Role for Rab7 in maturation of late autophagic vacuoles

Abstract

The small GTP binding protein Rab7 has a role in the late endocytic pathway and lysosome biogenesis. The role of mammalian Rab7 in autophagy is, however, unknown. We have addressed this by inhibiting Rab7 function with RNA interference and overexpression of dominant negative Rab7. We show here that Rab7 was needed for the formation of preferably perinuclear, large aggregates, where the autophagosome marker LC3 colocalised with Rab7 and late endosomal and lysosomal markers. By electron microscopy we showed that these large aggregates corresponded to autophagic vacuoles surrounding late endosomal or lysosomal vesicles. Our experiments with quantitative electron microscopy showed that Rab7 was not needed for the initial maturation of early autophagosomes to late autophagic vacuoles, but that it participated in the final maturation of late autophagic vacuoles. Finally, we showed that the recruitment of Rab7 to autophagic vacuoles was retarded in cells deficient in the lysosomal membrane proteins Lamp1 and Lamp2, which we have recently shown to accumulate late autophagic vacuoles during starvation. In conclusion, our results showed a role for Rab7 in the final maturation of late autophagic vacuoles.

Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B

“…S1-S3). Consistent with previous results, 35 RAB7 WT displayed a good colocalization rate with LC3B, which increased upon starvation (Fig. 1A; Fig.…”

“…The analysis of the autophagic flux, obtained by the ratio between the amount of autophagic vesicles in basal conditions (FM) and upon protease inhibition (Baf), 39 showed a reduced autophagic flux upon expression of the CMT2B-causing RAB7 mutants and, as expected, 35 of RAB7 T22N , as compared to RAB7 WT and RAB7 Q67L proteins (Fig. 1C).…”

“…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.…”

“…S1-S3), indicating that CMT2B-causing mutants are less recruited to autophagic vesicles when compared with RAB7 WT . Based on the established role of RAB7 in autophagy, 35 the reduced localization of this protein on autophagic vesicles suggests potential functional consequences of RAB7 mutant expression on this process.

10.1080/15548627.2017.1388475-F0001Figure 1.Analysis of autophagy in HeLa cells expressing CMT2B-causing RAB7 mutant proteins.

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“…33 , 34 Notably, RAB7 regulates autophagosomal maturation controlling the final step of maturation of late autophagic vesicles into autolysosomes, presumably involving fusion with lysosomes. 12 , 35–37 …”