Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport (original) (raw)
Fig 5
Worms overexpressing RAB-28(GTP) or RAB-28(GDP) display overlapping and distinct defects in sensory pore formation and function.
(A) Dye filling (DiI) of amphid neurons in non-transgenic (control) or transgenic wild type (N2) worms expressing GFP::RAB-28(WT), GFP::RAB-28(GTP) or GFP::RAB-28(GDP). All constructs driven by the endogenous rab-28 gene promoter and expressed to similar levels in transgenic animals (all injected at 5 ng/ul). For each amphid pore, the number of dye-filling neurons was scored. Each dataset represents mean ± standard deviation (error bars) from 3 independent experiments (at least 40 amphid pores scored for each strain per experiment). (B) Roaming scores for non-transgenic (control) and transgenic wild type (N2) worms expressing GFP-tagged RAB-28 variants. Scores normalised to non-transgenic N2 worms. For each strain, >45 worms were scored. *p<0.01 (unpaired Student t-test vs non-transgenic N2 controls). (C) Transmission electron microscopy images of the amphid pore from serial cross-section of wild type (N2) and N2 worms expressing GFP-tagged RAB-28(GTP) or RAB-28(GDP). Low magnification images (top rows) show the enlarged amphid channel (yellow outline and asterisk) in RAB-28(GTP)-expressing worms and dense matrix filled vesicles (mfv) in the amphid sheath cell of RAB-28(GDP) expressing worms (white arrows). High magnification images (bottom rows) display close ups of the amphid pore and ciliary axonemes, showing the distal segments (DS), middle segments (MS), transition zones (TZ) and periciliary membrane compartments (PCMC). Note the misplaced (disconnected) channel axoneme (white arrowhead) that fails to enter the channel of worms expressing RAB-28(GDP). Images representative of at least 4 analysed pores for each strain. Cartoons show the amphid channel in cross section and longitudinal orientations (only 3 of the 10 axonemes shown for simplicity in longitudinal cartoon), and indicate observed phenotypes. Numbers above images indicate the position of the section relative to the most anterior section (at ‘0’); section positions also indicated in cartoon. Scale bars; 1 μm (top rows); 200 nm (large images in bottom rows); 100 nm (small images in bottom rows).