Spatiotemporal Resolution of Rab9 and CI‐MPR Dynamics in the Endocytic Pathway

Abstract

Rab9 is a small GTPase that localizes to the trans-Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose-6-phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation-independent (CI-MPR) away from the Golgi yet, has no effect on the retrograde transport of CI-MPR. We also show that CI-MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5-positive, and late, Rab7a-positive, endosomes. CI-MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI-MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI-MPR to the endosomal pathway, entering the maturing endosome at the early-to-late transition.

Bidirectional traffic between the Golgi and the endosomes – machineries and regulation

“…Our recent findings now help to unify the discussion, as by using live imaging, we have been able to show that MPRs reach early endosomes just before their Rab5 (the Rab5a isoform) coat is lost and the Rab7a coat is acquired (Kucera et al, 2016). This is in line with earlier work that demonstrated that the recruitment of the retromer to endosomal membranes is regulated by the Rab5-to-Rab7a switch (Rojas et al, 2008).…”

“…Therefore, a new role of Rab9 as a mediator of the transport towards lysosomes or lysosome-related organelles, such as melanosomes, is now beginning to emerge. Indeed, our recent live-imaging studies have allowed us to detect Rab9 on maturing endosomes before they have lost Rab5 and gained a Rab7a coat, therefore suggesting an additional role for Rab9 in the transport from Golgi to maturing endosomes (Kucera et al, 2016). In agreement with this, it has been previously demonstrated that Rab9 binds to the Rab5 GAP SGSM3 (Gillingham et al, 2014).…”

“…The recent advances in microscopy, which allow the detection and the quantitative analysis of fast dynamic live events at high resolution, have provided new tools for the study of complex intracellular pathways, such as those occurring between the endosomes and TGN. The simultaneous use of different fluorescent proteins to visualize both cargo and endosomal compartments in live cells now allows experimenters to precisely define the timing and sequence of trafficking events and to therefore solve issues that for many years have been the subject of debates and controversies Kucera et al, 2016;van Weering et al, 2012). In addition, several high-throughput methods have been developed in the past few years.…”

“…Indeed, constitutively active mutants of Rab7b (Rab7bQ67L) and Rab9 (Rab9Q66L) not only localize to different target compartments (Golgi and late endosomes, respectively), but also induce opposite effects on carrier formation from the TGN with Rab9Q66L increasing and Rab7bQ67L decreasing the number of TGN-derived vesicles (Kucera et al, 2016;Progida et al, 2010Progida et al, , 2012. However, Rab7b has been much less characterized compared to Rab9, and only a few effectors have been identified, including sortilin and myosin II (Borg et al, 2014; Progida et al, 2012).…”

Parkinson’s Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation

“…The recruitment of this protein to mitochondria is actively inhibited by Parkin. Rab9, a late endosomal/Golgi guanosine triphosphatase (GTPase) that regulates vesicle release from late endosomes (Kucera et al, 2015), is also recruited to mitochondria for the formation of MDVs, while a second GTPase, Rab7, regulates the fusion of MDVs with endosomal compartments. These data identify an antigen presentation pathway regulated by PINK1 and Parkin, providing a link between mitochondrial dynamics and the potential engagement of autoimmune mechanisms in the etiology of PD.…”