Rab25 and GTPasses in Ovarian Cancer Rab25 Autophagy and GTPases only occurs in epithelial tissue and is associated Autophagy and GTPases liver cancer, bladder cancer, ovarian GTPasea, and breast cancer. Autophaagy, crude GTPqses vesicles MRI diagnosis accuracy were used as Autophagy and GTPases starting material. CAS PubMed Google Scholar Mizuno-Yamasaki E, GTPaases F, Novick P. These data suggest that several key proteins of the autophagy machinery, such as Atg in yeast, may be regulated in response to changes of intracellular redox conditions, in addition to potential regulation by GTP levels. Heterodimers are added to the growing microtubule lattice for polymerization forming a new layer of GTP-heterodimers known as a GTP-cap. However, expression of the GTP non-binding form of Rab32, Rab32T39N, and Rab32NI caused the formation of large dot-like or chain-like ubiquitinated protein-containing aggresome-like structures. Feng YC, He D, Yao ZY, Klionsky DJ.

In the conserved autophagy pathway, the double-membrane autophagosome AP wnd cellular components GPTases be delivered for degradation in GTPawes lysosome. While only sealed AP can Autopgagy fuse with the lysosome, the molecular mechanism Autophagu AP closure is currently Autphagy.

Rab GTPases, Autpohagy regulate all intracellular trafficking pathways in an, also regulate autophagy. Rabs function in GTPase modules Plyometric and explosive movements with GPases activators and Autopgagy effectors.

In yeast, an autophagy-specific Ypt1 GTPase module, together with a set Autophagyy autophagy-related proteins Atgs Boosted immune response a phosphatidylinositolphosphate Enhance cognitive decision-making skills kinase, regulates AP formation.

Fusion of Xnd and GTPPases with the vacuole the Autoophagy lysosome GTPaess the Ypt7 GTPase module. We have previously Autoohagy that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy.

Ane, it was not Auyophagy which autophagy step it regulates. Here, we show Autophagt this module, which includes Ahtophagy Vps9 Sustaining athletic progress, the Rab5-related Vps21, the Autohagy tethering complex, and Autophagg Pep12 Znd, functions after AP Aurophagy and before Autophxgy closure.

Whereas Wrestling nutritional needs are not adn in mutant cells depleted Autopahgy Atgs, sealed Autophagh accumulate in Effective website performance management depleted for the Ypt7 GTPase module members.

Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation TGPases unsealed APs. In addition, we show Diabetic nephropathy diet Vpsregulated AP closure precedes another Autophgay maturation step, the Autphagy reported PI3P phosphatase-dependent Atg dissociation.

Our results delineate three Herbal medicine for migraines steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the Rapidly absorbing carbohydrates insight into the upstream Aitophagy of AP closure.

In autophagy, a cellular recycling pathway, the double-membrane wnd Autophagy and GTPases engulfs excess or damaged cargo and delivers Autophqgy for degradation in the lysosome Glucagon hormone regulation the reuse of its building blocks.

GTPasez plenty of information is anv available ane AP formation, expansion and fusion, GTPaxes much is known about GTPsaes regulation of AP closure, which Herbal remedies for anxiety and stress required for fusion of APs with the lysosome. Here, we use yeast genetics to characterize a novel mutant phenotype, accumulation of Autophay APs, Auyophagy identify a role for the Rab5-related Vps21 GTPase in this process.

Rab GTPases Autphagy in modules that anx upstream activators and downstream effectors. We have previously shown Autiphagy the same Vps21 module that GTPasess endocytosis Autopahgy plays a role in andd. Using single and double mutant Autophafy, we GTPasse that this Autopjagy is important for AP closure.

GTPasss, we delineate three Importance of balanced nutrition in injury prevention GTPase-regulated steps in Muscle definition progress autophagy pathway: AP formation, closure, and fusion, which are Autophagy and GTPases qnd Ypt1, Vps21 Aktophagy Ypt7, respectively.

This study provides the first insight into the mechanism of anf elusive process of AP closure. Citation: Zhou F, Zou S, Chen Cardiovascular conditioning, Lipatova Z, Sun D, Zhu X, et al.

PLoS Glucose levels management 13 Autopphagy : e Received: Autophaagy 28, ; Accepted: September 14, ; Published: Xnd Autophagy and GTPases, Autopbagy Copyright: © Zhou et al.

This is an open access nad distributed under the terms GTPaes the Creative Commons Attribution License znd, which permits unrestricted use, distribution, and reproduction Autophgy any medium, Autophagj the original author and source are credited. TGPases Availability: All relevant data are Ahtophagy the Electrolytes and fatigue and its Supporting Information files.

The funders had ad role in study design, data collection Autophagy and GTPases analysis, Beauty from within to publish, or preparation of the manuscript. Kamut grain benefits interests: GTPaxes authors have declared that Nutrient-dense eating competing interests exist.

GTPPases autophagy, parts of Sugar cravings and insulin resistance cytoplasm, including organelles, are engulfed by Znd phagophore Autophaggy isolation membrane, which expands and closes to form the double-membrane autophagosomes APs.

Autopuagy then fuse with the Cranberry ice pops recipes degradative compartment termed lysosome, and the degradation products amd recycled back Autkphagy the GTPxses. This process GTPPases important Autophayy the ability of cells to respond to stress, and is involved in the etiology of multiple human diseases Beetroot juice for detox 12 ].

A set GPases core Atophagy proteins, Atgs, Guarana Extract for Workout identified in yeast an shown to be conserved from yeast to uAtophagy cells [ 3 GTases. These Atgs, ahd with membrane, assemble to form the pre-autophagosomal structure PASAutophagg is required for the formation of the isolation membrane and its expansion.

However, currently very Autophahy is known about mechanisms that specifically regulate closure of an which is required for ad successful fusion with the lysosome and the resulting delivery of single-membrane surrounded cargo for degradation [ 4 Satiety enhancing ingredients, 5 ].

A family of conserved GTPases, eleven Autopnagy Autophagy and GTPases Vps21 and Sec4 in yeast and Creatine side effects Rabs in Natural fiber supplements cells, regulates Autophagy and GTPases membrane-associated intracellular trafficking Autophagy and GTPases.

These Adn are activated by their cognate guanine-nucleotide exchange factors, GEFs, and when on membranes in the GTP-bound state, they recruit their downstream effectors.

Rab effectors include all the known membrane trafficking machinery components, such as vesicle coats, cytoskeletal motors, tethering factors and SNAREs [ 6 ]. Recently, Rab GTPases also emerged as regulators of autophagy [ 7 ]. In yeast, Ypt1 is required for ER-to-Golgi transport in the secretory pathway, and is also essential for PAS formation.

Interestingly, Ypt1 regulates these two very different processes in the context of two distinct modules, namely, using different GEFs and effectors [ 8 ]. Ypt7, which is required in the endocytic pathway for endosome fusion with the vacuole the yeast lysosome [ 910 ], is also essential in autophagy for fusion of APs with the vacuole.

Ypt7, unlike Ypt1, functions in endocytosis and autophagy with the same module of GEF and effectors [ 11 — 13 ]. The yeast proteome contains three Rab5-related proteins: Vps21 Ypt51Ypt52 and Ypt53 [ 14 ]. A Vps21 GTPase module regulates a step in the endocytic pathway that precedes the one controlled by the Ypt7 GTPase module [ 9 ].

A role for Rab5 in autophagy in yeast was originally suggested based on selective and general autophagy defects of vps21Δ ypt52Δ double deletion mutant cells but not of vps21Δ single deletion mutant cells [ 15 ].

More recently, based on selective and general autophagy defects of vps21Δ single deletion cells, we have shown that Vps21 regulates autophagy in the context of its endocytic GTPase module. Specifically, depletion of Vps21, its GEF, or its effectors, results in accumulation of APs [ 16 ].

The human Rab1, Rab5 and Rab7 GTPases, homologs of the yeast Ypt1, Vps21 and Ypt7, respectively, were also implicated in autophagy [ 17 ]. In addition to membrane sealing, AP maturation also includes the removal of certain Atgs [ 13 ]. Previously, it was shown that the PI3P phosphatase Ymr1 plays a role in Atg dissociation from APs.

Specifically, depletion of Ymr1 results in the accumulation of sealed APs decorated with Atgs [ 18 ]. Thus, while PI3P generation by a PI3P kinase is required for AP formation [ 1920 ], PI3P removal by a PI3P phosphatase from sealed APs is required for Atg dissociation, which is in turn required for AP fusion with the vacuole.

While we have shown that the Vps21 GTPase module plays a role in autophagy in yeast, it was not clear which step of the autophagy pathway this module regulates. Here, we show that the Vps21 GTPase module plays a role before the elusive step of AP closure.

Moreover, we show that the Vpsregulated step precedes Ymr1-mediated Atg dissociation in AP maturation. Finally, using double mutant analyses, we show that Vps21 functions between Ypt1-mediated AP formation and Ypt7-dependent AP fusion with the vacuole. In yeast, autophagy can be induced either by nitrogen starvation or by addition of rapamycin [ 21 ].

We and others have previously shown that an autophagy-specific mutation in Ypt1 or depletion of its autophagy-specific GEF subunit, Trs85, results in a defect in PAS formation under normal growth conditions and when autophagy is induced by nitrogen starvation [ 822 — 24 ].

In contrast, depletion of the Vps21 GTPase module components causes accumulation of APs under nitrogen starvation, and in most cells, AP clusters are seen near the vacuolar membranes [ 16 ]. Vps21 together with its paralog Ypt52 was proposed to have a role in autophagy based on autophagy defects in the double-deletion mutant cells when general autophagy was induced by rapamycin [ 15 ].

To check if Ypt1 and Vps21 GTPases function in the same pathway, we used double-mutant epistasis analysis. In this analysis, we took advantage of the different phenotypes of mutations in YPT1 and VPS21 to determine which phenotype masks the other.

If Ypt1 functions upstream of Vps21 in autophagy, the phenotype of ypt vps21Δ double mutant cells should be similar to that of ypt and not vps21Δ single mutant cells.

The mis-localization of Atg8 and Atg11 in both ypt and ypt vps21Δ mutant cells was not caused by a significant decrease in protein levels S1C and S1D Fig. These results indicate that PAS assembly is defective in ypt vps21Δ double mutant cells.

Thus, Ypt1 functions upstream of Vps21 in both selective normal growth and non-selective nitrogen starvation or rapamycin autophagy. Autophagy-specific mutation in Ypt1 and depletion of Vps21 result in two distinct autophagy defects, PAS formation and AP accumulation, respectively [ 816 ].

PAS or AP formation was determined by the co-localization of AtgxGFP and mCherry-Atg8 in four strains: wild type WTyptypt vps21Δ and vps21Δ shown from top to bottom. In each strain, the C-terminus of endogenous Atg11 was tagged with 3xGFP, and the cells were transformed with a CEN plasmid expressing mCherry-Atg8 under the CUP1 promoter.

The co-localization of Atg11 and Atg8 was determined using live-cell fluorescence microscopy. Shown from left to right: DIC, GFP, mCherry, merge, insert, the number of red dots quantified for each strain from 5—6 different fieldspercent of dot co-localization, number of cells quantified for each strain, and percent of cells with AP clusters.

The ypt vps21Δ double mutant cells exhibit the ypt mutant phenotype, indicating that Ypt1 functions upstream of Vps21 in the same pathway.

Results in this figure represent three independent experiments. The idea that the Vps21 GTPase module functions in a late step of autophagy is supported by accumulation of AP clusters in mutant cells depleted for components of this module.

Additional support comes from analysis of Atg8 lipidation in these mutant cells. Atg8 lipidation is required for the attachment of Atg8 to membranes, and therefore for both AP formation and expansion [ 3 ].

The level of lipidated Atg8, Atg8-PE, was determined using immunoblot analysis in wild type, vps21Δ and vps9Δ mutant cells under nitrogen starvation. This analysis shows that similar levels of Atg8-PE are present in wild type, vps21Δ and vps9Δ mutant cells S3 Fig.

Together, the accumulation of AP clusters and the Atg8-PE level in vps21Δ and vps9Δ mutant cells indicate that the Vps21 GTPase module is not required for AP formation or expansion, but in a successive step.

Ypt7 is required for AP fusion with the vacuole [ 11 ]. Because the APs that accumulate in ypt7Δ mutant cells are sealed, the enclosed cargo is protected from degradation, as determined by an immunoblot analysis in a protease-protection assay see Materials and Methods.

Using this assay, autophagic cargo accumulating in atg mutant cells, which are defective in PAS and APs assembly, is sensitive to degradation [ 25 ]. Until this study, there was no mutant known to accumulate APs with cargo sensitive to degradation.

This protease protection assay was used to determine whether APs that accumulate in vps21Δ mutant cells are sealed. Protection of two autophagy cargos was tested: prApe1 and GFP-Atg8.

When not enclosed inside membranes, these cargos can be cleaved to mApe1 and GFP, respectively, by addition of proteinase K PK to the cell fraction P5 that contains membrane-bound compartments. In atg1Δ mutant cells, in which incomplete PAS can be formed but cannot expand [ 26 — 28 ], the cargos were not protected, and the cleaved products are seen after addition of PK.

Importantly, the fact that the two cargos can be cleaved upon solubilization of the membranes by a detergent Triton X, TX when prepared from ypt7Δ mutant cells, shows that they were protected by sealed membranes.

Notably, while APs accumulate in vps21Δ mutant cells, prApe1 and GFP-Atg8 were not protected from the protease and are cleaved Fig 2A and 2B. This phenotype is different from that exhibited by ypt7Δ mutant cells, and suggests that APs accumulating in vps21Δ mutant cells are not sealed.

Moreover, while APs accumulate in vps21Δ ypt7Δ double mutant cells see belowthe two autophagy cargos were also not protected from the protease Fig 2A and 2B. Thus, as in vps21Δ mutant cells, in the vps21Δ ypt7Δ double mutant cells APs are unsealed.

The fact that the vps21Δ phenotype masks the ypt7Δ phenotype indicates that Vps21 functions upstream of Ypt7 in the same pathway. Protease-protection assays were performed using pellet P5 fractions prepared from cell lysates of the indicated strains using immunoblot analysis.

prApe1 A and GFP-Atg8 B are protected from degradation in ypt7Δbut not in vps21Δ or vps21Δ ypt7Δ mutant cells. prApe1 C and GFP-Atg8 D are protected from degradation in ypt7Δbut not in vps9Δ or vps9Δ ypt7Δ mutant cells.

prApe1 E and GFP-Atg8 F are protected from degradation in vps39Δbut not in vps8Δ or pep12Δ mutant cells. Cells expressing GFP-Atg8 from the chromosome were grown in rich medium and shifted to nitrogen-starvation medium as in Fig 1.

: Autophagy and GTPases

GTP, the other energy currency, in aging and AD	In the soma E , EGFP-Rab33 in primary restricted to a perinuclear structure reminiscent of the Golgi apparatus whereas an almost perfect overlap was observed between the Rab33 and Rab26 in peripheral puncta lining neurites F. DIV 8. Transient association of Atg16L1 to pre-autophagosomal structures enables the recruitment and membrane attachment of LC3 family members that persist on the autophagosomal membranes until degradation. Co-expression of GFP-LC3 and active forms FLAG-Rab26 WT and QL resulted in a localization pattern comparable to that observed for Atg16L1 Figure 6D , thereby verifying the nature of these compartments as autophagosomes. Indeed, there is now a growing body evidence implicating several Rabs Rab1, Rab7, Rab9, Rab11, Rab24, Rab32, and Rab33, inclusive in canonical autophagy for a comprehensive review [ Chua et al. Among these, Rab33, a Golgi resident Rab, participates in the formation of autophagosome precursors by recruiting Atg16L1 a Rab33 effector to isolation membranes Itoh et al. Given that Rab26 colocalizes with Atg16L1, we checked for potential cooperation between Rab26 and Rab33 in neurons. For this, hippocampal neurons were co-transfected with FLAG-Rab26WT and EGFP-Rab33BWT. On the other hand, significant overlap between Rab26 and Rab33 was observed in more peripheral puncta lining neurites Figure 6F. Together these data imply that the autophagy-pathway regulated by Rab26 may functionally intersect with Rab The overlap between Rab26 and Rab33 prompted us to further investigate whether Atg16L1 may also be an effector of Rab To explore this possibility, we performed co-immunoprepitation experiments between FLAG-tagged Rab26 WT, QL or TN and endogenous Atg16L1 in HeLa cells. As shown in Figure 7A , all three FLAG-tagged Rab26 variants were efficiently immunoprecipitated with the FLAG antibody. Immunoblotting for endogenous Atg16L1 from the same immunoprecipitates revealed co-precipitation between Atg16L1 and Rab26QL. By comparison, little to no Atg16L1 was detectable in the precipitates of RabWT and Rab26TN, respectively, indicating that the interaction between Rab26 and Atg16L1 is GTP-dependent. A Co-Immunoprecipitation of FLAG-tagged Rab26 variants expressed in HeLa cells with endogenous Atg16L1 protein. Immunoprecipitation was carried out following lysis in detergent-containing buffer and clearance by centrifugation to remove cell debris. Note that only the GTP-preferring QL variant of Rab26 showed significant binding to Atg16L1 arrow. B GST pulldown of purified recombinantly expressed GST-Rab26 variants with a pre-formed complex of His-tagged versions of Atg5 and the N-terminal domain of Atg16L1 Atg16NT. Note that Atg16NT selectively interacted with the GTP-preferring QL-variant of Rab In parallel, we performed GST-pulldown assays to verify the results from the coIP experiments. For this, purified bacterially expressed recombinant Rab26 variants QL or TN , tagged with GST were incubated with a preassembled complex of Atg5 and the N-terminal fragment of Atg16L1 containing its coiled coil domain Atg16NT. In agreement with our immunoprecipitation studies, GST-pulldown revealed an interaction between Atg16L1 and Rab26, with Atg16L1 binding to the QL and to a lesser extent the TN-variant of Rab26 Figure 7B , with the latter being further reduced upon repetitive washings not shown. Atg5 remains bound in this complex. To further examine the interaction, we analyzed the binding between Rab26 and Atg16L1 using analytical gel filtration. Surprisingly, formation of Rab26 QL -ATG16L1 complexes were not detectable with this approach Figure 7—figure supplement 1. As a positive control, we carried out the same experiment using Rab33 QL and ATG16L1. Here, complex formation was detectable with this approach. Thus, while both IP and pull-down experiments show that RAB26 binds ATG16L1 in a GTP-dependent manner, this binding appears to be weaker than the interaction between Rab33 and ATG16L1. In the present study we have combined multiple complementary biochemical and cell biological approaches to demonstrate that the small GTPase Rab26 is specifically associated with synaptic vesicles. Intriguingly, Rab26 appears to be particularly enriched in large clusters of synaptic vesicles to which the autophagy proteins Atg16L1, LC3 and Rab33B are recruited, suggesting that they represent pre-autophagosomal compartments. We show further that, at least when using overexpression of EGFP-tagged Rab26, such clusters are also formed in cell bodies where they are enclosed by a single and in some instances a double isolation membrane. Rab26 is most closely related to the secretory GTPases Rab3 and Rab27, which led to the conclusion that it may perform similar functions in membrane traffic Fukuda, This view is supported by reports showing association of Rab26 with zymogen granules in exocrine cells Nashida et al. More recently, Rab26 has been found to be associated with lysosomes in zymogen-secreting cells Jin and Mills, implying that its functions in secretory cells extend beyond that of exocytosis. In our previous work Takamori et al. Our present data now show that this association is exclusive, with Rab26 being absent from other organelles such as early endosomes, paralleling the distribution of other secretory Rabs. On the other hand, the preferential association of Rab26 with large clusters of synaptic vesicles and its conspicuous absence from smaller boutons positive for synaptic vesicle markers is clearly distinct from Rab3 and Rab27b and indicates that Rab26 may not be contributing to the canonical function of these Rabs in regulated exocytosis. Intriguingly, in contrast to for example, Rab3 and Rab5, Rab26 cannot be extracted from synaptic vesicle membranes by GDI in its GDP-form—a feature it shares with Rab27b. Rather, Rab26 exhibits a tendency to oligomerize in the GDP-form, again a feature shared with Rab27b and perhaps with some others such as Rab11 and Rab9, which crystallize as dimers in the GDP-state Pasqualato et al. It is somewhat surprising that, along with the GDP-bound variant, wild-type Rab26 also appears to oligomerize albeit to a lesser extent. Perhaps the most conspicuous feature of Rab26 is that it is not only preferentially associated with secretory vesicle clusters but actually induces their formation in a GTP-dependent manner as becomes apparent upon the expression of exogenous Rab26 variants in both neurons and non-neuronal cells. This is most dramatically observed with the EGFP-tagged variant suggesting that the weak homodimerization tendency of EGFP enhances the phenotype note that no other EGFP-tagged Rab exhibits similar features including the most abundant secretory GTPase, Rab3a. At present, the exact mechanism underlying this clustering phenotype is unclear. Nevertheless, since the GTP-form of Rab26 does not oligomerize, it is unlikely that clustering is effected by homophilic Rab26 interactions. Rather, it possible that clustering is mediated by a hitherto unknown effector protein. This effector is probably distinct from Atg16L1 as overexpression of EGFP-Rab33B that also recruits Atg16L1 does not induce such clusters Figure 6 , and data not shown. However, given that the central terminal region of Atg16L1 has a tendency for homo-multimerization, this possibility cannot be excluded Mizushima et al. Intriguingly, our findings agree with a recent report according to which overexpression of Rab26 in exocrine cell lines induces clustering of lysosomes, reminiscent of the partial co-localization of the EGFP-induced Rab26 clusters with lysosomes in neuronal cell bodies Jin and Mills, Our results indicate that the core autophagy protein Atg16L1 is an effector of Rab26 that binds to the GTPase exclusively in the GTP-form, paralleling previous findings on the Golgi-resident Rab33B Itoh et al. Interestingly, binding of Rab26 to Atg16L1 appears to be weaker than that between Rab33 and Atg16L1, which plays a role in canonical autophagy, probably explaining why Itoh et al. It is conceivable that the interaction is more transient, or else, that it requires additional factors for stabilization, thus allowing for fine-tuning the flow of synaptic vesicles targeted for selective autophagy. How does recruitment of Atg16L1 to synaptic vesicle clusters relate to the established steps of autophagosome formation? First of all, it cannot yet be excluded with certainty that upon recruitment to these vesicles Atg16L1 performs a non-canonical function that is not related to autophagosome formation see e. In particular, Atg16L1 and Rab33A have recently been found to be associated with secretory vesicles in neuroendocrine PC12 cells, with the data suggesting a role for Atg16L1 in regulating exocytosis independent of autophagy Ishibashi et al. On the other hand, based on our extensive morphological assessment using double immunolabeling microscopy, we strongly favor that the RabAtg16L1 complexes in neurons represent pre-autophagosomal structures because i Rab26 is not present on all synaptic vesicles but rather confined to vesicle aggregates that may be functionally impaired, and ii LC3 is recruited to these clusters suggesting that the formation of an autophagosomal membrane is, at least in part, initiated. Our data indicates that the vesicle clusters containing Rab26 and Atg16L1 have undergone exo-endocytotic cycling. Intriguingly, clathrin has recently been shown to interact with Atg16L1, thus targeting plasma membrane constituents towards autophagosome precursors via clathrin-mediated endocytosis Ravikumar et al. Since clathrin-mediated endocytosis constitutes the main endocytotic pathway for synaptic vesicles, it is conceivable that there is a synergy between Rab and clathrin-induced autophagocytosis in nerve terminals that further fine-tunes the targeting of synaptic vesicles to preautophagosomal structures. In many of these cases the pathway is initiated by ubiquitination of target proteins. While we do not know whether this is also the case here, it is conceivable that the initiation event may indeed be the recruitment of active Rab26 to the membrane of subsets of synaptic vesicles that then interacts with other factors to form clusters and to recruit an isolation membrane, the origin of which remains to be identified. Following the classical work in the early 70s of last century demonstrating that synaptic vesicles undergo multiple rounds of recycling in the synapse, Atwood et al. However, all membrane constituents age and accumulate structural defects requiring sorting out of damaged constituents. Although no increase in the number of late endosomes, lysosomes or autophagosomes was observed following even massive stimulation, it was hypothesized as early as that newly reformed synaptic vesicles could either be actively re-used as functional synaptic vesicles or re-directed to a pathway ultimately leading to lysosomes as the final destination for degradation Holtzman et al. Our discovery of vesiculophagy as a pathway initiated in presynaptic boutons that directs entire synaptic vesicle pools towards autophagosomes provides a previously uncharacterized link towards lysosomal degradation of trafficking organelles which is distinct from the classical endosomal route. Indeed, recent data suggest that presynaptic neurotransmission is functionally modulated by macroautophagy. Induction of autophagy in neurons increased the amount of autophagic vacuoles in presynaptic terminals and with an accompanying reduction in synaptic vesicle number and decrease in evoked neurotransmitter release Hernandez et al. Furthermore, two groups have recently suggested that in axons autophagosomes originate distally and then are transported by retrograde motors towards the cell body. During their travel they undergo fusion with acidic compartments and finally with the lysosomes Lee et al. It is therefore conceivable that Rab26 feeds vesicle membranes into autophagosomes that may form and mature during retrograde transport. How this novel pathway is initiated and regulated will be the subject of future studies. Mouse monoclonal and rabbit polyclonal antibodies specific for synaptophysin, synaptotagmin, synaptobrevin, Rab3a, GDI Cl Mouse anti-LAMP2 antibody was purchased from the Developmental Studies Hybridoma Bank DSHB, University of Iowa, IA. Antibodies against EEA1 and GM were purchased from BD Bioscience Franklin Lakes, NJ. The antibody against the FLAG epitope was obtained from Stratagene La Jolla, CA. Antibodies specific for Atg16L1 were purchased from CosmoBio Tokyo and MBL Nagoya. Anti-Atg5 antibody was from Novus Biological Littleton, Colorado. The antibody against secretogranin II was kindly provided by Sharon Tooze Cancer Research UK. Cells from knee lymph nodes were fused with the mouse myeloma cell line P3X63Ag. Cell culture supernatants obtained from individual clones were then screened using enzyme-linked immunosorbent assay ELISA , immunoblot assays and immunoflourescence. The final hybridoma used in this study was cloned two times by limiting dilution. The monoclonal antibody produced from this clone was determined to be of the IgG2a subclass and is specific for Rab26 Figure 1—figure supplement 1. The antibody is commercially available from Synaptic Systems Göttingen, Germany. Cy3-labeled goat anti-mouse or anti-rabbit and Alexa labeled goat anti-mouse secondary antibodies were purchased from Dianova Hamburg, Germany and used at a dilution of Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Bio-Rad Hercules, CA and used at a dilution of or Likewise, inserts encoding Rab26 QL, T77N or NI mutants were generated by recombinant PCR and similarly inserted into these vectors. For recombinant protein expression in bacteria, inserts for the Rab26 variants were inserted into pGEX-KG using EcoRI and BamHI while the insert encoding alpha-GDI was sub-cloned into pETa Novagen, Madison, WI. The sequence corresponding to murine Atg16L1 1— BC was cloned into pETa Novagen using NdeI and NotI restriction sites. Full-length murine Atg5 1— BC was cloned with an N-terminal thrombin cleavage site into the multiple cloning site 1 of pETDuet-1 Novagen using the SalI and NotI sites. The vector expressing neuropeptide Y NPY was generated by inserting the sequence encoding human pro-NPY into the pmRFP vector. Cloning was performed according to standard procedures Janssen, The plasmid expressing GFP-tagged human LC3B was a kind gift from Dr Zvulun Elazar Weizmann Institute, Israel. Culturing of the HEK and HeLa SS6 cell lines and the preparation of high density primary rat hippocampal neurons have been previously described Rosenmund and Stevens, ; Chua et al. Neurons were transfected between 7 to 12 days after seeding or, in the case of the cell lines, 1 day after seeding using Lipofectamine Invitrogen, Carlsbad, CA according to the manufacturer's protocol. Neurons in Figures 3F, 4 , Figure 3—figure supplement 1 and Figure 4—figure supplement 1 were transfected using calcium phosphate as previously described Pavlos et al. Immunostaining was then performed as described in Chua et al. Afterwards, cells were permeabilized with 0. Incubation with primary antibodies diluted in blocking solution was then carried out for 1 hr at room temperatures or overnight at 4°C. Subsequently, cells were exposed to secondary Cy3 or Alexafluor conjugated goat anti-rabbit and anti-mouse antibodies, respectively, for 1 hr at room temperature. After washing, cells were mounted on slides SuperFrost Plus, VWR International bvba, Leuven, Belgium and then imaged using a confocal microscope LSM , Zeiss, Germany or an epifluorescence microscope Axiovert M, Zeiss, Germany. Linescan analyses were performed using ImageJ or LAS AF Lite software. To visualize synaptic vesicles that have undergone recycling, live neurons transfected with EGFP-Rab26WT were incubated in culture for 24 hr with Oyster labeled anti-synaptotagmin-I antibodies Synaptic Systems that recognize its luminal domain Willig et al. The UAST-YFP. Rab26, UAST-YFP. Rab26QL, UAST-YFP. Rab26TN Zhang et al. Dissection and immunostaining of neuromuscular junctions from third instar larvae were performed as described Schmid and Sigrist, using the following antibodies: mouse Anti-Brp hybridoma clone nc82, DSHB; dilution , anti-Csp antibody hybridoma clone ab49, DSHB; dilution , the chicken anti-GFP antibody Abcam; dilution and the goat anti-HRP Sigma; dilution. Dylight labeled anti-goat and Alexa labeled anti-chicken secondary antibodies were purchased from Jackson ImmunoResearch Laboratories West Grove, PA. Alexa conjugated anti-mouse secondary antibodies were purchased from Invitrogen Carlsbad, CA. Images were acquired with a microscope DMR-E; Leica, Germany equipped with a scan head TCS SP2 AOBS; Leica, Germany and an oil-immersion 63 × 1. Biochemical isolation of synaptic vesicles from the rat brain was performed as described previously Huttner et al. Purified monoclonal antibodies directed against Rab26 described above and synaptophysin clone 7. The bound vesicles were subsequently analyzed by electron microscopy or eluted with 40 µl 2 × SDS sample buffer for immunoblots analysis. The RabGDI assay was performed as described in Pavlos et al. Briefly, crude synaptic vesicles LP2 were used as the starting material. The samples were then kept on ice and subsequently centrifuged for 20 min at ,× g , 4°C using a Beckman S AT3 rotor. The resulting pellet was re-suspended in 50 µl 2 × lithium dodecyl sulfate LDS sample buffer Invitrogen , boiled at 95°C for 5 min and analyzed by immunoblotting. Thin sections 80 nm were examined using a Philips CM BioTwin transmission electron microscope Philips Inc. Eindhoven, The Netherlands. Images were taken with a TemCam FA slow scan CCD camera TVIPS, Gauting, Germany. The evaluation of the samples was done using the iTEM software Olympus Soft Imaging Solutions GmbH, Münster, Germany. For immunogold electron microscopy, ultrathin cryosections of neuronal cultures Figure 5A and Figure 5—figure supplement 1 and HeLa cells Figure 6—figure supplement 1 transfected with EGFP-Rab26WT, were prepared as described previously Tokuyasu, , ; Zink et al. For the ultrastructural analyses of the Drosophila neuromuscular junction Figure 5D , a standard protocol was used. Images were taken with a TemCam F slow scan CMOS camera TVIPS, Gauting, Germany. Human GST-tagged Rab26WT, QL, T77N were expressed in Escherichia coli BL21 D3. The cultures were then incubated for 1 hr at 16°C. Induction was initiated by adding 1 mM IPTG to the cultures and the expression was carried out overnight at 16°C. Thereafter, cells were harvested by centrifugation at rpm for 10 min using a Beckman centrifuge. Pellets obtained from each 1 l culture flask were re-suspended in 25 ml of protein buffer containing 50 mM HEPES pH 7. The samples were left for 10—15 min at 4°C and subsequently sonicated four times for 30 s each, separated by a 1 min incubation on ice, using a Branson Sonifier The lysate was then cleared at 13, rpm using a SLA rotor for 40 min at 4°C. The resulting supernatant was collected and filtered using a 0. The filtrate was then loaded onto a GST-column GST Trap4B GE Healthcare, Germany and eluted using 30 mM glutathione in protein buffer. The eluted fractions were collected and dialyzed three times for 3 hr each using protein buffer to remove glutathione. The purified proteins were then used for GST pulldown assays. His-tagged murine Atg16L1 1— -pETa and His-tagged murine Atg5-pETDuet-1 were co-transformed into E. coli Rosetta 2 cells Merck Millipore, Germany. Cells were harvested by centrifugation at × g for 20 min. Pellets were resuspended in ml buffer A 0. Cells were lysed by sonication and centrifuged for 1 hr at 25,× g. Fractions containing the purified proteins were pooled and dialyzed overnight at 4°C in gel filtration buffer 0. Co-immunoprecipitation assays were performed as described in Chua et al. The lysate was then clarified by centrifugation at 10,× g for 10 min. The resulting supernatant was incubated for 2 hr with anti-Flag or anti-GFP antibodies. Subsequently, 30 µl of protein G-Sepharose beads GE Healthcare, Sweden were added to each sample and further incubated for 1 hr under constant rotation. The samples were then washed thrice with lysis buffer. Finally, 25—30 µl of 2 × LDS sample buffer were then added to the beads and the mixture was boiled at 95°C for 5 min. The beads were then washed three times with buffer. Human Rab26 54— QL was cloned into pETa using NdeI and XhoI cleavage sites. Murine Rab33B 30— Q92L BC was cloned into pETDuet-1 with BamHI and NotI restriction sites. Plasmids were transformed into E. coli BL21 DE3. Bacteria were grown in 3 l ZYM autoinducing medium supplemented with the appropriate antibiotic for 8 hr at 37°C. Cells were harvested by centrifugation and resuspended in ml buffer A 30 mM imidazole, 0. Bacteria were lysed with a microfluidizer and centrifuged for 1 hr at 25,× g. Fractions containing protein were pooled and diluted with gel filtration buffer 0. Proteins were kept at 4°C overnight. The gel filtration buffer was 0. eLife posts the editorial decision letter and author response on a selection of the published articles subject to the approval of the authors. An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent see review process. Similarly, the author response typically shows only responses to the major concerns raised by the reviewers. Your article has been favorably evaluated by Eve Marder Senior editor and 2 reviewers, one of whom is a member of our Board of Reviewing Editors. The Reviewing editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. All reviewers were generally enthusiastic about your work. They felt that the data presented are of very high quality and generally support your conclusions, especially with regard to the SV association of Rab They also see strong potential in this work to represent a novel secretory vesicle degeneration pathway. However, the link to autophagy was considered still rather tenuous. We leave it up to you either to tone down your claim Title, Discussion, etc or to provide further experimental data. Please comment. We agree that we do not have conclusive evidence showing directly that Rab26 activation triggers autophagy of synaptic vesicles. The problem, as far as we see it, is that in situ this pathway is probably not active or of only very low activity, making loss-of-function experiments rather difficult. We have tried various approaches but it became clear that considerable additional efforts including suitable animal models are required for obtaining air-tight evidence, and it will probably be more efficient to carry out these experiments with a laboratory having the required tools and experiences. We have therefore revised the Discussion as suggested in order to make sure we are not over-interpreting our results or draw conclusions that are not substantiated by the evidence. We have also revised the Abstract accordingly. Therefore, we have retained it except for a small change suggested by the eLife office, but we are open for a better suggestion. The human Rab1, Rab5 and Rab7 GTPases, homologs of the yeast Ypt1, Vps21 and Ypt7, respectively, were also implicated in autophagy [ 17 ]. In addition to membrane sealing, AP maturation also includes the removal of certain Atgs [ 13 ]. Previously, it was shown that the PI3P phosphatase Ymr1 plays a role in Atg dissociation from APs. Specifically, depletion of Ymr1 results in the accumulation of sealed APs decorated with Atgs [ 18 ]. Thus, while PI3P generation by a PI3P kinase is required for AP formation [ 19 , 20 ], PI3P removal by a PI3P phosphatase from sealed APs is required for Atg dissociation, which is in turn required for AP fusion with the vacuole. While we have shown that the Vps21 GTPase module plays a role in autophagy in yeast, it was not clear which step of the autophagy pathway this module regulates. Here, we show that the Vps21 GTPase module plays a role before the elusive step of AP closure. Moreover, we show that the Vpsregulated step precedes Ymr1-mediated Atg dissociation in AP maturation. Finally, using double mutant analyses, we show that Vps21 functions between Ypt1-mediated AP formation and Ypt7-dependent AP fusion with the vacuole. In yeast, autophagy can be induced either by nitrogen starvation or by addition of rapamycin [ 21 ]. We and others have previously shown that an autophagy-specific mutation in Ypt1 or depletion of its autophagy-specific GEF subunit, Trs85, results in a defect in PAS formation under normal growth conditions and when autophagy is induced by nitrogen starvation [ 8 , 22 — 24 ]. In contrast, depletion of the Vps21 GTPase module components causes accumulation of APs under nitrogen starvation, and in most cells, AP clusters are seen near the vacuolar membranes [ 16 ]. Vps21 together with its paralog Ypt52 was proposed to have a role in autophagy based on autophagy defects in the double-deletion mutant cells when general autophagy was induced by rapamycin [ 15 ]. To check if Ypt1 and Vps21 GTPases function in the same pathway, we used double-mutant epistasis analysis. In this analysis, we took advantage of the different phenotypes of mutations in YPT1 and VPS21 to determine which phenotype masks the other. If Ypt1 functions upstream of Vps21 in autophagy, the phenotype of ypt vps21Δ double mutant cells should be similar to that of ypt and not vps21Δ single mutant cells. The mis-localization of Atg8 and Atg11 in both ypt and ypt vps21Δ mutant cells was not caused by a significant decrease in protein levels S1C and S1D Fig. These results indicate that PAS assembly is defective in ypt vps21Δ double mutant cells. Thus, Ypt1 functions upstream of Vps21 in both selective normal growth and non-selective nitrogen starvation or rapamycin autophagy. Autophagy-specific mutation in Ypt1 and depletion of Vps21 result in two distinct autophagy defects, PAS formation and AP accumulation, respectively [ 8 , 16 ]. PAS or AP formation was determined by the co-localization of AtgxGFP and mCherry-Atg8 in four strains: wild type WT , ypt , ypt vps21Δ and vps21Δ shown from top to bottom. In each strain, the C-terminus of endogenous Atg11 was tagged with 3xGFP, and the cells were transformed with a CEN plasmid expressing mCherry-Atg8 under the CUP1 promoter. The co-localization of Atg11 and Atg8 was determined using live-cell fluorescence microscopy. Shown from left to right: DIC, GFP, mCherry, merge, insert, the number of red dots quantified for each strain from 5—6 different fields , percent of dot co-localization, number of cells quantified for each strain, and percent of cells with AP clusters. The ypt vps21Δ double mutant cells exhibit the ypt mutant phenotype, indicating that Ypt1 functions upstream of Vps21 in the same pathway. Results in this figure represent three independent experiments. The idea that the Vps21 GTPase module functions in a late step of autophagy is supported by accumulation of AP clusters in mutant cells depleted for components of this module. Additional support comes from analysis of Atg8 lipidation in these mutant cells. Atg8 lipidation is required for the attachment of Atg8 to membranes, and therefore for both AP formation and expansion [ 3 ]. The level of lipidated Atg8, Atg8-PE, was determined using immunoblot analysis in wild type, vps21Δ and vps9Δ mutant cells under nitrogen starvation. This analysis shows that similar levels of Atg8-PE are present in wild type, vps21Δ and vps9Δ mutant cells S3 Fig. Together, the accumulation of AP clusters and the Atg8-PE level in vps21Δ and vps9Δ mutant cells indicate that the Vps21 GTPase module is not required for AP formation or expansion, but in a successive step. Ypt7 is required for AP fusion with the vacuole [ 11 ]. Because the APs that accumulate in ypt7Δ mutant cells are sealed, the enclosed cargo is protected from degradation, as determined by an immunoblot analysis in a protease-protection assay see Materials and Methods. Using this assay, autophagic cargo accumulating in atg mutant cells, which are defective in PAS and APs assembly, is sensitive to degradation [ 25 ]. Until this study, there was no mutant known to accumulate APs with cargo sensitive to degradation. This protease protection assay was used to determine whether APs that accumulate in vps21Δ mutant cells are sealed. Protection of two autophagy cargos was tested: prApe1 and GFP-Atg8. When not enclosed inside membranes, these cargos can be cleaved to mApe1 and GFP, respectively, by addition of proteinase K PK to the cell fraction P5 that contains membrane-bound compartments. In atg1Δ mutant cells, in which incomplete PAS can be formed but cannot expand [ 26 — 28 ], the cargos were not protected, and the cleaved products are seen after addition of PK. Importantly, the fact that the two cargos can be cleaved upon solubilization of the membranes by a detergent Triton X, TX when prepared from ypt7Δ mutant cells, shows that they were protected by sealed membranes. Notably, while APs accumulate in vps21Δ mutant cells, prApe1 and GFP-Atg8 were not protected from the protease and are cleaved Fig 2A and 2B. This phenotype is different from that exhibited by ypt7Δ mutant cells, and suggests that APs accumulating in vps21Δ mutant cells are not sealed. Moreover, while APs accumulate in vps21Δ ypt7Δ double mutant cells see below , the two autophagy cargos were also not protected from the protease Fig 2A and 2B. Thus, as in vps21Δ mutant cells, in the vps21Δ ypt7Δ double mutant cells APs are unsealed. The fact that the vps21Δ phenotype masks the ypt7Δ phenotype indicates that Vps21 functions upstream of Ypt7 in the same pathway. Protease-protection assays were performed using pellet P5 fractions prepared from cell lysates of the indicated strains using immunoblot analysis. prApe1 A and GFP-Atg8 B are protected from degradation in ypt7Δ , but not in vps21Δ or vps21Δ ypt7Δ mutant cells. prApe1 C and GFP-Atg8 D are protected from degradation in ypt7Δ , but not in vps9Δ or vps9Δ ypt7Δ mutant cells. prApe1 E and GFP-Atg8 F are protected from degradation in vps39Δ , but not in vps8Δ or pep12Δ mutant cells. Cells expressing GFP-Atg8 from the chromosome were grown in rich medium and shifted to nitrogen-starvation medium as in Fig 1. The P5 fractions of the cell lysates see Materials and Methods were treated with proteinase K PK with or without detergent Triton X; TX. These fractions were examined by immunoblot analysis using anti-Ape1 or anti-GFP. For each strain, the three lanes show from left to right : without protease negative control , with protease experimental , with protease and detergent positive control, should be fully degraded. Only in cells defective in Ypt7 A-D or its effector Vps39 E-F , prApe1 and GFP-Atg8 are protected from degradation by PK, whereas in all other strains, only the degradation products are seen mApe1 and GFP even without the addition of detergent. We have previously shown that other known components of the endocytic Vps21 GTPase module, the Vps9 GEF, and two of its known effectors, Vps8, a subunit of the CORVET tethering factor, and the Pep12 SNARE, are also required for autophagy, and APs accumulate upon their depletion [ 16 ]. Here, the protease protection assay was used to determine whether, as in vps21Δ , the cargo in APs that accumulate in vps9Δ , vps8Δ and pep12Δ mutant cells is accessible to PK. Both prApe1 and GFP-Atg8 were not protected from degradation in cellular membranes P5 isolated from vps9Δ and vps9Δ ypt7Δ mutant cells Fig 2C and 2D , indicating that, like Vps21, its Vps9 GEF is also involved in AP sealing. Together, these results suggest that components of the Vps21 GTPase module are required for sealing of APs, and show that this step precedes Ypt7-dependent AP fusion with the vacuole. Using three different parameters, our previous studies provided evidence that the APs that accumulate next to the vacuolar membranes in vps21Δ mutant cells are outside the vacuole: 1 High magnification fluorescence microscopy; 2 EM; and 3 time-lapse microscopy which shows that individual APs move throughout the cytosol [ 16 ]. While we observed that multiple APs accumulate in clusters adjacent to the vacuolar membrane in vps21Δ mutant cells under autophagy-inducing conditions, Nickerson et al. A similar observation was made in Drosophila cells depleted for Rab5 d2 , which accumulate Atg8 near the lysosomal membrane, apparently inside the lysosome [ 29 ]. Additionally, it has been suggested that Rab5 plays a role in lysosomal function in murine liver cells [ 30 ]. Thus, the accumulation of APs in vps21Δ mutant cells could potentially happen adjacent to the vacuolar membrane inside the vacuole [ 15 ]. However, we find this unlikely because yeast cells defective in vacuolar proteases, e. Importantly, the AP localization and accumulation in vps21Δ looked different from the known vacuolar accumulation of ABs in pep4Δ mutant cells by either fluorescence microscopy observation or TEM S4A and S4B Fig and [ 16 ]. Furthermore, the results from the protease protection assay show that autophagy cargos in vps21Δ mutant and vps21Δ pep4Δ double mutant cell lysates were not protected from proteases, while they were protected in pep4Δ mutant cell lysates S4C and S4D Fig to a level similar to that found in ypt7Δ and vps39Δ mutant cell lysates Fig 2. These results imply that the accumulated APs in vps21Δ and vps21Δ pep4Δ mutant cells are unsealed and are outside the vacuole, whereas accumulated ABs in pep4Δ mutant cells are sealed and are inside vacuolar membranes. Taken together, the aforementioned results indicate that the function of Vps21 in autophagy precedes those of Ypt7 and Pep4, whose depletion results in AP accumulation in the cytoplasm and AB accumulation inside the vacuole, respectively. One hallmark of AP maturation is the dissociation of several Atgs from the AP, e. In contrast, some Atg8 remains attached to APs as they fuse with the vacuole [ 13 ]. We wished to determine whether Atgs are still present on unsealed APs that accumulate in cells depleted for Vps The co-localization of Atg2, Atg5 and Atg18 with the AP marker Atg8 was determined in vps21Δ , ypt7Δ , and vps21Δ ypt7Δ mutant cells under nitrogen starvation. In addition, Atg11 and Atg17 also remained on the majority of APs in vps9Δ , vps9Δ ypt7Δ , vps8Δ and pep12Δ mutant cells, but not in vps39Δ mutant cells Fig 4. Together, these results show that Atg dissociation is defective in mutant cells depleted for Vps21, its Vps9 GEF or its Vps8 and Pep12 effectors, but not in cells depleted for Ypt7 or its Vps39 effector. Moreover, double mutant analyses support the idea that both Vps9 and Vps21 function upstream of Ypt7 in autophagy. Endogenous Atg5 A , Atg2 B , or Atg18 C were tagged with GFP at their C-terminus in four strains: WT, vps21Δ , vps21Δ ypt7Δ , and ypt7Δ. The cells, which also express mCherry-Atg8 from a plasmid as an AP marker, were grown in YPD and shifted to SD-N as in Fig 1. The co-localization of the GFP-tagged AtgX with Atg8 was determined using live-cell fluorescence microscopy. Shown from top to bottom: DIC, GFP, mCherry, merge, insert, and the number of red puncta used for the quantification. Arrows indicate co-localizing puncta, arrowheads in ypt7Δ mutant cells point to mCherry-Atg8 puncta that do not co-localize with the other Atg; bar, 2 μm. In WT cells, a single dot per cell that contains both Atgs represents PAS or AP. Whereas Atg5, Atg2 and Atg18 are removed from most APs that accumulate in ypt7Δ mutant cells, they remain on most APs that accumulate in vps21Δ and vps21Δ ypt7Δ mutant cells. Endogenous Atg11 A , or Atg17 B was tagged with GFP at their C-terminus in indicated strains; from left-to-right: WT, vps21Δ , vps21Δ ypt7Δ , ypt7Δ , vps9Δ , vps9Δ ypt7Δ , vps8Δ , pep12Δ , and vps39Δ. The cells, which express mCherry-Atg8 from a plasmid as an AP marker, were grown in YPD and shifted to SD-N as in Fig 1. Arrows indicate co-localizing puncta, arrowheads in ypt7Δ and vps39Δ mutant cells point to mCherry-Atg8 puncta that do not co-localize with the other Atg; bar, 2 μm. Quantification of percent AtgX-Atg8 co-localization from panels A-B in the indicated strains: Atg11 left , and Atg17 right , strain color legend is shown on the right. In WT cells, a single dot per cell that contains both Atgs, represents PAS or AP. Whereas Atg11 and Atg17 are removed from most APs that accumulate in ypt7Δ and vps39Δ mutant cells, they remain on most APs that accumulate in mutant cells defective in the Vps21 module as well as in the double mutant cells. Results in this figure represent at least three independent experiments. The PI3P phosphatase Ymr1 was previously demonstrated to play a role in Atg dissociation during AP maturation. Specifically, upon addition of rapamycin, ymr1Δ exhibit a defect in general autophagy, accumulation of sealed APs in the cytoplasm, and a failure of Atg dissociation from these APs [ 18 ]. We wished to explore the relationship between the roles of Vps21 and Ymr1 in autophagy and to determine whether they function in the same pathway sequentially. This is especially important since the autophagic phenotypes of both vps21Δ and ymr1Δ are partial [ 16 , 18 ]. The autophagy defects of the two single-deletion mutant strains and those of the double-deletion mutant strain, were compared upon induction of generic autophagy under nitrogen starvation. Analyses of autophagy cargos processing during nitrogen starvation show that in the single mutant cells, some of the prApe1 and GFP-Atg8 is processed to mApe1 and GFP, respectively. Whereas ymr1Δ mutant cells exhibit a less severe mApe1 processing defect than vps21Δ mutant cells, GFP-Atg8 processing is similar in both mutant cells. Importantly, the autophagy phenotypes of the vps21Δ ymr1Δ double mutant cells do not exceed those of the single deletion strains S5 Fig. These results are consistent with the idea that Vps21 and Ymr1 function in the same pathway. We next explored AP accumulation under nitrogen starvation in cells depleted for Vps21, Ymr1, or both. We have previously shown that APs accumulate in clusters next to the vacuole in vps21Δ mutant cells [ 16 ]. AP accumulation was determined here using two approaches, live-cell fluorescence microscopy and electron microscopy EM. Interestingly, as seen by the FM staining, unlike in ypt7Δ , vacuoles in ymr1Δ mutant cells are not fragmented. This observation indicates that dispersal of APs in the cytoplasm observed in ymr1Δ and ypt7Δ mutant cells is not caused by vacuole fragmentation. Importantly, AP cluster accumulation in vps21Δ ymr1Δ double mutant cells is similar to that observed in vps21Δ , and not in ymr1Δ , single mutant cells in both assays Fig 5A—5C , supporting the idea that Vps21 functions upstream of Ymr1 in autophagy. GFP-Atg8-labled APs accumulate in clusters near the vacuoles of vps21Δ and vps21Δ ymr1Δ , but not of ymr1Δ or ypt7Δ , mutant cells. Cells expressing GFP-Atg8 from the chromosome, were grown in YPD and shifted to SD-N for 2 hours as in Fig 1 , and FM red dye was added for the second hour to visualize the vacuolar membrane. Cells were visualized by live-cell fluorescence microscopy FM. Shown from left to right: WT, vps21Δ , vps21Δ ymr1Δ , ymr1Δ and ypt7Δ. Shown from top to bottom: strains, DIC, GFP, FM, merge, insert, and number of cells visualized with GFP-Atg8. Arrows indicate Atg8 clusters co-localizing with FM; bar, 2 μm. Accumulation of AP clusters outside the vacuole of vps21Δ and vps21Δ ymr1Δ , but not ymr1Δ or ypt7Δ mutant cells. The ultra-structure of cells as starved in A was visualized by electron microscopy EM. Representative cells are shown. APs appear as clusters outside the vacuole of both vps21Δ and vps21Δ ymr1Δ mutant cells. Nuc, nucleus; Vac, vacuole; white asterisks mark individual APs. Quantification of results from A and B is shown as the percent of cells with GFP-Atg8 clusters by FM left and AP clusters by EM right in the indicated strains strain color legend is shown on the right. Columns represent mean, and error bars represent STD. Whereas sealed APs accumulate in ymr1Δ and ypt7Δ , unsealed APs accumulate in vps21Δ and vps21Δ ymr1Δ mutant cells. Protease protection analysis of Ape1 blot D and GFP blot E , was done as described in Fig 2 in the following strains from left-to-right : atg1Δ unprotected control , vps21Δ , vps21Δ ymr1Δ , ymr1Δ , and ypt7Δ. Both prApe1 and GFP-Atg8 are protected from degradation in AP fractions isolated from ymr1Δ and ypt7Δ , but not from vps21Δ and vps21Δ ymr1Δ mutant cells. Atg dissociation from APs is defective in vps21Δ , vps21Δ ymr1Δ and ymr1Δ , but not in ypt7Δ mutant cells strain color legend is shown on the right. Quantification of results from S6 Fig and S7 Fig showing the percent of co-localization of AtgX with Atg8; from left to right: Atg5, Atg2 S6 Fig , Atg18 and Atg11 S7 Fig. It was previously shown that some prApe1 is protected from proteases in APs isolated from ymr1Δ mutant cells, suggesting that APs that accumulate in these cells are sealed [ 18 ]. To further dissect the relationship between depletion of Vps21 and Ymr1, single and double mutant cells were tested by the protease protection assay using two cargos. In contrast, in vps21Δ ymr1Δ double mutant cells, as in vps21Δ , both autophagy cargos were not protected from the protease Fig 5D—5E. These results show that Vps21 functions prior to Ymr1. A defect in Atg dissociation from APs that accumulate in ymr1Δ mutant cells was previously reported [ 18 ]. Because we show here that vps21Δ mutant cells are also defective in Atg dissociation from APs, we expected that if Vps21 and Ymr1 function in the same pathway, vps21Δ ymr1Δ double mutant cells will show a phenotype similar to and not exceeding that of the single deletion strains. Indeed, in live-cell fluorescence microscopy analyses, Atg5, Atg2, Atg18 and Atg11 were co-localized with APs and AP clusters that accumulate in vps21Δ ymr1Δ mutant cells Fig 5F and S6 and S7 Figs. PI3P is required for early autophagy [ 32 ], and its removal in later steps was inferred from the role of the PI3P phosphatase Ymr1 after AP accumulation [ 18 ]. However, PI3P removal from APs was not shown directly in the later study. Here, we explored PI3P presence on Atg8-marked APs using the PI3P reporter DsRed-FYVE domain [ 33 ] and live-cell fluorescence microscopy. This is important since PI3P also decorates endosomes [ 34 ]. PI3P is present on APs that accumulate in vps21Δ , vps21Δ ymr1Δ , and ymr1Δ , but not in ypt7Δ , mutant cells. Co-localization of the PI3P-binding FYVE domain with Atg8-marked APs A. Indicated strains expressing endogenously tagged GFP-Atg8 and DsRed-FYVE from a 2μ plasmid, were grown in YPD and shifted to SD-N for 2 hours. Cells were visualized by live-cell fluorescence microscopy. Strains from left to right : WT, vps21Δ , vps21Δ ymr1Δ , ymr1Δ , and ypt7Δ. Shown from top to bottom: DIC, DsRed, GFP, merge, insert, and number of cells expressing the FYVE domain used for quantification. Arrows indicate co-localizing puncta or AP clusters , arrowheads point to GFP-Atg8 puncta that do not co-localize with the FYVE domain; bar, 2 μm. Quantification B of the percent of cells with FYVE domain co-localizing with Atg8-marked APs from panel A. Localization of Ymr1 to APs is defective in vps21Δ mutant cells. Ymr1-yEGFP and the AP marker mCherry-Ape1 were co-expressed in WT and vps21Δ mutant cells. After 2 hours of nitrogen starvation, co-localization of the two proteins was determined using live-cell fluorescence microscopy C. Strains: WT top and vps21Δ bottom. From left to right: PhC, GFP, mCherry, merge and number of cells used for quantification. Arrow indicates co-localizing puncta. Quantification D of the percent of cells with co-localizing Ymr1 and Ape1 from panel C. Results in panels A-D represent three independent experiments. Model for the regulation of three successive steps in autophagy by Ypt GTPases: Ypt1 regulates PAS formation [ 8 ]; the Rab5-like module—Vps9 GEF, Vps21, and Vps8 and Pep12 effectors—regulates AP maturation shown here , and Ypt7 together with its Vps39 effector controls mature AP fusion with the lysosome [ 11 , 13 ]. Moreover, the two successive steps in AP maturation are separated: Vps21 regulates AP closure directly or indirectly, and Ymr1 mediates PI3P hydrolysis and Atg removal from closed APs. The above mentioned double-mutant analyses suggest that the Vpsdependent step precedes the Ymr1-dependent step in the autophagy pathway. Therefore, we wished to determine whether Ymr1 localization to APs is dependent on Vps We compared this co-localization in wild type and vps21Δ mutant cells expressing Ymr1-yEGFP and mCherry-Ape1 as an AP marker. This result suggests that the localization of Ymr1 to APs is dependent on Vps21 and further supports the order of their function, which is based on double mutant analyses. Together, our results show that Vps21 and Ymr1 function sequentially in two separate steps of AP maturation: during or upstream of AP sealing, and PI3P hydrolysis-dependent Atg dissociation from closed APs, respectively. As for the order of function of Ymr1 and Ypt7, while ymr1Δ mutant cells accumulate dispersed APs like ypt7Δ mutant cells, unlike ypt7Δ mutant cells, they accumulate APs decorated with Atgs and PI3P. AP maturation, which is required for AP fusion with the vacuole, includes membrane sealing and removal of most Atgs. Results presented here suggest that the Rab5-related Vps21, in the context of its endocytic GEF-GTPase-effector module, regulates AP closure. Specifically, while cells depleted for members of the Vps21 GTPase module accumulate APs, the cargo enclosed in these APs is not protected from degradation by proteases, implying that these APs are not sealed. AP accumulation next to, but outside of, the vacuole, is supported by microscopy and epistasis analyses showing that Vps21 functions upstream of both Ymr1 and Ypt7, which accumulate sealed APs dispersed in the cytoplasm. Currently, the evidence that APs accumulating in vps21Δ mutant cells are unsealed is provided by one approach, the protease protection assay. Future higher-resolution morphological analyses should be performed to confirm this idea. Notably, the observation that Atg dissociation, which follows AP closure, is also defective in mutant cells depleted for members of the Vps21 GTPase module, further supports a role of this module in AP maturation. To our knowledge, this is the first report that identifies regulators that function between AP expansion and closure. In addition, using double mutant analyses, we demonstrate that Vps21 functions in autophagy downstream of Ypt1 and upstream of Ypt7. This establishes a cascade of three successive steps in autophagy regulated by Rab GTPases: Ypt1-mediated AP formation, Vpsregulated AP closure, and Ypt7-dependent AP fusion with the vacuole Fig 6E. Rab7 was found to be involved in GcAV formation and maturation, most probably by assisting in the recruitment of LC3 to GcAVs and inducing homotypic membrane fusion during the enlargement process of GcAVs Yamaguchi et al. Rab7, indeed, appears to participate in the coalescence of multiple small Atg5-positive membranes that localise around the GAS bacteria, suggesting it has a role in the early phase of GcAV formation Sakurai et al. In addition to Rab7, Rab9A has also been implicated in the enlargement of GcAVs and the fusion of GcAVs with lysosomes during GAS-induced autophagy. However, starvation-induced autophagy does not appear to be regulated by this small GTPase, suggesting that different types of autophagy require different factors that confer distinct specificities and functions to the different autophagy pathways Nozawa et al. The autophagy machinery is also involved in regulating the secretion of the contents of granules or secondary lysosomes, such as Acyl-CoA-binding protein Acb1 in yeast, and interleukin 1β and interleukin 18 IL1B and IL18, respectively in mammalian cells Deretic et al. This newly described form of secretion, which has been named autophagy-based unconventional secretion, is dependent on Rab8a, a small GTPase that is located at the Golgi and canonically involved in the polarised sorting from the Golgi to the plasma membrane. This autophagy-dependent secretory pathway enables cytosolic proteins to exit the cell without entering the conventional ER-to-Golgi secretory pathway. It remains unclear why cells would use one particular secretion system for specific proteins instead of other systems. However, this recently unveiled aspect of autophagy may be relevant in the context of inflammatory diseases, such as cystic fibrosis and Crohn disease Cadwell et al. Autophagy can regulate the unconventional trafficking of the cystic fibrosis transmembrane conductance regulator CFTR , the protein that is mutated in cystic fibrosis, from the ER to the plasma membrane without passing through the Golgi. The impaired conventional Golgi-mediated secretion and cell surface expression of the CFTRΔ mutant protein can be rescued by directing it to an unconventional Golgi reassembly stacking protein GRASP -dependent secretion pathway using autophagosomes as a vehicle Gee et al. In the context of Crohn disease, autophagy participates in the regulated secretion of lysozyme from Paneth cells Cadwell et al. With regard to bacterial infection, Rab8 has been shown to be involved in the autophagic elimination of Mycobacterium tuberculosis var. bovis BCG through its interaction with TANK-binding kinase 1 TBK1 , which is involved in the phosphorylation of the autophagic adaptor p62 and assembly of the autophagy machinery Pilli et al. The fusion of autophagosomes with the vacuole the lysosome in yeast is compromised in Saccharomyces cerevisiae mutants that lack the yeast Rab7 orthologue Ypt7, resulting in the accumulation of autophagosomes in the cytoplasm Kirisako et al. Many other studies that subsequently emerged aimed to characterise the mechanisms involved in the regulation of the fusion event by Rab7. Rab7 was found to regulate the maturation of late autophagic vacuoles and the formation of the autolysosome both under nutrient replete and starvation conditions through a mechanism that is dependent on the binding and hydrolysis of GTP Gutierrez et al. More recently, a role for Rab7 has also been reported in a process named autophagic lysosomal restoration ALR , which involves the termination of autophagy and formation of nascent lysosomes from autolysosomes in a mechanism that depends on the reactivation of mTOR. Treatment of cells with GTPγS, a non-hydrolysable analogue of GTP, completely inhibits ALR, and overexpression of constitutively active Rab7 that is permanently associated with membranes, also abrogates ALR, resulting in the accumulation of enlarged and long-lasting autolysosomes. Inhibition of ALR by rapamycin also inhibits the dissociation of Rab7 from autolysosomes, suggesting that mTOR and Rab7 together participate in the regulation of ALR Yu et al. Rab7- and Arl8-ancillary machinery involved in the positioning of lysosomes and autophagosomes, and their fusion. A Rab7 has been implicated in the fusion between autophagosomes and lysosomes through a mechanism that is dependent on the binding and hydrolysis of GTP. The UVRAG—C-Vps complex appears to activate Rab7 activity and stimulate autophagosome-lysosome fusion, whereas Rubicon inhibits this step. B The movement of autophagosomes has been suggested to rely on a precise balance between dynein- and kinesin-dependent mechanisms. Starvation conditions are likely to promote the binding of FYCO1 to kinesin, and to Rab7 and LC3-II in the membrane of autophagosomes, inducing a redistribution of the pre-autophagosomes throughout the cytosol. FYCO1 appears to compete with the RILP-dynactin-dynein complex for the binding to Rab7, providing means to regulate the bidirectional movement of autophagosomes along microtubules. RILP-Rab7 interaction is further controlled by the UVRAG—C-Vps complex. C Left: starvation conditions, characterised by an increase of the intracellular pH, inhibit the recruitment of Arl8B which is kept on the cytosol in a GDP-bound form and of the kinesin KIF2A to the lysosomal membrane; this favours the accumulation of lysosomes and the fusion between lysosomes and autophagosomes in a perinuclear region close to the MTOC. mTOR is also inhibited under starvation conditions, favouring the formation of new autophagosomes. Right: after nutrient replenishment characterised by a decrease of the intracellular pH , Arl8 bound to GTP is recruited to the lysosomal membrane in a complex with pleckstrin-homology-domain-containing family M member 2 PLEKHM2; here referred to as SKIP and kinesin, which binds to microtubules Korolchuk et al. Lysosomes are subsequently transported towards the cell periphery. The centrifugal movement of lysosomes also diminishes the encounter between lysosomes and autophagosomes, which interferes with their fusion and clearance of autophagic substrates. Several groups have been focusing their efforts on the understanding of the molecular mechanisms, as well as in the identification of the molecular complexes that are involved in the regulation of autophagy by Rab7. FYVE and coiled-coil- domain-containing protein FYCO1 has been identified in a recent study as a Rab7 effector protein that is able to bind LC3 and PtInsP 3 and mediate microtubule plus-end-directed transport of autophagic vesicles Pankiv et al. FYCO1 seems to have a role in the redistribution of Rab7- and LC3-positive vesicles to the cell periphery in a microtubule-dependent manner that might, for instance, interfere with the fusion of autophagosomes with lysosomes. Indeed, the authors identified a potential kinesin-binding site in the central part of the coiled-coil region of FYCO1 Pankiv et al. Although the physiological implications of this mechanism were not explored in depth, the authors propose a mechanism whereby FYCO1 preferentially localises at the ER in a conformation that prevents its binding to kinesins under nutrient-rich conditions. However, upon starvation, FYCO1 binds to the microtubule plus-end-directed motors and redistributes pre-autophagosomal membrane compartments to the sites of autophagosome formation throughout the cytosol. It is also proposed that, after the formation of autophagosomes, FYCO1 competes with the dynein recruitment complex for binding to Rab7, providing a regulated bidirectional transport of autophagosomes along microtubule tracks Pankiv et al. Another factor that is involved in the regulation of Rab7 is Rab7-interacting lysosomal protein RILP , a component of the complex that is responsible for the binding of Rab7 to dynactin—dynein1 Fig. Interestingly, the interaction between Rab7 and RILP is positively affected by the activation of Rab7 through the complex of class C-Vps also known as HOPS complex — a tethering protein complex that serves multiple membrane fusion events e. autophagosome fusion with late endosomes and lysosomes — and UVRAG, a protein known to induce autophagy and membrane curvature through a mechanism that is dependent on Beclin 1 and PtdIns 3-kinase class III Liang et al. Indeed, the interaction between C-Vps and UVRAG stimulates Rab7 activity and promotes autophagosome maturation and fusion with lysosomes Liang et al. The GDP-GTP exchange on Rab7 necessary for its activation is likely to be dependent on the GEF activity of the C-Vps complex Liang et al. The Rab7-RILP interaction is also regulated by the insulin-like growth factor 1 IGF1 —AKT pathway during neuronal autophagy Bains et al. An additional level of Rab7 regulation is also provided by rubicon Run domain beclininteracting and cysteine-rich-containing protein , another Beclin 1-binding protein. Rubicon has been shown to inhibit the maturation and fusion steps during autophagy Matsunaga et al. Interestingly, rubicon appears to sequester UVRAG from the C-Vps complex and block Rab7 activation Sun et al. This response appears to be independent of a functional ER stress response pathway, because thapsigargin also blocks autophagy in ER-stress inositol requiring enzyme IRE -null cells Ganley et al. In addition to Rab7, other trafficking-associated small GTPases probably also regulate the autophagosome-lysosome fusion step. For instance, Rab33B, a Golgi-resident Rab protein that is involved in retrograde transport, might have an indirect role in this process through the activity of its GAP OATL1 also known as TBC1D25 Itoh et al. It has been proposed that activated Rab33B recruits the Atg12—Atg5—Atg16L1 complex to pre-autophagosomal structures, thereby inducing the subsequent conjugation of LC3 to PtdEtn. Following this, OATL1 recognises LC3 in the autophagosomal membrane in proximity to Rab33B and inactivates Rab33B through its GAP activity, exerting a feedback loop. This mechanism appears to be involved in autophagosomal maturation and conversion of autophagosomes to autolysosomes, because inactivation of Rab33B through overexpression of OATL1 inhibits the encounter of autophagosomes and lysosomes, whereas overexpression of constitutively active Rab33B reduces the rate of fusion between autophagosomes and lysosomes Itoh et al. Another GTPase that indirectly regulates autophagosome—lysosome fusion is the Arf-like GTPase Arl8B. A decrease of the intracellular pH — induced, for example, through nutrient-rich conditions — increases recruitment of Arl8B and the kinesin KIF2A to lysosomes, which promotes their centrifugal movement along microtubules towards the cell periphery Korolchuk et al. This occurs concomitantly with the activation of mTORC1, which inhibits autophagosome formation and decreases autophagosome-lysosome fusion because encounters of autophagosomes and lysosomes in the perinuclear region are less likely Korolchuk et al. Activation of mTORC1 primarily occurs at the surface of lysosomes through a mechanism that depends on Rheb, ragulator and the Ras-related GTPases RagA or RagB, and RagC or RagD Sancak et al. In this Commentary, we have described how a number of small GTPases modulate different steps of autophagy, including autophagosome formation, autophagosome secretion, autophagosome trafficking and fusion with lysosomes. For instance, Arf6 and Rab33B are small GTPases implicated in Atg16L-mediated autophagosome formation, whereas the small GTPases Rab1, Rab11 and Sec4 are likely to be involved in the formation of Atg9-autophagosome precursors. Although the most-accepted role of Rab7 in autophagy refers to its ability to control the autophagosome—lysosome fusion step, it is also involved in the formation and maturation of autophagosomes during bacterial infection, as is the small GTPase Rab9A. In addition, RalB has been implicated in the regulation of ULK1-mediated formation of autophagosomes, whereas Rab8 controls a new and unconventional form of secretion that relies on autophagy. The trafficking of autophagosomes and lysosomes, as well as their fusion has been shown to mainly depend on Rab7, but also requires Arl8 and Rab33B Fig. Despite extensive research efforts, the molecular networks and complexes that support the functions of these small GTPases in autophagy, as well as their spatial and temporal control, are still not completely understood and important questions still need to be answered. In addition, it is still unclear how small GTPases reach the autophagosomal membrane. Understanding this step may shed some light into the mechanisms that underlie the intersections between endocytic, secretory and autophagic pathways. An interesting hypothesis is that a pool of Atg9-positive membranes can cycle dynamically between the endosomal compartment, the secretory pathway and the pre-autophagosomal structures Reggiori and Tooze, ; Young et al. However, one should not exclude the canonical process for the recruitment of small GTPases to membranes without the need of vesicular transport or fusion-dependent events, which relies on their direct recruitment from the cytoplasm to the membrane by a mechanism that is dependent on the exchange of GDP to GTP. One can, therefore, hypothesise that the primary location of these GTPases provides clues about the membrane sources for the formation of the autophagosome. Although autophagy was considered in the past as a nonselective process, several cargo-specific autophagic processes have been recently described, including xenophagy degradation of intracellular pathogens , aggrephagy clearance of certain protein aggregates , pexophagy elimination of peroxisomes , mitophagy removal of damaged mitochondria and ribophagy elimination of ribosomes , which assist the quality control of essential cellular components reviewed by Mizushima, Autophagy occurs under basal conditions and can be induced by certain environmental stresses, such as nutrient deprivation, some infections, oxidative stress and treatment with certain drugs e. Under starvation conditions, autophagy is induced and increases the availability of nutrients e. amino acids by releasing them from proteins and other macromolecules that are targeted for degradation. Indeed, autophagy has roles in both health and disease conditions. It regulates early embryonic development, neonatal starvation, clearance of pathogenic bacteria during infectious processes, cancer-associated mechanisms and degradation of misfolded and aggregation-prone proteins i. tau, mutant α-synuclein, polyglutamine-expanded huntingtin that are involved in neurodegeneration disorders, such as Alzheimer, Parkinson and Huntington diseases reviewed by Harris and Rubinsztein, ; Mizushima and Komatsu, An extensive array of signals regulates the formation of autophagosomes. Generally, they can be categorised into mTOR-dependent or mTOR-independent stimuli. The mTOR pathway is a classic negative regulator of autophagy that is conserved from yeast to mammals. mTOR activity is inhibited under starvation conditions and rapamycin treatment, which results in the partial dephosphorylation of its targets Atg13, ULK1 and ULK2; this activates ULK1 and ULK2 to phosphorylate FIP and, thereby, induces autophagy Hosokawa et al. In addition, mTOR is positively and negatively regulated by a plethora of other stimuli. For example, depending on the oncogenic or genotoxic stress, p53 can activate AMP-activated protein kinase AMPK , which directly activates ULK1 and also inhibits mTOR, or upregulate phosphatase and tensin homologue PTEN , which inhibits mTOR through inhibition of the Akt kinase reviewed by Ravikumar et al. In addition, AMPK can also inhibit mTOR activity through the tuberous sclerosis complex 1 Tsc1 , Tsc2 and Ras homology enriched in brain Rheb reviewed by Ravikumar et al. mTOR can also be regulated by GTPases that influence its lysosomal localisation and activity during starvation conditions Saci et al. Recent work has also described that the G-protein-coupled taste receptor complex T1R1—T1R3 acts as a sensor for amino acids, which then regulates mTOR activity and autophagy Wauson et al. AMPK can also regulate autophagy independently of mTOR. Another well-characterised mTOR-independent signal that regulates autophagy includes the inhibition of inositol monophosphatase IMPase , which reduces free inositol and inositol 1,4,5 -trisphosphate [Ins 1,4,5 P 3] levels, resulting in an upregulation of autophagy Sarkar et al. We are grateful to Fiona Menzies, Mariella Vicinanza and Maurizio Renna for critical reading of the manuscript. We thank the Wellcome Trust for a Principal Research Fellowship [to D. and a Strategic Grant to the Cambridge Institute for Medical Research. We are now welcoming submissions for our upcoming Special Issue: Imaging Cell Architecture and Dynamics. This issue will be coordinated by two Guest Editors: Lucy Collinson The Francis Crick Institute, UK and Guillaume Jacquemet University of Turku, Finland. Submission deadline: 1 March People who know JCS well will know that we're more than just a journal and that our community — the cell biology community — really is at the heart of everything we do. Read the full Editorial by Editor-in-Chief Michael Way and Executive Editor Seema Grewal. Registration is open for our Journal Meeting Diversity and Evolution in Cell Biology, which aims to bring together evolutionary biologists and cell biologists investigating diverse aspects of cellular physiology. Submit your abstract by 5 April. Final registration deadline: 3 May Early-career researchers interested in the roles of nuclear lipids, apply now for one of the ten funded places at this Workshop, which will take place October Application deadline: 19 April. There are many benefits to publishing in Journal of Cell Science - read more about why you should choose JCS or visit our submission page now. Sign In or Create an Account. Search Dropdown Menu. header search search input Search input auto suggest. filter your search All content All journals Journal of Cell Science. Advanced Search. User Tools Dropdown. Sign in. Toggle Menu Menu Articles Accepted manuscripts Latest complete issue Issue archive Archive by article type Special issues Subject collections Interviews Essay series Sign up for alerts About us About JCS Editors and Board Editor biographies Travelling Fellowships Grants and funding Journal Meetings Workshops The Company of Biologists The Forest of Biologists Journal news For authors Submit a manuscript Aims and scope Presubmission enquiries Fast-track manuscripts Article types Manuscript preparation Cover suggestions Editorial process Promoting your paper Open Access JCS Prize Manuscript transfer network Biology Open transfer Journal info Journal policies Rights and permissions Media policies Reviewer guide Sign up for alerts For librarians Contacts Contacts Subscriptions Advertising Feedback. Skip Nav Destination Close navigation menu Article navigation. Volume , Issue 5. Previous Article Next Article. Article contents. Small GTPases involved in the formation of autophagosomes. Small GTPases involved in autophagosome-mediated exocytosis. Small GTPases involved in the trafficking of autophagosomes and lysosomes and their fusion. Article Navigation. The role of membrane-trafficking small GTPases in the regulation of autophagy Carla F. Bento , Carla F. Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge. This site. Google Scholar. Claudia Puri , Claudia Puri. Kevin Moreau , Kevin Moreau. David C. Rubinsztein David C. Author and article information. Claudia Puri. Kevin Moreau. Online ISSN:
1. Introduction	Guerrero-Gomez D, Mora-Lorca JA, Saenz-Narciso B, Naranjo-Galindo FJ, Munoz-Lobato F, Parrado-Fernandez C, Goikolea J, Cedazo-Minguez A, Link CD, Neri C, Sequedo MD, Vazquez-Manrique RP, Fernandez-Suarez E, Goder V, Pane R, Cabiscol E, Askjaer P, Cabello J, Miranda-Vizuete A. Dev Cell. Key Role of Rab7 in Autophagosome Maturation Many studies have confirmed that Rab7 has a key role in autophagosome maturation. John JE Chua Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Contribution JJC, Conception and design, Analysis and interpretation of data, Drafting or revising the article For correspondence john. Autophagy in the pathogenesis of disease. Construction of the strains used for live-cell microscopy of Atg-GFP dissociation from mCherry-Atg8 marked APs or dsRed-FYVE on GFP-Atg8 marked APs: ORF of specific gene was deleted from AtgX-GFP X for 5, 2, 18, 11, 17 or GFP-Atg8 tagged wild type with a drug resistance cassette hygromycin or KanMX or the LYS2 gene to get different deletion mutants by PCR amplification and recombination.
Regulation of autophagy by the Rab GTPase network | Cell Death & Differentiation	It is GTPasees to note that GTPzses can regulate autophagy not only Autoophagy modulating Autophagy and GTPases, but Autophagy and GTPases by annd the membrane flow from compartments other than the plasma GTPazes, or by modulating phospholipase D activity, whose activity has been shown to regulate autophagy Dall'Armi et al. Article Google Scholar Bubber P, Haroutunian V, Fisch G, Blass JP, Gibson GE. Article Google Scholar Inoki K, Li Y, Xu T, Guan KL. Download asset Open asset. Dou Z, Chattopadhyay M, Pan JA, Guerriero JL, Jiang YP, Ballou LM, Yue Z, Lin RZ, Zong WX.

Video

Mechanism of Nuclear Transport - RAN GTPase Cycle Metrics details. Autophagy is a GTPaxes cellular Autophagy and GTPases process in eukaryotes that Aufophagy the recycling and reutilization GTPasex damaged organelles and compartments. Quick Reflexes Formula plays a pivotal role in cellular homeostasis, pathophysiological processes, and diverse diseases in humans. Autophagy involves dynamic crosstalk between different stages associated with intracellular vesicle trafficking. Golgi apparatus is the central organelle involved in intracellular vesicle trafficking where Golgi-associated Rab GTPases function as important mediators.

Autophagy and GTPases -

ULK1, FIP, Beclin1, and Vps34 but not Atg7, Atg12, Atg16, or Atg9 were involved in non-canonical autophagy. Moreover, Rab9 silencing decreased the quantity of autophagic vacuoles but increased the accumulation of isolation membranes, which represent the membrane origin of autophagic vacuoles.

These findings suggest that Rab9 is involved in non-canonical autophagosome generation. As a D2 dopamine receptor antagonist, raclopride induces autophagy in cardiac myocytes.

Downregulation of Rab9 in raclopride-treated cells caused reduced clearance of p62, which is a substrate of autophagy, and lipidation of LC3-I to LC3-II.

However, downregulation of Atg7 had no effect on LC3 lipidation or p62 clearance. These results suggest that raclopride-induced autophagy requires Rab9 but probably does not require Atg7. Many studies have confirmed that Rab7 has a key role in autophagosome maturation. Schematic diagram illustrating the regulation of autophagosome—lysosome fusion by Rab proteins.

In trophic factor withdrawal TFW -induced neuronal death, insulin-like growth factor-I IGF-I protects Purkinje neurons from cell death by increasing the rate of autophagosome-to-lysosome fusion.

Under TFW conditions, Rab7 bound to Rab7-interacting lysosomal protein RILP , which only binds to the active GTP-bound form of Rab7, at a lower level, and autophagosomes accumulated in neurons.

However, IGF-I treatment during TFW restored Rab-RILP binding to normal levels and reduced the accumulation of autophagosomes. Because the Rab7-RILP complex regulates the microtubule minus-end-direct transport of late endocytic vesicles and phagosomes by recruiting the dynein—dynactin motor complex, 85 , 86 , 87 IGF-I probably promotes Rab7-RILP binding under TFW conditions and, as a result, the Rab7-RILP complex recruits the dynein—dynactin motor complex to facilitate the transport of autophagosomes to lysosomes for fusion Figure 3.

The COP9 signalosome CSN comprises eight essential subunits CSN1—CSN8 and is responsible for the regulation of the ubiquitin proteasome system. Quantification of autophagosomes, lysosomes, and autolysosomes in CR-Csn8KO hearts revealed a decreased amount of autolysosomes relative to autophagosomes and lysosomes; therefore, the accumulation of autophagosomes in CR-Csn8KO hearts was attributed to the defective fusion of autophagosomes with lysosomes.

Furthermore, extensive accumulation of autophagosomes was accompanied by minimal Rab7 expression in CR-Csn8KO hearts. In cardiomyocytes, downregulation of Rab7 inhibited the fusion of autophagosomes with lysosomes and exacerbated cell necrosis or apoptosis.

Collectively, these results suggest that Csn8 likely downregulates Rab7 to impair autophagosome maturation that causes cardiomyocytes to undergo necrosis or apoptosis. Autophagy has been shown to confer resistance to Mycobacterium tuberculosis , 91 and a recent report 92 showed that Rab8B and TANK-binding kinase 1 TBK-1 , which is a downstream interaction partner of Rab8B and a pivotal innate immunity regulator, 93 , 94 participate in this autophagy-mediated antimicrobial defense by controlling autophagosome maturation.

tuberculosis var. bovis BCG infection, both knockdown of Rab8B or TBK-1 reduced autophagic killing of mycobacteria, which suggests that Rab8B regulates autophagic elimination of mycobacteria possibly through TBK In addition, TBK-1 knockdown increased the number of autophagosomes but decreased the number of autolysosomes with accumulation of LC3-II, which implies that TBK-1 is involved in autophagosome maturation.

In addition, Rab8B interacted with TBK-1 in LC3-labeled autophagic organelles, and some autophagic components, such as LC3-II, p62, and UVRAG, were cofractionated with TBK-1 and Rab8B. These findings suggest that Rab8B facilitates autophagic elimination of mycobacteria by regulating autophagosome maturation through TBK-1 Figure 3.

Rab24 is localized in the ER and is thought to primarily participate in autophagosome maturation. In contrast, in amino-acid starvation-induced autophagy, the distribution of Rab24 changes into punctate spots, larger dots, ring-shaped small vesicles, and some tubular-like structures, and these structures colocalize with markers for autophagic vacuoles, such as LC3 and monodansylcadaverine.

The overexpression of wt Rab24 caused an increase in autophagosomes, whereas in cells overexpressing the mutant Rab24S67L, the amount of autophagosomes decreased. These results demonstrated for the first time that Rab24 is involved in autophagy. The drs gene products suppress apoptosis-inducing tumors.

Peptide mass fingerprinting analyses and a pull-down assay revealed that Rab24 binds to drs and that the binding requires the transmembrane domain of drs. Under low serum level conditions, drs is colocalized with Rab24 in LC3-positive vesicle-like structures. These results suggest that drs interacts with Rab24 in the autophagosome and may regulate the fusion of autophagosomes with lysosomes through Rab Curcumin, a hydrophobic polyphenol of the golden spice turmeric, influences various cellular biochemical and molecular functions through effects on multiple targets.

However, when autophagy was inhibited, curcumin treatment did not result in improved survival. These results suggest that autophagy mediates the protective effect of curcumin.

Moreover, both mRNA and protein expression of Rab7 increased drastically during curcumin-induced autophagy, implying that curcumin may facilitate autophagy by increasing Rab7 expression.

Autophagy has long been considered to be an intracellular degradation system in mammalian cells. However, a recent study showed that in mammals, autophagy also contributes to the biogenesis and secretion of the proinflammatory cytokine IL-1 β.

Although previous studies , , showed that basal autophagy inhibits IL-1 β secretion, the study described above found that the inflammasome and autophagy apparatus were synergized to intensify IL-1 β secretion in cells stimulated to induce autophagy, and that knockdown of Rab8A or overexpression of the dominant-negative Rab8A mutant inhibited the secretion, which suggests that this autophagy-based unconventional secretory pathway for IL-1 β involves Rab8A.

Rab25 expression only occurs in epithelial tissue and is associated with liver cancer, bladder cancer, ovarian cancer, and breast cancer. The amount of intracellular acidic vesicle organelles, which are a characteristic of autophagic cell death, and the amount of GFP-LC3 puncta in Rabknockdown cells was noticeably higher than the respective amounts in the control.

In addition, knockdown of Rab25 promoted Beclin1 expression and LC3-I lipidation. In this review, we discussed in detail the role of Rab proteins in various stages of autophagy and the relevant molecular mechanisms Figure 4 , Table 1. At this stage, studies on the involvement of Rabs in autophagy focus more on discovering different functions of Rab proteins in autophagy and less on investigating the underlying molecular mechanisms.

Such studies have only clarified some molecular events related to the involvement of Rab1, Rab5, Rab11, Rab32, and Rab33B in autophagosome formation and Rab7, Rab8B, and Rab24 in autophagosome maturation; thus, an intensive study of detailed molecular mechanisms should be conducted in the future.

Many natural compounds, such as curcumin, cannabinoids, and resveratrol, exert biological or pharmacological effects through autophagy. Moreover, recent studies , , have shown that other vesicle-trafficking proteins, such as soluble NSF attachment protein receptors and Ras-like GTPase Ral , participate in autophagy, which suggests that, in addition to Rab proteins, other vesicle-trafficking proteins may have a role in autophagy.

Thus, the function of other vesicle-trafficking proteins in autophagy needs to be further explored. Autophagy has a key role in organism growth and development, cell differentiation, and the response to cellular stress, and also in many diseases and aging.

Therefore, comprehensive understanding of the role of autophagic membrane trafficking regulated by Rab proteins will not only provide knowledge regarding the mechanisms underlying autophagy regulation but will also contribute to the search for new therapeutic targets to cure diseases through regulation of autophagy.

Schematic diagram illustrating the regulation of autophagy by Rab proteins. The solid line shows the canonical autophagic process in which Rab1, Rab5, and Rab32 regulate autophagosome formation from the ER.

Rab33B is involved in autophagosome formation from the GA, and autophagosome maturation into autolysosome is mediated by Rab7, Rab8B, and Rab The dotted line points to the fusion of MVB and RE with the autophagosome to form an amphisome that is regulated by Rab The dashed line indicates the formation and maturation of GcAVs; the homotypic fusion mediated by Rab7, Rab9A, and Rab Rab9A also participates in the fusion of GcAVs with lysosomes.

ER, endoplasmic reticulum; GA, Golgi apparatus; GAS, Group A Streptococcus ; GcAVs, GAS-containing autophagosome-like vacuoles; MVB, multivesicular bodies; PM, plasma membrane; RE, recycling endosome. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation.

Science ; : — CAS PubMed PubMed Central Google Scholar. Chua CE, Gan BQ, Tang BL. Involvement of members of the Rab family and related small GTPases in autophagosome formation and maturation.

Cell Mol Life Sci ; 68 : — CAS PubMed Google Scholar. Tanida I. Autophagosome formation and molecular mechanism of autophagy. Antioxid Redox Signal ; 14 : — Mizushima N. Physiological functions of autophagy. Curr Top Microbiol Immunol ; : 71— Kroemer G, Marino G, Levine B.

Autophagy and the integrated stress response. Mol Cell ; 40 : — Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway with a central role in health and disease.

Biochim Biophys Acta ; : — Beau I, Mehrpour M, Codogno P. Autophagosomes and human diseases. Int J Biochem Cell Biol ; 43 : — Mathew R, White E. Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night. Curr Opin Genet Dev ; 21 : — Tung YT, Wang BJ, Hu MK, Hsu WM, Lee H, Huang WP et al.

Autophagy: a double-edged sword in Alzheimer's disease. J Biosci ; 37 : — Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell ; : 27— Article CAS PubMed PubMed Central Google Scholar. Rubinsztein DC, Shpilka T, Elazar Z.

Mechanisms of autophagosome biogenesis. Curr Biol ; 22 : R29—R Tooze SA, Yoshimori T. The origin of the autophagosomal membrane.

Nat Cell Biol ; 12 : — Weidberg H, Shvets E, Elazar Z. Biogenesis and cargo selectivity of autophagosomes. Annu Rev Biochem ; 80 : — Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum.

J Cell Biol ; : — Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett ; : — Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast.

Nat Rev Mol Cell Biol ; 10 : — Autophagy basics. Microbiol Immunol ; 55 : 1— Yamamoto H, Kakuta S, Watanabe TM, Kitamura A, Sekito T, Kondo-Kakuta C et al.

Atg9 vesicles are an important membrane source during early steps of autophagosome formation. Rieter E, Vinke F, Bakula D, Cebollero E, Ungermann C, Proikas-Cezanne T et al. Atg18 function in autophagy is regulated by specific sites within its beta-propeller.

J Cell Sci ; : — Watanabe Y, Kobayashi T, Yamamoto H, Hoshida H, Akada R, Inagaki F et al. Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg J Biol Chem ; : — Simonsen A, Tooze SA.

Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes. Lindmo K, Stenmark H. Regulation of membrane traffic by phosphoinositide 3-kinases. Homma K, Suzuki K, Sugawara H. The Autophagy Database: an all-inclusive information resource on autophagy that provides nourishment for research.

Nucleic Acids Res ; 39 : D—D Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J ; 19 : — Burman C, Ktistakis NT. Autophagosome formation in mammalian cells.

Semin Immunopathol ; 32 : — Otomo C, Metlagel Z, Takaesu G, Otomo T. Nat Struct Mol Biol ; 20 : 59— Cherfils J, Zeghouf M. Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev ; 93 : — Bos JL, Rehmann H, Wittinghofer A.

GEFs and GAPs: critical elements in the control of small G proteins. Cell ; : — Mizuno-Yamasaki E, Rivera-Molina F, Novick P. GTPase networks in membrane traffic. Annu Rev Biochem ; 81 : — Hutagalung AH, Novick PJ. Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev ; 91 : — Segev N.

Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein. Pind SN, Nuoffer C, McCaffery JM, Plutner H, Davidson HW, Farquhar MG et al.

De Antoni A, Schmitzova J, Trepte HH, Gallwitz D, Albert S. Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs.

Barrowman J, Bhandari D, Reinisch K, Ferro-Novick S. TRAPP complexes in membrane traffic: convergence through a common Rab. Nat Rev Mol Cell Biol ; 11 : — Zou S, Liu Y, Zhang XQ, Chen Y, Ye M, Zhu X et al.

Genetics ; : — Sacher M, Barrowman J, Schieltz D, Yates JR 3rd, Ferro-Novick S. Identification and characterization of five new subunits of TRAPP. Eur J Cell Biol ; 79 : 71— Meiling-Wesse K, Epple UD, Krick R, Barth H, Appelles A, Voss C et al. Trs85 Gsg1 , a component of the TRAPP complexes, is required for the organization of the preautophagosomal structure during selective autophagy via the Cvt pathway.

Nazarko TY, Huang J, Nicaud JM, Klionsky DJ, Sibirny AA. Trs85 is required for macroautophagy, pexophagy and cytoplasm to vacuole targeting in Yarrowia lipolytica and Saccharomyces cerevisiae. Autophagy ; 1 : 37— Suzuki K. Selective autophagy in budding yeast.

Cell Death Differ ; 20 : 43— Lynch-Day MA, Bhandari D, Menon S, Huang J, Cai H, Bartholomew CR et al. Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy.

Proc Natl Acad Sci USA ; : — Lipatova Z, Belogortseva N, Zhang XQ, Kim J, Taussig D, Segev N. He C, Song H, Yorimitsu T, Monastyrska I, Yen WL, Legakis JE et al. Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast.

Kim J, Kamada Y, Stromhaug PE, Guan J, Hefner-Gravink A, Baba M et al. Kakuta S, Yamamoto H, Negishi L, Kondo-Kakuta C, Hayashi N, Ohsumi Y. Atg9 vesicles recruit vesicle-tethering proteins Trs85 and Ypt1 to the autophagosome formation site. Zoppino FC, Militello RD, Slavin I, Alvarez C, Colombo MI.

Autophagosome formation depends on the small GTPase Rab1 and functional ER exit sites. Traffic ; 11 : — Olanow CW, Brundin P. Parkinson's disease and alpha synuclein: is Parkinson's disease a prion-like disorder?

Mov Disord ; 28 : 31— Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy.

Winslow AR, Chen CW, Corrochano S, Acevedo-Arozena A, Gordon DE, Peden AA et al. alpha-Synuclein impairs macroautophagy: implications for Parkinson's disease. Kuballa P, Nolte WM, Castoreno AB, Xavier RJ. Autophagy and the immune system. Annu Rev Immunol ; 30 : — Gong L, Devenish RJ, Prescott M.

Autophagy as a macrophage response to bacterial infection. IUBMB Life ; 64 : — Huang J, Birmingham CL, Shahnazari S, Shiu J, Zheng YT, Smith AC et al.

Antibacterial autophagy occurs at PI 3 P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase. Autophagy ; 7 : 17— Gabernet-Castello C, O'Reilly AJ, Dacks JB, Field MC. Mol Biol Cell ; 24 : — Uchiya K, Tobe T, Komatsu K, Suzuki T, Watarai M, Fukuda I et al.

Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading. Mol Microbiol ; 17 : — Dong N, Zhu Y, Lu Q, Hu L, Zheng Y, Shao F. Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses.

Joshi AD, Swanson MS. Secrets of a successful pathogen: legionella resistance to progression along the autophagic pathway. Front Microbiol ; 2 : Ohbayashi N, Fukuda M. Role of Rab family GTPases and their effectors in melanosomal logistics. J Biochem ; : — Ravikumar B, Imarisio S, Sarkar S, O'Kane CJ, Rubinsztein DC.

Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease. Dou Z, Chattopadhyay M, Pan JA, Guerriero JL, Jiang YP, Ballou LM et al.

The class IA phosphatidylinositol 3-kinase pbeta subunit is a positive regulator of autophagy. Dou Z, Pan JA, Dbouk HA, Ballou LM, Deleon JL, Fan Y et al. Class IA PI3K pbeta subunit promotes autophagy through Rab5 Small GTPase in response to growth factor limitation.

Mol Cell ; 50 : 29— Su WC, Chao TC, Huang YL, Weng SC, Jeng KS, Lai MM. Rab5 and class III phosphoinositide 3-kinase Vps34 are involved in hepatitis C virus NS4B-induced autophagy. J Virol ; 85 : — Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K et al. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons.

J Virol ; 77 : — Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol ; 5 : — Dwivedi M, Sung H, Shen H, Park BJ, Lee S. Disruption of endocytic pathway regulatory genes activates autophagy in C. Mol Cells ; 31 : — Yamaguchi H, Nakagawa I, Yamamoto A, Amano A, Noda T, Yoshimori T.

An initial step of GAS-containing autophagosome-like vacuoles formation requires Rab7. PLoS Pathog ; 5 : e PubMed PubMed Central Google Scholar. Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, Kamimoto T et al.

Autophagy defends cells against invading group A Streptococcus. Nozawa T, Aikawa C, Goda A, Maruyama F, Hamada S, Nakagawa I. The small GTPases Rab9A and Rab23 function at distinct steps in autophagy during Group A Streptococcus infection.

Cell Microbiol ; 14 : — Morvan J, Kochl R, Watson R, Collinson LM, Jefferies HB, Tooze SA. In vitro reconstitution of fusion between immature autophagosomes and endosomes. Autophagy ; 5 : — Fader CM, Sanchez DG, Mestre MB, Colombo MI.

Fader CM, Sanchez D, Furlan M, Colombo MI. Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k cells. Traffic ; 9 : — Fader CM, Savina A, Sanchez D, Colombo MI.

Exosome secretion and red cell maturation: Exploring molecular components involved in the docking and fusion of multivesicular bodies in K cells.

Blood Cells Mol Dis ; 35 : — Longatti A, Lamb CA, Razi M, Yoshimura S, Barr FA, Tooze SA. TBC1D14 regulates autophagosome formation via Rab and ULK1-positive recycling endosomes. Hirota Y, Tanaka Y. A small GTPase, human Rab32, is required for the formation of autophagic vacuoles under basal conditions.

Cell Mol Life Sci ; 66 : — Wang C, Liu Z, Huang X. Rab32 is important for autophagy and lipid storage in Drosophila.

PLoS One ; 7 : e Itoh T, Fujita N, Kanno E, Yamamoto A, Yoshimori T, Fukuda M. Golgi-resident small GTPase Rab33B interacts with Atg16L and modulates autophagosome formation. Mol Biol Cell ; 19 : — Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, Kanaseki T et al.

Nature ; : — Yan H, Li WL, Xu JJ, Zhu SQ, Long X, Che JP. D2 dopamine receptor antagonist raclopride induces non-canonical autophagy in cardiac myocytes. J Cell Biochem ; : — Hyttinen JM, Niittykoski M, Salminen A, Kaarniranta K. Maturation of autophagosomes and endosomes: a key role for Rab7.

Gutierrez MG, Munafo DB, Beron W, Colombo MI. Rab7 is required for the normal progression of the autophagic pathway in mammalian cells. Jager S, Bucci C, Tanida I, Ueno T, Kominami E, Saftig P et al. Role for Rab7 in maturation of late autophagic vacuoles.

PubMed Google Scholar. Ganley IG, Wong PM, Gammoh N, Jiang X. Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell ; 42 : — Li W, Zou W, Yang Y, Chai Y, Chen B, Cheng S et al.

Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell. J Cell Biol ; : 27— Pankiv S, Alemu EA, Brech A, Bruun JA, Lamark T, Overvatn A et al. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport.

Bains M, Florez-McClure ML, Heidenreich KA. Insulin-like growth factor-I prevents the accumulation of autophagic vesicles and cell death in Purkinje neurons by increasing the rate of autophagosome-to-lysosome fusion and degradation.

Bains M, Zaegel V, Mize-Berge J, Heidenreich KA. IGF-I stimulates Rab7-RILP interaction during neuronal autophagy. Neurosci Lett ; : — Harrison RE, Bucci C, Vieira OV, Schroer TA, Grinstein S.

Mol Cell Biol ; 23 : — Johansson M, Rocha N, Zwart W, Jordens I, Janssen L, Kuijl C et al. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-pGlued, ORP1L, and the receptor betalll spectrin.

Jordens I, Fernandez-Borja M, Marsman M, Dusseljee S, Janssen L, Calafat J et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr Biol ; 11 : — Wei N, Serino G, Deng XW.

The COP9 signalosome: more than a protease. Trends Biochem Sci ; 33 : — Su H, Li J, Menon S, Liu J, Kumarapeli AR, Wei N et al. Perturbation of cullin deneddylation via conditional Csn8 ablation impairs the ubiquitin-proteasome system and causes cardiomyocyte necrosis and dilated cardiomyopathy in mice.

Circ Res ; : 40— Su H, Li F, Ranek MJ, Wei N, Wang X. COP9 signalosome regulates autophagosome maturation. Circulation ; : — Ponpuak M, Davis AS, Roberts EA, Delgado MA, Dinkins C, Zhao Z et al.

Delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties. Immunity ; 32 : — Pilli M, Arko-Mensah J, Ponpuak M, Roberts E, Master S, Mandell MA et al.

TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity ; 37 : — Morton S, Hesson L, Peggie M, Cohen P. Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma. Ou YH, Torres M, Ram R, Formstecher E, Roland C, Cheng T et al.

Mol Cell ; 41 : — Munafo DB, Colombo MI. Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab Traffic ; 3 : — Egami Y, Kiryu-Seo S, Yoshimori T, Kiyama H. Induced expressions of Rab24 GTPase and LC3 in nerve-injured motor neurons.

Biochem Biophys Res Commun ; : — Kim CJ, Shimakage M, Kushima R, Mukaisho K, Shinka T, Okada Y et al. Down-regulation of drs mRNA in human prostate carcinomas. Hum Pathol ; 34 : — Tambe Y, Yamamoto A, Isono T, Chano T, Fukuda M, Inoue H. The drs tumor suppressor is involved in the maturation process of autophagy induced by low serum.

Cancer Lett ; : 74— Goel A, Kunnumakkara AB, Aggarwal BB. Curcumin as "Curecumin": from kitchen to clinic. Biochem Pharmacol ; 75 : — Han J, Pan XY, Xu Y, Xiao Y, An Y, Tie L et al. Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage.

Autophagy ; 8 : — Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1beta. EMBO J ; 30 : — The overlap between Rab26 and Rab33 prompted us to further investigate whether Atg16L1 may also be an effector of Rab To explore this possibility, we performed co-immunoprepitation experiments between FLAG-tagged Rab26 WT, QL or TN and endogenous Atg16L1 in HeLa cells.

As shown in Figure 7A , all three FLAG-tagged Rab26 variants were efficiently immunoprecipitated with the FLAG antibody. Immunoblotting for endogenous Atg16L1 from the same immunoprecipitates revealed co-precipitation between Atg16L1 and Rab26QL. By comparison, little to no Atg16L1 was detectable in the precipitates of RabWT and Rab26TN, respectively, indicating that the interaction between Rab26 and Atg16L1 is GTP-dependent.

A Co-Immunoprecipitation of FLAG-tagged Rab26 variants expressed in HeLa cells with endogenous Atg16L1 protein. Immunoprecipitation was carried out following lysis in detergent-containing buffer and clearance by centrifugation to remove cell debris. Note that only the GTP-preferring QL variant of Rab26 showed significant binding to Atg16L1 arrow.

B GST pulldown of purified recombinantly expressed GST-Rab26 variants with a pre-formed complex of His-tagged versions of Atg5 and the N-terminal domain of Atg16L1 Atg16NT.

Note that Atg16NT selectively interacted with the GTP-preferring QL-variant of Rab In parallel, we performed GST-pulldown assays to verify the results from the coIP experiments.

For this, purified bacterially expressed recombinant Rab26 variants QL or TN , tagged with GST were incubated with a preassembled complex of Atg5 and the N-terminal fragment of Atg16L1 containing its coiled coil domain Atg16NT. In agreement with our immunoprecipitation studies, GST-pulldown revealed an interaction between Atg16L1 and Rab26, with Atg16L1 binding to the QL and to a lesser extent the TN-variant of Rab26 Figure 7B , with the latter being further reduced upon repetitive washings not shown.

Atg5 remains bound in this complex. To further examine the interaction, we analyzed the binding between Rab26 and Atg16L1 using analytical gel filtration. Surprisingly, formation of Rab26 QL -ATG16L1 complexes were not detectable with this approach Figure 7—figure supplement 1.

As a positive control, we carried out the same experiment using Rab33 QL and ATG16L1. Here, complex formation was detectable with this approach.

Thus, while both IP and pull-down experiments show that RAB26 binds ATG16L1 in a GTP-dependent manner, this binding appears to be weaker than the interaction between Rab33 and ATG16L1. In the present study we have combined multiple complementary biochemical and cell biological approaches to demonstrate that the small GTPase Rab26 is specifically associated with synaptic vesicles.

Intriguingly, Rab26 appears to be particularly enriched in large clusters of synaptic vesicles to which the autophagy proteins Atg16L1, LC3 and Rab33B are recruited, suggesting that they represent pre-autophagosomal compartments.

We show further that, at least when using overexpression of EGFP-tagged Rab26, such clusters are also formed in cell bodies where they are enclosed by a single and in some instances a double isolation membrane.

Rab26 is most closely related to the secretory GTPases Rab3 and Rab27, which led to the conclusion that it may perform similar functions in membrane traffic Fukuda, This view is supported by reports showing association of Rab26 with zymogen granules in exocrine cells Nashida et al.

More recently, Rab26 has been found to be associated with lysosomes in zymogen-secreting cells Jin and Mills, implying that its functions in secretory cells extend beyond that of exocytosis.

In our previous work Takamori et al. Our present data now show that this association is exclusive, with Rab26 being absent from other organelles such as early endosomes, paralleling the distribution of other secretory Rabs.

On the other hand, the preferential association of Rab26 with large clusters of synaptic vesicles and its conspicuous absence from smaller boutons positive for synaptic vesicle markers is clearly distinct from Rab3 and Rab27b and indicates that Rab26 may not be contributing to the canonical function of these Rabs in regulated exocytosis.

Intriguingly, in contrast to for example, Rab3 and Rab5, Rab26 cannot be extracted from synaptic vesicle membranes by GDI in its GDP-form—a feature it shares with Rab27b.

Rather, Rab26 exhibits a tendency to oligomerize in the GDP-form, again a feature shared with Rab27b and perhaps with some others such as Rab11 and Rab9, which crystallize as dimers in the GDP-state Pasqualato et al.

It is somewhat surprising that, along with the GDP-bound variant, wild-type Rab26 also appears to oligomerize albeit to a lesser extent. Perhaps the most conspicuous feature of Rab26 is that it is not only preferentially associated with secretory vesicle clusters but actually induces their formation in a GTP-dependent manner as becomes apparent upon the expression of exogenous Rab26 variants in both neurons and non-neuronal cells.

This is most dramatically observed with the EGFP-tagged variant suggesting that the weak homodimerization tendency of EGFP enhances the phenotype note that no other EGFP-tagged Rab exhibits similar features including the most abundant secretory GTPase, Rab3a.

At present, the exact mechanism underlying this clustering phenotype is unclear. Nevertheless, since the GTP-form of Rab26 does not oligomerize, it is unlikely that clustering is effected by homophilic Rab26 interactions. Rather, it possible that clustering is mediated by a hitherto unknown effector protein.

This effector is probably distinct from Atg16L1 as overexpression of EGFP-Rab33B that also recruits Atg16L1 does not induce such clusters Figure 6 , and data not shown.

However, given that the central terminal region of Atg16L1 has a tendency for homo-multimerization, this possibility cannot be excluded Mizushima et al. Intriguingly, our findings agree with a recent report according to which overexpression of Rab26 in exocrine cell lines induces clustering of lysosomes, reminiscent of the partial co-localization of the EGFP-induced Rab26 clusters with lysosomes in neuronal cell bodies Jin and Mills, Our results indicate that the core autophagy protein Atg16L1 is an effector of Rab26 that binds to the GTPase exclusively in the GTP-form, paralleling previous findings on the Golgi-resident Rab33B Itoh et al.

Interestingly, binding of Rab26 to Atg16L1 appears to be weaker than that between Rab33 and Atg16L1, which plays a role in canonical autophagy, probably explaining why Itoh et al.

It is conceivable that the interaction is more transient, or else, that it requires additional factors for stabilization, thus allowing for fine-tuning the flow of synaptic vesicles targeted for selective autophagy.

How does recruitment of Atg16L1 to synaptic vesicle clusters relate to the established steps of autophagosome formation? First of all, it cannot yet be excluded with certainty that upon recruitment to these vesicles Atg16L1 performs a non-canonical function that is not related to autophagosome formation see e.

In particular, Atg16L1 and Rab33A have recently been found to be associated with secretory vesicles in neuroendocrine PC12 cells, with the data suggesting a role for Atg16L1 in regulating exocytosis independent of autophagy Ishibashi et al.

On the other hand, based on our extensive morphological assessment using double immunolabeling microscopy, we strongly favor that the RabAtg16L1 complexes in neurons represent pre-autophagosomal structures because i Rab26 is not present on all synaptic vesicles but rather confined to vesicle aggregates that may be functionally impaired, and ii LC3 is recruited to these clusters suggesting that the formation of an autophagosomal membrane is, at least in part, initiated.

Our data indicates that the vesicle clusters containing Rab26 and Atg16L1 have undergone exo-endocytotic cycling. Intriguingly, clathrin has recently been shown to interact with Atg16L1, thus targeting plasma membrane constituents towards autophagosome precursors via clathrin-mediated endocytosis Ravikumar et al.

Since clathrin-mediated endocytosis constitutes the main endocytotic pathway for synaptic vesicles, it is conceivable that there is a synergy between Rab and clathrin-induced autophagocytosis in nerve terminals that further fine-tunes the targeting of synaptic vesicles to preautophagosomal structures.

In many of these cases the pathway is initiated by ubiquitination of target proteins. While we do not know whether this is also the case here, it is conceivable that the initiation event may indeed be the recruitment of active Rab26 to the membrane of subsets of synaptic vesicles that then interacts with other factors to form clusters and to recruit an isolation membrane, the origin of which remains to be identified.

Following the classical work in the early 70s of last century demonstrating that synaptic vesicles undergo multiple rounds of recycling in the synapse, Atwood et al.

However, all membrane constituents age and accumulate structural defects requiring sorting out of damaged constituents.

Although no increase in the number of late endosomes, lysosomes or autophagosomes was observed following even massive stimulation, it was hypothesized as early as that newly reformed synaptic vesicles could either be actively re-used as functional synaptic vesicles or re-directed to a pathway ultimately leading to lysosomes as the final destination for degradation Holtzman et al.

Our discovery of vesiculophagy as a pathway initiated in presynaptic boutons that directs entire synaptic vesicle pools towards autophagosomes provides a previously uncharacterized link towards lysosomal degradation of trafficking organelles which is distinct from the classical endosomal route.

Indeed, recent data suggest that presynaptic neurotransmission is functionally modulated by macroautophagy.

Induction of autophagy in neurons increased the amount of autophagic vacuoles in presynaptic terminals and with an accompanying reduction in synaptic vesicle number and decrease in evoked neurotransmitter release Hernandez et al.

Furthermore, two groups have recently suggested that in axons autophagosomes originate distally and then are transported by retrograde motors towards the cell body.

During their travel they undergo fusion with acidic compartments and finally with the lysosomes Lee et al. It is therefore conceivable that Rab26 feeds vesicle membranes into autophagosomes that may form and mature during retrograde transport.

How this novel pathway is initiated and regulated will be the subject of future studies. Mouse monoclonal and rabbit polyclonal antibodies specific for synaptophysin, synaptotagmin, synaptobrevin, Rab3a, GDI Cl Mouse anti-LAMP2 antibody was purchased from the Developmental Studies Hybridoma Bank DSHB, University of Iowa, IA.

Antibodies against EEA1 and GM were purchased from BD Bioscience Franklin Lakes, NJ. The antibody against the FLAG epitope was obtained from Stratagene La Jolla, CA. Antibodies specific for Atg16L1 were purchased from CosmoBio Tokyo and MBL Nagoya.

Anti-Atg5 antibody was from Novus Biological Littleton, Colorado. The antibody against secretogranin II was kindly provided by Sharon Tooze Cancer Research UK. Cells from knee lymph nodes were fused with the mouse myeloma cell line P3X63Ag.

Cell culture supernatants obtained from individual clones were then screened using enzyme-linked immunosorbent assay ELISA , immunoblot assays and immunoflourescence. The final hybridoma used in this study was cloned two times by limiting dilution.

The monoclonal antibody produced from this clone was determined to be of the IgG2a subclass and is specific for Rab26 Figure 1—figure supplement 1. The antibody is commercially available from Synaptic Systems Göttingen, Germany. Cy3-labeled goat anti-mouse or anti-rabbit and Alexa labeled goat anti-mouse secondary antibodies were purchased from Dianova Hamburg, Germany and used at a dilution of Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Bio-Rad Hercules, CA and used at a dilution of or Likewise, inserts encoding Rab26 QL, T77N or NI mutants were generated by recombinant PCR and similarly inserted into these vectors.

For recombinant protein expression in bacteria, inserts for the Rab26 variants were inserted into pGEX-KG using EcoRI and BamHI while the insert encoding alpha-GDI was sub-cloned into pETa Novagen, Madison, WI.

The sequence corresponding to murine Atg16L1 1— BC was cloned into pETa Novagen using NdeI and NotI restriction sites. Full-length murine Atg5 1— BC was cloned with an N-terminal thrombin cleavage site into the multiple cloning site 1 of pETDuet-1 Novagen using the SalI and NotI sites.

The vector expressing neuropeptide Y NPY was generated by inserting the sequence encoding human pro-NPY into the pmRFP vector. Cloning was performed according to standard procedures Janssen, The plasmid expressing GFP-tagged human LC3B was a kind gift from Dr Zvulun Elazar Weizmann Institute, Israel.

Culturing of the HEK and HeLa SS6 cell lines and the preparation of high density primary rat hippocampal neurons have been previously described Rosenmund and Stevens, ; Chua et al. Neurons were transfected between 7 to 12 days after seeding or, in the case of the cell lines, 1 day after seeding using Lipofectamine Invitrogen, Carlsbad, CA according to the manufacturer's protocol.

Neurons in Figures 3F, 4 , Figure 3—figure supplement 1 and Figure 4—figure supplement 1 were transfected using calcium phosphate as previously described Pavlos et al.

Immunostaining was then performed as described in Chua et al. Afterwards, cells were permeabilized with 0. Incubation with primary antibodies diluted in blocking solution was then carried out for 1 hr at room temperatures or overnight at 4°C.

Subsequently, cells were exposed to secondary Cy3 or Alexafluor conjugated goat anti-rabbit and anti-mouse antibodies, respectively, for 1 hr at room temperature.

After washing, cells were mounted on slides SuperFrost Plus, VWR International bvba, Leuven, Belgium and then imaged using a confocal microscope LSM , Zeiss, Germany or an epifluorescence microscope Axiovert M, Zeiss, Germany.

Linescan analyses were performed using ImageJ or LAS AF Lite software. To visualize synaptic vesicles that have undergone recycling, live neurons transfected with EGFP-Rab26WT were incubated in culture for 24 hr with Oyster labeled anti-synaptotagmin-I antibodies Synaptic Systems that recognize its luminal domain Willig et al.

The UAST-YFP. Rab26, UAST-YFP. Rab26QL, UAST-YFP. Rab26TN Zhang et al. Dissection and immunostaining of neuromuscular junctions from third instar larvae were performed as described Schmid and Sigrist, using the following antibodies: mouse Anti-Brp hybridoma clone nc82, DSHB; dilution , anti-Csp antibody hybridoma clone ab49, DSHB; dilution , the chicken anti-GFP antibody Abcam; dilution and the goat anti-HRP Sigma; dilution.

Dylight labeled anti-goat and Alexa labeled anti-chicken secondary antibodies were purchased from Jackson ImmunoResearch Laboratories West Grove, PA.

Alexa conjugated anti-mouse secondary antibodies were purchased from Invitrogen Carlsbad, CA. Images were acquired with a microscope DMR-E; Leica, Germany equipped with a scan head TCS SP2 AOBS; Leica, Germany and an oil-immersion 63 × 1. Biochemical isolation of synaptic vesicles from the rat brain was performed as described previously Huttner et al.

Purified monoclonal antibodies directed against Rab26 described above and synaptophysin clone 7. The bound vesicles were subsequently analyzed by electron microscopy or eluted with 40 µl 2 × SDS sample buffer for immunoblots analysis.

The RabGDI assay was performed as described in Pavlos et al. Briefly, crude synaptic vesicles LP2 were used as the starting material. The samples were then kept on ice and subsequently centrifuged for 20 min at ,× g , 4°C using a Beckman S AT3 rotor.

The resulting pellet was re-suspended in 50 µl 2 × lithium dodecyl sulfate LDS sample buffer Invitrogen , boiled at 95°C for 5 min and analyzed by immunoblotting. Thin sections 80 nm were examined using a Philips CM BioTwin transmission electron microscope Philips Inc.

Eindhoven, The Netherlands. Images were taken with a TemCam FA slow scan CCD camera TVIPS, Gauting, Germany. The evaluation of the samples was done using the iTEM software Olympus Soft Imaging Solutions GmbH, Münster, Germany.

For immunogold electron microscopy, ultrathin cryosections of neuronal cultures Figure 5A and Figure 5—figure supplement 1 and HeLa cells Figure 6—figure supplement 1 transfected with EGFP-Rab26WT, were prepared as described previously Tokuyasu, , ; Zink et al.

For the ultrastructural analyses of the Drosophila neuromuscular junction Figure 5D , a standard protocol was used. Images were taken with a TemCam F slow scan CMOS camera TVIPS, Gauting, Germany. Human GST-tagged Rab26WT, QL, T77N were expressed in Escherichia coli BL21 D3.

The cultures were then incubated for 1 hr at 16°C. Induction was initiated by adding 1 mM IPTG to the cultures and the expression was carried out overnight at 16°C. Thereafter, cells were harvested by centrifugation at rpm for 10 min using a Beckman centrifuge.

Pellets obtained from each 1 l culture flask were re-suspended in 25 ml of protein buffer containing 50 mM HEPES pH 7. The samples were left for 10—15 min at 4°C and subsequently sonicated four times for 30 s each, separated by a 1 min incubation on ice, using a Branson Sonifier The lysate was then cleared at 13, rpm using a SLA rotor for 40 min at 4°C.

The resulting supernatant was collected and filtered using a 0. The filtrate was then loaded onto a GST-column GST Trap4B GE Healthcare, Germany and eluted using 30 mM glutathione in protein buffer.

The eluted fractions were collected and dialyzed three times for 3 hr each using protein buffer to remove glutathione. The purified proteins were then used for GST pulldown assays. His-tagged murine Atg16L1 1— -pETa and His-tagged murine Atg5-pETDuet-1 were co-transformed into E.

coli Rosetta 2 cells Merck Millipore, Germany. Cells were harvested by centrifugation at × g for 20 min. Pellets were resuspended in ml buffer A 0. Cells were lysed by sonication and centrifuged for 1 hr at 25,× g. Fractions containing the purified proteins were pooled and dialyzed overnight at 4°C in gel filtration buffer 0.

Co-immunoprecipitation assays were performed as described in Chua et al. The lysate was then clarified by centrifugation at 10,× g for 10 min. The resulting supernatant was incubated for 2 hr with anti-Flag or anti-GFP antibodies. Subsequently, 30 µl of protein G-Sepharose beads GE Healthcare, Sweden were added to each sample and further incubated for 1 hr under constant rotation.

The samples were then washed thrice with lysis buffer. Finally, 25—30 µl of 2 × LDS sample buffer were then added to the beads and the mixture was boiled at 95°C for 5 min. The beads were then washed three times with buffer. Human Rab26 54— QL was cloned into pETa using NdeI and XhoI cleavage sites.

Murine Rab33B 30— Q92L BC was cloned into pETDuet-1 with BamHI and NotI restriction sites. Plasmids were transformed into E. coli BL21 DE3. Bacteria were grown in 3 l ZYM autoinducing medium supplemented with the appropriate antibiotic for 8 hr at 37°C.

Cells were harvested by centrifugation and resuspended in ml buffer A 30 mM imidazole, 0. Bacteria were lysed with a microfluidizer and centrifuged for 1 hr at 25,× g. Fractions containing protein were pooled and diluted with gel filtration buffer 0.

Proteins were kept at 4°C overnight. The gel filtration buffer was 0. eLife posts the editorial decision letter and author response on a selection of the published articles subject to the approval of the authors. An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown.

Reviewers have the opportunity to discuss the decision before the letter is sent see review process. Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Your article has been favorably evaluated by Eve Marder Senior editor and 2 reviewers, one of whom is a member of our Board of Reviewing Editors. The Reviewing editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

All reviewers were generally enthusiastic about your work. They felt that the data presented are of very high quality and generally support your conclusions, especially with regard to the SV association of Rab They also see strong potential in this work to represent a novel secretory vesicle degeneration pathway.

However, the link to autophagy was considered still rather tenuous. We leave it up to you either to tone down your claim Title, Discussion, etc or to provide further experimental data. Please comment.

We agree that we do not have conclusive evidence showing directly that Rab26 activation triggers autophagy of synaptic vesicles. The problem, as far as we see it, is that in situ this pathway is probably not active or of only very low activity, making loss-of-function experiments rather difficult.

We have tried various approaches but it became clear that considerable additional efforts including suitable animal models are required for obtaining air-tight evidence, and it will probably be more efficient to carry out these experiments with a laboratory having the required tools and experiences.

We have therefore revised the Discussion as suggested in order to make sure we are not over-interpreting our results or draw conclusions that are not substantiated by the evidence.

We have also revised the Abstract accordingly. Therefore, we have retained it except for a small change suggested by the eLife office, but we are open for a better suggestion. It is surprising why EGFP-Rab26 is not LC3 positive: is this a question of weaker efficiency or eGFP-Rab26? We were also puzzled by the observation that EGFP-Rab26, unlike FLAG-Rab26, does not readily localize with LC3.

Possibly, the addition of the bulky EGFP-moiety which is of similar size as the GTPase itself interferes with the binding of additional factors needed for recruiting LC3 or for targeting vesicles to LC3 positive compartments, thereby trapping the vesicles at a step just prior to the recruitment of LC3.

While uncommon, there are notable examples of the bulky GFP moiety interfering with protein-protein interactions and thus with function such as in the ESCRT pathway Howard et al.

Cell Sci. Such a scenario would explain the accumulation of the enormous vesicle clusters observed within neuronal cell bodies that whilst retained Atg16L1, formed no or only incomplete isolation membranes. It may also account for the densely packed vesicle clusters lacking isolation membranes observed upon expression of EGFP-Rab26 in Drosophila neuromuscular synapses.

What role does Rab26 expression levels play in the morphological phenotype? A higher level of Rab26 WT expression in cells monitored by immunocytochemistry does increase the phenotype. Nevertheless, even though Rab26 WT and Rab26 QL both express at similar levels in these cells see e.

Figure 2B , the WT variant still exhibits the stronger morphological phenotype. Consequently, the functional state of Rab26 rather than its level alone is the dominant factor influencing the magnitude of the phenotype. Figure 5 : which synaptobrevin was used for colocalization with EGFP-Rab26?

Antibodies specific for synaptobrevin 2 were used. This information has been added to the legend. We thank Brigitte Barg-Kues and Sigrid Schmidt for excellent technical assistance. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.

Article citation count generated by polling the highest count across the following sources: Crossref , PubMed Central , Scopus. Neurostimulation of the hippocampal formation has shown promising results for modulating memory but the underlying mechanisms remain unclear.

In particular, the effects on hippocampal theta-nested gamma oscillations and theta phase reset, which are both crucial for memory processes, are unknown. Moreover, these effects cannot be investigated using current computational models, which consider theta oscillations with a fixed amplitude and phase velocity.

Here, we developed a novel computational model that includes the medial septum, represented as a set of abstract Kuramoto oscillators producing a dynamical theta rhythm with phase reset, and the hippocampal formation, composed of biophysically realistic neurons and able to generate theta-nested gamma oscillations under theta drive.

We showed that, for theta inputs just below the threshold to induce self-sustained theta-nested gamma oscillations, a single stimulation pulse could switch the network behavior from non-oscillatory to a state producing sustained oscillations.

Next, we demonstrated that, for a weaker theta input, pulse train stimulation at the theta frequency could transiently restore seemingly physiological oscillations. Importantly, the presence of phase reset influenced whether these two effects depended on the phase at which stimulation onset was delivered, which has practical implications for designing neurostimulation protocols that are triggered by the phase of ongoing theta oscillations.

This novel model opens new avenues for studying the effects of neurostimulation on the hippocampal formation. Furthermore, our hybrid approach that combines different levels of abstraction could be extended in future work to other neural circuits that produce dynamical brain rhythms.

org is a mature open-access knowledge base of the rodent hippocampal formation focusing on neuron types and their properties. Previously, Hippocampome. org v1. Releases v1. Those additional properties increased the online information content of this public resource over fold, enabling numerous independent discoveries by the scientific community.

org v2. In all cases, the freely downloadable model parameters are directly linked to the specific peer-reviewed empirical evidence from which they were derived. Possible research applications include quantitative, multiscale analyses of circuit connectivity and spiking neural network simulations of activity dynamics.

These advances can help generate precise, experimentally testable hypotheses and shed light on the neural mechanisms underlying associative memory and spatial navigation. CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues.

Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available.

Here, we report cryoEM structures of human CLC-2 at 2. AK binds within the extracellular entryway of the Cl — -permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains CTDs.

In the absence of CTD rotation, an intracellular N-terminal residue hairpin peptide nestles against the TM domain to physically occlude the Cl — -permeation pathway.

This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Through electrophysiological studies of an N-terminal deletion mutant lacking the residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl — -permeation pathway.

Share this article Doi. Cite this article Beyenech Binotti Nathan J Pavlos Dietmar Riedel Dirk Wenzel Gerd Vorbrüggen Amanda M Schalk Karin Kühnel Janina Boyken Christian Erck Henrik Martens John JE Chua Reinhard Jahn The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Material and methods References Decision letter Author response Article and author information Metrics.

Figure 1 with 1 supplement see all. Download asset Open asset. Figure 2. Figure 3 with 2 supplements see all. Figure 4 with 1 supplement see all.

Figure 5 with 1 supplement see all. Figure 6 with 1 supplement see all. Figure 7 with 1 supplement see all. Andres DA Seabra MC Brown MS Armstrong SA Smeland TE Cremers FP Goldstein JL cDNA cloning of component A of Rab geranylgeranyl transferase and demonstration of its role as a Rab escort protein Cell 73 — Araki S Kikuchi A Hata Y Isomura M Takai Y Regulation of reversible binding of smg p25A, a ras plike GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor.

Atwood HL Lang F Morin WA Synaptic vesicles: selective depletion in crayfish excitatory and inhibitory axons Science — Bedford FK Kittler JT Muller E Thomas P Uren JM Merlo D Wisden W Triller A Smart TG Moss SJ GABA A receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1 Nature Neuroscience 4 — Burger PM Mehl E Cameron PL Maycox PR Baumert M Lottspeich F De Camilli P Jahn R Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate Neuron 3 — Campbell DS Holt CE Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation Neuron 32 — Caroni P Donato F Muller D Structural plasticity upon learning: regulation and functions Nature Reviews Neuroscience 13 — Ceccarelli B Hurlbut WP Mauro A Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction The Journal of Cell Biology 57 — Chan CC Scoggin S Wang D Cherry S Dembo T Greenberg B Jin EJ Kuey C Lopez A Mehta SQ Perkins TJ Brankatschk M Rothenfluh A Buszczak M Hiesinger PR Systematic discovery of Rab GTPases with synaptic functions in Drosophila Current Biology 21 — Chavas LM Torii S Kamikubo H Kawasaki M Ihara K Kato R Kataoka M Izumi T Wakatsuki S Structure of the small GTPase Rab27b shows an unexpected swapped dimer Acta Crystallographica Section D, Biological Crystallography 63 — Chou JH Jahn R Binding of Rab3A to synaptic vesicles The Journal of Biological Chemistry — Chua CE Gan BQ Tang BL Involvement of members of the Rab family and related small GTPases in autophagosome formation and maturation Cellular and Molecular Life Sciences 68 — Chua JJ Butkevich E Worseck JM Kittelmann M Grønborg M Behrmann E Stelzl U Pavlos NJ Lalowski MM Eimer S Wanker EE Klopfenstein DR Jahn R Phosphorylation-regulated axonal dependent transport of syntaxin 1 is mediated by a Kinesin-1 adapter Proceedings of the National Academy of Sciences of USA — Chung WS Barres BA The role of glial cells in synapse elimination Current Opinion in Neurobiology 22 — Clarke LE Barres BA Emerging roles of astrocytes in neural circuit development Nature Reviews Neuroscience 14 — Darcy KJ Staras K Collinson LM Goda Y Constitutive sharing of recycling synaptic vesicles between presynaptic boutons Nature Neuroscience 9 — DiAntonio A Haghighi AP Portman SL Lee JD Amaranto AM Goodman CS Ubiquitination-dependent mechanisms regulate synaptic growth and function Nature — Ding M Shen K The role of the ubiquitin proteasome system in synapse remodeling and neurodegenerative diseases Bioessays 30 — Fernández-Alfonso T Kwan R Ryan TA Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling Neuron 51 — Fukuda M Regulation of secretory vesicle traffic by Rab small GTPases Cellular and Molecular Life Sciences 65 — Goody RS Rak A Alexandrov K The structural and mechanistic basis for recycling of Rab proteins between membrane compartments Cellular and Molecular Life Sciences 62 — Grosshans BL Ortiz D Novick P Rabs and their effectors: achieving specificity in membrane traffic Proceedings of the National Academy of Sciences — Hernandez D Torres CA Setlik W Cebrián C Mosharov EV Tang G Cheng HC Kholodilov N Yarygina O Burke RE Gershon M Sulzer D Regulation of presynaptic neurotransmission by macroautophagy Neuron 74 — Heuser JE Reese TS Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction The Journal of Cell Biology 57 — Hirokawa N Niwa S Tanaka Y Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease Neuron 68 — Holt CE Schuman EM The central dogma decentralized: new perspectives on RNA function and local translation in neurons Neuron 80 — Holtzman E Freeman AR Kashner LA Stimulation-dependent alterations in peroxidase uptake at lobster neuromuscular junctions Science — Huttner WB Schiebler W Greengard P De Camilli P Synapsin I protein I , a nerve terminal-specific phosphoprotein.

Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation The Journal of Cell Biology 96 — Ishibashi K Fujita N Kanno E Omori H Yoshimori T Itoh T Fukuda M Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an AtgL2 complex Autophagy 7 — Ishibashi K Uemura T Waguri S Fukuda M Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity Molecular Biology of the Cell 23 — Itoh T Fujita N Kanno E Yamamoto A Yoshimori T Fukuda M Golgi-resident small GTPase Rab33B interacts with Atg16L and modulates autophagosome formation Molecular Biology of the Cell 19 — Jahn R Schiebler W Ouimet C Greengard P A 38,dalton membrane protein p38 present in synaptic vesicles Proceedings of the National Academy of Sciences of USA 82 — Janssen KA Molecular cloning: a laboratory manual.

Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, volume 1—3. Jiang X Litkowski PE Taylor AA Lin Y Snider BJ Moulder KL A role for the ubiquitin-proteasome system in activity-dependent presynaptic silencing The Journal of Neuroscience 30 — Jin RU Mills JC RAB26 coordinates lysosome traffic and mitochondrial localization Journal of Cell Science — Kamin D Lauterbach MA Westphal V Keller J Schönle A Hell SW Rizzoli SO High- and low-mobility stages in the synaptic vesicle cycle Biophysical Journal 99 — Kato A Rouach N Nicoll RA Bredt DS Activity-dependent NMDA receptor degradation mediated by retrotranslocation and ubiquitination Proceedings of the National Academy of Sciences of USA — Katzmann DJ Odorizzi G Emr SD Receptor downregulation and multivesicular-body sorting Nature Reviews Molecular Cell Biology 3 — Kraszewski K Mundigl O Daniell L Verderio C Matteoli M De Camilli P Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin.

Lamb CA Yoshimori T Tooze SA The autophagosome: origins unknown, biogenesis complex Nature Reviews Molecular Cell Biology 14 — Lee S Sato Y Nixon RA Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy The Journal of Neuroscience 31 —

Small GTPases of the GTPsaes family Autophagy and GTPases only regulate target recognition Autophagy and GTPases membrane traffic but also control other cellular GTPasses such as cytoskeletal transport and autophagy. Here Autophagy and GTPases show Cognitive function training programs Rab26 is specifically associated with clusters GPases synaptic vesicles in Autophagg. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity.