InteractiveFly: GeneBrief

Rab7: Biological Overview | References


Gene name - Rab7

Synonyms -

Cytological map position - 95D5-95D5

Function - signaling protein

Keywords - endosomal transport, facilitates endosome maturation and fusion with lysosomes, required for autophagosome-lysosome fusion, neuromuscular junction

Symbol - Rab7

FlyBase ID: FBgn0015795

Genetic map position - chr3R:23,993,179-23,995,252

NCBI classification - Rab GTPase family 7

Cellular location - cytoplasmic



NCBI links: | EntrezGene
Recent literature
Court, H., Ahearn, I. M., Amoyel, M., Bach, E. A. and Philips, M. R. (2017). Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT. J Cell Biol 216(12): 4165-4182. PubMed ID: 29051265
Summary:
Isoprenylcysteine carboxyl methyltransferase (ICMT) methylesterifies C-terminal prenylcysteine residues of CaaX proteins and some RAB GTPases. Deficiency of either ICMT or NOTCH1 accelerates pancreatic neoplasia in Pdx1-Cre;LSL-Kras(G12D) mice, suggesting that ICMT is required for NOTCH signaling. This study used Drosophila melanogaster wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (Ste14 in flies). Rab7 and Rab8 were identified as ICMT substrates that when silenced phenocopy ste14 deficiency. ICMT, RAB7, and RAB8 were all required for efficient NOTCH1 signaling in mammalian cells. Overexpression of RAB8 rescued NOTCH activation after ICMT knockdown both in U2OS cells expressing NOTCH1 and in fly wing vein development. ICMT deficiency induced mislocalization of GFP-RAB7 and GFP-RAB8 from endomembrane to cytosol, enhanced binding to RABGDI, and decreased GTP loading of RAB7 and RAB8. Deficiency of ICMT, RAB7, or RAB8 led to mislocalization and diminished processing of NOTCH1-GFP. Thus, NOTCH signaling requires ICMT in part because it requires methylated RAB7 and RAB8.
BIOLOGICAL OVERVIEW

The small GTPase Rab5 promotes recruitment of the Ccz1-Mon1 guanosine exchange complex to endosomes to activate Rab7, which facilitates endosome maturation and fusion with lysosomes. How these factors function during autophagy is incompletely understood. This study shows that autophagosomes accumulate due to impaired fusion with lysosomes upon loss of the Ccz1-Mon1-Rab7 module in starved Drosophila fat cells. In contrast, autophagosomes generated in Rab5 null mutant cells normally fuse with lysosomes during the starvation response. Consistent with that, Rab5 is dispensable for the Ccz1-Mon1-dependent recruitment of Rab7 to PI3P-positive autophagosomes, which are generated by the action of the Atg14-containing Vps34 PI3 kinase complex. Finally, Rab5 was found to be required for proper lysosomal function. Thus, the Ccz1-Mon1-Rab7 module is required for autophagosome-lysosome fusion, whereas Rab5 loss interferes with a later step of autophagy: the breakdown of autophagic cargo within lysosomes (Hegedus, 2016).

Autophagy ensures the lysosomal degradation of self-material, including cytosol and organelles. During the main pathway, double-membrane autophagosomes serve as the transport vesicles. Endocytosis delivers plasma membrane, including transmembrane receptors, and exogenous substances taken up from the environment to lysosomes. Thus autophagy and endocytosis converge at the level of lysosomes, where degradation of cargo arriving from both routes takes place (Hegedus, 2016).

A critical event during these transport processes is vesicle maturation: how the newly formed vesicles acquire the molecular characteristics and protein complexes that establish their identity and determine the subsequent vesicle fusion events that often culminate in the lysosomal compartment. Several similarities between endosomes and autophagosomes are known. For example, both autophagosomes and endosomes are positive for phosphatidylinositol-3-phosphate (PI3P) due to localized vacuolar protein sorting 34 (Vps34) PI3 kinase activity, which has been showed to be required for the generation of both types of vesicles in Drosophila larvae (Lindmo, 2006; Juhasz et al., 2008; Dooley, 2014). Autophagosomes can also fuse with endosomes to give rise to hybrid organelles termed amphisomes, which then fuse with lysosomes (Hegedus, 2016).

Small GTPases of the Ras-related protein in brain (Rab) family are critical regulators of membrane trafficking in eukaryotic cells. An active, GTP-bound Rab protein binds to various effectors that usually regulate vesicle motility and fusion with the proper membrane compartment. In the endocytic pathway, Rab5 associates with early endosomes and activates a Vps34-containing phosphoinositide 3-kinase complex that generates PI3P on the surface of these vesicles. PI3P-binding domains such as the Fab-1, YGL023, Vps27, and EEA1 (FYVE) domain promote recruitment to early endosomes. Of importance, several proteins, including the vesicle tethers early endosomal antigen 1 (EEA1) and Rabenosin-5, have both FYVE and Rab5-binding domains, indicating that multiple interactions may play a role in the recruitment of effectors (Stenmark, 2009). Similarly, the Rab7 guanine nucleotide exchange factor (GEF) monensin sensitivity protein 1 (Mon1)-caffeine, calcium, and zinc 1 (Ccz1) complex binds to both the GTP-bound form of endosomal Rab5 and PI3P (Poteryaev, 2010; Cabrera, 2014; Cui, 2014). Rab7 is then activated by this complex and promotes fusion of late endosomes and lysosomes (Hegedus, 2016).

Recruitment of the soluble N-methylamaleimide-sensitive factor attachment protein receptor (SNARE) Syntaxin 17 is a critical step in autophagosome maturation because these vesicles acquire fusion competence this way. Interaction of Syntaxin 17 with the homotypic fusion and vacuole protein sorting (HOPS) tethering complex ensures efficient fusion between autophagosomes and lysosomes. HOPS is believed to be a Rab7 effector, and Rab7 was indeed found to promote the formation of degradative autolysosomes in cultured cells, although it remains to be established whether this protein is already present on autophagosomes before the fusion with lysosomes. In theory, the binding of HOPS to lysosomal Rab7 and autophagosomal Syntaxin 17 (and other factors, such as phospholipids) may be sufficient for its tethering activity. In addition, autophagy-related gene 14 (Atg14), a Vps34 kinase complex subunit that is important for autophagosome formation, also functions as a tether and promotes autophagosome-lysosome fusion by directly binding to Syntaxin 17 (Hegedus, 2016 and references therein).

In yeast, the fusion machinery differs somewhat from that of the animal cells because the SNAREs involved are not homologous. Still, autophagosome fusion with the vacuole (the equivalent of the lysosomal system in metazoan cells) requires HOPS, Ypt7/Rab7, and its GEF, the Mon1-Ccz1 complex, and more recently, autophagosome-like structures were found to accumulate in yeast cells lacking the major Rab5 homologue Vps21. Of interest, decreased Rab5 function attenuates the autophagic degradation of the pathogenic, mutant form of huntingtin in cultured human cells. This was attributed to impaired Vps34 lipid kinase activity and reduced formation of the Atg5-Atg12 conjugate, both of which are important for autophagosome formation (Hegedus, 2016).

Thus the role of the Rab5-Ccz1-Mon1-Rab7 axis during autophagy is not clear. This study set out to address this problem in the popular animal model Drosophila. Fruit flies offer certain advantages for such studies. First, there is only a single fly homologue of Rab5 (unlike in mammalian and yeast cells, which both have three different Rab5 proteins). Second, massive induction of autophagy is seen in the fat and liver tissue-like fat cells of starved larvae. Third, it is straightforward to carry out functional studies in mosaic animals, in which mutant cells are surrounded by control cells in the same tissue of the same animal, which reduces variability because one can compare the phenotype of neighboring control and loss-of-function cells. Using this system, Ccz1, Mon1, and Rab7 are shown to be required for autophagosome-lysosome fusion in fat cells of starved animals independent of Rab5. Of interest, Rab5 was found to function downstream of the Rab7 module by controlling a later step of autophagy: degradation of autophagic cargo within lysosomes (Hegedus, 2016).

This study showed that the Rab7 module and Rab5 control different steps of autophagy. Rab7 mediates autophagosome-lysosome fusion together with its GEF, the Ccz1-Mon1 complex. This is likely achieved by the recruitment of Rab7 to autophagosomes in a Ccz1-Mon1-dependent manner. Although Drosophila Mon1 binds to the active, GTP-locked form of Rab5 as in other organisms, Rab5 is dispensable for the fusion of autophagosomes with lysosomes and for Rab7 localization to autophagosomes and autolysosomes. The question is then: what is the signal that recruits Ccz1-Mon1 and Rab7 to autophagic structures? (Hegedus, 2016).

Mon1 and Ccz1 bind to phospholipids, including PI3P, in yeast, and this study found that Drosophila Mon1 has similar features. This raises the possibility that the Ccz1-Mon1 complex is recruited to the PI3P-positive surface of autophagosomes through this interaction. Vps34-dependent PI3P generation is required for autophagosome formation and endosome maturation. Vps34 is activated by Rab5 (Stenmark, 2009). Of interest, the current data suggest that loss of Rab5 inhibits PI3P generation only on endosomes but not on autophagosomes. Loss of UVRAG but not Atg14 inhibits PI3P generation on endosomes, whereas loss of Atg14 leads to complete inhibition of PI3P-positive autophagosome biogenesis. Thus UVRAG is dispensable for Vps34 activity during autophagosome formation, and its loss causes a defect in autolysosomal degradation. Similarly, Rab5 mutant cells showed accumulation of autophagic cargo due to impaired lysosomal degradation (Hegedus, 2016).

Recently the Rab5-related Vps21 small GTPase was suggested to control the fusion of autophagosome with the vacuole (lysosome) in yeast cells. In this study, clusters of autophagic structures were found to accumulate near the vacuole. However, these vesicles were positive for both the autophagy marker GFP-Atg8 and the vacuolar marker FM4-64, suggesting that some sort of fusion must have occurred in this case, too (Hegedus, 2016).

On the basis of the current results, the following model is proposed of autolysosome formation in fat cells of starved Drosophila larvae (see The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy). PI3P-positive autophagosomes are generated through the action of an Atg14-containing Vps34 PI3 kinase complex. PI3P attracts Ccz1-Mon1, which promotes Rab7 recruitment to autophagosomes. Both PI3P and Rab7 bind to the HOPS tethering complex, and thus these factors promote the tethering of autophagosomes with late endosomes and lysosomes. The membrane fusion is then executed by the Syx17-Snap29-Vamp7 SNARE complex. Autophagic cargo is broken down in autolysosomes, and their full degradative capacity requires the function of Rab5 and the UVRAG-containing Vps34 complex for the proper delivery of lysosomal proteins, likely including both acidic hydrolases and membrane proteins. This is in line with the finding that simultaneous knockdown of all three Rab5 homologues leads to a collapse of the endolysosomal system in mouse liver cells (Hegedus, 2016).

It has already been demonstrated that autophagosome-lysosome fusion is mediated by the HOPS tethering complex and the SNAREs Syx17, Snap29, and Vamp7/8. It is not yet clear how these fusion factors are recruited to the autophagosomal membrane. HOPS is known as a Rab7 effector (Stenmark, 2009), and according to the current findings, Rab7 is present on autophagosomes. It is proposed that autophagosomal PI3P recruits the Ccz1-Mon1-Rab7 module to facilitate the loading of HOPS and subsequent tethering of vesicles (Hegedus, 2016).

Vps34 is considered as a bona fide Rab5 effector (Stenmark, 2009). Surprisingly, this study found that whereas Rab5 mediates only the generation of PI3P on endosomes mainly through the action of a UVRAG-containing Vps34 complex, it is dispensable for PI3P-positive autophagosome biogenesis, which depends on the Atg14-containing Vps34 complex. Thus the current concept that Vps34 is a Rab5 effector must be revisited: it is true for endocytosis but not applicable for autophagy in fat cells of starved Drosophila larvae (Hegedus, 2016).

A previous study showed that Rab5 promotes autophagy-mediated huntingtin clearance in cultured human cells and Drosophila eyes. Simultaneous small interfering RNA knockdown of all three Rab5 genes (Rab5a, Rab5b, Rab5c) reduced the level of Atg5-Atg12 conjugate and autophagosome formation. Although no perturbations of autophagosome biogenesis and fusion were seen in Rab5 mutant fat cells, these discrepancies may be due to the different models used. In the current experiments, starvation induces a massive wave of autophagy in larval Drosophila fat cells that entirely relies on the activity of the Rab5-independent Atg14-Vps34 PI3 kinase complex. It is possible that during the basal, nonstarved conditions in a previous study, Rab5 can contribute to autophagosome formation. In fact, UVRAG has also been suggested to control autophagosome formation in cultured cells, which is compatible with this model (Hegedus, 2016).

In summary, Rab7 is recruited to autophagosomes by the Ccz1-Mon1 complex to promote autophagosome-lysosome fusion. This study show that autophagosome formation and fusion is independent of Rab5 and the UVRAG-containing Vps34 PI3 kinase complex but requires the action of the Atg14-Vps34 complex. Rab5, similar to UVRAG, is necessary for proper lysosomal function by promoting the trafficking of lysosomal proteins (Hegedus, 2016).

Rab2 promotes autophagic and endocytic lysosomal degradation

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. This study shows that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. This study demonstrates that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. RAB2A is also required for autophagosome clearance in human breast cancer cells. In conclusion, Rab2 has been identified as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes (Lorincz, 2017).

The two main pathways of lysosomal degradation are endocytosis and autophagy. Double-membrane autophagosomes (generated in the main pathway of autophagy) and endosomes can fuse with each other to generate amphisomes, and mature into degradative endo- and autolysosomes, respectively, by ultimately fusing with lysosomes. One of the main regulators of intracellular trafficking and vesicle fusions are Rab small GTPases. Active, GTP-bound Rab proteins recruit various effectors including tethers and molecular motors, of which Rab7 is the only known direct regulator of both autophagosome-lysosome and endosome-lysosome fusions (Lorincz, 2017).

The tethering complex homotypic fusion and vacuole protein sorting (HOPS) was identified in yeast, and it simultaneously binds two yeast Rab7 (Ypt7) molecules on its opposing ends. In animal cells, Rab7 binds to RILP, ORPL1, FYCO1, and PLEKHM1 to recruit dyneins and HOPS and ensure the fusion of late endosomes and autophagosomes with lysosomes. This way, HOPS could cross-link two Rab7-positive membranes to prompt tethering and fusio. Rab7 is present on lysosomes, autophagosomes, and endosomes, but it is not clear whether another Rab is involved in degradative auto- and endolysosome formation, which also requires transport of hydrolases from the Golgi (Lorincz, 2017).

Rab2 is known to control anterograde and retrograde traffic between the ER and Golgi. A recent biochemical screen identified Rab2 as a direct binding partner of HOPS, and active Rab2 was found to localize to Rab7-positive vacuoles in cultured Drosophila melanogaster cells. This study proposes an updated model in which Rab7 and Rab2 coordinately promote the HOPS-dependent degradation of autophagosomes and endosomes via fusion of these as well as biosynthetic vesicles with lysosomes (Lorincz, 2017).

Rab2 is highly conserved among higher eukaryotes, including Drosophila melanogaster and humans. The HOPS subunits Vps39 and Vps41 directly bind to Ypt7/Rab7 in yeast, whereas their interaction may be indirect in mammalian cells. No binding was detected between Drosophila Rab7 and Vps39 or Vps41, whereas GTP-locked Rab7 bound to its known effector PLEKHM1 in yeast two-hybrid (Y2H) experiments. Vps39 directly bound Rab2GTP in both Y2H and recombinant protein pull-down experiments, and Rab2GTP immunoprecipitated endogenous Vps16A (another HOPS subunit) from fly lysates. Consistently, it has been reported that recombinant mammalian RAB2A pulls down Vps39 but not Vps41 from cell lysates, and human HOPS subunits did not show Rab7 binding in Y2H experiments (Lorincz, 2017).

To address whether Rab2 functions in autophagy and endocytosis, rab2 was knocked out by imprecise excision of a transposon from the 5' UTR. The resulting rab2d42 allele carries a 2,047-bp deletion, which removes most of the protein coding sequences of both predicted Rab2 isoforms and eliminates protein expression. Rab2 mutant animals die as L2/L3-stage larvae, and their viability is fully rescued by expression of YFP-Rab2 (Lorincz, 2017).

Larval fat cells are widely used for autophagy analyses because of their massive autophagic potential. Numerous Lysotracker Red (LTR)-positive vesicles appear upon starvation, which represent newly formed autolysosomes with likely increased v-ATPase-mediated acidification in these cells. LTR dot number and size (and signal intensity as a likely consequence) decreased in rab2-null cells compared with controls, which was rescued by expression of YFP-Rab2. RNAi knockdown of Rab2 in GFP-marked fat cell clones also impaired starvation-induced punctate LTR staining compared with surrounding GFP-negative cells (Lorincz, 2017).

A 3xmCherry-Atg8a reporter that labels all autophagic structures via retained fluorescence of mCherry inside autolysosomes revealed increased number and decreased size of such vesicles in both starved rab2 RNAi and mutant fat cells. A dLamp-3xmCherry reporter of late endosomes and lysosomes showed similar changes in rab2 RNAi or mutant fat cells of starved animals. Tandem tagged mCherry-GFP-Atg8a reporters are commonly used to follow autophagic flux, because GFP is quenched in lysosomes, whereas mCherry signal persists. Knockdown of rab2 prevented the quenching of GFP that is seen in starved control fat cells: dots positive for both GFP and mCherry accumulated, raising the possibility that Rab2 promotes autophagosome-lysosome fusion, similar to HOPS. Colocalization of 3xmCherry-Atg8a with the lysosomal hydrolase cathepsin L (CathL) was examined. The overlap of these markers of autophagic and lysosomal structures strongly decreased in rab2 mutant fat cells compared with controls, and rab2 RNAi also impaired endogenous CathL-positive vesicle formation, suggesting that formation of degradative autolysosomes requires Rab2 (Lorincz, 2017).

These phenotypes resembled the autophagosome-lysosome fusion defect of mutants for the autophagosomal SNARE syntaxin 17, HOPS, and Rab7. Accordingly, ultrastructural analysis of starved fat cells revealed accumulation of double-membrane autophagosomes and small dense structures likely representing amphisomes, similar to HOPS mutants. Recently, rab2 RNAi was reported to cause accumulation of autophagosomes in Drosophila muscles and enlarged amphisomes in fat cells. Autophagosome accumulation in our rab2-null mutant fat cells is likely caused by a complete loss-of-function condition (Lorincz, 2017).

Western blots detected increased levels of the selective autophagy cargo p62/Ref2p, along with both free and lipidated autophagosome-associated forms of Atg8a in starved rab2 mutants. Basal autophagic degradation was also impaired in rab2 mutants, based on increased numbers of endogenous Atg8a and p62 dots in well-fed conditions (Lorincz, 2017).

The importance of Rab2 for autophagic degradation was confirmed in human cells. Knockdown of RAB2B had no effect on endogenous LC3 structures in breast cancer cells, whereas RAB2A or combined siRNA treatment caused accumulation of autophagic vesicles. LC3 accumulated within Lamp1-positive structures upon RAB2A knockdown, which likely represent amphisomes unable to mature into autolysosomes in these cells, consistent with the recently reported role of Rab2 homologs for degradation of autophagic cargo in mouse embryonic fibroblasts (Lorincz, 2017 and references therein).

To analyze the possible involvement of Drosophila Rab2 in endosomal degradation, dissected nephrocytes were incubated with fluorescent avidin for 5 min. Trafficking of this endocytic tracer was clearly perturbed in rab2 mutant cells, similar to vps41/lt and rab7 mutants. Loss of HOPS leads to enlargement of late endosomes. Similarly, Rab7 endosomes are enlarged in rab2 mutant nephrocytes compared with control or rescued cells. Importantly, fluorescent avidin was trapped in Rab7 endosomes and failed to reach CathL-positive lysosomes after a 30-min chase in rab2 mutants. LTR staining showed the presence of acidic vacuoles in rab2 mutant nephrocytes, which probably include the enlarged late endosomes in rab2 mutant nephrocytes, based on ultrastructural analysis . Aberrant late endosomes accumulated in mutant cells, which were apparently unable to fuse with neighboring acid phosphatase-positive lysosomes. Of note, the number of acid phosphatase-positive lysosomes also decreased in mutant nephrocytes, suggesting that Rab2 promotes both endosome-lysosome fusion and biosynthetic transport to lysosomes (Lorincz, 2017).

GTP-locked, constitutively active Rab2GTP redistributes from the Golgi onto Rab7 vacuoles in cultured Drosophila cells. Similarly, Rab2GTP colocalized with endogenous Rab7 in starved fat cells, unlike wild-type Rab2. Rab2GTP appeared as large pronounced rings around LTR-positive autolysosomes in starved fat cells, unlike wild-type Rab2. Similarly, Rab2GTP formed rings around lysosomes and autophagic structures marked by dLamp-3xmCherry and 3xmCherry-Atg8a, respectively. Of note, small Rab2GTP dots often closely associated with large Rab2GTP rings in these experiments, raising the possibility that Rab2 vesicles fuse with autolysosomes. Finally, wild-type Rab2 or Rab2GTP modestly overlapped with autophagosomes marked by endogenous Atg8a (Lorincz, 2017).

These localization and loss-of-function data pointed to Rab2 as a positive regulator of autolysosome formation. Indeed, fat and midgut cells expressing Rab2GTP contained enlarged and brighter 3xmCherry-Atg8a autophagic structures and dLamp-3xmCherry lysosomes compared with surrounding control cells, suggesting that Rab2 controls autolysosome size. Increased lysosomal input or a block of degradation can cause enlargement of autolysosomes. Systemic expression of Rab2GTP did not impair the viability of animals, and Western blots of starved L3 larval lysates revealed no changes in p62 and Atg8a levels, suggesting that autophagic degradation proceeds normally in cells expressing Rab2GTP. Thus, Rab2GTP may increase autolysosome size by accelerating fusions with other vesicles. Importantly, expression of GTP-locked, active Rab7 did not increase the size of autophagic structures. Rab7 is required for autophagosome-lysosome fusion, and its knockdown prevents the formation of large, bright 3xmCherry-Atg8a-positive autolysosomes: these cells contain only small, faint autophagosomes. Similarly, only small, faint 3xmCherry-Atg8a dots appeared in Rab2GTP-expressing fat cells undergoing Rab7 RNAi, indicating that Rab2-dependent fusions also require Rab7 and there is no functional redundancy between them (Lorincz, 2017).

Eye pigment granules are lysosome-related organelles. Changes in lysosomal transport often lead to eye discoloration caused by pigment granule alterations, such as in HOPS mutants. Rab2GTP expression led to a slight darkening of eyes and appearance of enlarged pigment granules, consistent with the role of Rab2 in promoting lysosomal fusions (Lorincz, 2017).

Several homo- and hetero-typic fusions occur during endosome and autophagosome maturation into degradative lysosomes. Known metazoan factors acting at lysosomal fusions include HOPS and EPG5 tethers and Rab7 together with its effectors. Because biosynthetic transport to lysosomes also requires input from Golgi, the role of Golgi-associated Rab2 in various lysosomal fusions fits well into this picture. Consistently, Rab2 promotes breakdown of phagocytosed apoptotic bodies and lysosome-related acrosome biogenesis (Lorincz, 2017).

Accumulation of unfused autophagosomes and enlarged late endosomes in rab2 mutants resembles the fusion defect of rab7 mutant cells. The decreased function of lysosomes in rab2 mutants is unlikely to account for these fusion defects, because we have shown that autophagosome-lysosome fusion proceeds and gives rise to enlarged, nondegrading autolysosomes in fat cells with perturbed acidification or biosynthetic transport to lysosomes (Lorincz, 2017).

The role of Rab2 in the fusion of lysosomes with other vesicles is also supported by the autolysosomal localization of its active form and by its binding to the Vps39-containing end of HOPS, the tethering complex required for autophagosomal, endosomal, and biosynthetic transport to lysosomes. Consistently, Rab2 recruits HOPS to Rab7-positive vesicles in cultured Drosophila cells. Expression of Rab2GTP increases degradative autolysosome and pigment granule size, suggesting that it is rate limiting during these fusion reactions, unlike Rab7. This is supported by low levels of wild-type Rab2 on these organelles, unlike wild-type Rab7 that is abundant on autophagosomes, late endosomes, and lysosomes. Consistent with this, it has been recently shown that expression of RAB2AGTP also increases Rab7 vesicle size in human cells. Based on binding of Rab2 to one end of HOPS, an updated model is proposed of lysosomal fusions in animal cells. It is hypothesized that GTP-loaded Rab2 is transported on Golgi-derived carrier vesicles toward Rab7 positive vesicles, and its interaction with Vps39 promotes fusions. Vps41 located on the other end of HOPS may bind Rab7 vesicles via adaptors such as PLEKHM1. These interactions help the tethering and fusion of autophagic, endocytic, and lysosomal vesicles to generate degrading compartments. Lysosomal membranes may contain active Rab2 for only a short period of time, and it likely dissociates upon GTP hydrolysis to limit organelle size. Rab asymmetry is also observed during homotypic vacuole fusion in yeast: GTP-bound Ypt7/Rab7 is necessary on only one of the vesicles, and its nucleotide status is irrelevant on the opposing membrane. Importantly, Rab7 directly interacts with both ends of HOPS in the absence of a Rab2 homolog in yeast. This difference may explain why yeast cells contain one large vacuole instead of the many smaller lysosomes seen in animal cells. Collectively, these data indicate that Rab2 and Rab7 coordinately promote autophagic and endosomal degradation and lysosome function (Lorincz, 2017).

A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. The deubiquitinating enzyme UBPY/USP8 has been identified as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, it was hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. This study blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. Inhibition of endosomal trafficking at different steps in vivo was observed to be systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. These results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo (Jacomin, 2016).

A genome-wide RNAi screen for microtubule bundle formation and lysosome motility regulation in Drosophila S2 Cells

Long-distance intracellular transport of organelles, mRNA, and proteins ('cargo') occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. This study capitalized on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. This study used an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. Innate immunity genes, ion channels, and signaling proteins were identified having a role in lysosome motility regulation; an unexpected relationship was found between the dynein motor, Rab7a, and lysosome motility regulation (Jolly, 2016).

A pre-synaptic regulatory system acts trans-synaptically via Mon1 to regulate Glutamate receptor levels in Drosophila

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. This study has identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. Mutants were generated in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels suggesting its involvement in a pre-synaptic mechanism that regulates post-synaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants suggesting that homeostatic mechanisms are not affected in these mutants. It is proposed that DMon1 is part of a pre-synaptically regulated trans-synaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction (Deivasigamani, 2015).

Neurotransmitter release at the synapse is modulated by factors that control synaptic growth, synaptic vesicle recycling, and receptor turnover at postsynaptic sites. Endolysosomal trafficking modulates the function of these factors and therefore plays an important role in regulating synaptic development and function. Intracellular trafficking is regulated by Rabs, which are small GTPases. These proteins control specific steps in the trafficking process. A clear understanding of the role of Rabs at the synapse is still nascent. Drosophila has 31 Rabs, and most of these are expressed in the nervous system. Rab5 and Rab7, present on early and late endosomes, respectively, are critical regulators of endolysosomal trafficking and loss of this regulation affects neuronal viability underscored by the fact that mutations in Rab7 are associated with neurodegeneration (Verhoeven, 2003). Rab5 along with Rab3 is present on synaptic vesicles, and both play a role in regulating neurotransmitter release. In Drosophila, Rab3 is involved in the assembly of active zones by controlling the level of both Bruchpilot-a core active zone protein-and the calcium channels surrounding the active zone (Graf, 2009). In hippocampal and cortex neurons, Rab5 facilitates LTD through removal of AMPA receptors from the synapse. In Drosophila, Rab5 regulates neurotransmission; it also functions to maintain synaptic vesicle size by preventing homotypic fusion. Compared to Rab5 or Rab3, less is known about the roles of Rab7 at the synapse. In spinal cord motor neurons, Rab7 mediates sorting and retrograde transport of neurotrophin-carrying vesicles. In Drosophila, tbc1D17-a known GAP for Rab7-affects GluRIIA levels (J. Lee, 2013); the effect of this on neurotransmission has not been evaluated. Excessive trafficking via the endolysosomal pathway also affects neurotransmission. This has been observed in mutants for tbc1D24-a GAP for Rab35. A high rate of turnover of synaptic vesicle proteins in these mutants is seen to increase neurotransmitter release (Uytterhoeven, 2011; Fernandes, 2014; Deivasigamani, 2015 and references therein).

This study has examined the synaptic role of DMon1-a key regulator of endosomal maturation. Multiple synaptic phenotypes are found associated with Dmon1 loss of function, and one of these is altered synaptic morphology. Boutons in Dmon1 mutants are larger with more satellite or supernumerary boutons-a phenotype strongly associated with endocytic mutants (Dickman, 2006). Formation of satellite boutons is thought to occur due to loss of bouton maturation, with the initial step of bouton budding being controlled postsynaptically and the maturation step being regulated presynaptically (J. Lee, 2010). Supporting this, a recent study shows that miniature neurotransmission is required for bouton maturation. The presence of excess satellite boutons in Dmon1 mutants suggests that the number of 'miniature' events is likely to be affected in these mutants. The fact that this phenotype can be rescued upon expression of vGlut supports this possibility. However, this does not fit with the observed decrease in size and intensity of Brp positive puncta in these mutants. Active zones with low or nonfunctional Brp are known to be more strongly associated with increased spontaneous neurotransmission. Considering the involvement of postsynaptic signaling in initiating satellite bouton formation, it is thought that altered neurotransmission possibly together with impaired postsynaptic or retrograde signaling, contributes to the altered synaptic morphology in Dmon1 mutants. This may also explain why no satellite boutons are observed in neuronal RNAi animals (Deivasigamani, 2015).

A striking phenotype associated with loss of Dmon1 is the increase in GluRIIA levels. This phenotype seems presynaptic in origin since neuronal loss of Dmon1 is sufficient to increase GluRIIA levels. Is the increase in GluRIIA due to trafficking defects in the neuron? This seems unlikely for the following reasons: First, it has been shown that although neuronal overexpression of wild-type and dominant negative Rab5 alters evoked response in a reciprocal manner, there is no change in synaptic morphology, glutamate receptor localization and density, or change in synaptic vesicle size. The role of Rab7 at the synapse is less clear. In a recent study, loss of tbc1D15-17, which functions as a GAP for Rab7, was shown to increase GluRIIA levels at the synapse. Selective knockdown of the gene in muscles, and not neurons, was seen to increase GluRIIA levels, indicating that the function of the gene is primarily postsynaptic (J. Lee, 2013). These data are not consistent with the current results from neuronal knockdown of Dmon1, suggesting that the presynaptic role of Dmon1 in regulating GluRIIA levels is likely to be independent of Rab5 and Rab7 and therefore novel (Deivasigamani, 2015).

The current experiments to evaluate the postsynaptic role of Dmon1 have been less clear. Although a modest increase in GluRIIA levels are seen upon knockdown in muscles, the increase is not always significant when compared to controls. However, the fact that muscle expression of Dmon1 can rescue the GluRIIA phenotype in the mutant suggests that it is likely to be one of the players in regulating GluRIIA postsynaptically. Further, it is to be noted, that while overexpression of vGlut leads to down-regulation of the receptor at the synapse, the receptors do not seem to get trapped in the muscle, suggesting that multiple pathways are likely to be involved in regulating receptor turnover in the muscle, and the DMon1-Rab7-mediated pathway may be just one of them (Deivasigamani, 2015).

How might neuronal Dmon1 regulate receptor expression? One possibility is that the increase in receptor levels is a postsynaptic homeostatic response to defects in neurotransmission, given that Dmon1Δ181 mutants have smaller synaptic vesicles. However, in dvglut mutants, presence of smaller synaptic vesicles does not lead to any change in GluRIIA levels, given that receptors at the synapse are generally expressed at saturating levels. Therefore, it seems unlikely that the increase in GluRIIA is part of a homeostatic response, although one cannot rule this out completely. The other possibility is that DMon1 is part of a transsynaptic signaling mechanism that regulates GluRIIA levels in a post-transcriptional manner. The observation that presynaptically expressed DMon1 localizes to postsynaptic regions and the results from neuronal RNAi and rescue experiments support this possibility. The involvement of transsynaptic signaling in regulating synaptic growth and function has been demonstrated in the case of signaling molecules such as Ephrins, Wingless, and Syt4. In Drosophila, both Wingless and Syt4 are released by the presynaptic terminal via exosomes to mediate their effects in the postsynaptic compartment. It was hypothesized that DMon1 released from the boutons either directly regulates GluRIIA levels or facilitates the release of an unknown factor required to maintain receptor levels. The function of DMon1 in the muscle is likely to be more consistent with its role in cellular trafficking and may mediate one of the pathways regulating GluRIIA turnover. These possibilities will need to be tested to gain a mechanistic understanding of receptor regulation by Dmon1 (Deivasigamani, 2015).

Dmon1 controls recruitment of Rab7 to maturing endosomes in Drosophila

The small GTPases Rab5 and Rab7 are important organisers of endosome formation and maturation. In addition, they orchestrate the trafficking of cargo through the endosomal pathway. A crucial event during maturation of endosomes is the replacement of the early organiser Rab5 with the late organiser Rab7 in a process called Rab conversion. Rab conversion is a prerequisite for late events, chief among them the fusion of matured endosomes with the lysosome. Recent work identifies members of the Sand1/Mon1 protein family as crucial factors during this process. This study presents an analysis of the function of the Drosophila ortholog of mon1/sand1, Dmon1. Loss of function of Dmon1 results in an enlargement of maturing endosomes and loss of their association with Rab7. The enlarged endosomes contain Notch and other trans-membrane proteins as cargo. This study reports an electron microscopy analysis of Dmon1 cells and extends the analysis of the endosomes in mutant cells. The results suggest that the phenotype can be explained by the loss of function of Rab7. Moreover, the endosomes of Dmon1 cells mature normally in many aspects, despite the loss of association with Rab7. Surprisingly, overactive or ectopic signalling through receptors such as Notch and RTKs was not observed in Dmon1 mutant cells, as would have been expected because of the accumulation of receptors in the maturing endosomes of these cells. This was the case even when receptor uptake into intraluminal vesicles was suppressed (Yousefian, 2013).

Tbc1d15-17 regulates synaptic development at the Drosophila neuromuscular junction

Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. This paper provides genetic evidence that Tbc1d15-17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15-17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15-17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15-17. Presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15-17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, these findings establish a novel role for Tbc1d15-17 and its potential substrate Rab7 in regulating synaptic development (Lee, 2013).

Eiger-induced cell death relies on Rac1-dependent endocytosis

Signaling via tumor necrosis factor receptor (TNFR) superfamily members regulates cellular life and death decisions. A subset of mammalian TNFR proteins, most notably the p75 neurotrophin receptor (p75NTR), induces cell death through a pathway that requires activation of c-Jun N-terminal kinases (JNKs). However the receptor-proximal signaling events that mediate this remain unclear. Drosophila express a single tumor necrosis factor (TNF) ligand termed Eiger (Egr) that activates JNK-dependent cell death. This model was exploited to identify phylogenetically conserved signaling events that allow Egr to induce JNK activation and cell death in vivo. This study reports that Rac1, a small GTPase, is specifically required in Egr-mediated cell death. rac1 loss of function blocks Egr-induced cell death, whereas Rac1 overexpression enhances Egr-induced killing. Vav was identified as a GEF for Rac1 in this pathway, and dLRRK functions were identified as a negative regulator of Rac1 that normally acts to constrain Egr-induced death. Thus dLRRK loss of function increases Egr-induced cell death in the fly. Rac1-dependent entry of Egr into early endosomes was shown to be a crucial prerequisite for JNK activation and for cell death and show that this entry requires the activity of Rab21 and Rab7. These findings reveal novel regulatory mechanisms that allow Rac1 to contribute to Egr-induced JNK activation and cell death (Ruan, 2016).

Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning

LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. This study shows that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2G2019S allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD (Dodson, 2016).

This study used Drosophila as an in vivo system to dissect the cell biological functions of lrrk by exploring the previously uncharacterized role of lrrk in oogenesis. The studies point to a crucial role for lrrk in regulating rab7 -dependent lysosomal positioning, and identify alterations in rab7 -dependent lysosomal positioning and lysosome function as potential pathogenic mechanisms in LRRK2-mediated PD. Interestingly, lysosome dysfunction has been demonstrated in PD patient brains, and two strong genetic risk factors for PD, mutations in β-glucocerebrosidase and ATP13A2, are associated with lysosomal dysfunction, suggesting an important role for lysosome dysfunction in the pathogenesis of PD (Dodson, 2016).

Mammalian LRRK2 has previously been found to localize to Rab5-positive early endosomes and to physically interact with Rab5, with knockdown of LRRK2 causing impairments in Rab5-dependent synaptic vesicle endocytosis. However, an interaction between LRRK2 and Rab7 had not been previously reported. This study likewise detected a physical interaction between Drosophila Lrrk and Rab5, however localization of Lrrk to early endosomes was not see, nor was evidence seen of early endosomal defects with genetic manipulation of lrrk. Rather, it was found that Lrrk binds to Rab7, and localizes predominately to Rab7-positive late endosomes and lysosomes. Furthermore, alterations were seen in the morphology and distribution of late endosomal and lysosomal compartments associated with either loss of lrrk function or with expression of lrrkGS (the G2019S mutation is the most common pathogenic LRRK2 mutation). While it cannot be rule out Lrrk also has early endosomal functions, the data clearly point to Rab7-positive late endosomes as a major site of Lrrk function in Drosophila. It is important to point out that lysosome dysfunction has been reported with genetic manipulation of LRRK2 in mammalian systems, as overexpression of mutant forms of LRRK2 in cultured neurons results in neurite morphology defects associated with the formation of lysosome inclusions, and LRRK2 knockout mice accumulate lipofuscin in the kidney, suggestive of lysosome dysfunction. Thus, the role of lrrk in regulating lysosomal processes is likely conserved in mammals. Further work is required to clarify whether LRRK2 has distinct functions at early endosomes and lysosomes (Dodson, 2016).

What is the relationship between Lrrk and Rab7? lrrk loss-of-function enhances while lrrk overexpression suppresses the increased perinuclear positioning of lysosomes due to rab7 CA expression, consistent with the role for lrrk as a negative regulator of rab7. However, no significant effect is seen on lysosome position with either lrrk loss- or gain-of-function alone. Thus, uncovering the role of lrrk as a negative regulator of rab7 activity in this assay requires a sensitized genetic background in which rab7 activity is augmented. This suggests that the significance of lrrk's role as a negative regulator of rab7 may vary with rab7 activity, such as during periods of high flux through late endosomes. Interestingly, it was found that wild-type Lrrk preferentially binds to dominant-negative Rab7. While more work is required to dissect the molecular basis of the Lrrk/Rab7 interaction, it is tempting to speculate based on these data that Lrrk might exert its negative regulation of Rab7 by preferentially stabilizing its inactive GDP-bound form (Dodson, 2016).

Activated Rab7 induces perinuclear lysosome clustering by recruiting its binding partner Rab7-interacting lysosomal protein (RILP) to the vesicle membrane, which in turn recruits the dynein motor via an interaction between RILP and the dynactin/p150glued subunit, with the help of an additional Rab7-binding partner oxysterol binding protein-related protein 1. The net result is increased localization of the dynein motor to lysosome membranes, and therefore increased transport of lysosomes toward microtubule minus ends in the perinuclear region. This study has also shown that the perinuclear clustering of lysosomes mediated by LrrkGS also requires dynein- and microtubule-based transport, suggesting a similar mechanism. Moreover, LrrkGS-induced lysosome clustering is inhibited by expression of dominant-negative Rab7, suggesting that LrrkGS may in fact induce lysosome clustering by acting through Rab7 (Dodson, 2016).

In addition to its effects on lysosome positioning, Rab7 plays a crucial role in regulating the maturation of late endosomes to lysosomes. This point is underscored by the fact that the dispersed lysosomes in the context of dominant-negative Rab7 expression are inaccessible to endocytosed substrates. Among other Rab7 functions that are crucial for late endosome to lysosome maturation, Rab7 mediates membrane tethering and fusion events between late endosomes and lysosomes via its interaction with the homotypic fusion and vacuole protein sorting complex. The current experiments suggest that in addition to promoting microtubule-based perinuclear transport, LrrkGS also promotes lysosome membrane tethering, as lysosomes remain clustered in the context of LrrkGS expression even when microtubules are destabilized. These data suggest that LrrkGS may also promote other Rab7 functions in addition to perinuclear positioning. Whether endogenous Lrrk likewise acts as a general regulator of Rab7 activity, or rather plays a specific role in regulating Rab7-dependent lysosome positioning, remains to be seen. However, it is interesting to note that lrrk null mutants accumulate enlarged Rab7-positive late endosomes that aberrantly accumulate an endocytic tracer, suggesting that lrrk loss-of-function may also disrupt aspects of Rab7-dependent late endosome to lysosome maturation (Dodson, 2016).

lrrk and its mouse homolog play important roles in neuronal process morphology, and the Caenorhabditis elegans lrrk mutant causes defects in axonal-dendritic polarity. In Drosophila, lrrk mutants show defects in the NMJ, which are due in part to defects in microtubule dynamics (S. Lee, 2010). Moreover, expression of human LRRK2GS in Drosophila dopaminergic neurons causes dendrite degeneration associated with fragmentation of the microtubule network and mislocalization of the microtubule-associated protein Tau. LRRK2 has been shown to bind to tubulin in vitro, and the current data demonstrate that microtubules are required for the effects of lrrkGS on lysosome positioning. Taken together, these findings raise the intriguing possibility that lrrk/LRRK2 might regulate neurite morphology and/or polarity through effects on microtubule-based transport of lysosomes and/or other vesicular compartments in the endolysosomal pathway. Interestingly, Rab7 has well-characterized roles in vesicle trafficking in neurons and the regulation of neuronal process morphology. Knockdown of rab7 in mouse cortical neurons impairs neuronal migration and neurite morphology, and rab7-dependent vesicle trafficking has been shown to be required for the intracellular transport of neuritogenic growth factors and their receptors. Interestingly, dominant mutations in rab7 cause an inherited form of neuropathy, and expression of these dominant mutant forms of rab7 in cultured mammalian neurons impair neurite outgrowth. Thus, it is hypothesized that LRRK2 and rab7 may cooperate in the maintenance of neuritic processes by linking effects on microtubule dynamics to the trafficking of endolysosomal structures (Dodson, 2016).

A key goal in the search for novel therapies for PD is the development of compounds that target mutant LRRK2 alleles. Such an approach requires an understanding of the mechanisms by which the mutant protein exerts toxicity. A point mutation can reduce or abolish the endogenous functions of the protein (hypomorph/amporh), inhibit the endogenous functions of the remaining wild-type copy (dominant-negative) or exert gain-of-function effects that reflect either an increase in the normal functions of the protein (hypermorph) or novel functions not shared with the wild-type protein (neomorph). lrrkGS retains at least some endogenous functions of wild-type lrrk, as evidenced by the ability of lrrkGS to restore female fertility in lrrk null mutants. However, the GS mutation appears to cause at least partial loss of lrrk functions since lrrkGS, unlike lrrkWT, is unable to antagonize the activity of rab7 in regulating lysosome positioning. In fact, lrrkGS causes a phenotype consistent with rab7 activation, thus behaving precisely opposite to lrrkWT with respect to the genetic relationship with rab7. However, this is not a dominant-negative effect of lrrkGS, as lrrk loss-of-function on its own has no effect on lysosome distribution. Thus, it is concluded that the GS mutation causes both partial loss-of-function and neomorphic effects with respect to rab7. Interestingly, this altered functional relationship is accompanied by altered binding to Rab7, in which LrrkWT, but not LrrkGS, binds preferentially to the inactive form of Rab7. These data suggest the possibility that this differential binding may at least partially explain the different genetic relationships that LrrkWT and LrrkGS have with Rab7 (i.e. LrrkWT negatively regulates Rab7, while LrrkGS promotes Rab7-dependent functions). One intriguing hypothesis that this result suggests is that LrrkWT may preferentially stabilize the inactive form of Rab7 by direct binding, but that LrrkGS may lack this ability. These results have important implications for the development of PD therapies targeting this allele (Dodson, 2016).

Components of the engulfment machinery have distinct roles in corpse processing

This study used the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. Engulfed material was shown to be processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, the roles of Draper, integrins, and downstream signaling components in corpse processing were also investigated. It was found that some proteins are required for internalization only, while others have defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. By performing double mutant analysis, it was found that combined loss of draper and αPS3 still results in a small number of engulfed vesicles. Next, another known engulfment receptor, Crq, was investigated. It was found that loss of all three receptors does not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells (Meehan, 2016).

Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling

During illumination, the light sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. This study found that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition (Thakur, 2016).

Rab7 induces clearance of α-synuclein aggregates

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, it was hypothesized that overexpressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis this study expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila. In HEK293 cells, EGFP-Rab7 decorated vesicles contain α-synuclein. Rab7 overexpression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 overexpression, presumably representing autolysosomes. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. Rab7 might be involved in the biogenesis of protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease (Dinter, 2016).

FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, Drosophila models were generated of FIG4-related diseases. Fig4 null mutant flies are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS (Bharadwaj, 2015).

Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo

Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. This study analyzed the in vivo movement of 16 Rab GTPases within Drosophila larval axons and showed that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, these findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease (White, 2015).

Stem cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

UVRAG is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. This study shows that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. These data raise the possibility that endocytic but not autophagic defects contribute to UVRAG deficient colorectal cancer development in human patients (Deivasigamani, 2015).

Toward a comprehensive map of the effectors of Rab GTPases

The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, a comprehensive set of Drosophila Rabs was used for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. Over 25 interactions were confirmed for Rab2, Rab4, Rab5, Rab6, Rab7, Rab9, Rab18, Rab19, Rab30, and Rab39. These include tethering complexes, coiled-coiled proteins, motor linkers, Rab regulators, and several proteins linked to human disease (Gillingham, 2014).

Rab7 is conserved in most eukaryotes and is localized to late endosomes. A duplication event during the emergence of metazoans created Rab9, which is also on endosomes. Previously reported effectors of Rab7 include the dynein adaptor RILP and the two related proteins PLEKHM1 and Rubicon. Of these, Drosophila PLEKHM1 (CG6613) was found in both data sets, and Rubicon (CG12772) was found in one, suggesting that the GST-Rab7 was functional, and it was confirmed that the interaction with CG6613 was GTP specific. There were not many other high-scoring hits shared by both data sets, although one was CG12132, a HEAT repeat protein with several mammalian paralogs of unknown function. A GFP-tagged form of CG12132 localized to both the Golgi and Rab7-positive late endosomes, but not early endosomes, and a GTP-specific interaction was detected by yeast two-hybrid, although not by affinity chromatography. The putative Rab7 interactors that we did not test include Drosophila orthologs of Spg11 and Spg15, human spastic paraplegia proteins that form a complex on late endosomes (Gillingham, 2014).

Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment

The morphogenetic gradient of Hh is tightly regulated for correct patterning in Drosophila and vertebrates. The Patched (Ptc) receptor is required for restricting Hh long-range activity in the imaginal discs. In this study, the different types of Hh accretion that can be observed in the Drosophila embryonic epithelial cells were investigated. In receiving cells, large apical punctate structures of Hh (Hh-LPSs) are not depending on the Ptc receptor-dependent internalization of Hh but rather reflect Hh gradient. By analyzing the dynamic of the Hh-LPS gradient formation, it was demonstrated that Hh distribution is strongly restricted during late embryonic stages compared to earlier stages. The up-regulation of Ptc is required for the temporal regulation of the Hh gradient. Dynamin-dependent internalization of Hh does not regulate Hh spreading but is involved in shaping Hh gradient. Hh gradient modulation is directly related to the dynamic expression of the ventral Hh target gene serrate (ser) and with the Hh-dependent dorsal cell fate determination. Finally, this study shows that, in vivo, the Hh/Ptc complex is internalized in the Rab7-enriched lysosomal compartment in a Ptc-dependent manner without the co-receptor Smoothened (Smo). It is proposed that controlled degradation is an active mechanism important for Hh gradient formation (Gallet, 2005).

To confirm that Ptc is involved in the temporal restriction of Hh movement, Hh distribution was analyzed in ptc mutant embryos and in embryos expressing ptc in en/hh cells. In ptc null embryos, the Hh gradient is impaired and Hh-LPSs distribution was found to be extended throughout the entire segment without restriction. In such embryos, ser expression is totally repressed in a manner similar to that seen under ubiquitous Hh expression. Note that the ectopic hh expressing source present in ptc mutant might also contribute to the broad distribution of Hh. When Ptc is expressed in en/hh expressing cells, the range of Hh-LPSs movement is limited to the vicinity of the producing cells. This effect is not due to a diminution of hh expression since it has been shown that Ptc does not affect hh transcription. Hence, Ptc might directly affect Hh-LPS range of action. Indeed, the slope of the Hh-LPSs gradient decreased sharply compared to wild-type stage 11 embryos. Interestingly, in these embryos, ser expression was extended correlating with the absence of Hh-LPS away from the source (Gallet, 2005).

Temporal regulation of Hh gradient is necessary because signaling requirements for Hh change in a time-dependent manner. One can suggest that, during early development, Hh acts at long range due to moderate levels of Ptc. Hh would easily overcome repression by the low concentrations of Ptc protein to prime a subset of ectodermal cells at a long distance to make them competent to respond to other signals. At later stages, Hh distribution is restricted and allows expression of ser and acts over a short range to induce rhomboid, both genes being necessary for ventral denticle specification (Gallet, 2005).


REFERENCES

Search PubMed for articles about Drosophila Rab7

Bharadwaj, R., Cunningham, K. M., Zhang, K. and Lloyd, T. E. (2015). FIG4 regulates lysosome membrane homeostasis independent of phosphatase function. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26662798

Cabrera, M., Nordmann, M., Perz, A., Schmedt, D., Gerondopoulos, A., Barr, F., Piehler, J., Engelbrecht-Vandre, S. and Ungermann, C. (2014). The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longin-fold-Rab interface and association with PI3P-positive membranes. J Cell Sci 127(Pt 5): 1043-1051. PubMed ID: 24413168

Deivasigamani, S., Basargekar, A., Shweta, K., Sonavane, P., Ratnaparkhi, G. S. and Ratnaparkhi, A. (2015). A pre-synaptic regulatory system acts trans-synaptically via Mon1 to regulate Glutamate receptor levels in Drosophila. Genetics 201(2): 651-64. PubMed ID: 26290519

Dickman, D. K., Lu, Z., Meinertzhagen, I. A. and Schwarz, T. L. (2006). Altered synaptic development and active zone spacing in endocytosis mutants. Curr Biol 16(6): 591-598. PubMed ID: 16546084

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Biological Overview

date revised: 20 July 2017

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