Abstract:The endocytic Rab5 effectors Ccz1-Mon1 complex and Rab7 promote autophagosome-lysosome fusion independent of Rab5, which facilitates a later step of autophagy: degradation of cargo within lysosomes.
“…Confirming these results, TEM myofiber transverse sections (Figure 2B) revealed Rab2 , Rab7 or Stx17 RNAi-depleted IOMs similarly and uniformly filled with thousands of accumulated autophagosomes carrying nondegraded cytoplasm and organelles (Figure 4E–F). Thus, similar to previous reports of Rab7, Stx17, SNAP29 and Vamp7/8 functions in other Drosophila tissues (Hegedűs et al, 2016; Takáts et al, 2013), with myofiber remodeling, Rab2 also is required for autophagosome-lysosome fusion.10.7554/eLife.23367.009Figure 4.Autophagosomes accumulate in IOMs with Rab2, Rab7 or Stx17 knockdown.All IOMs imaged at 4d APF. ( A ) Autophagic flux assay using tandem-tagged mCherry:GFP:Atg8 (mCherry (C), pink; GFP (G), green; colocalization, white).…”
Section: Resultssupporting
confidence: 85%
“…Indeed, it has been shown that four of the five genes play known roles together in lysosome fusion: Stx17, SNAP29, and Vamp7/8 form a trans-SNARE complex involved in autophagosome-lysosome fusion (Itakura et al, 2012; Takáts et al, 2013), while Rab7 functions in late endosome-lysosome fusion as well as late steps in autophagy (Gutierrez et al, 2004; Jäger et al, 2004; Hegedűs et al, 2016). It was unexpected, however, to find a shared RNAi phenotype between this set of known functions and Rab2, which had been implicated with functions at the ER and Golgi (Saraste, 2016).…”
Section: Resultsmentioning
confidence: 99%
“…Interestingly, this disparity in autophagy phenotypes across species is also seen with Rab7. In flies and yeast, Rab7/Ypt7 is essential for autophagosome-lyososome/vacuole fusion (Kirisako et al, 1999; Hegedűs et al, 2016), while mammalian Rab7 knockdowns more clearly indicate a required role in autolysosome maturation (Gutierrez et al, 2004; Jäger et al, 2004). Other examples indicate that the autophagosome-lysosome fusion machinery is not highly evolutionarily conserved.…”
Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.DOI:
http://dx.doi.org/10.7554/eLife.23367.001
“…Confirming these results, TEM myofiber transverse sections (Figure 2B) revealed Rab2 , Rab7 or Stx17 RNAi-depleted IOMs similarly and uniformly filled with thousands of accumulated autophagosomes carrying nondegraded cytoplasm and organelles (Figure 4E–F). Thus, similar to previous reports of Rab7, Stx17, SNAP29 and Vamp7/8 functions in other Drosophila tissues (Hegedűs et al, 2016; Takáts et al, 2013), with myofiber remodeling, Rab2 also is required for autophagosome-lysosome fusion.10.7554/eLife.23367.009Figure 4.Autophagosomes accumulate in IOMs with Rab2, Rab7 or Stx17 knockdown.All IOMs imaged at 4d APF. ( A ) Autophagic flux assay using tandem-tagged mCherry:GFP:Atg8 (mCherry (C), pink; GFP (G), green; colocalization, white).…”
Section: Resultssupporting
confidence: 85%
“…Indeed, it has been shown that four of the five genes play known roles together in lysosome fusion: Stx17, SNAP29, and Vamp7/8 form a trans-SNARE complex involved in autophagosome-lysosome fusion (Itakura et al, 2012; Takáts et al, 2013), while Rab7 functions in late endosome-lysosome fusion as well as late steps in autophagy (Gutierrez et al, 2004; Jäger et al, 2004; Hegedűs et al, 2016). It was unexpected, however, to find a shared RNAi phenotype between this set of known functions and Rab2, which had been implicated with functions at the ER and Golgi (Saraste, 2016).…”
Section: Resultsmentioning
confidence: 99%
“…Interestingly, this disparity in autophagy phenotypes across species is also seen with Rab7. In flies and yeast, Rab7/Ypt7 is essential for autophagosome-lyososome/vacuole fusion (Kirisako et al, 1999; Hegedűs et al, 2016), while mammalian Rab7 knockdowns more clearly indicate a required role in autolysosome maturation (Gutierrez et al, 2004; Jäger et al, 2004). Other examples indicate that the autophagosome-lysosome fusion machinery is not highly evolutionarily conserved.…”
Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.DOI:
http://dx.doi.org/10.7554/eLife.23367.001
“…35 , 42 , 46–48 Indeed, by first using a HeLa model cell system, we demonstrate that, different from the wild-type protein, these mutants fail to increase the number of autophagosomes both in basal conditions and after stimulation, behaving similarly to the dominant negative and functionally impaired RAB7 T22N mutant. Still, while model cell lines are widely used as easily manageable tools to understand molecular mechanisms involved in a wide variety of cellular phenomena, they do not necessarily fully recapitulate the biological conditions of affected cells in vivo.…”
Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7AWT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7AT22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7AV162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.
“…We therefore tested if autophagy is also differentially regulated by the nutrient level in C4da neurons and epidermal cells. To examine the autophagy level, we used an mCherry-Atg8a reporter under the control of the endogenous Atg8a regulatory sequence, which labels autophagic structures (Hegedus et al, 2016). As expected, the autophagosome level in epidermal cells was low under the HY condition (Figure 3A) but increased 5 folds in the LY diet (Figures 3B and 3C).…”
17During prolonged nutrient restriction, developing animals redistribute vital nutrients to favor brain 18 growth at the expense of other organs. In Drosophila, such brain sparing relies on a glia-derived growth 19 factor to sustain proliferation of neural stem cells. However, whether other aspects of neural 20 development are also spared under nutrient restriction is unknown. Here we show that dynamically 21 growing somatosensory neurons in the Drosophila peripheral nervous system exhibit organ sparing at 22 the level of arbor growth: Under nutrient stress, sensory dendrites preferentially grow as compared to 23 neighboring non-neural tissues, resulting in dendrite overgrowth. Underlying this neuronal nutrient-24 insensitivity is the lower expression of the stress sensor FoxO in neurons. Consequently, nutrient 25 restriction suppresses Tor signaling less and does not induce autophagy in neurons. Preferential dendrite 26 growth is functional desirable because it results in heightened animal responses to sensory stimuli, 27 indicative of a potential survival advantage under environmental challenges.28
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