Rab-protein 11


Bchs, a BEACH domain protein, antagonizes Rab11 in synapse morphogenesis and other developmental events

BEACH proteins, an evolutionarily conserved family characterized by the presence of a BEACH (Beige and Chédiak-Higashi) domain, have been implicated in membrane trafficking, but how they interact with the membrane trafficking machinery is unknown. This study shows that the Drosophila BEACH protein Bchs (Blue cheese) acts during development as an antagonist of Rab11, a small GTPase involved in vesicle trafficking. Reduction in, or loss of, bchs function restores viability and normal bristle development in animals with reduced rab11 function, while reductions in rab11 function exacerbate defects caused by bchs overexpression in the eye. Consistent with a role for Bchs in modulating Rab11-dependent trafficking, Bchs protein is associated with vesicles and extensively colocalized with Rab11 at the neuromuscular junction (NMJ). At the NMJ, rab11 is found to be important for synaptic morphogenesis; reductions in rab11 function cause increases in bouton density and branching. These defects are also suppressed by loss of bchs. Taken together, these data identify Bchs as an antagonist of Rab11 during development and uncover a role for these regulators of vesicle trafficking in synaptic morphogenesis. This raises the interesting possibility that Bchs and other BEACH proteins may regulate vesicle traffic via interactions with Rab GTPases (Khodosh, 2006; full text of article)

While several BEACH-family proteins have been implicated in vesicle trafficking, the mechanisms through which they may regulate this process are unknown. Bchs, the Drosophila relative of the human Alfy protein, is a functional antagonist of the vesicle-trafficking regulator Rab11. In particular, reduction in bchs strongly suppresses the defects in viability, synaptic morphogenesis and bristle extension exhibited by rab11 loss-of-function mutants. Additionally, reduction in rab11 activity strongly enhances the eye phenotype caused by bchs gain of function. The reciprocity of genetic interactions between these two genes provides compelling evidence that Bchs participates in many of the same processes as Rab11 and, thus, suggests that Bchs, like Rab11, is a regulator of vesicle trafficking (Khodosh, 2006).

The subcellular localization of Bchs also supports the hypothesis that this protein functions in membrane traffic. Bchs was present exclusively in membrane fractions and exhibited punctate staining in the presynaptic motoneuron terminals and in the muscles at the NMJ. This pattern is consistent with the localization of Bchs to a membrane-bound organelle. Furthermore, in line with a functional relationship between Bchs and Rab11, significant subcellular co-localization of Bchs and Rab11 was observed at the NMJ, as well as partial overlap in the distribution of Bchs and Rab11 within membrane fractions. These data further support the hypothesis that Bchs regulates vesicle trafficking and that it may do so via an interaction with the Rab11 GTPase (Khodosh, 2006).

A prominent bchs phenotype was the suppression of the rab11 sensory bristle defects, which entails both shortened and missing bristles. These defects probably arise from alterations in membrane traffic. The extension of sensory bristles involves several vesicle trafficking steps, including membrane addition at the tip of the growing bristle and the secretion of cuticle to support the bristle cell. The complete loss of mechanosensory bristles could result from extremely impaired bristle growth. Alternatively, bristle loss could arise from a cell fate transformation that prevents the specification of the bristle-producing cell, since Rab11-mediated vesicle trafficking has also been implicated in the asymmetric cell divisions of the precursors that give rise to these cells. The ability of mutations in bchs to strongly suppress all rab11 bristle defects implicates Bchs in bristle morphogenesis and is consistent with it playing a role in Rab11-mediated vesicle trafficking (Khodosh, 2006).

A crucial role for rab11 was uncovered in the formation of the Drosophila NMJ: rab11 mutants exhibit an increase in the density and branching of synaptic boutons and a decrease in the size of the muscles. Vesicle trafficking is important in determining the number and morphology of boutons at the NMJ. In sculpting the synapse, membrane traffic is needed not only for the addition of new membrane and active zone proteins, but also for the insertion, removal and signaling of regulatory molecules at the cell surface. Furthermore, exocyst-dependent membrane addition is required for the expansion of NMJs, and Rab11 is a known regulator of exocyst function. Thus, Rab11 is involved in synaptic morphogenesis at the NMJ, probably via regulation of vesicle trafficking (Khodosh, 2006).

By virtue of suppressing rab11 NMJ phenotypes, Bchs is also implicated in a membrane-trafficking aspect of synaptic morphogenesis. Consistent with such a model, both Bchs and Rab11 showed punctate localization and partial overlap at the NMJ. A functional role of Bchs in presynaptic development may explain its concentration in the axonal rather than dendritic compartment of ellipsoid body neurons (Khodosh, 2006).

The link between Bchs and Rab11 function provides initial mechanistic insights into the trafficking pathways that may involve Bchs. Rab11 is involved in both biosynthetic exocytic traffic and membrane traffic through the recycling endosome. As the loss of bchs suppresses lethality of rab11 alleles, it is likely to be involved in all the essential functions of Rab11 (Khodosh, 2006).

The partial colocalization of Bchs and Rab11 suggests candidate sites for the function of Bchs. In particular, Rab11 has been observed on the trans-Golgi network, post-Golgi vesicles, recycling endosomes and vesicles that travel from the recycling endosome to the plasma membrane. In regulating traffic to the plasma membrane, Rab11 has been shown to physically interact with members of the exocyst complex. The distribution of Bchs is highly polarized in neurons and enriched at synaptic endings, not cell bodies or dendrites. This suggests that interactions between Bchs and Rab11 may occur in a compartment adjacent to the presynaptic plasma membrane, rather than near the trans-Golgi network, the perinuclear recycling endosome or dendritic endosomes (Khodosh, 2006).

At the synapse, the most prominent membrane trafficking pathway is the synaptic vesicle cycle. However, Bchs immunoreactivity did not colocalize with synaptic vesicles or co-migrate with them in cell fractionation. These findings, together with the fact that loss of bchs does not alter the viability of the mutants, suggests that Bchs does not play a major role in the release of neurotransmitter. In this regard, Bchs is clearly distinct from Neurobeachin, a Beach-domain protein that is essential for transmitter release at the mouse NMJ. Bchs did not colocalize with early endosomes, marked with either 2XFYVE-GFP or Rab5-GFP, through which at least some synaptic vesicles are thought to cycle, nor did Bchs immunoreactivity resemble the distribution of endocytic vesicles identified by clathrin-GFP. The former was perhaps surprising, given that Bchs possesses a FYVE domain. These data suggest that Bchs is unlikely to be involved in early endocytic events. The BEACH domain protein Lyst has been implicated in lysosomal trafficking; however, no overlap of Bchs immunoreactivity with lysotracker was observed. Bchs is, therefore, not a lysosomal protein. Rather, Bchs appears to reside on a novel synaptic compartment adjacent to or overlapping a Rab11-containing organelle. It is speculated that this may be a previously unappreciated presynaptic sorting endosome through which either recycled or Golgi-derived proteins are trafficked (Khodosh, 2006).

What cellular and molecular mechanisms underlie the extensive genetic interactions between bchs and rab11? In one scenario, Bchs could negatively regulate Rab11 activity, perhaps by promoting a Rab11-GAP that restricts Rab11 function. Bchs could modulate the efficacy of Rab11 function, but might be only one of several negative regulators of Rab11. Such a model would be consistent with the genetic studies, since loss of bchs might not cause defects on its own, but bchs overexpression would shut down the Rab11 pathway. Alternatively, rab11 and bchs could be involved in competing intracellular pathways. For example, Rab11 might direct endosomal cargos toward the plasma membrane, while Bchs diverts these cargos elsewhere. This hypothesis receives support from the observation that Rab11 and Bchs appear to concentrate in partially distinct subcompartments of those organelles on which they both reside. This pattern of partially overlapping localizations is reminiscent of other pairs of regulators of membrane traffic, including Rab4 and Rab5, and Rab4 and Rab11 on two sequential, yet distinct, populations of endosomes. The distribution of these Rabs reflects their participation in linked steps of cargo transport along the recycling pathway, which may also explain the localization pattern of Bchs and Rab11 (Khodosh, 2006).

In addition to Bchs, other BEACH family proteins, such as Lyst and Neurobeachin, have been implicated in vesicle trafficking. Mutations in Lyst result in the accumulation of giant lysosomes in the cells of both beige mutant mice and Chédiak-Higashi syndrome patients, suggesting that Lyst is involved in lysosome trafficking, fusion or formation. Mouse Neurobeachin mutants lack evoked synaptic transmission at the NMJ, implicating neurobeachin in neurotransmitter release. The finding that Bchs antagonizes Rab11 raises the intriguing possibility that other BEACH proteins might also interact with Rab family members. It will be interesting to determine whether the defects observed in Lyst and neurobeachin mutants reflect the altered activity of particular Rabs and whether alterations in Rab function could ameliorate defects caused by the absence of Lyst or Neurobeachin (Khodosh, 2006).

The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation

Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, this study demonstrates that microtubules are required for proper metaphase furrow formation. It is proposed that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows (Riggs, 2007; full text of article).

Microtubule-based motility has been implicated in many steps in endocytosis, and there is increasing evidence that it influences the distribution and activity of endocytic organelles. The work presented in this study suggests that motor-based movement of Rab effectors may be another means of regulating endosomal activity. Previous studies have shown that the Drosophila Rab11 effector, Nuf, is required for stable Rab11 localization at the RE and thus RE activity. Nuf concentrates at the MTOC during interphase through prophase and disperses into the cytoplasm at metaphase. This study demonstrates that Nuf relies on microtubules and minus-end microtubule motor Dynein both for its accumulation and maintenance at the MTOC. This raises the possibility that the Dynein-dependent delivery of Nuf to the RE may play a role in regulating Rab11 activity at the RE. Significantly maximal localization of Nuf at the MTOC-associated RE occurs during late interphase and prophase. This is the time of the establishment and formation of the metaphase furrows, which rely on RE-based vesicle delivery (Riggs, 2007).

Immunoprecipitation data demonstrates a physical interaction between Nuf and Dynein. This raises the possibility that the cell cycle-regulated localization of Nuf at the MTOC is mediated by a corresponding cell cycle-regulated interaction between Nuf and Dynein. Support for this idea comes from a study in vertebrate cells, demonstrating that Polo-like kinase (Plk) mediated phosphorylation of Ninein-like protein (Nlp), a microtubule-nucleating protein, directly determines its cell cycle-regulated localization at the centrosome. Like Nuf, Nlp localizes to the centrosome by associating with the minus-end-directed motor protein Dynein. As cells progress into metaphase, Plk is activated and phosphorylates Nlp on sites that are required for its association with Dynein. This disrupts Nlp ability to associate with Dynein and results in loss of Nlp from the centrosome (Riggs, 2007).

There is a strong correlation between maximal Nuf localization at the MTOC and furrow invagination. During the cortical divisions, furrow invagination and maximal Nuf concentration at the MTOC occurs during prophase. During cellularization, furrow invagination and maximal Nuf concentration at the MTOC occurs during interphase. Stable localization of Nuf and Rab11 at the MTOC during cellularization enabled a demonstration that microtubules are continuously required for maintaining Nuf and Rab11 at the MTOC. Colchicine-induced disruption of the interphase microtubules results in the rapid loss of Nuf from the MTOC. One interpretation of this result is that colchicine disrupts MTOC organization, which is required for maintaining Nuf at the MTOC. In contrast to the colchicine injections, injecting anti-Dynein antibody does not alter microtubule organization and results in a slow steady decrease of Nuf at the MTOC. This result suggests that the steady-state level of Nuf at the MTOC is maintained by continuous Dynein-dependent recruitment of Nuf to the MTOC. This also implies that Nuf is continuously released from the MTOC as well. The mechanism driving the release is unclear. Previous live analysis revealed vectorial movement of Nuf away from the centrosome, suggesting that it may rely on a kinesin, a plus-end-directed microtubule motor. If kinesin is involved, this implies that the balance between plus- and minus-end motor activities dictates whether Nuf is concentrated at the MTOC or dispersed in the cytoplasm. Recent work indicates that the positioning and activity of the early endosome is mediated through a balance of plus- and minus-end motor activities. In addition, investigations into cellular furrow elongation demonstrated that Lava lamp, a Golgi-associated protein, is complexed with Dynein and is responsible for Golgi-based movements necessary for latter half of furrow elongation (Riggs, 2007 and references therein).

The above studies demonstrate that microtubules are continuously required for proper Nuf localization at the MTOC. This raises the possibility that microtubule-based localization of Nuf at the MTOC is necessary for its association with the Rab11 and proper RE function. Because RE function is necessary for metaphase furrow formation, this predicts that microtubules are required for proper metaphase furrow formation. However previous studies did not observe defects in furrow formation when embryos were treated with microtubule inhibitors. It has been concluded that microtubules are dispensable for proper metaphase furrow formation in the early embryo. This issue was reexamined by injecting microtubule inhibitors at precise times throughout the cell cycle during the syncytial divisions. Because disrupting the microtubules at metaphase activates the spindle assembly checkpoint, the embryos were injected immediately after entry into anaphase. In these experiments, the nuclear cycle progressed normally but formation of the metaphase furrows were profoundly disrupted. Incorporation of GFP-tagged Moesin into the furrows that form at the next prophase completely fails. Thus these experiments define anaphase as a key time in which microtubules are required for recruiting actin to the furrows that form in the following prophase. The previous study failed to appreciate the role of microtubules in metaphase furrow formation because it was not possible to produce disruptions in the microtubule network at defined stages of the cell cycle (Riggs, 2007).

These studies also revealed that injecting colchicine at telophase produced no defects in actin recruitment. Similar injections at interphase through prophase also produced no defects in actin recruitment to the metaphase furrows. One interpretation of these results is that microtubules are specifically required during anaphase but not telophase or later for furrow formation in the next prophase. However it must be pointed the different classes of microtubules are differentially sensitive to microtubule inhibitors. Thus this differential sensitivity may contribute to the observed cell phase sensitivity of metaphase furrow formation to colchicine (Riggs, 2007).

That microtubules are required during anaphase for metaphase furrow formation in the following prophase is significant for a number of reasons. First, these studies support, although certainly do not prove, a model in which microtubule-based transport of Nuf to the MTOC is necessary for normal metaphase furrow formation. Second, anaphase/telophase is the point at which the metaphase furrows begin to regress. Thus the timing of furrow regression corresponds to the time at which microtubules are involved in establishing the next round of furrow formation. This indicates that the speed of the cortical divisions is not only achieved by an accelerated nuclear cycle but also by overlapping furrow regression with furrow formation. During anaphase, the replicated centrosomes possess robust astral arrays and the midbody has not yet fully formed. It is hypothesized that the plus ends of these overlapping arrays from neighboring centrosomes define the position of the metaphase furrow in the next cell cycle. This readily explains why furrows encompass the spindle and do not form at the midzone microtubules. Finally, although the furrows form at prophase, these studies identify anaphase as a critical time in which furrow is established. This also corresponds to the time at which microtubules are required during conventional furrow formation (Riggs, 2007).

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

Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. This study reports that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated Mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, these results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insigfhts into the pathogenesis of ACSL4-related mental retardation (Liu, 2014).

The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11

Myosin-5 (DmM5; Didum) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. This study overexpressed DmM5 in Sf9 cells and investigated its regulation using purified proteins. The actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid and dRab11 respectively. GTP-bound dRab11, but not Lightoid, significantly activates DmM5 actin-activated ATPase activity. Moreover, Gln1689 in the GTD was identified as the critical residue for the interaction with dRab11. Based on those results, it is proposed that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx (Ji, 2021).

Three mechanisms control E-cadherin localization to the zonula adherens

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

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

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

Non-autonomous role of Cdc42 in cell-cell communication during collective migration

Collective cell migration is involved in numerous processes both physiological, such as embryonic development, and pathological such as metastasis. Compared to single cell migration, collective motion requires cell behaviour coordination through an as-yet poorly understood but critical cell-cell communication mechanism. Using Drosophila border cell migration, this study shows that the small Rho GTPase Cdc42 regulates cell-cell communication. Indeed, Cdc42 controls protrusion formation in a cell non-autonomous manner. Moreover, the endocytic small GTPase Rab11 was found to control Cdc42 localisation to the periphery of migrating border cell clusters. Accordingly, over-expression of Cdc42 in border cells rescues the loss of Rab11 function. Thus, this study positions Cdc42 as a new key player in cell-cell communication, acting downstream of Rab11 (Colombie, 2017).

dOCRL maintains immune cell quiescence by regulating endosomal traffic

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. This study shows that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that identified in docrl mutant hemocytes, the cause of innate immune cell activation was pinpointed to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spatzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome (Del Signore, 2017).

Protein Interactions

The p150-Spire protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spire proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spire encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spire family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as 'Spir-box'. The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of Drosophila p150-Spire and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spire protein colocalizes specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spire localization pattern is dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibits the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein arrests in membrane structures, largely colocalizing with the TGN marker TGN46. The findings that the Spire actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport (Kerkhoff, 2001).

Actin cytoskeleton remodeling during early Drosophila furrow formation requires interacting endosomal components Nuclear-fallout and Rab11

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition via vesicle fusion. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two remodeling processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homolog of arfophilin-2, an ADP ribosylation factor (ARF; see InterPro's ADP-ribosylation factor) effector that binds Rab11 (and Arf5) and influences recycling endosome (RE) organization. Nuf has been shown to be an important component of the RE; these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component implicated in vesicle targeting. Tests for protein interaction and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation. Membrane addition is mediated via endosomal-mediated membrane delivery to the site of furrow formation (Riggs, 2003).

It has been proposed that endosomes are organized into distinct domains defined by combinations of Rab proteins (Zerial, 2001). These provide a platform for regulatory/effector proteins to create a distinct fusion-competent domain. The proteins are thought to act cooperatively, and loss of one may destabilize the domain. Nuf and Rab11 may be mutually required for the stable formation of such a domain at the RE of the Drosophila embryo (Riggs, 2003).

The production of two daughter cells at the end of mitosis is accomplished through a dramatic constriction of the plasma membrane. This is known as cytokinesis and involves the formation of an actin/myosin-based contractile ring that forms perpendicular to and midway between the anaphase spindle. In animal cells, the position of the mitotic spindle largely determines the position and orientation of the contractile ring. Actin, myosin II, and other furrow components (such as anillin and the septins) are recruited to this site and form the contractile ring. Once the contractile ring forms, constriction of the plasma membrane occurs (Riggs, 2003 and references therein).

Although the mechanism of constriction is contractile, recent reports have begun to define the role of membrane addition in this process. A cell undergoing cytokinesis requires significant additional membrane to accommodate the increased surface area of producing two daughter cells. Work in Xenopus relying on a variety of surface-marking techniques indicates that the additional membrane has a different composition from the original membrane. This suggests that the membrane is not derived from the expansion of preexisting surface membrane, but instead forms through insertion of membrane from internal stores. In plant cells, it is well established that the additional membrane necessary for cytokinesis is provided through a Golgi-based delivery system. In Caenorhabditis elegans ovaries, RNA interference inhibition of Rab11, the small GTPase required for vesicle transport through the recycling endosome, causes cytokinesis defects, including furrow regression and scission (Skop, 2001). Mutation and RNA interference analyses demonstrate that the t-SNARE syntaxin 1 is required for cytokinesis during early embryogenesis. Lamellar bodies, the ER, and internal lipid stores may also prove important in providing membrane for cytokinesis furrows (Riggs, 2003 and references therein).

The rapid and simultaneous formation of thousands of furrows during early Drosophila embryogenesis makes this system particularly valuable for studying the recruitment of membrane and other furrow components during cytokinesis. Drosophila development begins with 13 synchronous, rapid, syncytial nuclear divisions. After nine divisions in the interior of the embryo, divisions 10-13 occur in the actin-rich cortex, just beneath the plasma membrane. The nuclei and their associated centrosomes induce a dramatic redistribution of the cortical actin. During interphase, actin concentrates into caps centered above each cortical nucleus and its apically positioned centrosomes. As the nuclei progress into prophase, the centrosomes migrate toward opposite poles and the actin caps undergo a dramatic redistribution to form an oblong ring outlining each nucleus and its associated separated centrosome pair. These rings are equivalent in composition to conventional cytokinesis contractile rings and include actin, myosin II, spectrins, cofilin, ARP, anillin, septins, and formins. In addition, these components are closely associated with the plasma membrane and are required for the invagination of these rings around the spindles. These rings are referred to as metaphase or pseudocleavage furrows. At metaphase, the furrows invaginate to a depth of ~8 microm to form a half shell that encompasses each spindle. During late anaphase and telophase, the metaphase furrows rapidly regress. Centrosome duplication occurs during late anaphase, and the newly formed centrosome pairs locate apically. The actin caps reform directly above the centrosome pairs in the next interphase. This alternation between interphase actin caps and metaphase furrows occurs until interphase of nuclear cycle 14. At this point, the nuclei remain in interphase and an inverted microtubule basket, which originates from an apically positioned centrosome pair, guides invagination of the cellularization furrows. At a depth of micro5 µm, the furrows pinch off at their base to form individual mononucleate cells (Riggs, 2003 and references therein).

Genetic and biochemical analyses indicate that vesicle fusion plays an important role in furrow formation in early Drosophila embryogenesis. Mutations in dynamin, a GTPase involved in endocytic vesicle formation, disrupt cellular furrow formation and result in an abnormal accumulation of vesicles in the cytoplasm. Unconventional myosin VI has been shown to be involved in the transport of cytoplasmic particles in the Drosophila embryo, and mutations in this gene cause defects in formation of the metaphase furrows. alpha-Adaptin, a coated vesicle component necessary for receptor-mediated endocytosis, is concentrated apically and laterally around the metaphase and cellularization furrows. Syntaxin 1, a t-SNARE involved in vesicle targeting, is also required for cellularization in Drosophila. Inhibition of Golgi-based vesicle transport inhibits progression of the cellularization furrow front. In addition, a major source of this membrane necessary for the cellularization furrows is derived internally rather than from the plasma membrane (Riggs, 2003 and references therein).

Activities associated with the centrosome are also important for vesicle-mediated metaphase and cellular furrow formation. Insights into the centrosome-associated activities directing these rearrangements have come from the analysis of the maternal effect mutation, nuclear fallout (nuf). Nuf encodes a pericentrosomal protein that is essential for normal metaphase and cellularization furrow formation. Nuf concentrates at the centrosomes during prophase, when metaphase furrows are forming (Rothwell, 1998). In the nuf mutation, microtubule dynamics and distribution appear normal, but remodeling and recruitment of actin to the furrows is disrupted and actin remains abnormally concentrated around the centrosomes. Vesicle-based membrane recruitment to the furrows is also disrupted in nuf-derived embryos (Rothwell, 1999; Zhang, 2000). These phenotypes lead to the intriguing suggestion that a common mechanism mediates actin remodeling and membrane addition during cytokinesis (Riggs, 2003 and references therein).

Additional insight into these two processes is provided by demonstrating that Nuf is a component of the RE, and nuf phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a member of the Rab family of small GTPases specific to the RE. In addition, Rab11 and Nuf exhibit a mutual dependence for their normal localization to the RE. Rab11-deficient embryos produce metaphase and cellular furrow defects strikingly similar to those observed in nuf-derived embryos. In accord with these results, recent reports demonstrate that Nuf is a homolog of arfophilin-2 (Arfo2), an ADP ribosylation factor (Arf) effector that also binds Rab11 and influences RE organization (Hickson, 2003). Together, these reports suggest that actin remodeling during the initial stages of cytokinesis may in part rely on endosomal-mediated membrane delivery to the site of furrow formation (Riggs, 2003).

Nuf and Arfo2 are functionally as well as structurally related. In HeLa cells, Arfo2 localizes to the perinuclear TGN with staining also observed at the centrosomes and focal adhesions (Hickson, 2003). In Drosophila, Nuf has a similar localization at the centrosomes (Rothwell, 1998). Overexpression of either Drosophila Nuf or human Arfo2 in mammalian cells results in a collapse of the late RE to a pericentrosomal region (Hickson, 2003). These observations suggest that Nuf and Arfo2 are functionally similar and play a role in maintaining the integrity of the RE (Riggs, 2003).

Nuf is a structural and functional homolog of Arfo2 (Hickson, 2003) and contains a highly conserved 20-aa Rab11-binding site. This binding domain was first identified by Prekeris (2001) and Hales (2001) as important for the interaction between Rab11 and a novel family of putative Rab11 effector proteins. Within this domain, Nuf and Arfo2 contain eight identical and six conserved amino acids. Nuf and hRip11, a mammalian Rab11 effector protein, contain ten identical and three conserved amino acids. This sequence conservation, combined with the colocalization results, prompted an examination of whether Nuf and Rab11 physically interact. Bacterially expressed GST-Rab11 was mixed with CHO cells transiently expressing GFP-Nuf. GTPgammaS and GDPßS were added to the buffer to test the nucleotide specificity of the interaction. GFP-Nuf is effectively pulled down by both GST-Rab11+GTPgammaS and GST-Rab11+GDPßS, indicating that the interaction is not tightly linked to the state of the nucleotide. GST-Rab11+GDPßS pulls down Nuf to a lesser extent than GST-Rab11+GTPgammaS. Nucleotide-independent binding has also been observed with other Rab11 effectors, Rab11-FIP2 (Hales, 2001) and Arfo2, the mammalian homolog of Nuf (Hickson, 2003). To test the specificity of the interaction, similar pull-down experiments were performed with Rab5, a component of the early endosome. Unlike the results with GST-Rab11, GFP-Nuf is not pulled down by GST-Rab5 in either the activated or unactivated form (Riggs, 2003).

To determine if Nuf is required for pericentriolar Rab11 localization, Rab11 localization was examined in nuf-derived embryos. Rab11 exhibits a concentrated punctate distribution around the centrosome during prophase. In nuf-derived embryos, both the punctate distribution and concentration of Rab11 around the centrosomes is completely abolished. Although levels of Nuf at the centrosome are greatly reduced during metaphase, Nuf is required for Rab11 centrosome localization at this stage as well. Nuf is also required for Rab11 localization during cellularization. The robust tight localization of Rab11 around the centrosome during cellularization is absent in nuf-derived embryos. It is believed that mislocalization of Rab11 in nuf is not a result of a general disruption of the intracellular transport pathway, since staining with Golgi marker Lava-lamp revealed normal Golgi distribution throughout the cell cycle in wild-type and nuf-derived embryos. From this analysis, it cannot be determined whether levels of Rab11 protein are reduced in nuf-derived embryos (Riggs, 2003).

Whether Rab11 is required for normal pericentriolar Nuf localization was also examined. Because Rab11 is an essential gene, a combination of hypomorphic rab11 alleles were used that permitted normal zygotic development (Jankovics, 2001). However, these transheterozygote females produced embryos with reduced levels of maternally supplied Rab11 and showed a reduced hatch rate. Wild-type and rab11-derived embryos were double stained for Nuf and DNA, and were examined during the syncytial divisions and cellularization. During prophase, while the pericentriolar localization of Nuf was robust in control embryos, pericentriolar Nuf levels were absent in rab11-derived embryos. The same result was obtained when cellularizing rab11-derived embryos were examined; the normal pericentriolar localization of Nuf is completely abolished. From this analysis, it cannot be determined whether levels of Nuf protein are reduced in rab11-derived embryos. These experiments demonstrate that Nuf and Rab11 are mutually dependent on one another for their localization to the RE (Riggs, 2003).

The nuf maternal-effect mutation specifically disrupts syncytial nuclear divisions only after the nuclei migrate to the cortex (Sullivan, 1993). These nuclear defects are a consequence of incomplete metaphase furrow formation, which allows inappropriate fusions between nonsister nuclei (Rothwell, 1998). Although the interphase actin caps form normally, large gaps are present in the metaphase and cellularization furrows. The gaps are observed in the earliest stages of furrow formation, suggesting that Nuf disrupts recruitment of actin to the furrows rather than in stabilization of actin once at the furrows. To determine if reduced maternal supplies of Rab11 produce cortical phenotypes similar to those observed in nuf mutations, rab11 transheterozygotes were used. The nuclear phenotype is equivalent to nuf. In rab11-derived embryos, nuclear distribution and morphology is normal in premigration and early cortical blastoderm embryos. However, during the late cortical divisions when the nuclei are more densely packed, the nuclear distribution and morphology is disrupted. In premigration and early cortical embryos, 8% (2/23) exhibit disrupted nuclear morphology. During the late cortical divisions, 65% (31/48) exhibit severely disrupted nuclear morphology. This is indicative of defects in the metaphase furrows that serve to separate neighboring nonsister nuclei (Riggs, 2003).

To examine the role of Rab11 in organizing the cortical cytoskeleton and metaphase furrows, wild-type, nuf-derived, and rab11-derived cortical nuclear cycle 12 embryos were double stained for DNA and actin. During interphase, actin organizes into caps apically positioned above each nucleus. In nuf- and rab11-derived embryos, actin cap formation occurs normally. As the embryos progress into prophase, the actin caps are dismantled and actin reorganizes into furrows encompassing each prophase nucleus and its developing spindle. As the nuclei progress into metaphase, these furrows become more pronounced and tightly focused. The actin-based furrow defects in rab11-derived embryos are strikingly similar to those observed in nuf-derived embryos. In both, the hexagonal furrow network is riddled with gaps. The gaps are present at prophase during the initial stages of furrow formation, suggesting defects in the initial actin recruitment. nuf and rab11 mutations also produce similar defects during cellularization at nuclear cycle 14, although defects in nuf-derived embryos are much more extensive than observed in rab11-derived embryos. This difference may be a result of partial zygotic rescue by the paternally supplied rab+ allele (Riggs, 2003).

nuf-derived embryos disrupt recruitment of membrane components during furrow invagination. The Drosophila protein Discontinuous actin hexagon (Dah) tightly associates with the plasma membrane as well as actin, and is thought to link cortical microfilaments to the plasma membrane (Zhang, 1996). In cortical Drosophila embryos, Dah localizes to the plasma membrane as well as to vesicles that concentrate at the leading edge of the invaginating furrows. Analysis of Dah mutations indicates that incorporation of these vesicles into the plasma membrane contributes to furrow invagination (Rothwell, 1999). To determine the role of Rab11 and Nuf in Dah-associated vesicle delivery, wild-type, nuf-derived, and rab11-derived embryos were double stained for actin and Dah. In nuf-derived embryos, incorporation of Dah into the metaphase furrows is dramatically reduced. Although Dah vesicles are observed, they are more randomly distributed throughout the cytoplasm. A similar defect is observed in rab11-derived embryos; incorporation of Dah into the invaginating metaphase furrows is disrupted. However, in contrast to nuf, Dah staining is not observed in the furrow regions and few Dah-staining vesicles are visible (Riggs, 2003).

The fact that both Nuf and Arfo2 contain a conserved Rab11-binding domain provides additional support for a common function at the RE. Similar to Arfs, Rabs are members of a large family of small GTPases involved in the regulation of vesicle-trafficking pathways (Segev, 2001). However, unlike Arfs, Rabs are thought to be involved in vesicle targeting rather than vesicle biogenesis. Rab11 is primarily localized at the RE and plays an essential role in receptor-mediated recycling to the plasma membrane (Ullrich, 1996; Sheff, 2002). In addition, the Rab11 GTPase cycle is essential for normal RE organization and function (Ullrich, 1996). Sequence analysis of Arfo2 and Nuf reveals a common conserved 20-aa Rab11-binding domain originally identified among members of the Rab11-interacting protein family (Hales, 2001; Prekeris, 2001). In accord with this observation, Arfo2 and Nuf physically interact with Rab11 (Riggs, 2003).

Nuf is primarily associated with the RE in the early Drosophila embryo. Nuf shows extensive colocalization with Rab11. The most significant difference between the distribution of Rab11 and Nuf in the early embryo is that the former maintains a constant level of pericentriolar staining, whereas levels of the latter oscillate with the cell cycle. During the cortical syncytial divisions, pericentriolar Nuf staining is at its highest levels at prophase and negligible during metaphase and anaphase. It is not known whether this is a result of cycling of Nuf levels, subcellular location, or both. At nuclear cycle 14, Nuf levels are highest during interphase as the cellularization furrows are forming. Thus, maximal pericentriolar levels of Nuf are correlated with metaphase and cellular furrow formation and invagination. Nuf is highly phosphorylated (Rothwell, 1998), raising the possibility that its localization and/or levels may be regulated by cell cycle–dependent kinases (Riggs, 2003).

Further evidence that Nuf is intimately associated with pericentriolar endosomal material comes from live analysis of Nuf dynamics in the early embryo. This analysis reveals a dynamic punctate distribution of Nuf rapidly moving to and from the centrosome. Dual imaging reveals that these puncta are closely associated with astral microtubules, and disruption of the microtubule network severely disrupts GFP-Nuf distribution and movement (unpublished data). This colocalization and dependency of the microtubule network has also been demonstrated for Rab11 and GFP-Arfo2 (Mammoto, 1999; Hickson, 2003). In comparison with live fluorescent analysis of GFP-Rab11 in mammalian systems (Sonnichsen, 2000), GFP-Nuf shows a similar localization, distribution, and movement pattern. This supports the view that Nuf localizes to the RE and that these images reflect RE dynamics in the Drosophila embryo (Riggs, 2003).

The results also demonstrate a mutual dependence of Nuf and Rab11 for their localization to the RE. In nuf-derived embryos, the robust Rab11 pericentriolar distribution is completely disrupted. Whether Nuf is specifically disrupting Rab11 localization to the RE or more globally disrupting RE integrity is not known. However, the effect of Nuf is believed to be specific to the RE, since Golgi morphology and distribution is normal in nuf-derived embryos. The effect of nuf mutations on Rab11 localization is consistent with reports (Hickson, 2003) demonstrating that overexpression of GFP-Arfo2 alters the organization of Rab11 in mammalian cells. Conversely, Nuf pericentriolar localization fails in embryos with reduced levels of Rab11 (Riggs, 2003).

Analysis of nuclear and cortical cytoskeletal defects in nuf- and rab11-derived embryos supports the idea that Nuf and Rab11 are involved in a similar function at the RE. As observed in the nuf mutation, embryos with reduced levels of Rab11 disrupt the syncytial nuclear divisions only after the nuclei reach the cortex. This phenotype indicates that Rab11 is involved in a process specific to the cortical divisions such as cytoskeletal rearrangements or furrow formation. Also like nuf, rab11-derived embryos exhibit fusions between nonsister nuclei, a hallmark of defective furrow formation (Riggs, 2003).

Previous analysis of nuf-derived embryos has revealed normal actin organization during interphase, but gaps occur in the actin network early in the process of furrow formation (Rothwell, 1998). Analysis of rab11-derived embryos reveals an equivalent phenotype with respect to actin; the interphase actin caps form normally, but the actin-based metaphase furrows are disrupted. Previous analysis of actin dynamics in the nuf-derived embryos revealed that actin recruitment during the initial stages of furrow formation is compromised (Rothwell, 1999). Fixed analysis of actin defects in rab11-derived embryos reveals actin gaps at the initial stages of furrow formation. Therefore, the rab11 furrow defects are likely the result of defects in the initial recruitment of actin to the furrows (Riggs, 2003).

Although the nuf mutation only partially disrupts actin recruitment to the invaginating furrows, it has a much more severe effect on membrane recruitment. The Drosophila homolog of the dystrobrevins, Dah, was used as a marker for furrow membrane (Zhang, 1996). Biochemical analysis demonstrates that this protein associates tightly with actin and membrane, suggesting it is involved in linking the cortical cytoskeleton and the plasma membrane (Zhang, 2000). Immunofluorescent analysis reveals that it localizes to the plasma membrane and invaginating furrows, as well as vesicles that accumulate at furrow formation sites (Rothwell, 1999). These vesicles are often associated with actin, suggesting that they incorporate as a unit into the growing furrow. In nuf-derived embryos, there is some localization of Dah at the furrows; however, most remain in vesicles widely dispersed throughout the cortex (Riggs, 2003; Rothwell, 1999). The effect of the rab11 mutation on Dah localization is even more severe. There is no Dah localization at the furrows, and few Dah-containing vesicles are seen throughout the cortex (Riggs, 2003).

nuf and rab11 mutations disrupt membrane recruitment and actin remodeling during the early stages of furrow formation, supporting the argument that these proteins function in a common process at the RE. Analysis of Rab11 function in C. elegans reveals that it also is important for normal furrow progression during cytokinesis (Skop, 2001). However, this analysis showed varying degrees of defects during furrow invagination, suggesting a role for Rab11 during either the initial stages or latter stages (or both) of cytokinesis. In the Drosophila embryo, Rab11 appears to be involved in the initial stages of furrow formation when actin is being recruited to the invaginating furrow (Riggs, 2003).

These analyses indicate that activities of Nuf and Rab11 at the RE influence cortical actin dynamics. Specifically, they direct the recruitment of actin to the sites of metaphase furrow formation. One explanation for this linkage between the endosome and cortical actin dynamics is that membrane and actin are recruited as a unit to the metaphase furrows (Rothwell, 1999). Immunofluorescent analysis reveals that Dah-containing vesicles are often tightly associated with actin at the leading edge of the invaginating furrows. Therefore, disrupting membrane recruitment would also disrupt actin recruitment (Riggs, 2003).

An intriguing alternative explanation for trafficking activities at the RE influencing actin recruitment during the initial stages of furrow formation comes from reports that Rac GTPases are positioned in the cell through the endosomal recycling pathway. For example, Arf6 GTPase regulates an endosomal recycling pathway and cortical actin remodeling at the plasma membrane. In HeLa cells, ARF6 and Rac1, a potent actin organizer, colocalize at the plasma membrane as well as the RE. Mutational analysis and drug analyses indicate that ARF6 influences actin dynamics by regulating the trafficking of Rac1 to the plasma membrane. This latter model readily explains the effects of Rab11 and Nuf mutations on both actin recruitment and membrane delivery. These proteins are not only required at the RE for membrane delivery to the metaphase and cellularization furrows, but they are also required for the delivery of actin-remodeling proteins, such as Rac, to the plasma membrane (Riggs, 2003 and references therein).

Cortical actin remodeling and localized plasma membrane expansion not only mediate cytokinetic furrow formation, but also are involved in cell motility, lamellipodia formation, and phagocytosis. Phagocytosis is particularly interesting because recent work has shown that it occurs through targeted delivery of vesicles from the RE. Accumulation of RE-derived VAMP3-containing vesicles occurs at the site of phagosome formation, and disruption of VAMP3 with tetanus toxin prevents phagosome formation. As has been demonstrated for metaphase and cellular furrow formation, activity at the RE may also mediate cortical actin cytoskeletal remodeling during phagocytosis (Riggs, 2003 and references therein).

Asymmetric Rab11 endosomes regulate Delta recycling and specify cell fate in the Drosophila nervous system

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. This study describes a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. Trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, it was demonstrated that recycling endosomes are essential for Delta activity. These results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments (Emery, 2005).

To test whether Rab11 asymmetry is important for cell-fate specification, Rab11 accumulation in the pIIa cell was induced by nuf expression. Postorbital ES organs, which can easily be scored in fairly high numbers, were used. Cell-fate transformations upon nuf overexpression have been described (Abdelilah-Seyfried, 2000), but surprisingly, they do not occur at high frequency. Such transformations can, however, be observed upon coexpression of constitutively active Rab11. Upon expression of nonphosphorylatable lgl, Numb and Neuralized asymmetry are disrupted, but most ES organs still develop normally. When both pathways are disrupted by coexpression of lgl3A and nuf, however, a large fraction of ES organs shows cell-fate transformations that are consistent with a higher level of Delta activity in pIIa. Lineage analysis shows cell-fate transformations in 44% of postorbital ES organs, and in 18% of these, pIIb cells are transformed into pIIa cells (6% in ES organs expressing lgl3A alone). Twenty-five percent of the cell fate transformations affect the first (SOP) while 75% affect the second (pIIa) division, indicating that Rab11 asymmetry also plays a role in other divisions of the SOP lineage. Taken together, these results suggest that two partially redundant pathways exist to generate asymmetry in the SOP lineage: the Par proteins phosphorylate Lgl to direct Numb and Neuralized into the pIIb cell where they repress Notch or activate Delta, respectively. In the pIIa cell, inhibition of Nuf and Rab11 inhibits Delta by preventing its trafficking through the recycling endosome (Emery, 2005).

These results suggest that cells can also regulate signal transduction pathways by controlling the formation or distribution of whole endocytic compartments. After SOP division, Rab11-positive vesicles accumulate around the centrosome in this cell but not in pIIa. Rab11 plays a well-documented role in controlling vesicular protein transport through recycling endosomes to the plasma membrane (Zerial, 2001). Dominant-negative forms of Rab11 inhibit the recycling of endocytosed Transferrin receptors or recruitment of H+-K+-ATPase to the plasma membrane suggesting that Rab11 regulates trafficking of vesicular cargo through the recycling endosomal compartment. In SOP cells, the asymmetric localization of Rab11 reflects a different ability of pIIa and pIIb cells to recycle the Notch ligand Delta. Rab11 asymmetry is observed 3.5 min after cytokinesis but Delta is in recycling endosomes only 15 min after endocytosis. Thus, the protein is endocytosed before mitosis and recycles back to the plasma membrane in pIIb but not in pIIa. In pIIa, more Delta/Hrs double-positive vesicles are observed, indicating that the protein enters a late-endosomal pathway (Emery, 2005).

Several observations indicate that passage through recycling endosomes is essential for Delta to signal. In a marrow stromal cell line, OP9, inhibition of recycling endosomes dramatically reduces Delta signaling capacity. Similarly, blocking the recycling pathway by overexpression of a dominant-negative form of Rab11 in SOP cells causes relocalization of Delta into enlarged late endosomes. In Drosophila wing discs, Delta has been postulated to pass through a specific endocytic recycling pathway to acquire signaling capacity (Wang, 2004). Finally, Jafar-Nejad (2005) demonstrates that the Rab11 binding partner Sec15 is required both for Delta trafficking and Notch activation in the SOP lineage. Sec15 is a component of the exocyst and is a Rab11 effector (Zhang, 2004). Although Sec15 is not asymmetric itself, it is conceivable that the higher amounts of GTP bound Rab11 in pIIb increase its activity in delivering Delta to the plasma membrane. A difference between Delta trafficking in pIIa and pIIb has been observed previously (Le Borgne, 2003), but both Delta/Hrs vesicles and total number of Delta vesicles were actually higher in pIIb in these previous experiments. While these earlier experiments analyzed the whole two cell stage, this study focusses on the short time interval right after mitosis where Rab11 is asymmetric. This explains the different outcome and might in fact indicate that pIIb cells switch from an initial phase where Delta is recycled to a later phase where trafficking is regulated by neuralized-dependent endocytosis (Emery, 2005).

Although many cell types in different organisms undergo asymmetric cell division, only one mechanism has been identified so far that directs this important biological process in animals. This mechanism involves the Par proteins, which phosphorylate Lgl on one side and direct cell fate determinants to the opposite side of the cell cortex. Several results indicate that other pathways might exist: in dividing progenitor cells of the mammalian brain, Numb segregates into one of the two daughter cells and is required for lineage specification. However, some of these divisions are asymmetric, although their orientation predicts that Numb would be inherited by both daughter cells. In Drosophila SOP cells, lgl3A overexpression affects both Numb and Neuralized localization but has only a minor influence on the asymmetric outcome of the division. The results indicate that the asymmetric distribution of Rab11 is established through a distinct pathway: (1) Rab11 asymmetry is unaffected in SOP cells overexpressing lgl3A; (2) Rab11 is still asymmetric in dlg mutants where Par proteins do not localize and Numb and Neuralized segregate into both daughter cells; (3) Rab11 asymmetry can be uncoupled from Numb and Neuralized localization by the expression of inscuteable; (4) the events responsible for Rab11 asymmetry seem to occur in the pIIa cell, but none of the known determinants is inherited by this daughter cell. Although the observations could also be explained if Numb or Neuralized would relieve a general suppression of recycling endosome formation in the SOP lineage, this is unlikely since Rab11 asymmetry is unaffected in numb or neuralized mutants. More likely, an unknown factor could act on Nuf or the centrosome in the pIIa cell to prevent Rab11 accumulation. Nuf localization is cell cycle regulated, and a key regulatory component could be missing in pIIa. For example, Nuf is highly phosphorylated and differential activity of a kinase or phosphatase could prevent its pericentriolar localization in the pIIa cell. Homologs of Nuf exist and bind to Rab11 in vertebrates. Their expression pattern has not yet been described but it will be interesting to determine whether these homologs regulate Notch signaling in vertebrates and are responsible for asymmetric cell division in the mammalian brain (Emery, 2005).

Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo

Sec15, a component of the exocyst, recognizes vesicle-associated Rab GTPases, helps target transport vesicles to the budding sites in yeast and is thought to recruit other exocyst proteins. This study reports the characterization of a 35-kDa fragment that comprises most of the C-terminal half of Drosophila Sec15. This C-terminal domain binds a subset of Rab GTPases, especially Rab11, in a GTP-dependent manner. Evidence is provided that in fly photoreceptors Sec15 colocalizes with Rab11 and that loss of Sec15 affects rhabdomere morphology. Determination of the 2.5-Å crystal structure of the C-terminal domain revealed a novel fold consisting of ten alpha-helices equally distributed between two subdomains (N and C subdomains). The C subdomain, mainly via a single helix, is sufficient for Rab binding (Wu, 2005).

Sec15 plays multiple key roles in a variety of processes in exocytosis by interacting with vesicle-associated small Rab GTPases, assisting in targeting vesicles to budding sites and recruiting, by way of Sec10, other components of the exocyst. in vitro and in vivo studies have shed light on some of these roles. The relatively large size (85 kDa) of Sec15 ensures that there are enough docking sites, each probably residing in either a single domain or a combination of domains, for other protein components necessary for its diverse functions. These studies revealed a segment named the C-terminal domain, containing nearly the entire C-terminal half of the protein, possesses several interesting properties. This domain binds to a set of Rabs in a GTP-dependent manner. Its atomic structure is composed of two distinct subdomains, only one of which (the C subdomain) harbors the Rab-binding site. The finding of a bipartite C-terminal domain was unexpected; there was no hint of this feature even from the BLAST Conserved Domain Database search for domains. The all-helical structure of the domain, with its two different subdomains, has a novel fold. A search for overall structural similarities of the whole domain and of the N and C subdomains separately against the DALI database, a network tool for protein structure comparison, did not find any substantial matches (Wu, 2005).

Binding studies further show that Sec15 lacks stringent substrate specificity in vitro. The domain binds Rab11, Rab3, Rab8 and Rab27, but the binding data suggest that Rab11 is the major target. The ability of an effector protein to interact with different Rab proteins has been observed previously: rabphilin-3, originally identified as a Rab3-binding protein, has also been shown to interact with Rab8 and Rab27 in a cotransfection assay in COS-7 cells. In addition, Sec15 and Sec5 apparently do not have significant roles in regulating neurotransmitter release, a process in which Rab3 has been implicated. This suggests that the weaker in vitro binding of Sec15 with Rab3, Rab8 and Rab27 may have in vivo consequences. It remains to be investigated whether these interactions have any role in vivo (Wu, 2005).

The Rab-binding site is apparently confined mainly to the exposed middle three-fourths of one helix (alpha9) of the C subdomain, which contains mostly hydrophobic residues. The participation of hydrophobic residues in binding is a common feature observed in several crystal structures of complexes of effectors with their cognate small GTPases. The in vivo consequence of abolishing the Sec15-Rab11 interaction by using the Sec15 mutants is under investigation (Wu, 2005).

These studies further raise questions about the role of the ~15-kDa N subdomain, which is also composed entirely of helices, but helices with various lengths and with topology and geometry different from those in the C subdomain. The same questions could also apply to the function(s) of the ~40-kDa N-terminal half of Sec15. The combination of the N subdomain and the N-terminal half, which is approximately two-thirds of the entire Sec15 protein, seems too large for binding only the Sec10 component of the exocyst. This suggests that Sec15 may have additional partners that have yet to be identified (Wu, 2005).

There are informative differences between the phenotypes in rhodopsin1 trafficking and rhabdomere morphogenesis of rab11 and sec15 mutants. A defect in the initial delivery of rhodopsin in rab11 mutants has been associated with accumulations of rhodopsin outside the rhabdomere. No noticeable amounts of rhodopsin were observed outside the rhabdomeres in sec15 mutants. This observation, combined with the normal photoreceptor depolarization measured by electroretinography, suggests that initial delivery of rhodopsin occurs in sec15 mutants. However, there is also a significant defect in rhabdomere morphology in rab11 mutant photoreceptors. Hence, the photoreceptor staining data suggest that although targeted membrane delivery to the rhabdomere might occur in Sec15-mutant cells, defects in membrane recycling (evidenced by the strong accumulation of Rab11 in the rhabdomere) probably lead to disruptions in rhabdomere morphology over time. Together, these data suggest that the rab11 mutant phenotype is more severe than the sec15 mutant phenotype but that the two show some similarities. In summary, the interaction of Rab11 and Sec15 has functional consequences in vivo, in that Rab11 trafficking is disrupted in sec15 mutants and at least one aspect of the sec15 mutant phenotype, namely abnormal rhabdomere morphology, can be explained by loss of Rab11 function. An interaction between Sec15 and Rab11 has also been shown to have an important role in the asymmetric division of sensory organ precursors in Drosophila, in that Sec15 promotes Notch signaling during the asymmetric division of sensory organs, suggesting that Sec15/Rab11 interaction is required not only in rhodopsin targeting but also during cell-fate specification (Wu, 2005).

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate E-Cadherin trafficking from recycling endosomes to the plasma membrane

Loss of function of the Drosophila exocyst components in epithelial cells results in E-Cadherin (Shotgun) accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits Shotgun delivery to the membrane. Rab11 and Armadillo interact with Sec15 and Sec10, respectively. These results support a model whereby the exocyst regulates E-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located (Langevin, 2005).

In budding yeast, the exocyst has been proposed to tether post-Golgi vesicles to the membrane of the growing bud prior to fusion. This model is supported by several observations. (1) Exocyst components localize both on post-Golgi vesicles and on the bud membrane (Boyd, 2004). Analogously in Drosophila, Sec5 and Sec15 localize along the lateral membrane and on the REs. (2) Mutations in genes encoding components of the exocyst complex lead to the accumulation of post-Golgi vesicles (Novick, 1980). Analogously, Sec5, Sec6, and Sec15 loss of function leads to an enlargement of the recycling endosome (RE) compartment; this enlargement interpreted as an accumulation of RE vesicles. (3) The localization of Sec8p and Exo70p at the growing bud, i.e., the site of polarized exocytosis, depends on the function of the other exocyst components. Analogously, Sec5 is localized along the lateral membrane, where E-Cadherin delivery is affected, and its localization along the cortex depends on Sec6. It is therefore proposed that in Drosophila epithelial cells, Sec5, Sec6, and Sec15 act by tethering vesicles originating from the recycling endosomal compartment to the lateral membrane of epithelial cells, as a prerequisite for their exocytosis (Langevin, 2005).

In epithelial cells, Arm and E-Cadherin colocalize to the AJs of the ZA as well as along the lateral membrane. In the absence of Sec5, Sec6, and Sec15 function, E-Cadherin trafficking is affected and E-Cadherin accumulates in the RE. Similarly, in the absence of arm, E-Cadherin fails to localize at the membrane and localizes in the RE. The identification of an interaction between Arm and Sec10 is therefore consistent with a model whereby this interaction provides a landmark at the site where Arm is enriched in order to deliver E-Cadherin from the recycling endosomes. Nevertheless, Arm may play an additional role in stabilizing E-Cadherin at the AJs. A direct demonstration of the function of Arm in regulating the delivery of E-Cadherin will therefore require the identification of arm mutant alleles that do not perturb its function as a regulator of E-Cadherin stabilization and only affects its interaction with Sec10 (Langevin, 2005).

In the absence of Sec5, Sec6, or Sec15 function, E-Cadherin delivery to the lateral membrane is inhibited and E-Cadherin accumulates in the REs. Furthermore, E-Cadherin was found to transcytose in a Sec5-dependent manner from the lateral membrane of epithelial cells to the apical AJs. Therefore, this study reveals at least a role of the exocyst in the recycling of E-Cadherin from the lateral membrane to the apical AJs. Furthermore, the strong reduction of E-Cadherin present on the lateral membrane is interpreted as a failure to recycle E-Cadherin from the lateral membrane back to the lateral membrane, which cannot be compensated for by the delivery of newly synthesized E-Cadherin to the lateral membrane. The loss of E-Cadherin on the lateral membrane may also lead to a reduction of E-Cadherin delivery at the AJs. This may have also contributed to the loss of epithelial cell polarity observed in some of the sec5 mutant epithelial cells (Langevin, 2005).

In polarized MDCK cells, the apical REs are well known as a site of sorting during endocytic and transcytotic transport. The REs have also been shown to serve as an intermediate during the transport of newly synthesized proteins from the Golgi to the plasma membrane in nonpolarized MDCK cells. Similarly, upon overexpression of GFP-E-Cad in HeLa cells, E-Cad transits from the Golgi to the Rab11 endosomes. Nevertheless, the existence of such a pathway remains to be established in polarized MDCK cells. In fact, the overexpression of a dominant-negative form of Rab11 leads to sequestration of E-Cadherin in the REs, but whether sequestered E-Cadherin represented newly synthesized or recycled E-Cadherin was not determined. The existence of such a Golgi-to-RE pathway also remains to be established in Drosophila epithelial cells. If so, a role of the exocyst in regulating the delivery of newly synthesized E-Cadherin from the Golgi to the lateral membrane via the REs remains plausible (Langevin, 2005).

Whether the exocyst regulates E-Cadherin localization in mammalian cells has not been directly analyzed. However, E-Cadherin is proposed to act as a regulator of the localization of the exocyst complex in polarizing mammalian cells since E-Cad- and Nectin-2α-dependent cell-cell contacts were proposed to recruit the exocyst complex in order to promote the growth of the lateral epithelial cell domain. The current study suggests that upon the recruitment of the exocyst complex by E-Cadherin, the exocyst promotes the delivery of more E-Cadherin to the lateral membrane during the establishment of apico-basal polarity. In fact, several reports can be reconciled with a function of the exocyst in regulating the transport of E-Cadherin in mammalian cells. Thus, polarized exocytosis of E-Cad to the lateral membrane is dependent upon its interaction with Arm. And, as stated above, REs have shown to serve as an intermediate during the transport of E-Cad from the Golgi to the lateral membrane where E-Cadherin, β-Catenin, and α-Catenin form the AJs. Furthermore, the overexpression of a dominant-negative form of Rab11 impairs the delivery of E-Cadherin to the lateral membrane. Consistent with the exocyst regulating trafficking from the REs, exocyst components also localize on the REs, and Sec15 is an effector of Rab11. Finally, E-Cadherin and catenins are associated with exocyst components (Langevin, 2005 and references therein).

In conclusion, this work provides evidence for a conserved role of the exocyst in regulating the delivery of E-Cadherin from REs to sites on the plasma membrane and in thereby contributing to the maintenance of epithelial cell polarity (Langevin, 2005).

Myosin V, Rab11 and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors

Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and it was asked if myosin V (Didum) and the Drosophila Rab11 interacting protein, dRip11 (lethal (1) G0003), also participate in secretory transport. Reduction of either protein impairs rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin. MyoV-reduced photoreceptors also develop ectopic rhabdomeres inappropriately located in basolateral membrane, indicating a role for MyoV in photoreceptor polarity. Binary yeast two hybrids and in vitro protein-protein interaction predict a ternary complex assembled by independent dRip11 and MyoV binding to Rab11. It is proposed that this complex delivers morphogenic secretory traffic along polarized actin filaments of the subcortical terminal web to the exocytic plasma membrane target, the rhabdomere base. A protein trio conserved across eukaryotes thus mediates normal, in vivo sensory neuron morphogenesis (Li, 2007).

Across eukaryotes, a protein trio comprising a Rab protein, a member of the family of small GTPases that regulate exchange between membrane compartments, a myosin motor, notably myosin V (MyoV), and a linker/adaptor protein, powers organelle motility and polarized secretion. For example, HeLa and MDCK cells recycle endocytosed cell surface receptors through a recycling endosome, the return leg mediated by Rab11 together with MyoV and the Rab11 adaptor/linker protein, family interacting protein 2 (FIP2). Drosophila photoreceptors are typical polarized epithelial cells and morphogenesis of their photosensory membrane organelles, rhabdomeres, is driven by a late-pupal surge of secretory traffic that greatly expands the apical plasma membrane in a column of closely packed, rhodopsin-rich photosensitive microvilli. It was recently found that Rab11 mediates membrane transport to developing rhabdomeres, prompting an investigation to see if Drosophila MyoV and dRip11, and Drosophila FIP2 also participate in morphogenic secretory transport (Li, 2007).

Numerous observations link MyoV to polarized membrane transport. Budding yeast lacking essential MyoV, Myo2p, accumulate cytoplasmic post-Golgi secretory vesicles; secretion continues in mutants, but is not correctly targeted to the growing bud. Melanocytes of mouse dilute mutants lacking MyoVa fail to properly localize melanosome pigment organelles to the actin-rich cell periphery; expression of a MyoVa C-terminal fragment (MyoVa-CT) that displaces endogenous MyoVa from melanosomes mimics MyoVa loss. Expression of MyoVa-CT similarly inhibits Xenopus melanosome motility and HeLa cell transferrin receptor recycling. Notably, in polarized MDCK cells, MyoVb-CT selectively disrupts Rab11-dependent apical, but not basolateral, membrane recycling (Li, 2007).

Parallel loss-of-function phenotypes suggest MyoV and Rab11 cooperate in membrane transport. Loss of either activity inhibits recycling of CXCR2 chemokine and M4 muscarinic acetylcholine receptors. Similarly, MyoV or Rab11 reduction prevents biogenesis of apical cannicular membranes in polarized hepatocytes and decreases glutamate receptor 1 (GluR1) subunit delivery to developing synapses of hippocampal cells in culture (Li, 2007).

Direct interaction between rabbit Rab11a and MyoVb is detected in yeast two-hybrid screens, and deletion of MyoVb-CT's Rab11 binding sequence neutralizes its dominant-negative impact on GluR1 delivery in hippocampal neurons, suggesting MyoVb binds Rab11 in GluR1 trafficking. Genetic interaction between Saccharomyces cerevisiae Myo2p and Sec4p mutants is consistent with direct or close cooperation (Li, 2007).

In addition to MyoV, Rab11 interacts with Rab11-FIPs at a signature Rab11 binding domain (RBD). Class I FIPs contain a C2 domain that targets recycling vesicles to the plasma membrane, and truncated FIPs lacking the C2 domain inhibit receptor recycling. Drosophila encodes a single class I FIP, dRip11, but its function has not been reported (Li, 2007 and references therein).

The Drosophila genome includes a single MyoV gene, myoV (didum) (Bonafe, 1998; MacIver, 1998). Drosophila embryos receive substantial maternal MyoV and the protein is ubiquitously expressed throughout development, including the adult retina, where it localizes to the base of the rhabdomere. Mutants lacking MyoV show strong developmental delays and substantial late larval lethality. Surprisingly, rare homozygous mutant escapers showed normal embryogenesis and cellular architecture, suggesting MyoV is dispensable for the wide range of membrane trafficking that supports normal development. Actin staining of myoV mutant eyes showed apparently normal rhabdomeres and adult mutants were normally phototaxic, suggesting that MyoV does not play an obvious role in rhabdomere development or photoreception (Li, 2007 and references therein).

This paper investigated the role of MyoV and dRip11 in the polarized membrane transport that builds Drosophila rhabdomeres. Both were found to be essential. In MyoV mutants, rhodopsin 1 (Rh1) is not delivered to the growing rhabdomere, but instead accumulates in photoreceptor cytoplasm; rhodopsin-bearing vesicles, and the Rab11 and dRip11 they carry, do not approach the rhabdomere base. dRip11 loss similarly impairs secretory transport, delocalizing MyoV and Rab11 and promoting cytoplasmic Rh1. MyoV mutant photoreceptors also develop supernumerary rhabdomeres ectopically positioned within basolateral plasma membrane, suggesting MyoV-mediated transport suppresses formation of inappropriate rhabdomere primordia. Drosophila photoreceptors harness an evolutionarily conserved protein trio to deliver polarized apical membrane traffic in cellular morphogenesis (Li, 2007).

Drosophila photoreceptors, like many polarized epithelial cells, greatly amplify their apical membranes during terminal differentiation via targeted membrane delivery. This study shows that a protein trio (Rab11, dRip11, and MyoV) mediates this morphogenic secretory traffic. MyoV normally concentrates at the base and its loss causes three notable phenotypes of compromised apical transport: Rab11 and dRip11 delocalize from the base, Rh1 accumulates in photoreceptor cytoplasm, and ectopic rhabdomeres are formed. dRip11, the sole Drosophila class I Rab-FIP, is also required for normal Rh1 transport; its loss delocalizes Rab11 and MyoV. Together with the demonstration that Rab11 is essential for photoreceptor secretory traffic, it is proposed MyoV pulls post-Golgi secretory vesicles, marked for rhabdomere delivery by Rab11 and dRip11, through an exclusionary subcortical cytoskeletal web along polarized microfilaments leading directly to the exocytic targeting patch at the rhabdomere base (Li, 2007).

Cytoplasm adjacent to the rhabdomere base is permeated by a dense microfilament brush, the rhabdomere terminal web (RTW), which extends from the rhabdomere base deep into photoreceptor cytoplasm. Microfilaments are poorly preserved in chemically fixed tissue, but distinct 'RTW cytoplasm' is manifest as organelle-poor cytoplasm behind the rhabdomere. RTW cytoplasm excludes even ribosomes, whose absence contributes to the light, clear appearance of RTW cytoplasm. Biosynthetic ER and Golgi are distributed the length of the cell, in close proximity to the RTW's cytoplasmic terminus (Li, 2007).

The rhabdomere base differentiates in mid-pupal photoreceptors as the photoreceptor apical membrane resolves to a central Moesin-rich rhabdomere primordium surrounded by a Crumbs-rich supporting domain. Once founded, the rhabdomere base organizes the RTW and receives morphogenic traffic. The stalk accepts little traffic, focusing exocytosis to the rhabdomere. The stalk links the rhabdomere to the retina's junctional network and projects it into an apical lumen, the IRS, aligned to the eye's optical axis (Li, 2007).

Membrane transport in light-adapted late pupal photoreceptors is dynamic, with biosynthetic and endocytic traffic reflected in numerous, complex membrane compartments. Post-Golgi secretory traffic is carried in tubular vesicles, approximately 100 nm across; endocytosed membrane gathers in multivesicular bodies. Complex membrane forms are common at the rhabdomere base, likely a consequence of extensive membrane fusion (Li, 2007).

Confocal immunofluorescence localizes Rab11 to puncta throughout photoreceptor cytoplasm, with a prominent concentration at the rhabdomere base . dRip11 immunolocalization resembled Rab11, with cytoplasmic puncta and localization at the rhabdomere base. Note that Rab11 and dRip11 lie within RTW cytoplasm, overlapping the actin brush extending from the rhabdomere's curving base. MyoV concentrates across the rhabdomere base of late pupal photoreceptors, often appearing strongest at the sides. Like Rab11 and dRip11, MyoV staining is strongly within RTW cytoplasm. Cytoplasmic MyoV is lightly diffuse with scattered brighter puncta (Li, 2007).

The RTW's role as both a barrier and a carrier for morphogenic traffic is an instance of a general theme of a dynamic regulatory role of the subcortical cytoskeleton in secretion. Myosin S1 decoration shows RTW filaments are oriented with plus-ends at the membrane, a correct orientation for MyoV-based secretory transport (Arikawa, 1990), and disruption of the actin cytoskeleton prevents the morphogenic traffic that rebuilds crab rhabdomeres at dusk. The RTW's strong polarization and anchorage to a secretory targeting patch resembles the polarized actin cables that mediate budding yeast secretory traffic (Li, 2007 and references therein).

Absorptive and secretory epithelial cell specialists often regulate apical membrane activity by dynamic, Rab11-dependent exchange of plasma membrane with recycling endosomes. For example, gastric parietal cells meet demand for additional acid secretion by Rab11-, Rab11-FIP2-, and MyoV-dependent delivery of additional H+/K+ ATPase pumps to the cell surface from a recycling endosome. Like GPCRs generally, Drosophila Rh1 is endocytosed upon stimulation but appears to be degraded rather than recycled back to the rhabdomere. Drosophila photoreceptor Rab11-dependent transport appears to be principally devoted to delivery of newly synthesized cargo from the TGN to the plasma membrane, a conserved Rab11 activity (Chen, 1998) now seen to further parallel recycling transport. Ectopic rhabdomeres in hypomorphs suggest MyoV normally suppresses the establishment of inappropriate rhabdomere primordia; once-founded ectopic rhabdomeres develop in concert with principal rhabdomeres, presumably drawing from the same secretory traffic. It is speculated that MyoV normally drives traffic to the differentiating rhabdomere primordium and that positive feedback driven by the incorporation of morphogenic determinants, perhaps proteins that anchor and promote RTW development, gives the original, 'true' apical membrane an overwhelming growth advantage, starving weak, inappropriate sites. MyoV reduction might diminish this advantage, allowing ectopic foci to capture sufficient morphogenic traffic to assemble a rhabdomere patch (Li, 2007).

The observation that MyoV is required for normal rhabdomere development differs from Mermall's report of normal rhabdomeres in MyoVQ1052st mutant eyes (Mermall, 2005). However, long ribbons of the principal rhabdomeres dominate phalloidin-stained longitudinal sections, and ectopic rhabdomeres, often patches a few microns across, are not prominent. Mermall's supplementary Fig. 1 L, a tangential section, shows actin-bright profiles apart from the principal rhabdomeres - potentially ectopic rhabdomeres. Massive biosynthetic traffic in late pupal photoreceptors sensitizes cells to compromise of efficient, accurate transport and accumulation of cytoplasmic Rh1 reflects an inability of transport to keep pace with biosynthesis (Li, 2007).

dRip11 loss inhibits secretory transport and misolcalizes Rab11 and MyoV. It is suggested that dRip11 couples two broad streams of membrane transport, Rab11- and MyoV-dependent activities, to drive morphogenic secretory traffic. The results are consistent with previously demonstrated roles for FIPs as contributors to membrane targeting, and as scaffolds for the growing Rab11 effector ensemble. Similar to chromaffin cells, where MyoV only partially overlaps with secretory vesicles, MyoV and Rab11 only partially overlap in developing photoreceptors, and it is likely MyoV transports multiple and changing cargoes (Li, 2007).

Rab11 participates in both constitutive and Ca2+-regulated secretion, and both cargo binding and Ca2+ regulate MyoV activity. Rhabdomere morphogenesis utilizes constitutive exocytosis, with substantial rhabdomere growth before Rh1 expression and photoresponse Ca2+ influx. Rhabdomeres likewise develop normally in the dark, indicating light-dependent Ca2+ elevation is not required for MyoV morphogenic transport. It is proposed that dRip11, in proximity to MyoV via their mutual binding to Rab11 on post-Golgi secretory vesicles, interprets or conveys non-Ca2+-stimulated MyoV activation, promoting developmental MyoV secretory transport (Li, 2007).

Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis

Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. This study identified the phosphatidylinositol (PI) 4-kinase III beta Four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila spermatocytes. Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. These data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis (Polevoy, 2009).

The discovery that Drosophila PI4Kβ Fwd and fission yeast PI4P 5-kinase Its3 are required for cytokinesis provided the first genetic evidence that phosphoinositides play a critical role in this process. Consistent with this, the phosphatidylinositol transfer proteins Gio and Nir2 are also required for cytokinesis, and may serve in part to provide the PI precursor for PI4P. In addition, the pool of PI4P synthesized by PI4Kβ may serve as a precursor to PIP2, which is also required for cytokinesis. Nonetheless, individual phosphoinositides and their regulatory enzymes likely play unique roles, regulating distinct steps of the process. Importantly, a role for PI4Kβ -- and therefore PI4P -- in cytokinesis appears conserved (Polevoy, 2009).

Experiments reveal that Fwd is required for synthesis of PI4P on Golgi membranes and for formation of PI4P- and Rab11-associated secretory organelles at the midzone. On the surface, this result appears at odds with previous observations suggesting that Fwd and Gio function at a later step to promote fusion of Lva-containing Golgi-derived vesicles with the cleavage furrow. However, because Lva serves as a Golgi scaffold, accumulation of Lva at the midzone in fwd and gio mutant cells may reveal a defect in segregation of a subset of Golgi membranes to the poles of the cell rather than a defect in vesicle fusion (Polevoy, 2009).

Although Rab11 has been shown to traffic to the midzone during cytokinesis, the membrane composition of Rab11-containing organelles was previously unknown. The current finding that PI4P is present on these organelles is consistent with proteomic analyses demonstrating an enrichment of Rab11 and PI4Kβ on PI4P-containing liposomes. Interestingly, these liposomes were also enriched in actin regulatory factors such as Rac1 and Wave/Scar. As actin is transported on vesicles to the midzone in Drosophila embryos, and the Rab11 effector Nuf promotes actin polymerization at the furrow, PI4P-dependent organelles may concentrate or recruit factors such as Nuf that contribute to maintenance of F-actin in the contractile ring. Consistent with this idea, mutations in fwd, like mutations in nuf and rab11, are associated with failure to maintain proper actin organization during cytokinesis (Polevoy, 2009).

The regulatory relationship between Fwd and Rab11 is evolutionarily conserved. In budding yeast, the Rab11 homologues Ypt31/32 act downstream of Pik1 to regulate post-Golgi trafficking. The two Arabidopsis thaliana PI4Kβs, PI-4Kβ1 and PI-4Kβ2, show genetic interactions with the Rab11 homologue Rab4Ab in root hair development and colocalize with RabA4b on root hair tip-associated membranes, and PI-4Kβ1 binds GTP-bound RabA4b in vitro. Moreover, RabA4b-containing membranes exhibit altered morphology in PI-4Kβ1/β2 double mutants, suggesting RabA4b may act downstream of PI4Kβs in this process. Mammalian PI4Kβ binds activated Rab11, and is thought to recruit Rab11 to Golgi membranes to promote post-Golgi secretory trafficking. The results of this study demonstrate that Fwd acts upstream of Rab11 during cytokinesis, and that bovine and human PI4Kβ can fully substitute for Fwd in vivo (Polevoy, 2009).

PI4Kβ and PI4P participate in vesicular and nonvesicular trafficking of cellular membranes and their lipid constituents, suggesting that, in addition to its role in formation of secretory organelles, Fwd may direct other trafficking pathways. For example, several conserved lipid transport proteins bind PI4P and depend on PI4Kβ for their localization and function in yeast and mammalian cells. PI4P is also found at ER exit sites (also called transitional ER, or tER). Intriguingly, tER was recently shown to accumulate at the midzone of dividing S. pombe cells, and normal ER morphology in dividing Caenorhabditis elegans embryos was found to require Rab11. Future experiments will be required to determine if Fwd-dependent tER or nonvesicular trafficking pathways actively participate in cytokinesis (Polevoy, 2009).

Despite strong parallels between cytokinesis in mammalian cells and in Drosophila, the mechanism by which Rab11 affects completion of cytokinesis is not entirely conserved. In mammalian cells, Rab11 associates indirectly with the plasma membrane regulator Arf6 via FIP3, a Rab11-binding protein with homology to Nuf. Both Rab11 and Arf6 bind members of the exocyst complex, which in turn mediates targeting of endosomes to the midzone. In contrast, in Drosophila, Arf6 and Rab11 appear to function in separate pathways. Nuf binds and colocalizes with Rab11, yet fails to bind Arf6. Consistent with this, Rab11 is essential and has specific functions at multiple stages of development, whereas Arf6 is required only for spermatocyte cytokinesis. Even in spermatocytes, Arf6 promotes trafficking of Rab4-positive but not Rab11-positive vesicles. Thus, in spermatocytes, Arf6/Rab4 and Fwd/Rab11 appear to constitute nonredundant membrane trafficking pathways required for completion of meiotic cytokinesis (Polevoy, 2009).

Despite its vital role in spermatocyte cytokinesis, Fwd is dispensable for normal development and female fertility. Drosophila tissue culture cells show only a weak requirement for fwd during cytokinesis, with knockdown of fwd by RNAi resulting in a small increase in binucleate cells. This is particularly surprising given that yeast PIK1 is required for post-Golgi secretory trafficking and endocytosis. As secretion and endocytosis are essential processes, it is hypothesized that fwd is redundant with other genes for carrying out these functions outside of the male germline. Future investigations will determine the identity of these fwd-interacting genes (Polevoy, 2009).

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. This study shows that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, it was demonstrated that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, these data demonstrate a fundamental role for a plasma membrane-endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration (Assaker, 2010).

During migration, the cell needs to rearrange its cytoskeleton, its plasma membrane content, and its interaction with other cells. Many of these features can be controlled by vesicular trafficking. For example, Integrins, Cadherins, and other cell-cell or cell-matrix attachment proteins are transmembrane proteins tightly regulated by trafficking. Furthermore, the distribution of proteins and lipids at the plasma membrane is directly controlled by vesicular trafficking, as well as the localization of some actin remodeling proteins. During the process of border cell migration, endocytosis has been shown to regulate the polarity of RTK activity. This paper shows that the endocytic process plays a role in regulating the spatial localization of RTK activity by trafficking through the recycling endosome and by the polarized redelivery of endocytosed material to the plasma membrane (Assaker, 2010).

The key endocytic proteins previously involved in border cell migration - Sprint, Cbl, and Shibire - regulate the polarization of RTK activity during border cell migration. Different possible mechanisms are proposed to explain their action and have not been addressed in this landmark article (Jékely, 2005). Recently, it was shown that both the degradative pathway and the recycling pathway might be involved in this process. Thus, at least two models, which are not mutually exclusive, could explain the role of endocytosis in establishing this polarity. First, active RTKs could be endocytosed and degraded when diffusing away from the leading edge. Second, polarized recycling of endocytosed active RTKs could concentrate these active receptors at the leading edge. From the current experiments it can be concluded that the recycling of active RTKs or of a cofactor at the plasma membrane is necessary for border cell migration. Furthermore, it was demonstrated that the slow recycling route, through the recycling endosome, is used and that polarized redelivery at the plasma membrane is mediated by the exocyst subunit sec15 (Assaker, 2010).

It seems logical to think that RTKs, or active RTKs, are the cargo transported through this endocytic cycle. However, the identity of the protein being recycled remains to be determined. Indeed, there is no indication that RTKs are recycled. Immunofluorescence staining of the EGFR was performed. The signal obtained was diffuse and inconclusive in both control and rab11SN-expressing border cells. In addition, if active RTKs were trafficking through the recycling endosome, the pTyr in endocytic vesicles would be expected to be marked by Rab11. However, such a colocalization has never been observed. These data do not rule out a potential recycling of RTKs, because they could be present in the recycling endosome in quantities below detection levels or in an inactive form. However, the data suggest that the main cargo of this trafficking cycle is of another nature. This cargo could be a plasma membrane diffusion barrier, because polarized cells, such as epithelial cells and neurons, maintain different membrane domains, which rely on such barriers: the tight junctions and the axon hillock, respectively. Moreover, diffusion barriers have been proposed to define plasma membrane domains in migrating cells. These diffusion barriers appear to be linked to the actin cytoskeleton, but their exact nature is unknown. Because E-cadherin is involved in cell migration, it would have been an ideal candidate to play such a role, but it is unaffected by rab11SN expression (Assaker, 2010).

Another possibility is that endocytosis acts indirectly. For example, it might regulate key components of the plasma membrane or of the cytoskeleton. Recent evidence has shown that endocytosis and recycling can play a critical role in creating a positive feedback loop during polarity establishment in the budding yeast. In this particular case, endocytosis is critical for the localization of regulators of small GTPases of the Rho family. Furthermore, mammalian Tiam1, a GDP-to-GTP exchange factor (GEF) for Rac, has been shown to localize to endosomes, leading to the loading of active Rac at the plasma membrane through an endocytic-recycling cycle. Interestingly, a robust genetic interaction has been found between Rab11 and Rac1, which directs border cell migration (Wang, 2010). Until now, two Rac-GEFs, Myoblast city and Elmo, have been involved in border cell migration, but not the Drosophila Tiam1 homolog still life. Further studies will be necessary to determine if Rac1 is the main cargo of the endocytic-recycling cycle that regulates border cell migration (Assaker, 2010).

In the past few years, trafficking via the recycling endosome has been involved in the establishment or rearrangement of cell polarity in various events. In particular, a role for the recycling endosome has been observed when a rapid and dramatic rearrangement of the cell organization is required, including cellularization, cell-cell boundary rearrangement, asymmetric cell division, and cell migration. Trafficking through the recycling endosome is an ideal mechanism to polarize a cell rapidly, because it hijacks material already available in the cell at a new location. Furthermore, it is a very efficient mechanism to reinforce polarity by feedback loops. Similarly the exocyst plays a key role in the majority of these cell polarizations. In the case of cell migration, the recycling endosome may transform the diffuse extracellular gradient of RTK ligand into a robust intracellular polarization of RTK activity that is crucial for directed migration (Assaker, 2010).

There is much evidence that the function of the recycling endosome in the regulation of directed migration is conserved in mammals. The mammalian homologs of Drosophila Rab11 are Rab11A and -B and Rab25. They have been directly implicated in the migration of cancerous cells and in the formation of metastasis, a cell migration event resembling border cell migration. Rab11 effectors are also involved in mammalian cell migration. More specifically, PDGF receptor-dependent cell migration has been shown to be regulated by endocytosis in a mammalian cell culture assay and the recycling endosome has been indirectly implicated in the regulation of migration guided by the EGFR. Given the involvement of the recycling endosome in so many processes, targeting its function to reduce metastasis is unlikely to be efficient. However, identifying the main cargo of this recycling cycle could help identify more specific targets for drugs blocking the formation of metastasis (Assaker, 2010).

Evi5 promotes collective cell migration through its Rab-GAP activity

Membrane trafficking has well-defined roles during cell migration. However, its regulation is poorly characterized. This paper describes a screen for putative Rab-GTPase-activating proteins (GAPs) during collective cell migration of Drosophila melanogaster border cells (BCs). The uncharacterized Drosophila protein Evi5 was identified as an essential membrane trafficking regulator, and the molecular mechanism by which Evi5 regulates BC migration is described. Evi5 requires its Rab-GAP activity to fulfill its functions during migration and acts as a GAP protein for Rab11. Both loss and gain of Evi5 function blocked BC migration by disrupting the Rab11-dependent polarization of active guidance receptors. Altogether, these findings deepen the understanding of the molecular machinery regulating endocytosis and subsequently cell signaling during migration (Laflamme, 2012).

To achieve their directed collective cell migration toward the oocyte, BCs need to polarize their guidance receptors, which consist of RTKs, at their leading edge (Jékely, 2005). Rab11 is involved in restricting the activation of the RTKs at the front of the cell cluster (Assaker, 2010). Thus, it was hypothesized that Evi5 might also be important for the polarization of active RTKs. To test this, the distribution of pTyr (phosphorylated tyrosine), which was previously used as a marker of RTK activity in BCs (Jékely, 2005; Assaker, 2010), was assessed. A strong polarization was observed of the pTyr signal at the leading edge of control BCs, particularly within membrane protrusions. Either overexpression or depletion of Evi5 abolishes the enrichment of the pTyr signal at the leading edge, as shown by the ratio of posterior over anterior pTyr signal in BCs . These data demonstrate that a strict regulation of the recycling endosome is necessary for the proper spatial restriction of RTK activity (Laflamme, 2012).

Most of the previous studies focusing on GAP proteins were performed in cell culture or in vitro. Furthermore, only fragments of the proteins, such as the TBC domain, were used in most cases because GAP proteins are frequently large and insoluble. To circumvent these limitations, this study has combined in vitro and in vivo approaches to demonstrate that the Rab-GAP Evi5 regulates Rab11 in Drosophila. Furthermore, this work identifies Evi5 as a new regulator of collective cell migration, necessary for the maintenance of active RTK at the leading edge (Laflamme, 2012).

dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions

Notch signaling governs binary cell fate determination in asymmetrically dividing cells. A forward genetic screen identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. It is proposed that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells (Giagtzoglou, 2012).

This study describes the identification of dEHBP1 as a novel, positive regulator of Notch signaling in asymmetrically dividing cells in the ESO lineage in Drosophila. In the absence of dEHBP1, external cell types, such as socket and shaft cells, are transformed into internal cell types, i.e., neuron and sheath cells, one of the hallmarks of loss of Notch signaling. EHBP1 has been previously studied in mammalian cell culture systems and in vivo in C. elegans. In mammalian adipocytes, EHBP1 affects endocytosis and recycling of the glucose transporter GLUT4 in the context of insulin signaling, depending on its interaction via the NPF motifs present in its N-terminal region with EHD2 or EHD1, respectively. However, the fly and worm EHBP1 lack the NPF motifs, suggesting that the EHD-EHBP1 interaction may have emerged later in evolution. In C. elegans, EHBP1 was shown to impair rab10-mediated endocytic recycling of clathrin-independent endocytosed cargoes, such GLR-1 glutamate receptor. This study shows that dEHBP1 is required in the exocytosis and recycling of Delta, a ligand of the Notch receptor. Notch signaling defects were not reported in C. elegans ehbp1 mutants. Therefore, it would be interesting to investigate whether EHBP1 and its homologues play an evolutionarily conserved role of EHBP1 in Notch signaling (Giagtzoglou, 2012).

dEHBP1 is a ubiquitous protein that is associated with the plasma membrane, enriched at the lateral and basal surface of pII cells, where it colocalizes with F-actin. Live imaging with mCherry-dEHBP1 and immunofluorescent stainings with anti-dEHBP1 antisera also reveal dEHBP1-positive, punctate, intracellular structures within ESO lineages. An extensive analysis with a diverse array of intracellular markers revealed that these punctae colocalize with Rab8, indicating their exocytic nature. Importantly, in C. elegans, EHBP1 physically interacts and colocalizes with Rab8 and Rab10, and controls the recruitment of Rab10 in recycling endosomal structures. However, in the current studies, overexpression of dominant-negative forms of Rab10 or Rab8 in the ESO lineages as well as thoracic clones of a newly identified Rab8 loss-of-function allele do not confer any cell fate phenotypes. Furthermore, no interaction was detected between dEHBP1 and Rab8 or Rab10 in a yeast two-hybrid analysis. Therefore, it is believed that loss of either Rab8 or Rab10 function does not underlie the dEHBP1 mutant phenotypes that are describe (Giagtzoglou, 2012).

Notably, many key players that affect cell polarity or mark subcellular compartments, including Arm, Rab11, Sec15, and F-actin, are not affected by the loss of dEHBP1. In addition, cell fate determinants Numb and Neuralized are correctly segregated upon asymmetric cell division in dEHBP1 mutant cells. However, loss of dEHBP1 specifically affects the abundance and localization of Spdo, a regulator of Notch signaling in asymmetrically dividing ESO cells, and the exocytosis and trafficking of Delta (Giagtzoglou, 2012).

Spdo facilitates reception of Notch signal at the plasma membrane of the signal-receiving cell. Therefore, accumulation of Spdo in dEHBP1−/− ESO clusters and its presence in the plasma membrane should result in a Notch gain of function, instead of the loss-of-function phenotype that was observed. No effects have been observed of Spdo overexpression upon cell fate acquisition in the ESO lineage. Alternatively, the accumulation of Spdo in the absence of dEHBP1 in these cells may reflect defects in its trafficking and membrane localization, which render the activation of Notch signaling more difficult (Giagtzoglou, 2012).

dEHBP1 mutations cannot suppress the gain of function phenotype of overexpressed ligand-independent, activated Notch intracellular domain. In addition, dEHBP1 does not affect the steady-state levels of Notch protein, as well as its endocytosis. Therefore, it is concluded that dEHBP1 functions at a level upstream of presenilin-mediated S3 cleavage of Notch during reception of the signal. Although it cannot be excluded that dEHBP1 functions in the signal-receiving cell, where it may control the trafficking and localization of Spdo, it is concluded that dEHBP1 also functions in the sending of the signal. This conclusion is based on the fact that dEHBP1 mutations are able to suppress the gain of function of Notch phenotype conferred by the overexpression of DaPKCΔN. Overexpressed constitutively active DaPKCΔN places Spdo at the plasma membrane, enabling the activation of Notch signaling. This study found that upon loss of dEHBP1, Spdo is still found at the plasma membrane under conditions of overexpression of DaPKCΔN. Therefore, the suppression of the overexpression phenotype of DaPKCΔN by loss of dEHBP1 may be because of other defects, such as loss of the ability of Delta to signal. Furthermore, loss of dEHBP1 leads to development of additional neurons despite the concomitant ectopic expression of DeltaR+, a variant of Delta, in clones within pupal nota at 36 h APF. Because the steady-state levels of Delta are not affected in dEHBP1−/− ESO lineages, whether dEHBP1 affects Delta trafficking in the signal-sending cell was examined. Upon loss of dEHBP1, the abundance of Delta at the cell surface is significantly reduced, suggesting that exocytosis is defective. Importantly, most of the remaining extracellular Delta protein localizes at the basal side of the signal-sending cell. This suggests that in addition to affecting exocytosis of Delta, dEHBP1 may also play a role in basal-to-apical trafficking of Delta. This leads to a reduced level of Delta at the signaling interface, which interferes with proper Notch signaling in the cell receiving the signal. Although the results do not exclude a possible role of dEHBP1 in other aspects of Delta trafficking, such as endocytosis, reduced exocytosis of Delta should mask an endocytic defect in the assays. The enrichment of dEHBP1 in the basal and lateral area of the plasma membrane, its colocalization with F-actin at the actin-rich structure at the interface of the pIIa and pIIb cells, the reduction of Delta exocytosis in mutant cells, and the absence of Delta at the interface and the apical surface of the ESO cluster in mutant cells indicate a role of dEHBP1 in the Sec15/Rab11 recycling pathway. Indeed, the colocalization of dEHBP1 and Delta in sec15−/− ESO lineages implies that the exocyst component, Sec15, controls exocytosis of Delta, Spdo, and dEHBP1 to the apical plasma membrane through a common compartment. Because loss of dEHBP1 does not affect the localization of either Rab11 or Sec15, it is concluded that sec15 lies more upstream in the trafficking pathway regulating the localization of multiple components, while dEHBP1 functions during the later stages of intracellular trafficking. Furthermore, the physical interaction between dEHBP1 and Sec15 as well as Rab11 suggest a mechanism how dEHBP1 may regulate the membrane localization of Delta via its interaction with Sec15 and Rab11 at the pII cells interface, even though such interaction was detected under transient overexpression conditions. It is proposed (see Model of dEHBP1 function) that dEHBP1 is an adaptor of the Rab11/Sec15-positive, Delta-bearing vesicles required for exocytosis (Giagtzoglou, 2012).

The identification of dEHBP1 provides further compelling evidence that the exocytosis and recycling pathway of Delta during asymmetric divisions is tightly regulated. The recycling pathway of Delta appears to be context dependent, i.e., it is not required in all cells that use Notch signaling. Still, the discovery of dEHBP1 as a novel player in Notch signaling provides the opportunity to test its role in Notch-related neurobiological behaviors, such as sleep and addiction, as well as in Notch-related diseases, as for example in Wiskott-Aldrich syndrome, an immunodeficiency characterized by abnormal differentiation and function of T cell lineages. Furthermore, because the anthrax toxins lethal factor (LF) and edema factor (EF) inhibit the Sec15/Rab11-dependent Delta-recycling pathway in flies and endothelial cells, it would be interesting to hypothesize whether they target dEHBP1 to mediate their toxicity (Giagtzoglou, 2012).

Rab11 modulates alpha-synuclein mediated defects in synaptic transmission and behaviour

A central pathological hallmark of Parkinson's disease (see Drosophila as a Model for Human Diseases: Parkinson's disease) is the presence of proteinaceous depositions known as Lewy bodies, which consist largely of the protein α-synuclein (αSyn). Mutations, multiplications, and polymorphisms in the gene encoding αSyn are associated with familial forms of PD and susceptibility to idiopathic PD. Alterations in αSyn impair neuronal vesicle formation/transport, and likely contribute to PD pathogenesis by neuronal dysfunction and degeneration. αSyn is functionally associated with several Rab family GTPases, which perform various roles in vesicle trafficking. This study explored the role of the endosomal recycling factor Rab11 in the pathogenesis of PD using Drosophila models of aSyn toxicity. αSyn induces synaptic potentiation at the larval neuromuscular junction by increasing synaptic vesicle size, and that these alterations are reversed by Rab11 overexpression. Furthermore, Rab11 decreases aSyn aggregation and ameliorates several αSyn-dependent phenotypes in both larvae and adult fruit flies, including locomotor activity, degeneration of dopaminergic neurons, and shortened lifespan. This work highlights the importance of Rab11 in the modulation of synaptic vesicle size and consequent enhancement of synaptic function. The results suggest that targeting Rab11 activity could have therapeutic value in PD (Breda, 2014).

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

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