Rab5 and Rab7 Control Endocytic Sorting along the Axonal Retrograde Transport Pathway

Abstract

Vesicular pathways coupling the neuromuscular junction with the motor neuron soma are essential for neuronal function and survival. To characterize the organelles responsible for this long-distance crosstalk, we developed a purification strategy based on a fragment of tetanus neurotoxin (TeNT H(C)) conjugated to paramagnetic beads. This approach enabled us to identify, among other factors, the small GTPase Rab7 as a functional marker of a specific pool of axonal retrograde carriers, which transport neurotrophins and their receptors. Furthermore, Rab5 is essential for an early step in TeNT H(C) sorting but is absent from axonally transported vesicles. Our data demonstrate that TeNT H(C) uses a retrograde transport pathway shared with p75(NTR), TrkB, and BDNF, which is strictly dependent on the activities of both Rab5 and Rab7. Therefore, Rab7 plays an essential role in axonal retrograde transport by controlling a vesicular compartment implicated in neurotrophin traffic.

The Rabies Virus Glycoprotein Receptor p75 NTR Is Not Essential for Rabies Virus Infection

“…Briefly, coverslips were transferred into extracellular fluid (ECF) (145 mM NaCl, 5 mM KCl, 10 mM HEPES 10, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM D-glucose) buffered to pH 7.4 and containing 2 M Fura-2 acetoxymethyl ester (derived from a stock solution of 2 mM Fura dissolved in dimethyl sulfoxide) and 80 M Pluronic (F-127; both from Molecular Probes) for 30 min. Coverslips were subsequently placed for 10 min in ECF containing a TRITC (tetramethyl rhodamine isothiocyanate)-tagged antibody directed against the extracellular domain of p75 NTR (a generous gift from Gipi Schiavo) (15) to allow vital staining of cells and de-esterization of Fura-AM. Afterwards, the coverslips were transferred to a recording chamber, and pairs of images were collected at intervals of 2 s with alternating exposure of 340 and 380 nm each for 50 to 200 ms using a Polychrome IV and an air-cooled Imago charge-coupled device camera (640-by 480-pixel resolution) that were controlled by TILLviSION 4.0 software (all from Till Photonics, Gräfeling, Germany).…”

Apical and lateral cell protrusions interconnect epithelial cells in live Drosophila wing imaginal discs

“…One way by which cellular protrusions convey instructive cues is by endocytosis and trafficking of signaling complexes on endosomes. The early endosomal protein Rab5a localizes, for example, to axons and dendrites of neurons (de Hoop et al, 1994) and is important for coupling clathrin-dependent endocytosis to axonal retrograde transport (Deinhardt et al, 2006). Furthermore, several signaling molecules are associated with endosomes (reviewed in Miaczynska et al, 2004).…”

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

“…Taken together, our data suggest that Rab7-endosomes are the primary carriers for the long-distance retrograde transport of the P2X 3 receptor. Nevertheless, Rab5-endosomes appear to be required during a step that precedes the long-distance axonal transport, as proposed by Deinhardt et al [47], given that a dominant-negative Rab5 mutant (Rab5 S34N ) also inhibited the movement of ~60% of P2X 3 receptorpositive puncta. P2X 3 receptor-positive puncta containing GFP-Rab5 WT displayed low mobility and should belong to the slowly maturing early endosome, as previously reported [48].…”

“…NGF signals appear to be carried by both Rab5-containing early endosomes [6,45] and Rab7-containing late endosomes [46], while tetanus neurotoxin (TeNT H C ) and brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) are retrogradely transported by late endosomes [47]. We used live cell imaging to show that ~55% of puncta co-expressing the P2X 3 receptor and Rab7 moved retrogradely at a fast transport speed, compared to ~30% of puncta co-expressing the P2X 3 receptor and Rab5, which traveled at a much slower average velocity.…”