Figure 5. RAB-28 and BBS-8 are negative regulators of EV shedding.

(A) Cartoon of the ultrastructure of the cephalic sensory organ reproduced from Wang et al. (2014a). EVs (magenta spheres) are ‘shed’ from the PCMC/ciliary base into the lumen and ‘released’ into the environment outside of the worm. (B) Transmission electron micrographs of the cephalic lumen surrounding the distal region of CEM cilia. Black arrows point to the CEM and magenta arrows to EVs. rab-28(tm2636) accumulate significantly more EVs in the lumen distal to the singlet region of CEM compared to control males. Scale bars; 100 nm (C) Transmission electron micrographs of the cephalic organ at the level of the CEM cilium transition zone. Scale bars; 100 nm. A subset of the rab-28(tm2636) animals accumulate EVs in the cephalic lumen surrounding the TZ. (D) TEM cross sections of the proximal and distal regions for the cephalic lumen of bbs-8 mutant males. Black arrows point to the CEM cilium. Ectopic EVs (magenta arrows) are observed at distal and proximal regions of the lumen. Matrix filled vesicles (MFVs) in cephalic sheath are marked by yellow arrowheads. Scale bar; 200 nm. (E) Cartoon depictions of the lumen surrounding the cilia in the male cephalic sensillum in control, rab-28(tm2636), and bbs-8 mutants. Color scheme is the same as the cartoon in (A). Brackets enclose the cephalic sensory organ pore region. rab-28 mutant males accumulate an excess of EVs (labeled by magenta spheres and pointed to by arrows) in the lumen surrounding the more distal portion of the CEM axoneme whereas bbs-8 mutant males accumulate excessive EVs at all levels of the cephalic lumen. rab-28 and bbs-8 mutants also have an enlarged cephalic pore/opening of the sensory organ.

Figure 5—figure supplement 1. rab-28(tm2636) negatively regulates extracellular vesicle numbers in cephalic lumens.

(A, E) Cartoon of the ultrastructure of the cephalic sensory organ reproduced from Wang et al. (2014a). Color scheme is same as that in Figure 5. The distal and proximal cephalic lumens (A) and PCMC (E) are marked. (B, C, F) Scatter plots showing the mean number of EVs in the distal (B), proximal (surrounding the TZ region of the CEM cilium) (C), and PCMC (F) regions of the cephalic lumen in control (him-5(e1490)) and rab-28(tm2636) mutant males. Error bars depict SEM. p values determined by unpaired t-tests with Welch’s correction. The rab-28 mutant accumulates significantly more EVs in the cephalic lumen distal to the singlet region of the CEM cilium (B); in a subset of animals, EVs accumulate in regions surrounding the CEM cilium TZ (C) and PCMC (F). Numbers in brackets under genotypes represent the number of cilia. All measurements are from at least 2 animals. Error bars depict SEM. p values calculated by unpaired t-test with Welch’s correction. The variances for the data in panel C are significantly different as assessed by a F-test p=0.0098. (D) TEM of control CEM ciliary tip (left) and slice view of an electron tomogram of the rab-28(tm2636) CEM ciliary tip (right). Arrows point to the CEM ciliary tips, its neighboring CEP cilium, the cuticle overlying the CEM cilium, and the nearby OLQ cilium. Note that the CEM ciliary tip is open and exposed to the environment in both genotypes. Scale bar is 200 nm. (E) TEM of control (him-5) and rab-28(tm2636); him-5 cephalic lumens at the level of the CEM cilium PCMC. White arrows point to the CEM neurons. A subset of rab-28 sensory organs accumulate more EVs (magenta arrowheads) in the cephalic lumen. Scale bar is 100 nm.