very large dynamic range, whether one considers data from Drosophila or human NMJ or mammalian central synapses. The homeostatic control of presynaptic release can achieve a 7-fold change in synaptic gain, and yet retains the ability to offset even small changes in postsynaptic neurotransmitter receptor function. Thus, it is expected that the regulatory systems that achieve PHP will be complex and have a profound impact on brain function. This study has defined a postsynaptic signaling system responsible for the rapid expression of PHP and a novel, albeit speculative, model is proposed for the postsynaptic control of PHP, taking into account the need for signaling specificity. The identification of this pathway paves the way for future advances in understanding how homeostatic signaling is designed and implemented at a cellular and molecular level (Hauswirth, 2018).


cDNA clone length - 1531

Bases in 5' UTR - 120

Exons - 4

Bases in 3' UTR - 766


Amino Acids - 214

Structural Domains

See NCBI Conserved Domains for information on Rab subfamily of small GTPases.

Rab-protein 11: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 May 2005

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.