Rabs on the fly: Functions of Rab GTPases during development

Abstract

The organization of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiologic requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo.

Keywords: Rab GTPases; Staccato/Munc13-4; cell polarity; lysosome related organelles (LROs).

Figure 1.

Rab3 expression in embryonic neurons. Rab3 and Rab27 are pan-neuronal presynaptic vesicle identifiers. In contrast to Rab27 (no detectable YRab27 in embryonic neurons, data not shown), Rab3 is expressed in a subset of wiring neurons (before synapse formation). Interestingly, after axogenesis all interconnected neurons show Rab3 and Rab27 restricted to presynaptic membranes. A-C Cartoons (adapted from Fly atlas (Hartenstein, V. (1993) Atlas of Drosophila development and flybase.org) depicting different developmental stages of Drosophila embryos. (A’-C’) Images from corresponding Yrab3 embryonic ventral nerve cords. Of note, YRab3 (green) is detectable in neural cell bodies (A’-C’, white arrowheads) and projections (A’-C’, white arrows). HRP (magenta) labels all neuronal membranes, developmental stages are indicated (A-C, upper left corner), scale bars indicate 20µm.

Figure 2.

ER associated Rab proteins in salivary gland cells. Rab1 and Rab2 are canonical Rab proteins that regulate traffic between ER and the Golgi apparatus. We show here that Rab7 and 8, but not Rab6 (trans-Golgi Rab protein) are present on Rab1 membranes in salivary gland cells. It remains to be shown what role Rab7 and Rab8 may play in ER/Golgi related transport. Of note, all used Yrab and Crab alleles reflect the endogenous expression of respective Rab proteins. A,A’ Confocal micrograph shows wild-type salivary gland (A) stained with phalloidin (F-actin, white) and DAPI (nuclei, blue). Cells in boxed region are magnified in (A’). Scale bars indicate 5µm. B-E” Confocal cross-sections of salivary gland cells from YFPrab6;CFPrab1 (B-B″), YFPrab8;CFPrab1 (C-C″), YFPrab2;CFPrab1 (D-D″) and YFPrab7;CFPrab1 (E-E″) larvae probed for direct YFP fluorescence (B-E, in gray and B”-E” in magenta) or stained against CFP-HA (B’-E’, in gray and B″-E″ in green). Arrows and arrowheads point to individual Rab domains; in B″-E″ dashed lines indicate cell nucleus (N) and dotted lines mark the apical domain; scale bars indicate 5µm. Note that Rab6 accumulates apically and in a domain adjacent to Rab1 basally, whereas Rab2, Rab7 and Rab8 partially overlap with Rab1.

Figure 3.

Overview of membrane trafficking steps during tracheal tube fusion. (A) Scheme depicting a fusion tip cell (blue) invaded by a stalk cell (gray). The primary (1°) and secondary (2°) apical membranes are in magenta, adherens junctions in bright green, actin/microtubule track in dark green, ER in blue, Golgi apparatus in light green, LROs in orange, late endosomes in dark red, early endosomes in light red and recycling endosomes in yellow. Broken lines represent hypothetical trafficking steps; continuous lines represent experimentally validated steps. Endocytic compartments (a) donate membrane material to apical membranes (b) or mature to give rise to late endosomes (c). Arl3 is recruited to Rab7- and Lamp1- positive compartments, and Arl3 effectors recruit Munc13–4/Staccato and Rab39. Dynein and kinesin (KIF) motor proteins are effectors of Rab7 and/or Rab39, and link LROs to the actin/microtubule track (d). Once the apical plasma membrane domains are reached, Ca²⁺-bound Munc13–4/Staccato promotes SNARE-dependent membrane fusion (e). Ca²⁺-enriched microdomains are established by localized release from Sec 31-positive ER portions close to the fusing membranes. (B) Stages of lumen fusion in the wild type (I-III). (C) In the absence of Stac or Rab39, LROs fail to form. As a consequence the 2 apposed apical membranes fail to fuse, although fusion cells are still able to expand their apical domains. (Modified after Caviglia et al. NCB, 2016).

Figure 4.

(A) novel strategy to impede intracellular protein/membrane traffic. A,B Homologous recombination strategy for generating TevP-rab and TEV-rab alleles. A and B are the genomic regions with the recombination points and the exogenous DNA inserted, which is indicated in each case. A' and B' are the proteins generated. TevP-rab1 (A,A’) and TEV-rab1 (B,B’) are shown as an example. 5′ and 3′ UTRs, gray; rab1 coding regions, green; TevP, pink; YFP, yellow; V5, magenta; MYC, cyan; HR stands for ‘Homology Region’. C,D Sketch depicts endosomal maturation without processing (C) and upon TEV-Rab7 cleavage by TevP-Rab5 (D). TevP cleavage of TEV-Rabs disconnects the Rab-GTPase domain from membranes. Early endosomes (EE) are in light-orange, intermediate endosomes (IE) in orange and late endosomes (LE) in red. TevP (pink star) tagged Rab5, and YFP (yellow) tagged TEV-Rab7 are depicted. E Larval fat body protein lysates from wild type (lanes 1 and 2), TEV-Rab1 (lane 3), TEV-Rab1;TevP-Rab5 (lane 4), TEV-Rab5 (lane 5), TEV-Rab5;TevP-Rab5 (lane 6), TEV-Rab9 (lane 7), TEV-Rab9;TevP-Rab5 (lane 8) probed for MYC (top), V5 (middle) and Tubulin (bottom). Note, TEV-Rab1 and TEV-Rab5 are cleaved by TevP-Rab5 whereas TEV-Rab9 is not. This result indicates that one traffic step requires Rab1 and Rab5 in close vicinity. Note that expression of TevP-Rab5 in a TEV-Rab5 background leads to complete degradation of TEV-Rab5 (lane 6).