Tbc1d15–17 regulates synaptic development at the Drosophila neuromuscular junction

Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15–17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15–17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15–17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15–17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15–17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15–17 and its potential substrate Rab7 in regulating synaptic development.     

Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles

Defects in lysosomal trafficking pathways lead to decreased cell viability and are associated with progressive disorders in humans. Previously we have found that loss-of-function (LOF) mutations in the Drosophila gene blue cheese (bchs) lead to reduced adult life span, increased neuronal death, and widespread CNS degeneration that is associated with the formation of ubiquitinated-protein aggregates. To identify potential genes that participate in the bchs functional pathway, we conducted a genetic modifier screen based on alterations of an eye phenotype that arises from high-level overexpression of Bchs. We found that mutations in select autophagic and endocytic trafficking genes, defects in cytoskeletal and motor proteins, as well as mutations in the SUMO and ubiquitin signaling pathways behave as modifiers of the Bchs gain-of-function (GOF) eye phenotype. Individual mutant alleles that produced viable adults were further examined for bchs-like phenotypes. Mutations in several lysosomal trafficking genes resulted in significantly decreased adult life spans and several mutants showed changes in ubiquitinated protein profiles as young adults. This work represents a novel approach to examine the role that lysosomal transport and function have on adult viability. The genes characterized in this study have direct human homologs, suggesting that similar defects in lysosomal transport may play a role in human health and age-related processes.     

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.     

Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.     

Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, we have tested 162 new mutations for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. We show that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. We then provide evidence that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity.     

Rab proteins in gastric parietal cells: evidence for the membrane recycling hypothesis.

The gastric parietal cell secretes large quantities of HCl into the lumen of the gastric gland in response to secretagogues such as histamine. In the membrane recycling hypothesis, this secretory activity requires the trafficking of the gastric H+/K(+)-ATPase to the cell surface from intracellular tubulovesicles. The Rab subclass of small GTP-binding proteins is thought to confer specificity to vesicle transport throughout the secretory pathway, and previous investigations established that Rab11 is highly expressed in gastric parietal cells. Recent discoveries in intra-Golgi transport and neuronal synaptic vesicle fusion have fortuitously converged on an evolutionarily conserved protein complex involved in vesicle docking and fusion. Recent results indicate that Rab11 is involved in the apical targeting of vesicles in parietal cells and other epithelial cells throughout the gastrointestinal tract. In support of the membrane recycling hypothesis, Rab co-segregates with H+/K(+)-ATPase in parietal cells. The presence of Rab11 on tubulovesicles supports a role for this Rab protein in recycling vesicle trafficking.     

Drosophila CK1-γ, gilgamesh, controls PCP-mediated morphogenesis through regulation of vesicle trafficking

Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.     

Exocyst-mediated membrane trafficking is required for branch outgrowth in Drosophila tracheal terminal cells

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increase surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs at the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find that the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.     

Rab11 regulates recycling through the pericentriolar recycling endosome

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11- positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.     

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.     

Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

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

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

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

Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.     

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.     

Characterization of a Rab11 homologue in Trypanosoma cruzi

Vesicle trafficking between organelles occurs through fusion of donor and specific acceptor membranes. This process is highly regulated and ensures proper direction in sorting and packaging of a number of molecules in eukaryotic cells. Monomeric GTPases of the Rab family play a pivotal role in the control of membrane fusion and vesicle traffic. In this paper, we characterize a Trypanosoma cruzi Rab 11 homologue (TcRab11) that shares at, the amino acid level, 40% similarity with human rab11, Arabdopsis thaliana rab11 and yeast rab11 homologue genes. Western blot analysis, using a polyclonal rabbit antiserum raised against a synthetic peptide derived from the COOH-terminus of predicted the TcRab11 protein, reacted to a 26kDa protein. In immunofluorescence assays, TcRab 11, was shown to be expressed in epimastigote and amastigote forms, but it was absent in trypomastigotes. Interestingly, the TcRab11 product seems to be located at the reservosome complex, a site of active endocytosis and vesicle fusion present only in the epimastigote stage. Therefore, TcRab11 may represent the first molecular marker of this peculiar organelle.     

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

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

Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release

During constitutive endocytosis, internalized membrane traffics through endosomal compartments. At synapses, endocytosis of vesicular membrane is temporally coupled to action potential-induced exocytosis of synaptic vesicles. Endocytosed membrane may immediately be reused for a new round of neurotransmitter release without trafficking through an endosomal compartment. Using GFP-tagged endosomal markers, we monitored an endosomal compartment in Drosophila neuromuscular synapses. We showed that in conditions in which the synaptic vesicles pool is depleted, the endosome is also drastically reduced and only recovers from membrane derived by dynamin-mediated endocytosis. This suggests that membrane exchange takes place between the vesicle pool and the synaptic endosome. We demonstrate that the small GTPase Rab5 is required for endosome integrity in the presynaptic terminal. Impaired Rab5 function affects endo- and exocytosis rates and decreases the evoked neurotransmitter release probability. Conversely, Rab5 overexpression increases the release efficacy. Therefore, the Rab5-dependent trafficking pathway plays an important role for synaptic performance.     

Molecular characterization of Rab11 interactions with members of the family of Rab11-interacting proteins

The Rab11 subfamily of GTPases plays an important role in vesicle trafficking from endosomes to the plasma membrane. At least six Rab11 effectors (family of Rab11-interacting proteins (FIPs)) have been shown to interact with Rab11 and are hypothesized to regulate various membrane trafficking pathways such as transferrin recycling, cytokinesis, and epidermal growth factor trafficking. In this study, we characterized interactions of FIPs with the Rab11 GTPase using isothermal titration calorimetric studies and mutational analysis. Our data suggest that FIPs cannot differentiate between GTP-bound Rab11a and Rab11b in vitro (50-100 nm affinity) and in vivo. We also show that, although FIPs interact with the GDP-bound form of Rab11 in vitro, the binding affinity (>1000 nm) is not sufficient for FIP and GDP-bound Rab11 interactions to occur in vivo. Mutational analysis revealed that both the conserved hydrophobic patch and Tyr628 are important for the GTP-dependent binding of Rab11 to FIPs. The entropy and enthalpy analyses suggest that binding to Rab11a/b may induce conformational changes in FIPs.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.     

The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.