Rab-protein 11


The mammalian homologue of Nuclear fallout, Arfo2 physically associates and colocalizes with Rab11, a key component of the RE (Hickson, 2003). Rab11 is required for the integrity of the RE, and is believed to mediate transport of vesicles from the RE to the TGN, early endosome, and plasma membrane via a "slow" recycling route (Ullrich, 1996; Ren, 1998). Dollar (2002) characterized the pattern of Rab11 localization in the developing Drosophila oocyte. Rab11 was shown to localize at the posterior pole and is necessary for proper microtubule organization and Oskar mRNA localization. The pattern of Rab11 localization during the cortical divisions was examined in the early Drosophila embryo. During interphase, Rab11 exhibits a diffuse punctate localization that concentrates around the nuclei. As the embryos progress into prophase, Rab11 maintains its punctate morphology, but exhibits significantly increased concentration at the centrosomes. During metaphase, the centrosomal concentration of Rab11 decreases and there is a concomitant dispersal of Rab11 throughout the cytoplasm encompassing each chromosomeƐspindle complex. This trend continues as the nuclei enter anaphase. Even though the nuclear envelope is substantially broken down during metaphase and anaphase, Rab11 does not enter the interior nuclear space. During telophase, Rab11 puncta concentrate around the newly formed nuclear envelope. There is a slight increase in the concentration of Rab11 puncta at the centrosomes. Cellularization occurs during the prolonged interphase of nuclear cycle 14. At this time, Rab11 is highly concentrated around the pair of apically located sister centrosomes (Riggs, 2003).

The pericentriolar concentration of Rab11 in Drosophila embryos is equivalent to Rab11 localization observed in mammalian cells. In CHO cells, Rab11 is primarily localized to a discrete pericentriolar region with a lower concentration of puncta distributed throughout the cell (Ullrich, 1996). Colocalization experiments with internalized transferrin indicated that Rab11 localizes to the pericentriolar RE (Ullrich, 1996; Sheff, 2002). GFP-Rab11 also exhibits a pericentriolar localization and colocalizes with the transferrin receptor (Sonnichsen, 2000). Given the equivalent staining patterns, it is concluded that Rab11 also localizes to the RE in syncytial and cellularized Drosophila embryos (Riggs, 2003).

Immunofluorescent analyses was performed using anti-Nuf and anti-Rab11 antibody. During prophase, when both antigens are highly concentrated in the pericentriolar region, areas of maximal Nuf localization correspond to areas of maximal Rab11 localization. Almost without exception, Nuf colocalizes with Rab11. However, the converse is not true, and in regions more distal from the centrosome, Rab11, but not Nuf, is present. During cellularization at interphase of nuclear cycle 14, Nuf and Rab11 exhibit high pericentriolar concentrations and extensive colocalization. As observed for prophase of the cortical divisions, Nuf always colocalizes with Rab11, but there are regions of Rab11 localization in which Nuf is not present. Given that Rab11 is an excellent marker of the RE (Ullrich, 1996; Ren, 1998), these results support the notion that Nuf localizes to the RE during cortical syncytial divisions and during cellularization at interphase of nuclear cycle 14 (Riggs, 2003).

Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth

Regulation of synaptic morphology depends on endocytosis of activated growth signal receptors, but the mechanisms regulating this membrane-trafficking event are unclear. Actin polymerization mediated by Wiskott-Aldrich syndrome protein (WASp) and the actin-related protein 2/3 complex generates forces at multiple stages of endocytosis. FCH-BIN amphiphysin RVS (F-BAR)/SH3 domain proteins play key roles in this process by coordinating membrane deformation with WASp-dependent actin polymerization. However, it is not known how other WASp ligands, such as the small GTPase Cdc42, coordinate with F-BAR/SH3 proteins to regulate actin polymerization at membranes. Nervous Wreck (Nwk) is a conserved neuronal F-BAR/SH3 protein that localizes to periactive zones at the Drosophila larval neuromuscular junction (NMJ) and is required for regulation of synaptic growth via bone morphogenic protein signaling. This study shows that Nwk interacts with the endocytic proteins dynamin and Dynamin associated protein 160 (Dap160) and functions together with Cdc42 to promote WASp-mediated actin polymerization in vitro and to regulate synaptic growth in vivo. Cdc42 function is associated with Rab11-dependent recycling endosomes, and this study shows that Rab11 colocalizes with Nwk at the NMJ. Together, these results suggest that synaptic growth activated by growth factor signaling is controlled at an endosomal compartment via coordinated Nwk and Cdc42-dependent actin assembly (Rodal, 2008).

Nwk interacts with the endocytic machinery and activates Wsp/Arp2/3 actin polymerization together with Cdc42 to regulate synaptic growth upstream of growth factor signaling. Mapping these interactions and activities provides a critical framework for determining the mechanism by which endocytic accessory proteins and the cytoskeleton control membrane deformation during endocytosis (Rodal, 2008).

Nwk activates Wsp/Arp2/3 actin polymerization via its SH3a domain, and Nwk-SH3b is not required for Wsp binding or activation, but is required for the residual Wsp-inhibitory activity of Nwk when SH3a function is abolished. This activity may be more pronounced on endogenous Wsp, which is more tightly autoinhibited than recombinant WASp, raising the possibility that Nwk-SH3b could potently regulate Nwk-SH3a-dependent activation of Wsp. Thus, ligands of Nwk-SH3b are in a position to serve as activators of Nwk and Wsp/Arp2/3 actin polymerization. Nwk-SH3b is required for interactions between Nwk and Dap160, which is an excellent candidate for acting upstream of Nwk, because dap160 mutants exhibit synaptic overgrowth and temperature-sensitive seizures like those of nwk mutants, and Nwk is mislocalized in dap160 NMJs. Recently, it was reported that the fragment of Dap160 containing its last two SH3 domains is required for interaction with full-length Nwk in Drosophila extracts, leading to the hypothesis that the C terminal proline-rich region of Nwk mediates these interactions (O'Connor-Giles, 2008). The current results show instead that interactions between purified Nwk{Delta}C (i.e., Nwk lacking the C terminus) and both endogenous full-length Dap160 as well as purified Dap160 SH3 domain-containing fragment depend on Nwk SH3b. Two possible interpretations can reconcile these results. Nwk SH3b may interact with a noncanonical SH3 domain-binding site in the intervening sequences between the Dap160 SH3 domains. Alternatively, Nwk SH3b may function in an intramolecular interaction within Nwk that is required to expose one of several proline-rich sequences in the N-terminal region Nwk for interaction with Dap160 SH3 domains. Thus, it is concluded that Nwk SH3b is important for Dap160-Nwk interactions via an indirect or noncanonical mechanism. Further experiments will be needed to identify the Nwk-binding site on Dap160 and to confirm activity of Dap160 on Nwk in vitro (Rodal, 2008).

Nwk-SH3a is required for interactions of Nwk with both dynamin and Wsp. Other F-BAR/SH3 family members have been postulated to link dynamin and Wsp by multimerization via their F-BAR domains (Itoh, 2006; Tsujita, 2006; Shimada, 2007), but endogenous complexes containing Wsp and dynamin have only been demonstrated for the F-BAR/SH3 protein syndapin (Kessels, 2006). Nwk could thus be in a position to bring dynamin and Wsp together. It has not been possible to coimmunoprecipitate endogenous Wsp and Nwk using the available antibodies. However, dynamin immunoprecipitates contain Nwk but not Wsp, suggesting that Nwk-SH3a may switch associations between dynamin and Wsp. Another interpretation is that Wsp and dynamin binding are restricted to separate populations of Nwk molecules, and that the SH3a domain thus acts in two parallel biochemical pathways (Rodal, 2008).

In vivo analysis reflects the complexity of these SH3 domain interactions. SH3a and SH3b of Nwk have both separate and overlapping functions in regulating synaptic growth, perhaps reflecting the multivalent nature of interactions in the Nwk network. [In addition to binding Nwk, Dap160 binds to both dynamin and to Wsp.] Furthermore, the fact that mutation of both SH3 domains together (Nwk-SH3a*b*) produces additional dominant effects suggests that a non-SH3 ligand of Nwk is inappropriately titrated away from its function after mutation of Nwk SH3 domains. An excellent candidate ligand is the membrane itself, because the Nwk F-BAR domain has the potential to bind to and tubulate phospholipid bilayers. Determining the specific order and regulation of F-BAR/SH3 domain protein interactions with competing SH3 domain ligands and with the membrane will be important for uncovering the molecular mechanisms of these proteins during endocytosis (Rodal, 2008).

NMJ overgrowth with an excess of satellite boutons is a hallmark of endocytic mutants. Nwk interacts with the endocytic machinery and cdc42 and nwk mutants exhibit overproliferation of satellite boutons. A prominent function of endocytosis in nerve terminals is the recycling of synaptic vesicles. However, nwk single mutants and cdc42; nwk double mutants show no detectable defect in endocytosis of synaptic vesicles. One interpretation of this result is that receptor endocytosis is more sensitive to perturbation than synaptic vesicle recycling. However, given the documented function of Cdc42 and Wsp in endosomes, it is more likely that Nwk functions in a later step of endocytic traffic. Importantly, although the synaptic vesicle endocytosis defects in shi (dynamin) and dap160 reflect the function of these molecules in the internalization step of endocytosis, synaptic overgrowth in these mutants could arise from defects at later steps of endocytic traffic, because dynamin functions in a variety of membrane-trafficking events, ranging from Golgi traffic to endosome traffic (van Dam and Stoorvogel, 2002Go; Kessels et al., 2006Go) (Rodal, 2008).

The endosomal system is organized into subdomains defined by specific members of the Rab GTPase family and adopts distinct morphology and ultrastructure in different cell types. Thus, functionally conserved Rab subdomains provide a unifying approach to understanding structurally diverse membrane systems. Rab11 controls the function of the recycling endosome in directing traffic to the cell surface and colocalizes with Nwk in periactive zones at the Drosophila NMJ [although it can occasionally be observed in larger puncta]. Like cdc42 and nwk mutants, rab11 mutants have a profound defect in synaptic growth, exhibiting excessive satellite boutons. Cdc42 and WASp have recently been implicated in recycling endosome function. Thus, periactive zones may be the synaptic representation of the recycling endosome, with Cdc42 and Nwk controlling actin polymerization-dependent traffic of signaling complexes at this Rab11-positive compartment. Whether Cdc42 functions as a signal-responsive element in this compartment or forms part of the constitutive machinery for membrane traffic remains uncertain (Rodal, 2008).

The TGF-β/BMP family member Gbb activates downstream signals that may be the critical targets of Nwk/Cdc42-mediated endocytosis in synaptic growth. Indeed, recent work has shown that Gbb signaling is required for synaptic overgrowth in nwk mutants, phosphorylation of the Gbb signaling target Mothers against decapentaplegic (Mad) is upregulated in nwk mutants, and Nwk biochemically interacts with the intracellular domain of the Gbb receptor Tkv. However, other signaling pathways could equally be regulated by Nwk/Cdc42-mediated endocytosis, lead to upregulation of phosphorylated Mad, and contribute to the synaptic overgrowth in cdc42; nwk mutants. One candidate pathway is the presynaptic component of the Wnt/Wg cascade, which may converge on Gbb/Mad regulation in the synapse as observed in other tissues. It has not been possible to detect any change in the steady-state localization of candidate cargoes in synaptic boutons in nwk or cdc42 mutants, suggesting that Nwk and Cdc42 are not required for the gross morphology of endosomes, but instead contribute to the rate of cargo trafficking through this compartment. Determining the specific signaling pathways, receptors, and their activation states in recycling endosomes will require tools to measure the activity and rates of traffic of specific receptors in situ (Rodal, 2008).

Nwk is conserved from insects to higher vertebrates, and the mammalian genome encodes two Nwk homologs, which have not yet been characterized. However, Cdc42 and WASp-induced actin polymerization have been implicated in synapse formation in Aplysia sensory neurons and in mammalian hippocampal cultures. These reports suggest that the direct consequence of activating these proteins was the formation of filopodia that mature into synapses. An alternative hypothesis, consistent with the established function of Cdc42 and WASp family members in generating force for intracellular membrane traffic rather than in filopodial formation, is that synaptic growth regulatory functions of Cdc42 and WASp depend on endosomal traffic of signaling complexes by a similar mechanism to Drosophila Nwk-Wsp-induced synapse formation (Rodal, 2008).

Rab11 accumulates at the posterior pole of the oocyte during mid-oogenesis and is required for its own localization

To gain insight into the role of Rab11 in the generation of oocyte polarity, its subcellular distribution was determined in developing oocytes by staining fixed ovaries with Rab11 antisera. The distribution of green fluorescent protein (GFP)-tagged Rab11 was examined in living tissues. Both approaches revealed a similar Rab11 expression pattern (Dollar, 2003).

Rab11 is abundant in stage 1-10 oocytes. Detection of the protein in later stage oocytes is problematic due to the deposition of the chorion and extrachorionic membranes. The protein is also expressed in follicle cells and nurse cells, but at reduced levels compared with oocytes. In stage 1-7 oocytes, Rab11 accumulates in a distinct perinuclear compartment and is abundantly distributed in a thick crescent along the lateral and posterior cortexes. In stage 8-10 oocytes, Rab11 continues to accumulate in the perinuclear compartment, but the thick cortical crescent is gradually replaced by a small cap of protein at the extreme posterior pole of the oocyte. Double label experiments showed near perfect colocalization of Rab11 with Osk in stage 9 and 10 oocytes (Dollar, 2003).

In situ hybridization for Rab11 mRNA revealed no specific accumulation of the transcript at the posterior pole of the oocyte. Rather, the mRNA is uniformly dispersed throughout the oocyte through at least stage 9. Thus, in contrast to Osk, Rab11 localization would appear to be mediated by a protein-based localization machinery (Dollar, 2003).

Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche

According to the stem cell niche synapse hypothesis postulated for the mammalian haematopoietic system, spatial specificity of niche signals is maximized by subcellularly restricting signalling to cadherin-based adherens junctions between individual niche and stem cells. However, such a synapse has never been observed directly, in part, because tools to detect active growth factor receptors with subcellular resolution were not available. This study describes a novel fluorescence-based reporter that directly visualizes bone morphogenetic protein (BMP) receptor activation and shows that in the Drosophila testis a BMP niche signal is transmitted preferentially at adherens junctions between hub and germline stem cells, resembling the proposed synapse organization. Ligand secretion involves the exocyst complex and the Rap activator Gef26, both of which are also required for Cadherin trafficking towards adherens junctions. It is therefore proposed that local generation of the BMP signal is achieved through shared use of the Cadherin transport machinery (Michel, 2011).

In keeping with the stem cell niche synapse hypothesis, a BMP niche signal in the Drosophila testis is transduced at subcellularly confined sites associated with adherens junctions between hub cells and GSCs. Although BMP ligands are also produced by the somatic CySCs, BMP receptor activation is not detected at the GSC surfaces facing the CySCs. There are several nonexclusive explanations that may contribute to this observation. Either, niche signalling is indeed dominated by the homodimeric Dpp or heterodimeric Dpp/Gbb ligands that are produced preferentially by the hub cells. In support of this idea, Dpp but not Gbb can fully suppress bam transcription upon ectopic expression, and is, at least in the wing, thought to have higher signalling activity. Alternatively, signalling from the CySCs may occur diffusely over the entire GSC surface and thus become diluted below the detection threshold of the reporter. Finally, based on the expression profile of the BMP ligands signalling from the CySCs is presumably dominated by Gbb and may therefore preferentially act through the alternative type I BMP receptor Saxophone, thus avoiding detection by a Tkv-specific reporter (Michel, 2011).

However, without artificial Jak/Stat pathway over-activation in the somatic cells of the testis, the CySC-derived BMP signal is by itself not sufficient to maintain GSC fate. Consequently, GSC detachment form the hub induces Bam derepression6 indicating a loss of BMP pathway activation. The junction-associated BMP signal from the hub to the germline, described in this study, is therefore essential for GSC maintenance (Michel, 2011).

In addition, this study shows that trafficking of both Dpp and DE-Cadherin in the hub cells involves the exocyst complex and the Rab11-positive recycling compartment. It is proposed that the local release of the junctional BMP signal is achieved through this shared use of intracellular machinery. Admittedly, RNAi-mediated inactivation of the exocyst complex is bound to have pleiotropic effects, and it cannot be excluded that the secretion of Upd or other growth factors may not also be affected. Can the observed loss of GSC stemness following exocyst knockdown therefore be directly attributed to a loss of BMP signalling from the hub? This is believed to be the case, because loss of Jak/Stat signalling in the germline would primarily be expected to affect adhesion of the GSCs to the hub. Although this loss of contact secondarily causes Bam derepression, Bam expression was also observed in GSCs still adhering to the hub. As Bam expression indicates a loss of BMP signalling also in the testis, this is attributed directly to the loss of the junction-associated BMP signal that is directly detected using a reporter that detects BMP receptor activation (Michel, 2011).

Future studies are required to address what directs Dpp secretion within the hub cells towards the adherens junctions with the overlying GSCs rather than towards those facing the adjacent hub cells. In addition, how the BMP ligands are confined after secretion to prevent lateral diffusion away from the site of release can now be studied. It is likely that for the latter proteoglycans has an essential role (Michel, 2011).

Finally, it was shown that the exocyst is also required for generation of the Dpp signal in the wing disc, where it forms a long-range morphogen gradient rather than a contact-dependent niche signal. It will be interesting to test whether this reflects a specific requirement of planar transcytosis, with the junctions forming a two dimensional network of signalling synapses. Alternatively, as suggested by zebrafish experiments, subcellularly restricted signal transduction at intercellular junctions may be a more general mechanism operating also in systems where BMP ligands spread through extracellular diffusion (Michel, 2011).

Guidance receptor promotes the asymmetric distribution of exocyst and recycling endosome during collective cell migration>

During collective migration, guidance receptors signal downstream to result in a polarized distribution of molecules, including cytoskeletal regulators and guidance receptors themselves, in response to an extracellular gradient of chemotactic factors. However, the underlying mechanism of asymmetry generation in the context of the migration of a group of cells is not well understood. Using border cells in the Drosophila ovary as a model system for collective migration, this study found that the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) is required for a polarized distribution of recycling endosome and exocyst in the leading cells of the border cell cluster. Interestingly, PVR signaled through the small GTPase Rac to positively affect the levels of Rab11-labeled recycling endosomes, probably in an F-actin-dependent manner. Conversely, the exocyst complex component Sec3 was required for the asymmetric localization of RTK activity and F-actin, similar to that previously reported for the function of Rab11. Together, these results suggested a positive-feedback loop in border cells, in which RTKs such as PVR act to induce a higher level of vesicle recycling and tethering activity in the leading cells, which in turn enables RTK activity to be distributed in a more polarized fashion at the front. Evidence is also provided that E-cadherin, the major adhesion molecule for border cell migration, is a specific cargo in the Rab11-labeled recycling endosomes and that Sec3 is required for the delivery of the E-cadherin-containing vesicles to the membrane (Wan, 2013).

It has been proposed that repeated cycles of endocytosis of RTKs (or active RTKs) and recycling of them back to the membrane would effectively concentrate active RTK in the front of the migrating border cells. However, if the levels of endocytic recycling remain uniform in all the outer border cells during migration, a fast amplification of RTK activity levels between front and back would be difficult to achieve. This study shows that there is a polarized endogenous distribution of the recycling endosome and exocyst in the leading border cells within the migrating cluster, which could conceivably make such amplification faster and more efficient in the leading cells. It was also shown previously that Sec15-GFP has an asymmetric localization at the front, when it is overexpressed in border cells. Along their migrating route, the border cells often tumble or rotate as a cluster, resulting in position changes such as front cells becoming lateral and back cells and vice versa. In such a scenario, a fast and robust amplification process would be essential to relocalize active RTKs. Indeed, this study found that overexpressing Sec3 or Rab11-GFP, but not Sec5-GFP, in a single cell clone within a mosaic border cell cluster significantly promotes the likelihood of such a cell being positioned at the leading position, suggesting that this cell utilizes its increased recycling and tethering to amplify and relocalize active RTKs faster and more efficiently than other wild-type neighbor cells. The difference in promoting effect from Sec3 and Sec5 is interesting, suggesting that when overexpressed the Sec3 subunit is more able to enhance the overall exocyst function than Sec5. This is consistent with a Sec3 study in budding yeast, which shows that as a unique subunit of exocyst Sec3 serves as a spatial landmark on the bud tip to recruit a subcomplex (comprising seven subunits) of exocyst containing all subunits but Sec3. Only when the subcomplex along with the associated vesicle arrives at the bud tip, can Sec3 be joined with it to form a fully functional tethering complex (Wan, 2013).

The next question is how the polarized distribution of recycling and tethering activity is initiated in border cells. This study demonstrated that this was likely to be induced by the guidance receptors in response to the external gradient of guidance cues, as removing guidance signaling by DN-PVR and DN-EGFR expression abolished Rab11 and Sec5 polarized distribution, and DN-PVR expression alone markedly reduced the polarization. These data suggested the presence of a positive feedback loop of active RTKs-endocytic recycling-active RTKs in border cells, as Rab11 and exocyst components (Sec3 and Sec15) were shown to be conversely required for polarized pTyr or active RTK localization at the front. Interestingly, this study found that PVR signals downstream through Rac and then polymerized actin to promote recycling endosome levels, providing mechanistic details to this feedback loop. Interestingly, it was recently shown that Rab11 interacts with Rac and actin cytoskeleton regulator moesin during border cell migration. Furthermore, this study found that strong Rab11 stainings were proximal to or partially overlapping with strong F-actin staining in the leading edge of wild-type border cells and around the ectopic F-actin regions in the λ-PVR, RacV12 or twinstar- RNAi expressing follicle cells and border cells. F-actin appears to be the direct cause rather than the effect of recycling endosome accumulation, because manipulating its levels by Lat-A or twinstar RNAi leads to either up- or downregulation of the levels of recycling endosome. However, the possibility cannot be ruled out that Rac can somehow act on recycling endosome-associated regulators directly (independently of F-actin) to affect their function. It was previously shown that actin polymerization is required for recycling of cargo back to plasma membrane, possibly through F-actin serving as a track for the movement of vesicles. However, how F-actin induces recycling endosome formation and organization is not clear and remains to be elucidated (Wan, 2013).

It was previously proposed that recycling of active RTKs needs to be directional (toward the front) to achieve polarized RTK activity. If active RTKs in the leading edge are endocytosed and then recycled to new regions in the membrane, RTK activity would be delocalized. What causes the recycling to be directed toward the front membrane is not clear. The proposed feedback loop via F-actin suggests that the active PVR (RTK) in the leading edge could locally induce higher levels of recycling endosome through Rac and enhanced actin polymerization (by Rac). As a result, the directional recycling could be achieved with the localized actin filaments serving both as a recycling endosome inducing agent and as tracks for movement of vesicles (carrying active RTKs) toward the front membrane, which prevents the active RTKs from being recycled to elsewhere and becoming delocalized. Indeed, inhibiting actin polymerization in the border cells by Lat-A treatment abolished both the polarized F-actin and the elevated Rab11 stainings proximal to F-actin, which are normally present in the leading edge of the wild-type cluster. Lastly, this work also provides some insight into the kinds of cargo that are recycled during border cell migration. E-cadherin is a specific cargo. E-cadherin is the major adhesion molecule required for border cell migration, whereas integrin plays only a minor role and is not required in border cells (Wan, 2013).

These finds suggests that cycles of endocytosis and recycling of E-cadherin could promote the dynamic assembly and disassembly of E-cadherin-mediated adhesion on the substrate (nurse cell E-cadherin), similar to how the turnover of integrin at the focal adhesion is regulated by endocytic recycling in mammalian cells. Interestingly, elevated intracellular E-cad stainings tended to be localized below the cell membrane that juxtaposes nurse cell membrane, suggesting that E-cadherin is normally delivered to or recycled back to this membrane region by Rab11 and exocyst during adhesion and migration. Another important candidate cargo to be determined is PVR. However, no significant colocalization was detected between Rab11 with PVR or active PVR with the previously reported PVR or pPVR antibody. Therefore, the definitive role of PVR or active PVR as a cargo for recycling still awaits further determination (Wan, 2013).

Effects of Mutation or Deletion

Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors

To evaluate the role of Rab11 in Rh1 transport, Rh1 transport was investigated in Rab11 mutants. Since animals lacking Rab11 die as embryos (Dollar, 2002; Jankovics, 2001), mosaic animals, with eyes containing a mixture of normal photoreceptors and photoreceptors with severely reduced Rab11, were made. Comparatively few Rab11 mutant photoreceptors were observed in mosaic eyes, probably reflecting a cell-essential role for the protein. Rab11-reduced photoreceptors fail to transport Rh1 to the rhabdomere. Mutant rhabdomeres are reduced in size and a profusion of vesicles fills the photoreceptor cytoplasm. These cells lack normal globular MVBs, but contain infrequent, irregular vesicular organelles resembling defective MVBs. Other vesicular compartments, including ER and Golgi, retain normal appearance (Satoh, 2005).

Rab11 reduction via dsRNA expression in developing eyes similarly blocks Rh1 delivery to the rhabdomere and disperses it throughout photoreceptor cytoplasm. Electron microscopy shows MVBs are lost and irregular vesicles fill the cytoplasm. Rab11 activity was also reduced via expression of a dominant-negative Rab11, Rab11N124I, a GTP-binding mutant (Duman, 1999). Rab11N124I expression during the time when TRP is normally delivered to the rhabdomere blocks TRP delivery with a concomitant accumulation of TRP in photoreceptor cytoplasm. Expression during the Rh1 delivery period recapitulates genetic Rab11 reduction: Rh1 fails to reach the rhabdomere and instead accumulates in vesicles dispersed throughout the cytoplasm. Confocal and electron microscope examination of Rab11N124I photoreceptors shows a parallel loss of RLVs and MVBs (Satoh, 2005).

These results above show that loss of Rab11 activity results in the accumulation of Rh1-containing vesicles in the cytoplasm and the absence of MVBs. The possibility is considered that the Rh1 vesicles may originate from a defective endocytic pathway, such that the cytoplasm accumulates early endocytic vesicles unable to consolidate into MVBs. To address this possibility, the effect was examined of a dominant-negative form of Rab5, Rab5N142I, on the process of rhodopsin transport and MVB accumulation. This inhibits endocytosis and prevents MVB formation (Shimizu, 2003). Indeed, MVBs containing both TRP and Rh1 are lost in flies expressing Rab5N142I. The same images show that Rab5N142I does not inhibit TRP and rhodopsin delivery to the rhabdomere, confirming that MVBs are not required for transport to the rhabdomere (Satoh, 2005).

Simultaneous expression of Rab11N124I and Rab5N142I allowed a determination of whether the Rab11N124I phenotype is the result of absence of Rab11 activity prior to TRP and Rh1 delivery to the rhabdomere, or after endocytic removal of these two proteins from the rhabdomere. If the TRP and Rh1 vesicles accumulating in cytoplasm upon Rab11N124I expression are endocytosed from the rhabdomere, Rab5N142I expression should inhibit their biogenesis. This is not the case; numerous cytoplasmic TRP- or Rh1-bearing vesicles accumulate and rhabdomeres do not stain for TRP and Rh1 (Satoh, 2005).

The interpretation of these results could be complicated by consideration that guanine-nucleotide-deficient small GTPase dominant negatives sequester activating GEF proteins, and crosstalk may exist among RabGEF signaling pathways. However, the fidelity with which Rab11N124I recapitulates genetic and RNAi Rab11 loss, the observation that Rab5N142I mutant shows the expected endocytic defect, and the marked contrast in the Rab11N124I and Rab5N142I individual phenotypes, suggest these dominant negatives do generate a specific loss of function for each of these genes. From this perspective, the failure of Rab5N142I expression to impact the Rab11N124I phenotype argues Rab11 is required upstream of Rab5 and prior to the initial delivery of TRP and Rh1 to the rhabdomere (Satoh, 2005).

It was also observed that Golgi morphology visualized by CFP-galactosyl transferase in confocal microscopy is unaffected in Rab11N124I photoreceptors. These data, in agreement with the localization studies, suggest Rab11 is required for a post-Golgi step in rhodopsin and TRP movement to the rhabdomere (Satoh, 2005).

The proposed role of Rab11 in delivery of membrane proteins to the rhabdomere does not account for the loss of MVBs in the Rab11 mutant photoreceptors. It is possible that loss of Rab11 activity depletes the rhabdomere of Rh1 and other membrane proteins, and the lack of protein in these membranes limits the rate of endocytosis and MVB formation. To investigate this possibility more directly, the effect was examined of Rab11N124I on uptake of an endocytic tracer, Texas Red-conjugated avidin (TR-avidin), by larval Garland cells, large and easily accessible endocytic specialists (the Garland cell is a nephrocyte which takes up waste products from the haemolymph). In normal cells, internalized TR-avidin could be seen in peripheral, vesicular structures 10 minutes after exposure to TR-avidin. In Garland cells expressing Rab11N124I, however, TR-avidin is not internalized (Satoh, 2005).

Electron microscopic observations provided insight into this defect. In wild-type Garland cells, numerous labyrinthine channels invaginate deeply from plasma membrane. These channels are the sites of active endocytosis; clathrin-coated buds mark the tips of the channels and the channels elongate when endocytosis is inhibited in the dynamin mutant shibirets. The labyrinthine channels are absent in Garland cells expressing Rab11N124I. Thus, the labyrinthine channels do not form correctly in the absence of Rab11 activity. These results are consistent with the view that membrane components essential to sustaining vigorous endocytosis are lost when Rab11-dependent apical delivery is compromised (Satoh, 2005).

Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. Functional evidence is provided for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, electron microscopy demonstrates the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, Rab11 is shown to be essential for membrane growth and invagination during cellularization. Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. Rab11 endosomes are key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain (Pelissier, 2003).

Epithelia separate different environments within an organism. This property relies on the existence of different membrane domains, the apical surface that forms the lumen of most organs, and the basal-lateral surface. Epithelia can form via two cellular pathways: the mesenchyme-to-epithelium transition as studied in Madin Darby canine kidney (MDCK) cells is the most common pathway, and the other is embryonic cleavage, which leads to the formation of primary epithelial tissues in a variety of organisms. Although the processes are different, the columnerization of flat squamous epithelial cells and embryonic cleavage are both accompanied by the growth of a basal lateral membrane domain. The extent of membrane growth varies between tissues. The cellular mechanisms underlying polarized membrane growth remain poorly understood (Pelissier, 2003).

In MDCK cells, the process depends mostly on vesicular sorting along the secretory pathway from the trans Golgi network (TGN) and on vesicular insertion at adherens junctions via the recruitment of the multiprotein complex called exocyst. In other cases, such as cultured hepatocytes, however, the process relies on vesicular sorting through endocytic trafficking pathways. In this case, called transcytosis, all proteins are first targeted to the future apical surface and endocytic targeting from the apical domain to another membrane domain ensures the formation of the basal-lateral membrane surface. The contribution of membrane trafficking pathways to the growth and polarization of the cell surface during embryonic cleavage has not been thoroughly addressed yet (Pelissier, 2003).

Drosophila cellularization, a specialized form of embryonic cleavage, is a very good system for addressing this problem, although the process may in part rely on specific mechanisms that may not apply to other cases of epithelial morphogenesis. In the early syncytial embryo, the nuclei undergo 13 synchronous division cycles in a common cytoplasm. Upon entry into cycle 14, the cell surface increases, and the plasma membrane invaginates between the nuclei. This invagination results in a columnar epithelium. Cellularization proceeds in two distinct phases, slow phase and fast phase. The process is characterized by the polarized growth of the cell surface as revealed by surface-labeling experiments in living embryos. Distinct membrane behaviors are observed in slow phase and fast phase. When the plasma membrane is labeled at the onset of cellularization, the surface marker gradually disappears during slow phase from the apical surface rich in microvilli and accumulates instead along the growing basal-lateral surface. If the surface is labeled instead at the onset of fast phase, a patch of unlabeled membrane appears in fast phase in the apical-lateral membrane. Two different mechanisms could underlie the disappearance of apical surface marker specific to the slow phase and its basal-lateral accumulation. The process could depend on the direct apical insertion of intracellular unlabeled membrane and a sorting between the newly inserted and recipient membranes within the plane of the epithelium. Conversely, the process could stem from an endocytic-based membrane transfer from the apical to the basal-lateral surface in a manner akin to transcytosis (Pelissier, 2003).

The transport of vesicles along the endocytic pathway proceeds via a series of organelles defined in part by the transport of cargo molecules (e.g., the transferrin receptor) and in part by the specific localization of regulatory molecules. Internalized vesicles at the plasma membrane are transported to early endosomes, also called sorting endosomes. This step is dependent on the activity of the small GTPase Rab5 as well as on Dynamin. Dynamin is encoded by shibire in Drosophila and controls vesicle budding and internalization in a large number of tissues. Vesicles are then transported back to the plasma membrane, destined for the late endosome and lysosome degradation pathway, or transported to the pericentriolar recycling endosome. The recycling endosome is involved in the recycling of vesicles back to the plasma membrane. In polarized epithelial cells, the apical recycling endosome is required for cell polarization as well as for transcytosis. Trafficking through recycling endosomes is dependent on the small GTPase Rab11 in mammals as well as in Drosophila. Rab11 localizes to this organelle in both vertebrates and invertebrate. Dynamin was also shown to control vesicle budding from recycling endosomes in mammalian cells.In order to address the implication of endocytic trafficking in plasma membrane growth during cellularization, the roles of key effectors of these pathways were tested. The requirements for Dynamin, Rab5, and Rab11 were tested (Pelissier, 2003).

The overexpression of Rab5S43N, a dominant-negative variant of Rab5, has been shown to block endocytosis in a number of Drosophila tissues. Because cellularization coincides with the zygotic induction of gene expression, high enough levels of the UAS-Rab5S43N transgene cannot be expressed. To circumvent this problem, GST+Rab5S43N was prepared in vitro and the proteins were injected into embryos during cycle 13, 10-15 min before cellularization. Injection of GST+Rab5S43N reduces the speed of membrane invagination near and opposite to the site of injection during slow phase:this is in contrast to the injection of GST alone and of GST+Rab5WT. The delay in membrane invagination can be seen via a comparison of the relative positions of the membrane front and of the nuclei at the end of slow phase (+26'), and in fast phase (+29' and +32'). Confocal images show the different positions of the membrane front and of the plasma membrane within the injected and the control area. Similar results are obtained with another dominant-negative variant of Rab5, Rab5N142I. However, no invagination defect was observed after injection of a dominant-negative form of Rab1, Rab1N124I, which is involved in ER-to-Golgi transport. These observations support the fact that the injection of Rab5S43N has a specific effect during cellularization (Pelissier, 2003).

The role of endocytosis from the plasma membrane can be examined by using the temperature-sensitive allele of shibire/dynamin. The timing of gene inactivation can be precisely controlled by shifting embryos to the restrictive temperature at 32°C. In shibire-ts mutant embryos this causes the total inhibition of membrane invagination in slow phase, unlike in control embryos, where cellularization proceeds normally. In contrast, both control and shibire-ts mutant embryos cellularize when the embryos are shifted to the restrictive temperature at the beginning of fast phase, albeit at a slightly slower rate in shibire-ts mutants. The specific requirement for shibire during slow phase is remarkable in light of the fact that, in wild-type embryos, apical membrane-labeled material is cleared from the plasma membrane specifically in slow phase. In agreement with the known role of shibire/dynamin in the internalization of plasma membrane vesicles, this study shows that apically labeled membrane is no longer endocytosed in a shibire mutant, as opposed to wild-type control embryos. Together, the data so far suggest that endocytosis from the apical plasma membrane is required throughout slow phase in order for the basal-lateral surface to grow (Pelissier, 2003).

Although the results are consistent with the known role of shibire in plasma membrane endocytosis, Dynamin has also been implicated in vesicle budding and trafficking from the TGN and from recycling endosomes. The possibility that other trafficking defects might account for the role of shibire during cellularization was also examined. Neurotactin is a transmembrane protein synthesized de novo during slow phase. In control embryos, the protein traffics through the secretory pathway and, at steady state, Nrt is predominantly localized at the plasma membrane. Small vesicular staining is also detected in the Golgi. shibire mutant embryos at the restrictive temperature were shifted at the onset of cellularization (e.g., when Nrt is not yet synthesized), incubated for 20 min during slow phase, and fixed to monitor the localization of Nrt. The striking result is that Nrt is not properly inserted in the plasma membrane and accumulates instead in a large subapical compartment. This compartment is not the Golgi apparatus, which can be detected with the middle-trans Golgi marker p120. In control and shibire mutant embryos, p120 is indeed mostly concentrated in the basal cytoplasm in the form of small (<2microm) vesicular structures. The very different distribution of these two proteins suggests that Nrt follows the secretory pathway normally but fails to be inserted in the plasma membrane apically. Note that Toll, a maternally provided transmembrane protein that is already present at the plasma membrane at the onset of cellularization, remains in the plasma membrane, unlike Nrt in a shibire mutant. The trafficking defects observed with Nrt, a newly synthesized protein en route to the plasma membrane, suggest that shibire controls another step in vesicular trafficking (Pelissier, 2003).

Rab11 was found in the search for markers of intracellular compartments in which Nrt accumulates. In Drosophila, during slow phase, Rab11 accumulates mostly in the subapical cytoplasm in two large pericentriolar endocytic compartments. An additional staining is seen in small puncta (<1microm) concentrated mostly in the basal cytoplasm and, in some cases, in the apical cytoplasm. These small puncta colocalize with Golgi markers, unlike the large apical staining. At steady state, Nrt is virtually absent from the large apical Rab11 endosomes, although some colocalization is occasionally detected in the small Rab11-positive Golgi puncta. However, in a shibire mutant, the subapical accumulation of Nrt predominantly colocalizes with Rab11 in the large subapical pericentriolar endosomes (Pelissier, 2003).

The sequestration of Nrt in Rab11 apical endosomes is unlikely to be the indirect effect of perturbing globally endocytic traffic in a shibire mutant. Indeed, an antibody to Shibire was used to detect the protein and a clear enrichment of Shibire was found in Rab11 endosomes, although the colocalization is not total. Shibire may mark a subdomain of recycling endosomes, as reported for other endosomal proteins. Moreover, the localization of Rab11 and of Shibire follows a similar developmental regulation. The proteins colocalize in subapical endosomes during slow phase but not in fast phase, when shibire is in fact no longer required for cellularization (Pelissier, 2003).

These results argue that shibire is also required for vesicular budding from Rab11 recycling endosomes during cellularization. In support of this conclusion, very large amounts of intracellular subapical coated pits were found in shibire mutant embryos, by using transmission electron microscopy. Large subapical endocytic structures, all of which contain one or more dark uncleaved vesicle, were found. In wild-type embryos, however, no such vesicular structures were detected in the subapical cytoplasm, suggesting that these structures may form very rapidly and transiently in the wild-type. Given the role of Dynamin/Shibire in severing vesicles, these observations support the role of shibire in vesicular traffic from subapical endosomes in addition to its role in plasma membrane endocytosis (Pelissier, 2003).

The role of trafficking through recycling endosomes was further tested by using a dominant-negative form of Rab11, Rab11S25N, which is an effector of this process. Injection of GST+Rab11S25N during cycle 12-13 causes a striking inhibition of membrane invagination during slow phase. Such an effect is not detected when either GST or GST+Rab11WT are injected in embryos at the same stages (Pelissier, 2003).

In vertebrates, Rab11 interacts with members of the Arfophilin family of proteins. Drosophila nuclear-fallout (nuf) encodes a centrosomal protein that is a member of the Arfophilin family, suggesting that, in Drosophila, rab11 and nuf may function in a similar functional pathway. In support of this proposal, nuf is required for the formation of membrane furrows prior to cellularization as well as during slow phase. In addition, the apical Rab11 endosomes are assembled at the centrosomes, as revealed by the centrosomal protein gamma-Tubulin. Interestingly, on top of the cellularization defects, Rab11S25N-injected embryos show a distinct 'nuclear fallout' phenotype reminiscent of nuf mutant embryos. Instead of being regularly aligned at the cortex, the nuclei are disorganized and occasionally 'fall' inside the embryo (Pelissier, 2003).

Two classes of phenotypes, distinguished by their severity, are described. In both cases, the phenotype is concentrated in the area of the embryo that was injected, at the injection site, and opposite to it. Class 1 (weak) represents 50% of the injected embryos (n = 56) and shows weak nuclear defects and membrane invagination defects. Of the injected embryos, 37.5% fall in class 2 (strong) and have strong nuclear and membrane invagination defects in slow phase. These data further support the role of trafficking through Rab11 endosomes to ensure membrane growth during cellularization (Pelissier, 2003).

The identification of Rab11 endosomes as a key intermediate in the trafficking of vesicles necessary for lateral membrane growth raises two questions. Where does the membrane come from? Where is it eventually inserted? The facts that shibire and rab5 are both required for basal-lateral membrane growth and that in a shibire mutant apically labeled membrane is not internalized suggests that the plasma membrane present in the villous projections accounts in part for the growth of the lateral surface. The plasma membrane is indeed a huge reservoir whose capacity largely exceeds the membrane needed for lateral membrane growth during slow phase. Trafficking through Rab11 endosomes appears to also be essential for cellularization, as assayed by the injection of a dominant-negative form of Rab11 and as revealed by the role of shibire in vesicular trafficking through this compartment. In a shibire mutant, the transmembrane protein Nrt, which is de novo synthesized and traffics normally through the Golgi, accumulates in part in Rab11 endosomes. This suggests that secretory material might also contribute to the growth of the basal-lateral surface. This is supported by the fact that the injection of the drug Brefeldin A, an inhibitor of Endoplasmic Reticulum (ER) to Golgi transport, blocks cellularization and that the Golgi structural protein Lava-Lamp is required for cellularization. Note that the failure to detect any invagination defect after injection of dominant-negative Rab1 could stem from the fact that Rab2 may compensate for ER-to-Golgi vesicular transport. Together, the data suggest that Rab11 endosomes constitute a point of integration of vesicles from the secretory and endocytic pathways necessary for the rapid exocytosis of vesicles required for the growth of the lateral membrane. Note that the ER has been suggested to be a membrane reservoir required in part for the formation of pseudopods, another example of rapid membrane growth during phagocytosis. In this process, an early step called 'focal exocytosis' is controlled in part by Dynamin-2 (Pelissier, 2003).

After trafficking through Rab11 endosomes, where might the vesicles be finally inserted? The sites of membrane insertion are likely to dictate how polarity arises. In light of the published data, the most likely proposal would state that vesicles are inserted at the basal junctions, a transient adherens junction that forms between the membrane front called 'furrow canal' and the lateral surface. The adherens junction is indirectly required for lateral membrane growth and basal-lateral targeting in polarizing MDCK cells through the recruitment of Sec6 and Sec8, key components of the exocyst that localize in part at adherens junctions and control vesicle insertion, presumably from the Golgi apparatus. Consistent with a role of junctions in regulating vesicular insertion, when basal adherens junctions fail to form, as in slam mutant embryos, membrane invagination is blocked and Nrt is less efficiently inserted in the plasma membrane (Pelissier, 2003).

Why does cellularization involve such a pathway to ensure the growth and invagination of the plasma membrane? The comparison with other examples of cells in which the plasma membrane is rapidly remodeled provides insight into this question. Endocytic trafficking is usually much more efficient than secretory trafficking at remobilizing membrane pools. Antigen-presenting cells and migrating Dictyostelium ameobae can, for instance, recycle their whole surface within a few minutes. In a situation where the membrane grows rapidly, remobilizing preexisting membrane pools at the plasma membrane and in the secretory pathway through recycling endosomes might be a very efficient way to transfer membrane 'en masse' toward a defined site of the plasma membrane. Note that endocytic recycling is indeed involved in the formation of membrane protrusions, ruffles, and lamellipodesa but also in other cases in which invaginations form, for example during cytokinesis. rab11 is required for late stages of cytokinesis in the C. elegans embryo (Skop, 2001). Endobrevin/VAMP8, a regulator of endosome trafficking, is also required for cytokinesis in mammalian cells, together with Syntaxin 2. Trafficking through recycling endosomes may also provide an efficient way to couple surface polarization to membrane growth. The current findings are consistent with the similarities between cellularization and cytokinesis. Through the identification of a trafficking pathway involved in the growth of the lateral surface during cellularization, this work may also provide insight into the formation of an epithelial sheet during embryonic cleavage (Pelissier, 2003).

Rab11 is involved in the localization of Oskar mRNA

Abdomen and germ cell development of the Drosophila embryo requires proper localization of Oskar mRNA to the posterior pole of the developing oocyte. Oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, a novel interaction type of genetic screen was developed by which 14 dominant enhancers were isolated of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. The detailed analysis of two allelic modifiers is described that identify Drosophila Rab11, a gene encoding small monomeric GTPase. Mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of Oskar mRNA, whereas localization of Gurken and Bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. The ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. These results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in Oskar RNA localization (Jankovics, 2001).

Whatever the role of the Drosophila Rab11 gene in the Osk localization pathway, it must be indirect and act in the reorientation of the oocyte microtubule network, as seen by the Tau:GFP microtubule visualization and by mislocalization of the Kinesin:ß-galactosidase fusion protein to the center in the Rab11 mutants. Central mislocalization of the Kinesin:ß-galactosidase protein has been observed in mutants that impair any step of the reciprocal signaling events between the oocyte and the posterior follicle cells. Given that the best-characterized role of the Rab11 proteins is the targeting of recycling endosomes or trans-Golgi vesicles to the plasma membrane, it seemed to be plausible that Rab11 mutants would exert their phenotypes by blocking signaling events between the oocyte and the follicular cells. However, two types of evidence suggest that both the oocyte-to-follicle cells and the follicle cells-to-oocyte signals are functional in the Rab 11 mutants: (1) absence of expression of an enhancer trap in follicle cells at the posterior cap indicates that the posterior polar follicle cell fate is properly adopted; (2) a focus of microtubules at the posterior pole was never observed by Tau:GFP labeling, indicating that posterior MTOC disassembles. Consistently, mislocalization of the Bcd mRNA to the posterior pole was never observed, indicating again that the back signaling from the posterior polar follicle cells is received, and the MTOC and the minus ends of the microtubules disappear from the posterior pole. However, working with hypomorphic allele combinations, the possibility that mislocalization of Bcd mRNA was not detected because of its relative insensitivity to the microtubule reorientation cannot be excluded. The analysis of the hypomorphic phenotypes also supports this interpretation. An intermediate Osk mislocalization phenotype occurs in Rab11 mutants, when Osk mRNA is detected in the center and simultaneously at the normal posterior position in the same oocyte. This indicates that in such mutant oocytes the MTOC does indeed disappear and the minus ends of the microtubules are replaced by plus ends at the posterior. Rab11 phenotypes are reminiscent of that of par1. In par1 mutant oocytes, the posterior MTOC also disappears but central osk mRNA localization is observed. It is therefore concluded that even though the posterior MTOC normally disassembles, the reorientation of the oocyte microtubule network is incomplete in Rab11 mutants. It is proposed that instead of having reverse polarity when the microtubules are nucleated predominantly from the anterior, in Rab11 mutants only a subset of microtubules, which are nucleated over the entire cortex of the oocyte, is intact driving Kinesin:ß-galactosidase motor protein and Osk mRNA to the center of the oocyte. In Rab11 mutants, Osk mRNA mislocalization phenotype is not fully penetrant and transient, and by stage 10-11, egg chambers exhibit wild-type Osk mRNA localization. It is suggested that the Rab11-dependent localization pathway for Osk RNA itself is redundant and the recovery observed in later stages is due to an alternative, Rab11-independent Osk localization mechanism when cytoplasmic streaming, which begins at stage 10, directs the Osk mRNA to the posterior pole. These results indicate that posterior MTOC breakdown may not be sufficient for reorientation of the microtubule network during stages 6-8 of the Drosophila oocyte; rather, the reorientation process depends on other factors too, like internal membrane functions. The precise mechanism by which Rab11 contributes to the microtubule reorientation is still unclear. A similar phenotype, characterized by Osk mislocalization to the center of the oocyte, is observed in Ter94 mutant ovaries. Ter94 encodes an AAA type ATPase that is also responsible for internal membrane trafficking, namely, for homotypic fusion of endoplasmic reticulum vesicles. Rab11 and Ter94 phenotypes reveal that the internal membranous structures and the cytoskeleton of the Drosophila oocyte have a functional connection to conduct cytoplasmic mRNA localization (Jankovics, 2001).

Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation

The Drosophila embryonic body plan is specified by asymmetries that arise in the oocyte during oogenesis. These asymmetries are apparent in the subcellular distribution of key mRNAs and proteins and in the organization of the microtubule cytoskeleton. Evidence suggests that the Drosophila oocyte also contains important asymmetries in its membrane trafficking pathways. Specifically, alpha-adaptin and Rab11, which function critically in the endocytic pathways of animal cells, are localized to neighboring compartments at the posterior pole of stage 8-10 oocytes. Rab11 and alpha-adaptin localization occurs in the absence of a polarized microtubule cytoskeleton, i.e. in grk null mutants, but is later reinforced and/or refined by Osk, the localization of which is microtubule dependent. Analyses of germline clones of a rab11 partial loss-of-function mutation reveal a requirement for Rab11 in endocytic recycling and in the organization of posterior membrane compartments. Such analyses also reveal a requirement for Rab11 in the organization of microtubule plus ends and osk mRNA localization and translation. 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).

The best evidence that the plasma membrane of the oocyte and membrane trafficking pathways are polarized in the Drosophila oocyte comes from the expression pattern of human transferrin receptor (Htr) in transgenic flies. During stages 8-10, the posterior pole of the oocyte becomes enriched with Htr, both along the plasma membrane and in vesicles. Htr rapidly disappears from vesicles upon inhibition of endocytosis, indicating that Htr is actively internalized and recycled in Drosophila oocytes as it is in many other examined cells. Together with the observation that Htr is restricted to the posterior plasma membrane, its active internalization and recycling strongly suggests that the membrane recycling pathway of the oocyte is polarized towards the posterior pole (Dollar, 2002 and references therein).

To investigate the role of Rab11 in the organization of the posterior pole of the oocyte, the distribution of osk mRNA and protein was examined in rab11P2148 germ-line clones (GLCs). In wild-type oocytes, osk mRNA is transported to the posterior pole during stages 8 and 9, coincident with the polarization of the microtubule cytoskeleton. During transport, and in the initial hours following transport, osk mRNA is seen as a large ball. By the end of stage 9, the ball resolves into a thin cap along the posterior cortex, which persists through the end of oogenesis. The nature of the transition from the ball to the cap is not clear, but coincides with the activation of osk translation. The cap structure is much more resistant to disruption with colchicine than is the ball and appears, then, to represent the binding of the mRNA to an anchor, which might be Osk. rab11P2148 germ-line clones (GLCs) are defective in the transport of osk mRNA to the posterior pole, and in its subsequent translation and anchoring. The transport defect is temporal in nature. Thus, while most osk transcripts reach the posterior pole of wild-type oocytes during stage 8, only a small fraction of transcripts reach the posterior pole of rab11 oocytes during stage 8. Typically, the lagging transcripts are aggregated into a mass near the center of the cell, possibly representing stalled transport at an intermediate step. Although most osk transcripts eventually reach the posterior pole of rab11 oocytes, two observations suggest that they are never anchored: (1) no Osk protein was ever detected in rab11 oocytes; (2) the osk transcripts of rab11P2148 GLCs never formed the characteristic cap at the posterior pole, but instead remained as a ball. Moreover, during late stages of oogenesis (e.g. when microtubules are bundled along the entire egg cortex), the ball of osk mRNA appears to drift away from the posterior pole and is often fragmented into several smaller balls. It is concluded that Rab11 is required for the efficient transport of osk mRNA to the posterior pole of the oocyte and for its subsequent translation and anchoring (Dollar, 2002).

Rab11 and alpha-adaptin are localized to the posterior pole of mid-stage oocytes and such localization does not require a polarized microtubule cytoskeleton or grk signaling. A reduction of rab11 activity in the oocyte alters the subcellular distribution of Rab11, alpha-adaptin and internalized transferrin. These alterations indicate that Rab11 is required for endocytic recycling and to organize posterior membrane compartments. A reduction of rab11 activity in the oocyte also causes defects in Kin:ß-gal localization, osk mRNA transport, and osk mRNA translation and anchoring. These latter defects suggest that the posterior membrane compartments established by Rab11 organize microtubule plus ends and, possibly, the translation factors and/or anchors for osk mRNA (Dollar, 2002).

The expression pattern of Htr in transgenic flies shows clearly that the oocyte establishes a posterior plasma membrane domain (PMD). Further evidence for such a domain comes from the finding that Rab11 localizes to the posterior pole of wild-type oocytes. Interestingly, the PMD is established independently of microtubule polarity or grk signaling, since Rab11 is localized normally in grk null mutants. Given the rapid rate at which Htr is internalized and recycled, it is likely that the maintenance, if not also the initial specification, of the PMD requires polarized endocytic recycling directed towards the posterior pole. The data presented indicate that Rab11 is responsible for such recycling: Rab11 is localized to the posterior pole of the oocyte and is required for the recycling of internalized transferrin (the ligand for transferrin receptor) to the plasma membrane of cultured oocytes. Independent evidence that Rab11 mediates polarized endocytic recycling comes from studies with vertebrates, where Rab11 recycles internalized molecules to the apical surface of polarized epithelial cells (Dollar, 2002).

What polarizes endocytic recycling to the posterior pole of the oocyte? The polarization of the endocytic pathways of other cells is triggered by Rho GTPase family members (i.e. Rho, Cdc42 and Rac), which are activated at specific regions of the cell cortex by a variety of intrinsic and extrinsic cues. Rho GTPases have also been strongly implicated in the polarization of exocytosis. Specifically, they have been shown to recruit the 'exocyst' to specific sites of the plasma membrane. The exocyst is a conserved complex of proteins to which vesicles of the secretory pathway fuse. Thus, through local activation of Rho GTPases, secretory vesicles are targeted to specific regions of the plasma membrane. By analogy, the Rho GTPases could localize Rab11 and polarize receptor recycling through local recruitment of an exocyst-like complex for Rab11-containing vesicles. Because Drosophila Rho GTPases are required for progression through early oogenesis, the analysis of their role in Rab11 localization and other aspects of oocyte polarization must await the identification of conditional mutants (Dollar, 2002).

Kinesin:ß-gal expression studies indicate that microtubule plus ends are not sharply focused onto the posterior pole of the oocyte in rab11P2148 mutant oocytes. The simplest interpretation of this finding is that microtubule plus ends are attached to the PMD, or to a neighboring membrane domain whose identity is established and/or maintained by Rab11. In wild-type oocytes, this domain is tightly defined such that Kin:ß-gal is concentrated at the posterior tip of the oocyte, while in rab11P2148 mutant oocytes, the domain is poorly defined and the Kin:ß-gal expression pattern is expanded. The slight enrichment of Kin:ß-gal at the posterior tip of rab11P2148 oocytes could reflect partial Rab11 activity and/or the polarizing activities of membrane trafficking pathways that may not rely on Rab11 (e.g. the secretory pathway), which targets newly synthesized molecules from the Golgi to the plasma membrane. Recent studies have identified two types of protein-protein interactions (CLIP-CLASP and APC-EB1) responsible for the stable association of microtubule plus ends with membranes. While the CLIP, CLASP, APC and EB1 protein families are all well-represented in the Drosophila genome, their role in the establishment of oocyte polarity has not yet been investigated (Dollar, 2002).

Apart from Rab11, the only protein known to play a specific role in microtubule plus end organization in Drosophila oocytes is Par-1, a kinase, whose suspected targets include the microtubule associated protein Tau. In strong par-1 mutants, microtubule plus ends, as revealed by Kin:ß-gal expression patterns, are not enriched at the posterior pole of the oocyte, but instead are concentrated tightly, forming a dot at the center of the cell. In weak par-1 mutants, a small amount of Kin:ß-gal is also found at the posterior pole. This small amount of Kin:ß-gal is always tightly localized to the cell tip, suggesting that Par-1 is not required for the specification of the PMD, but rather only for the efficient movement of already focused microtubule plus ends from the cell center to the PMD. Consistent with the idea that microtubule plus ends initially focus to a sharp point at the center of the cell and then move to the posterior pole, Kin:ß-gal and oskar mRNA show transient concentration at the center of the cell in wild-type oocytes. How Par-1 might promote the movement of microtubule plus ends from the cell center to the posterior pole is not clear. One possibility is that it promotes attachment of microtubule plus ends to a structure that is then moved to the posterior pole. Alternatively, Par-1 might stimulate a burst of microtubule growth, forcing growth toward the posterior end of the cell (Dollar, 2002).

The observation that osk mRNA transport to the posterior pole is delayed in rab11P2148 mutant oocytes suggests that Rab11 might also have a role in the movement of microtubule plus ends from the cell center to the posterior pole, and therefore, that such movement is membrane dependent. For example, microtubule plus ends could become attached to membrane compartments or vesicles at the cell center, and the vesicles may then be targeted to the posterior pole in a Rab11-dependent manner. Because osk mRNA arrives at the posterior pole as a fairly well-defined ball in rab11P2148 oocytes, Rab11 does not appear to be required for focusing microtubule plus ends at the cell center, but rather only for their timely movement and attachment to the posterior membrane domain (Dollar, 2002).

Although most osk transcripts are eventually transported to the posterior pole in rab11P2148 oocytes, they are not translated. Since Osk is required to anchor oskar mRNA at the posterior pole, the lack of oskar translation in rab11P2148 GLCs could explain the inability of the oskar mRNA ball to resolve into the thin posterior crescent. The nature of the osk translation block in rab11P2148 oocytes is not clear. One possibility is that key osk translation factors are localized to the posterior membrane domain established by Rab11. In rab11P2148 oocytes, this domain may be too poorly defined to support assembly of such factors into an active translation complex (Dollar, 2002).

Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary

All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. This study shows that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (ß-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs, through which E-cadherin transits, are tightly associated with the fusome. It is proposed that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology (Bogard, 2007).

The first clue that Rab11 plays important roles in early oogenesis in Drosophila came from immunostaining experiments that revealed strong expression of endogenous Rab11 and a fully functional Rab11::GFP in GSCs, cystoblasts and young (2-4- and 8-cell) germline cysts. Strikingly, the proteins were concentrated as discrete dots on the fusome, which electron microscopy and photobleaching studies have shown is highly vesicular and rapidly exchanged with other membrane stores. Triple-stain experiments showed that some of these dots also contained E-cadherin, which has been shown to transit though Rab11-positive recycling endosomes (REs) en route to the plasma membrane in some cells. High-magnification images showed that the Rab11 (and, more rarely, E-cadherin) dots were often nestled into cavities within the fusome. Such Rab11-harboring cavities are visible in the fusomes of all examined GSCs, cystoblasts and young germline cysts, not only in the ovary but also in the testes. In view of the well-described enrichment of Rab11 in REs, it is proposed that these Rab11- and E-cadherin-harboring cavities are REs and are therefore referred to as FREs (fusome-associated REs) (Bogard, 2007).

These studies indicate that Rab11 maintains GSC identity through polarized trafficking of E-cadherin and, possibly, other cargoes that reinforce essential GSC-niche contacts. These studies further indicate that Rab11 is required for fusome localization and asymmetric GSC division and suggest a feedback linkage between these events and E-cadherin trafficking. Although Rab11 has been implicated in the trafficking of E-cadherin in other cells, there are no other cases in which such trafficking has been correlated with a biological response. It will be of interest to determine whether Rab11 is required for the maintenance of stem cells in other systems and whether such maintenance involves E-cadherin trafficking or the trafficking of other adhesion molecules. It will also be of interest to determine the role of Rab11 in other E-cadherin-dependent cell behaviors, particularly as Rab11, at least in Drosophila, is expressed in only a small subset of E-cadherin-expressing cells (Bogard, 2007).

New components of the Drosophila fusome suggest it plays novel roles in signaling and transport

The fusome, an organelle highly enriched with small vesicles and without a delimiting membrane, plays an essential role in prefollicular germ cell development within insects such as Drosophila. Alpha-spectrin and the adducin-like protein Hu-li tai shao (Hts) are required to maintain fusome integrity, synchronize asymmetric cystocyte mitoses, form interconnected 16-cell germline cysts, and specify the initial cell as the oocyte. By screening a library of protein trap lines, 14 new fusome-enriched proteins were identified, including many associated with its characteristic vesicles. These studies reveal that fusomes change during development and contain recycling endosomal and lysosomal compartments in females but not males. A significant number of fusome components are dispensable, because genetic disruption of tropomodulin, ferritin-1 heavy chain, or scribble, does not alter fusome structure or female fertility. In contrast, rab11 is required to maintain the germline stem cells, and to maintain the vesicle content of the spectrosome, suggesting that the fusome mediates intercellular signals that depend on the recycling endosome (Lighthouse, 2008).

The first molecular component of the fusome (Hts) was identified as a mutation in an unbiased forward genetic screen, and several related fusome components were subsequently identified by antibody staining. Recently, an increasing number of Drosophila proteins have been fused to GFP in vivo in protein trap lines. Screening such lines provides a general method of identifying the molecular components of any subcellular structure that can be visualized in the fluorescence microscope. Several new fusome proteins have been discovered using this approach. This study screened the largest collection of protein trap lines currently available, and as a result, has more than doubled the previously known number of fusome proteins. The high frequency with which trapped proteins demonstrate fusome enrichment, 20/243 (8%), contrasts with the rarity of mutations affecting the fusome, but suggests that this organelle is more complex than previously supposed, and likely contains hundreds of different proteins. As expected from the rarity of fusome mutations, only a small fraction of the identified fusome proteins were found to be essential for fusome structure and/or function (Lighthouse, 2008).

Previous studies of the fusome have focused on its critical roles in germline cyst formation, cell synchronization, cytoskeletal polarization, and oocyte determination. Events crucial to all these processes occur while the fusome is still growing in region 1 of the ovariole. The current studies support the view that growing and completed fusomes differ in structure. Proteins likely to be involved in the cytoskeletal and microtubule-organizing activities of the fusome are expressed early, while proteins associated with proteolysis and oocyte determination begin to be enriched in the fusomes of newly completed cysts. This suggests that the fusome plays distinct roles during cyst formation and during subsequent stages of germ cell development (Lighthouse, 2008).

The upregulation of lysosomal proteases such as Sap-r, a homolog of a saposin class lysosomal protein, and cathepsin F in the fusome following cyst completion argues that developmentally regulated proteolysis mediates at least some of these changes in fusome structure. First, key components of the fusome and ring canals, such as Hts protein isoforms, are proteolytically processed from a large precursor in a manner that changes after cyst completion. Second, partial degradation of the fusome, especially in the vicinity of the ring canals, may open these intercellular bridges and allow specific molecular cargos and organelles to begin flowing toward the oocyte. This process may not only remove proteins blocking the ring canals, but may also enable microtubule motors to access the underlying core of stable, polarized microtubules within the fusome (Lighthouse, 2008).

The protein constituents of multiple vesicular compartments were observed within the fusome. These include transmembrane and luminal markers for ER membranes, lysosomes, secretory vesicles, and for recycling endosome components. The fusome may be a locus of post-Golgi vesicle trafficking within early germ cells, perhaps analogous to the subapical compartment of epithelia. Golgi elements themselves are located near but are excluded from the fusome (Lighthouse, 2008).

Experiments suggest two possible reasons for the non-random localization of vesicle compartments to the fusome. First, these vesicles may participate in carrying out the functions associated with the fusome during germ cell development. An intact fusome is required for cell cycle synchrony, and localization might ensure that lyosomal, endocytic, and secretory events are synchronized throughout the cyst. While the onset of pre-meiotic S phase in the newly formed 16-cell cyst may be synchronous, subsequent cell cycle events do not appear to be as tightly coordinated. The way synchrony is lost may correspond to the changes in fusome structure that take place following cyst completion (Lighthouse, 2008).

The second reason why multiple vesicle compartments are associated with the fusome may be related to the role of the later fusome as a major pathway for transporting materials to the oocyte. Many vesicular components, especially those enriched at later stages, may simply be in transit. Centrosomes, mitochondria, and Golgi elements are all transported along the fusome in region 2 cysts. Studies now suggest that post-Golgi endocytic compartments are also in transit, and many of these are found to be not essential for the completion of oogenesis. The identification of Fer1HCH within the fusome, a conserved protein involved in iron storage and transport, generally supports such an interpretation. Rather than functioning in the fusome, it may sequester iron for eventual storage and use within the oocyte (Lighthouse, 2008).

A molecular model of the vertebrate erythrocyte membrane skeleton has long served as a starting point for thinking about the structure of the fusome's cytoskeletal component. Transmembrane proteins associate with both the plasma membrane and with the membrane skeleton, linking the two together and allowing the underlying protein skeleton to shape the plasma membrane and provide rigidity. The same mechanism is thought to control the shape of Golgi elements. Consequently, it was postulated that a fusome skeleton exists and that individual vesicular elements might be linked to these proteins in a similar manner. The drastic disruption of fusome integrity caused by mutations in genes encoding membrane skeleton orthologs encouraged this view. Studies on the role of tropomodulin now call this model into question (Lighthouse, 2008).

Tropomodulin, like Adducin, is an actin capping protein. While Adducin caps the barbed ends of actin filaments in the erythrocyte cortical cytoskeleton, Tropomodulin localizes to and binds the pointed ends. Knocking out tmod greatly reduces membrane stability in mouse erythrocyte precursor cells. In contrast, loss of Tmod from the fusome resulted in no detectable changes in fusome stability or in other aspects of cystocyte and follicle development (Lighthouse, 2008).

There are several ways to rationalize these differences. The role of Tmod may differ in Drosophila and mammals. Perhaps proteins other than Tmod regulate actin pointed ends in junctional complexes in Drosophila. Alternatively, the basic organization of membrane skeleton proteins in the fusome may differ from that in the mammalian erythrocyte. Whatever the reason, it is likely to apply not only to the fusome, but also to the membrane skeleton of epithelial cells. Disrupting tmod within follicle cells did not affect their membrane skeletons as evidenced by anti-Hts staining, or compromise cell viability. Despite its dispensability in both germ cells and the follicular epithelia, tmod mutations in Drosophila are lethal, indicating that this protein plays an essential role in other tissues (Lighthouse, 2008).

These studies also revealed that male and female fusomes differ significantly in composition. All known early components of the female fusome are also found in males, including the newly identified proteins in that category described here (Tmod, Scribble, and Shaggy). This presumably reflects the similar mechanisms by which male and female fusomes are formed during synchronous cystocyte divisions and the strong similarity of their initial structure and branching pattern (Lighthouse, 2008).

Male fusomes also express proteins characteristic of endoplasmic reticulum. Markers for rough endoplasmic reticulum such as the translocon components Sec61alpha and Sec63 are enriched, as are ER luminal proteins involved in protein folding such as Protein disulfide isomerase and Calreticulin. However, not all ER-localized proteins found within female fusomes were found in the male version of the organelle. Reticulon I, a protein associated with smooth ER, is absent from male fusomes. Rtnl1 is highly enriched in the female fusome and later accumulates heavily in the oocyte. This also is true of Trailer hitch, which along with Cup, Me31B and other proteins is involved in translationally regulating mRNAs in transit to the oocyte. Loss of Rtnl1 in mutant clones has no effect on fusome structure or fertility. Male cyst cells have equivalent fates, whereas female cystocytes produce one oocyte and 15 nurse cells. The differential composition of the fusomes in male and female cysts is likely a reflection of this fundamental difference. Therefore, it is suggested that fusome constituents in the female that are simply in transit to the oocyte are likely to be absent in males, where such transport appears absent. Such proteins are also frequently unnecessary to complete egg formation (Lighthouse, 2008).

Endosomal and lysosomal proteins found in the female fusome were also absent from the male organelle. These components become prominent in mid- to late-fusomes, and may remodel fusome structure in order to activate directional transport. The absence of lysosomal components within male fusomes may ensure that it is not chewed back to expose its underlying microtubules. Instead, a different process of modification may occur in males, as it was observed that lysosomal proteases are located very close to the male fusome at some stages (Lighthouse, 2008).

Experiments suggest that the recycling endosome functions within the fusome. Rab11 has been considered necessary for proper oocyte polarization in maturing stage 8-10 follicles. This study found that Rab11 is required to maintain the normal vesicular structure of the fusome. Previously, only strong bam mutations had been observed to deplete spectrosomal membranes. The current observations suggest that Bam might normally function to promote endocytic recycling within the fusome (Lighthouse, 2008).

Recently, Rab11 was localized to the fusome and a role in GSC maintenance was detected. In contrast, this study did not observe a concomitant loss of cell adhesion molecules at the GSC-cap cell border, despite the fact that the alleles tested in both studies reduced GSC lifetime by a similar amount (fourfold). Changes in adhesion may be secondary to reduced endosome recycling within the fusome, but the most significant effect of lowering Rab11 may be on signaling. Rab11 plays an essential role in Notch signaling in the sensory organ precursors of Drosophila, and the Notch pathway is required both for GSC niche formation, and GSC maintenance. JAK/STAT signaling is also under endocytic control, but this pathway requires trafficking mediated by Rab5, rather than Rab11. The current results suggest that the fusome normally mediates a Notch signal that is essential for GSC maintenance (Lighthouse, 2008).

The fusome may operate as a locus of membrane recycling that allows it to function as a regulatory center for multiple events important for early oogenesis, in addition to GSC maintenance. The recycling endosome is thought to control important aspects of mitosis, such as plasma membrane growth. The completion of cytokinesis depends on the Rab11-mediated transport of endocytic vesicles to the cleavage furrow. Additionally, the recycling endosome, like the fusome, is frequently associated with the cell's microtubule organizing center. Therefore, the defects observed in rab11 germline cyst formation may represent defects in fusome-mediated membrane growth during cystocyte divisions, or from defects in MT organization (Lighthouse, 2008).

The recycling endosome is also critical to generate signals that can act within or between cells. Signaling pathways such as Notch, that are dependent on endocytic recycling, may accelerate G1/S transitions. Fusome-mediated signals might synchronize the cystocyte cell cycles and coordinate microtubule organization and other aspects of cystocyte development. Such a mechanism might explain how the amount of fusome within a cell could influence its developmental fate (Lighthouse, 2008).

The functions of auxilin and rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. This study use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, it was found that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, Drosophila female germline cells, the one cell type known to signal without clathrin, was found to not require auxilin to signal. Third, it was fond that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling (Banks, 2011).

There are three major results of this work. First, it was found that Rab11 is not required for several Notch signaling events in the developing Drosophila eye that require epsin and auxilin. Thus, as in the female germline cells, ligand recycling, at least via a Rab11-dependent pathway, is not necessary for Notch signaling in the eye disc. Second, the one Notch signaling event presently known to be clathrin-independent is also auxilin-independent. This result reinforces the idea that rather than performing some obscure function, the role of auxilin in Notch signaling cells is to regulate clathrin dynamics. Finally, overexpression of both clathrin heavy chain and epsin were found to rescue to nearly normal the severely malformed eyes and semi-lethality of aux hypomorphs. Presumably, vesicles uncoated of clathrin fuse with the sorting endosome, and so it seems reasonable to assume that uncoating clathrin-coated vesicles containing ligand is preprequisite for trafficking ligand through endosomal pathways. Thus, if ligand endocytosis is prerequisite to recycling, efficient production of uncoated vesicles would be required. In aux mutants with severe Notch-like mutant phenotypes, clathrin vesicle uncoating is inefficient. It is presumed that this remains so even when clathrin and epsin are overexpressed, yet the eye defects and lethality are nearly absent. Thus, it is reasoned that auxilin is required not for efficient production of uncoated vesicles per se, but for the other product of auxilin activity -- free clathrin (and possibly also free epsin). Taken together, these results argue strongly that at least in some cell types, the fundamental role of Notch ligand endocytosis is not ligand recycling (Banks, 2011).

Is it possible that the fundamental mechanism of Notch signaling is so completely distinct in different cell types, that ligand endocytosis serves only to activate ligand via recycling in some cellular contexts, and only for exerting mechanical force on the Notch receptor in others? While formally possible, this is not parsimonious. Thus, a model is favored where the fundamental role of ligand endocytosis is to exert mechanical force on the Notch receptor. In addition, some cell types will also require ligand recycling. As no altered, activated form of ligand has yet been identified, while ligand transcytosis has been well-documented, the most likely role of recycling is to relocalize ligand on the plasma membrane prior to Notch receptor binding (Banks, 2011).

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).

Rab11 facilitates crosstalk between autophagy and endosomal pathway through regulation of Hook localization

During autophagy, double-membrane autophagosomes deliver sequestered cytoplasmic content to late endosomes and lysosomes for degradation. The molecular mechanism of autophagosome maturation is still poorly characterized. The small GTPase Rab11 regulates endosomal traffic, and is thought to function at the level of recycling endosomes. This study shows that loss of Rab11 leads to accumulation of autophagosomes and late endosomes in Drosophila melanogaster. Rab11 translocates from recycling endosomes to autophagosomes in response to autophagy induction, and physically interacts with Hook, a negative regulator of endosome maturation. Hook anchors endosomes to microtubules, and Rab11 is shown to facilitate the fusion of endosomes and autophagosomes by removing Hook from mature late endosomes and inhibiting its homodimerization. Thus, induction of autophagy appears to promote autophagic flux by increased convergence with the endosomal pathway (Szatmari, 2013).


Abdelilah-Seyfried, S., et al. (2000). A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ. Genetics 155: 733-752. 10835395

Assaker, G., et al. (2010). Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration. Proc. Natl. Acad. Sci. 107: 22558-22563. PubMed Citation: 21149700

Banks, S. M., et al. (2011). The functions of auxilin and rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling. PLoS One. 6(3): e18259. PubMed Citation: 21448287

Bogard, N., Lan, L., Xu, J. and Cohen, R. S. (2007). Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary. Development 134(19): 3413-8. PubMed citation; Online text

Bonafe, N., and Sellers, J. R. (1998). Molecular characterization of myosin V from Drosophila melanogaster. J. Muscle Res. Cell Motil. 19: 129-141. PubMed Citation: 9536440

Breda, C., Nugent, M. L., Estranero, J. G., Kyriacou, C. P., Outeiro, T. F., Steinert, J. R. and Giorgini, F. (2014). Rab11 modulates alpha-synuclein mediated defects in synaptic transmission and behaviour. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25305083

Brill, J. A., Hime, G. R., Scharer-Schuksz, M., and Fuller, M. T. (2000). A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis. Development 127: 3855-3864. PubMed citation: 10934029

Chang, H. Y. and Ready, D. F. (2000). Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1. Science 290: 1978-1980. 11110667

Chen, W., Feng, Y., Chen, D. and Wandinger-Ness, A. (1998). Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. Mol. Biol. Cell 9: 3241-3257. 9802909

Cullis, D. N., Philip, B., Baleja, J. D. and Feig, L. A. (2002). Rab11-FIP2, an adaptor protein connecting cellular components involved in internalization and recycling of epidermal growth factor receptors. J. Biol. Chem. 277(51): 49158-66. 12364336

de Graaf, P., et al. (2004). Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex. Mol. Biol. Cell 15(4): 2038-47. 14767056

Deneka, M., Neeft, M. and van der Sluijs, P. (2003). Regulation of membrane transport by rab GTPases. Crit. Rev. Biochem. Mol. Biol. 38: 121-142. 12749696

Deretic, D., Huber, L. A., Ransom, N., Mancini, M., Simons, K. and Papermaster, D. S. (1995). rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis. J. Cell Sci. 108: 215-224. 7738098

Deretic, D. (1997). Rab proteins and post-Golgi trafficking of rhodopsin in photoreceptor cells. Electrophoresis 18: 2537-2541. 9527482

Dollar, G., Struckhoff, E., Michaud, J. and Cohen. R. S. (2002). Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation. Development. 129: 517-526. 11807042

Duman, J. G., Tyagarajan, K., Kolsi, M. S., Moore, H. P. and Forte, J. G. (1999). Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase. Am. J. Physiol. 277: 361-372. 10484323

Emery, G., et al. (2005). Asymmetric Rab11 endosomes regulate Delta recycling and specify cell fate in the Drosophila nervous system. Cell 122: 763-773. 16137758

Farkas, R. M., Giansanti, M. G., Gatti, M., and Fuller, M. T. (2003). The Drosophila Cog5 homologue is required for cytokinesis, cell elongation, and assembly of specialized Golgi architecture during spermatogenesis. Mol. Biol. Cell 14: 190-200. PubMed citation: 12529436

Giansanti, M. G., Farkas, R. M., Bonaccorsi, S., Lindsley, D. L., Wakimoto, B. T., Fuller, M. T., and Gatti, M. (2004). Genetic dissection of meiotic cytokinesis in Drosophila males. Mol. Biol. Cell 15: 2509-2522. PubMed citation: 15004238

Giansanti, M. G., et al. (2006). The class I PITP Giotto is required for Drosophila cytokinesis. Curr. Biol 16: 195-201. PubMed citation; Online text

Giansanti, M. G., Belloni, G. and Gatti, M. (2007). Rab11 is required for membrane trafficking and actomyosin ring constriction in meiotic cytokinesis of Drosophila males. Mol. Biol. Cell 18(12): 5034-47 . PubMed citation; Online text

Giagtzoglou, N., Yamamoto, S., Zitserman, D., Graves, H. K., Schulze, K. L., Wang, H., Klein, H., Roegiers, F. and Bellen, H. J. (2012). dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions. J Cell Biol 196: 65-83. PubMed ID: 22213802

Hales, C. M., et al. (2001). Identification and characterization of a family of Rab11-interacting proteins. J. Biol. Chem. 276: 39067-39075. 11495908

Hehnly, H. and Doxsey, S. (2014). Rab11 endosomes contribute to mitotic spindle organization and orientation. Dev Cell 28: 497-507. PubMed ID: 24561039

Hickson, G., et al. (2003). Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout. Mol. Biol. Cell. 14: 2908-2920. 12857874

Itoh, T., et al. (2005). Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9: 791-804. PubMed Citation: 16326391

Jafar-Nejad, H., Andrews, H. K., Acar, M., Bayat, V., Wirtz-Peitz, F., Mehta, S. Q., Knoblich, J. A. and Bellen, H. J. (2005). Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors. Dev. Cell 9(3): 351-63. 16137928

Jankovics, F., Sinka, R. and Erdelyi, M. (2001). An interaction type of genetic screen reveals a role of the Rab11 gene in oskar mRNA localization in the developing Drosophila melanogaster oocyte. Genetics. 158: 1177-1188. 11454766

Jékely, G., Sung, H. H., Luque, C. M. and Rørth, P. (2005). Regulators of endocytosis maintain localized receptor tyrosine kinase signaling in guided migration. Dev. Cell. 9: 197-207. PubMed Citation: 16054027

Junutula, J. R., et al. (2004). Molecular characterization of Rab11 interactions with members of the family of Rab11-interacting proteins. J. Biol. Chem. 279(32): 33430-7. 15173169

Karagiosis, S. A. and Ready, D. F. (2004). Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis. Development 131: 725-732. 14724125

Kerkhoff, E., et al. (2001). The Spir actin organizers are involved in vesicle transport processes. Curr. Biol. 11: 1963-1968. 11747823

Kessels, M. M. and Qualmann, B. (2006). Syndapin oligomers interconnect the machineries for endocytic vesicle formation and actin polymerization. J. Biol. Chem. 281: 13285-13299. PubMed Citation: 16540475

Khodosh, R., Augsburger, A., Schwarz, T. L. and Garrity, P. A. (2006). Bchs, a BEACH domain protein, antagonizes Rab11 in synapse morphogenesis and other developmental events. Development 133(23): 4655-65. Medline abstract: 17079274

Laflamme, C., et al. (2012). Evi5 promotes collective cell migration through its Rab-GAP activity. J. Cell Biol. 198(1): 57-67. PubMed Citation: 22778279

Langevin, J., et al. (2005). Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane. Dev. Cell 9(3): 355-76. 16224820

Lapierre, L. A., Kumar, R., Hales, C. M., Navarre, J., Bhartur, S. G., Burnette, J. O., Provance, D. W., Jr, Mercer, J. A., Bahler, M. and Goldenring, J. R. (2001). Myosin vb is associated with plasma membrane recycling systems. Mol. Biol. Cell 12: 1843-1857. 11408590

Le Borgne, R. and Schweisguth, F. (2003). Unequal segregation of Neuralized biases Notch activation during asymmetric cell division, Dev. Cell 5: 139-148. 12852858

Li, B. X., Satoh, A. K. and Ready, D. F. (2007). Myosin V, Rab11 and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors. J. Cell Biol. 177(4): 659-69. PubMed citation; Online text

Lighthouse, D. V., Buszczak, M. and Spradling, A. C. (2008). New components of the Drosophila fusome suggest it plays novel roles in signaling and transport. Dev. Biol. 317(1): 59-71. PubMed Citation: 18355804

Lindsay, A. J., et al. (2002a). Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein. J. Biol. Chem. 277(14): 12190-9. 11786538

Lindsay, A. J. and McCaffrey, M. W. (2002b). Rab11-FIP2 functions in transferrin recycling and associates with endosomal membranes via its COOH-terminal domain. J. Biol. Chem. 277(30): 27193-9. 11994279

Lindsay, A. J. and McCaffrey, M. W. (2004). The C2 domains of the class I Rab11 family of interacting proteins target recycling vesicles to the plasma membrane. J. Cell Sci. 117(Pt 19): 4365-75. 15304524

Liu, Z., Huang, Y., Hu, W., Huang, S., Wang, Q., Han, J. and Zhang, Y. Q. (2014). dAcsl, the Drosophila Ortholog of Acyl-CoA Synthetase Long-Chain Family Member 3 and 4, Inhibits Synapse Growth by Attenuating Bone Morphogenetic Protein Signaling via Endocytic Recycling. J Neurosci 34: 2785-2796. PubMed ID: 24553921

Lock, J. G. and Stow, J. L. (2005). Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin. Mol. Biol. Cell. 16(4): 1744-55. 15689490

Longley, R. L., Jr and Ready, D. F. (1995). Integrins and the development of three-dimensional structure in the Drosophila compound eye. Dev. Biol. 171: 415-433. 7556924

MacIver, B., McCormack, A., Slee, R. and Bownes, M. (1998). Identification of an essential gene encoding a class-V unconventional myosin in Drosophila melanogaster. Eur. J. Biochem. 257: 529-537. PubMed Citation: 9839940

Mammoto, A., et al. (1999). Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling. J. Biol. Chem. 274: 25517-25524. 10464283

Mermall, V., et al. (2005). Drosophila myosin V is required for larval development and spermatid individualization. Dev. Biol. 286: 238-255. PubMed citation: 16126191

Michel, M., et al. (2011). Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche. Nat. Commun. 2: 415. PubMed Citation: 21811244

Mitchell, H., Choudhury, A., Pagano, R. E. and Leof, E. B. (2004). Ligand-dependent and -independent transforming growth factor-beta receptor recycling regulated by clathrin-mediated endocytosis and Rab11. Mol. Biol. Cell. 15(9): 4166-78. 15229286

Moore, R. H., Millman, E. E., Alpizar-Foster, E., Dai, W. and Knoll, B. J. (2004). Rab11 regulates the recycling and lysosome targeting of beta2-adrenergic receptors. J. Cell Sci. 117(Pt 15): 3107-17. 15190120

Moritz, O. L., Tam, B. M., Hurd, L. L., Peranen, J., Deretic, D. and Papermaster, D. S. (2001). Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. Mol. Biol. Cell 12: 2341-2351. 7738098

O'Connor-Giles, K. M., Ho, L. L. and Ganetzky, B. (2008). Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth. Neuron 58(4): 507-18. PubMed Citation: 18498733

Pasqualato, S., Senic-Matuglia, F., Renault, L., Goud, B., Salamero, J. and Cherfils, J. (2004). The structural GDP/GTP cycle of Rab11 reveals a novel interface involved in the dynamics of recycling endosomes. J. Biol. Chem. 279(12): 11480-8. 14699104

Peden, A. A., Schonteich, E., Chun, J., Junutula, J. R., Scheller, R. H. and Prekeris, R. (2004). The RCP-Rab11 complex regulates endocytic protein sorting. Mol. Biol. Cell 15(8): 3530-41. 15181150

Pelissier, A., Chauvin, J. P. and Lecuit, T. (2003). Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis. Curr. Biol. 13: 1848-1857. 14588240

Polevoy, G., et al. (2009). Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis. J. Cell Biol. 187(6): 847-58. PubMed Citation: 19995935

Prekeris, R., Davies, J. M. and Scheller, R. H. (2001). Identification of a novel Rab11/25 binding domain present in Eferin and Rip proteins. J. Biol. Chem. 276(42): 38966-70. 11481332

Pylypenko, O., et al. (2016). Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes. Elife 5 [Epub ahead of print]. PubMed ID: 27623148

Ren, M., et al. (1998). Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc. Natl. Acad. Sci. 95: 6187-6192. 9600939

Riggs, B., et al. (2003). Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11. J. Cell Biol. 163: 143-154. 14530382

Riggs, B., et al. (2007). The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation. Mol. Biol. Cell 18(9): 3313-22 . PubMed citation; Online text

Riggs, B., et al. (2007). The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation. Mol. Biol. Cell 18(9): 3313-22. PubMed Citation: 17581858

Rodal, A. A., Motola-Barnes, R. N. and Littleton J. T. (2008). Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth. J. Neurosci. 28(33): 8316-25. PubMed Citation: 18701694

Rothwell, W. F., Fogarty, P., Field, C. M. and Sullivan, W. (1998). Nuclear-fallout, a Drosophila protein that cycles from the cytoplasm to the centrosomes, regulates cortical microfilament organization. Development. 125: 1295-1303. 9477328

Rothwell, W. F., Zhang C. X., Zelano, C., Hsieh T. S. and Sullivan. W (1999). The Drosophila centrosomal protein Nuf is required for recruiting Dah, a membrane associated protein, to furrows in the early embryo. J. Cell Sci. 112: 2885-2893. 10444383

Roosterman, D., Schmidlin, F. and Bunnett, N. W. (2003). Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2. Am. J. Physiol. Cell Physiol. 284: C1319-C1329. 12540381

Satoh, A. K., Tokunaga, F., Kawamura, S. and Ozaki, K. (1997). In situ inhibition of vesicle transport and protein processing in the dominant negative Rab1 mutant of Drosophila. J. Cell Sci. 110: 2943-2953. 9359879

Satoh, A. K. (1998). Rab proteins involved in the rhodopsin transport in Drosophila. In Department of Biology, pp. 124. Osaka: Osaka University.

Satoh, A. K., O'Tousa, J. E., Ozaki, K. and Ready, D. F. (2005). Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors. Development 132(7): 1487-97. 15728675

Savina, A., Vidal, M. and Colombo, M. I. (2002). The exosome pathway in K562 cells is regulated by Rab11. J. Cell Sci. 115: 2505-2515.

Savina, A., Fader, C. M., Damiani, M. T. and Colombo, M. I. (2005). Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner. Traffic 6(2): 131-43. 15634213

Segev, N. (2001). Ypt/rab GTPases: regulators of protein trafficking. Sci. STKE. 2001:RE11. 11579231

Sheff, D., Pelletier, L., O'Connell, C. B., Warren, G. and Mellman, I. (2002). Transferrin receptor recycling in the absence of perinuclear recycling endosomes. J. Cell Biol. 156: 797-804. 11877458

Shetty, K. M., Kurada, P. and O'Tousa, J. E. (1998). Rab6 regulation of rhodopsin transport in Drosophila. J. Biol. Chem. 273: 20425-20430. 9685396

Shimada, A., et al. (2007). Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129: 761-772. PubMed Citation: 17512409

Shimizu, H., Kawamura, S. and Ozaki, K. (2003). An essential role of Rab5 in uniformity of synaptic vesicle size. J. Cell Sci. 116: 3583-3590. 12876219

Skop, A. R., et al. (2001). Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex. Curr. Biol. 11: 735-746. 11378383

Sonnichsen, B., et al. (2000). Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. 149: 901-914. 10811830

Sullivan, W., Fogarty, P. and Theurkauf, W. (1993). Mutations affecting the cytoskeletal organization of syncytial Drosophila embryos. Development. 118: 1245-1254. 8269851

Szatmari, Z., Kis, V., Lippai, M., Hegedus, K., Farago, T., Lorincz, P., Tanaka, T., Juhasz, G. and Sass, M. (2013). Rab11 facilitates crosstalk between autophagy and endosomal pathway through regulation of Hook localization. Mol Biol Cell. [Epub ahead of print]. PubMed ID: 24356450

Tsujita, K., et al. (2006). Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J. Cell Biol. 172: 269-279. PubMed Citation: 16418535

Udayar, V., Buggia-Prevot, V., Guerreiro, R. L., Siegel, G., Rambabu, N., Soohoo, A. L., Ponnusamy, M., Siegenthaler, B., Bali, J., Aesg, Simons, M., Ries, J., Puthenveedu, M. A., Hardy, J., Thinakaran, G. and Rajendran, L. (2013). A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of β-Amyloid production. Cell Rep 5: 1536-1551. PubMed ID: 24373285; Graphical Abstract

Ullrich, O., Reinsch, S., Urbe, S., Zerial, M. and Parton, R. G. (1996). Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135: 913-924. 8922376

Urbe, S., Huber, L. A., Zerial, M., Tooze, S. A. and Parton, R. G. (1993). Rab11, a small GTPase associated with both constitutive and regulated secretory pathways in PC12 cells. FEBS Lett. 334: 175-182. 8224244

Wallace, D. M., Lindsay, A. J., Hendrick, A. G. and McCaffrey, M. W. (2002a). The novel Rab11-FIP/Rip/RCP family of proteins displays extensive homo- and hetero-interacting abilities. Biochem. Biophys. Res. Commun. 292(4): 909-15. 11944901

Wallace, D. M., Lindsay, A. J., Hendrick, A. G. and McCaffrey, M. W. (2002b). Rab11-FIP4 interacts with Rab11 in a GTP-dependent manner and its overexpression condenses the Rab11 positive compartment in HeLa cells. Biochem. Biophys. Res. Commun. 299(5): 770-9. 12470645

Wan, P., Wang, D., Luo, J., Chu, D., Wang, H., Zhang, L. and Chen, J. (2013). Guidance receptor promotes the asymmetric distribution of exocyst and recycling endosome during collective cell migration. Development 140(23): 4797-806. PubMed ID: 24198275

Wang, W. and Struhl, G. (2004). Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch. Development 131: 5367-5380. 15469974

Wang, X., Kumar, R., Navarre, J., Casanova, J. E. and Goldenring, J. R. (2000). Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25. J. Biol. Chem. 275: 29138-29146. 10869360

Wang, X., He, L., Wu, Y. I., Hahn, K. M. and Montell, D. J. (2010). Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat. Cell Biol. 12: 591-597. PubMed Citation: 20473296

Wilson, G. M., et al. (2005). The FIP3-Rab11 protein complex regulates recycling endosome targeting to the cleavage furrow during late cytokinesis. Mol. Biol. Cell 16(2): 849-60. 15601896

Wu, S., et al. (2005). Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo. Nat. Struct. Mol. Biol. 12(10): 879-85. 16155582

Xu, J., et al. (2011). Rab11 is required for epithelial cell viability, terminal differentiation, and suppression of tumor-like growth in the Drosophila egg chamber. PLoS One 6(5): e20180. PubMed Citation: 21629779

Xiong, B., Bayat, V., Jaiswal, M., Zhang, K., Sandoval, H., Charng, W. L., Li, T., David, G., Duraine, L., Lin, Y. Q., Neely, G. G., Yamamoto, S. and Bellen, H. J. (2012). Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells. PLoS Biol 10: e1001438. PubMed ID: 23226104

Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2: 107-117. 11252952

Zhang, C. X., et al. (1996). Isolation and characterization of a Drosophila gene essential for early embryonic development and formation of cortical cleavage furrows. J. Cell Biol. 134: 923-934. 8769417

Zhang, C. X., Rothwell, W. F., Sullivan, W. and Hsieh, T. S. (2000). Discontinuous actin hexagon, a protein essential for cortical furrow formation in Drosophila, is membrane associated and hyperphosphorylated. Mol. Biol. Cell. 11: 1011-1022. 10712516

Zhang, X. M., Ellis, S., Sriratana, A., Mitchell, C. A. and Rowe, T. (2004). Sec15 is an effector for the Rab11 GTPase in mammalian cells. J. Biol. Chem. 279(41): 43027-34. 15292201

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

date revised: 30 September 2016

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