Ciliary Rab28 and the BBSome negatively regulate extracellular vesicle shedding
- Department of Genetics and Human Genetics Institute of New Jersey, Rutgers University, United States
- School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland
- Center for C. elegans Anatomy, Albert Einstein College of Medicine, United States
- Department of Biology, University of Utah, United States
Figures
Figure 1 with 1 supplement
A BBSome-ARL-6-PDL-1 network targets RAB-28 to sensory cilia.
Representative confocal z-projection images of amphid (head) and phasmid (tail) neurons from hermaphrodites of the indicated genotype expressing GFP::RAB-28Q95L. Anterior is to the left; all images …
Figure 1—figure supplement 1
GFP::RAB-28 localization and IFT frequency analysis.
(A) Representative confocal z-projection images of amphid and phasmid neurons from worms of the indicated genotypes expressing GFP::RAB-28Q95L. Images are the same as those in Figure 1 except image …
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Figure 1—figure supplement 1—source data 1
Data for Figure 1 and Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/50580/elife-50580-fig1-figsupp1-data1-v1.xlsx
Figure 2
RAB-28 is expressed in and trafficked to EVN cilia via a modified BBSome-ARL-6-PDL-1 pathway.
(A) Epifluorescence z-projections of the heads and tails of C. elegans males expressing rab-28p::sfGFP and klp-6p::tdTomato (EVN cilia-specific reporter). Arrows denote the CEM neurons. Brightfield …
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Figure 2—source data 1
Data for Figure 2C.
- https://cdn.elifesciences.org/articles/50580/elife-50580-fig2-data1-v1.xlsx
Figure 3
BBSome and arl-6 mutant hermaphrodites display defects in amphid sensory organ structure and/or associated glia.
(A) Transmission electron microscopy (TEM) images showing the amphid channels of the indicated genotype in cross section, at the positions of ciliary distal segments (DS) and middle segments …
Figure 4 with 2 supplements
RAB-28 and BBSome components regulate the localization of ciliary EV cargoes in EV-releasing CEM cilia.
(A) Left- schematic showing the location of the deleted regions in the gk1040 and tm2636 mutant alleles of rab-28. Right- cartoon modified from Bae et al. (2006) depicting the morphology of the CEM …
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Figure 4—source data 1
Data for 4C-E, 4G-I, 4 K-N.
- https://cdn.elifesciences.org/articles/50580/elife-50580-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
tm2636 allele of rab-28.
Top schematic shows the location of the deleted region in the tm2636 mutant allele and the location of the primer pairs used to amplify full length cDNA sequence that was sequenced using the …
Figure 4—figure supplement 2
rab-28(gk1040) phenocopies the CIL-7 mislocalization phenotype of rab-28(tm2636) worms.
(A) Fluorescence micrographs of control and rab-28(gk1040) adult males expressing CIL-7::GFP. Close-ups of the male heads are shown on the left. Dotted white boxes mark one cilium. White arrowheads …
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Figure 4—figure supplement 2—source data 1
Data for Figure 4—figure supplement 2B,C.
- https://cdn.elifesciences.org/articles/50580/elife-50580-fig4-figsupp2-data1-v1.xlsx
Figure 5 with 1 supplement
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 …
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 …
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Figure 5—figure supplement 1—source data 1
Data for Figure 5—figure supplement 1B,C,F.
- https://cdn.elifesciences.org/articles/50580/elife-50580-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
BBS-8 suppresses ectopic EV shedding in amphid sensory organs.
(A) TEM cross sections of control, rab-28(tm2636), and bbs-8 amphid channel cilia at the middle segment (top) and at the transition zone (bottom). Yellow arrowheads in rab-28 indicate the occasional …
Figure 6—figure supplement 1
rab-28(tm2636) male amphid sensory organs have sheath cell defects.
Left- cartoon representation of the amphid sensory organ with the locations of the different ciliary segments marked. Right-Transmission electron micrographs of chemically frozen control (N2) adult …
Figure 7
Model Cartoon depictions of the cephalic sensory organs in wild-type, EV shedding defective rab-28 and bbs-8 mutants, and EV release defective IFT and axoneme mutants (ccpp-1, ttll-11, klp-6, tba-6).
Color scheme is the same as in Figure 5; magenta spheres represent shed EVs accumulating within sensory organs and EVs released into the environment, brown spheres represent secreted molecules …
Author response image 1
BBS-8::GFP localization in rab-28(tm2636) Confocal Z-projections of BBS-8:: GFP in rab-28(tm2636) in amphid cilia (A), phasmid cilia (B), and the EV releasing ray neuron cilia (C).
BBS-8:: GFP levels in rab-28(tm2636) are slightly reduced in ray neurons but, remain unaltered in amphid and phasmid cilia. Scale bar is 1μm for amphid and phasmid panels and 5μm for ray neuron cilia.
Videos
Video 1
Representative movies of GFP::RAB-28Q95L IFT behavior in the phasmid cilia of N2 (WT), arl-6 and pdl-1;arl-6 mutant hermaphrodites.
Anterior is to the left. Movies are played at 5 frames per second. Scale bars; 2 μm. PCMC: periciliary membrane compartment.
Video 2
Representative movies of GFP-RAB-28Q95L IFT behavior in CEM and RnB cilia of him-5(e1490) males.
A RAB-28-positive IFT particle can be seen in the bottom CEM and RnB cilia. Higher frequency IFT movement of RAB-28 is evident from the amphid cilia. PCMCs are marked by asterisks. Movies are played …
Video 3
Electron tomography and serial section TEM based model of the male cephalic sensory organ of control and rab-28(tm2636) respectively.
Models depict the CEM cilium (gold), and EVs (magenta spheres). Dotted white line in movie shows position of the TZ. rab-28 mutants ectopically accumulate excess EVs in distal regions of the …
Tables
Table 1
Table summarizing the amphid and cephalic sensory organ phenotypes of strains used in this study.
| Amphid sensory organ TEM phenotypes | |||||
|---|---|---|---|---|---|
| Mutant | Sex | Sensory compartment size | Matrix filled vesicles in sheath | EVs in pore | Reference |
| rab-28(tm2636) | Male | Not enlarged | Yes | Few vesicles/odd shaped particles observed surrounding MS | This work |
| rab-28(gk1040) | Hermaphrodite | Not enlarged | No | ND | Jensen et al., 2016 |
| RAB-28Q95L | Hermaphrodite | Enlarged | No | ND | Jensen et al., 2016 |
| RAB-28T49N | Hermaphrodite | Not enlarged | Yes | ND | Jensen et al., 2016 |
| bbs-8 | Male and hermaphrodite | Enlarged | Yes | Lots of EVs (in male) | This work |
| arl-6 | Hermaphrodite | Not enlarged | Yes | ND | This work |
| Male cephalic sensory organ TEM phenotypes | |||||
| Mutant | Sensory organ size | EVs in pore | Reference | ||
| rab-28(tm2636) | Enlarged | Yes, especially at the distal regions | This work | ||
| rab-28(gk1040) | ND | ND | N/A | ||
| RAB-28Q95L | ND | ND | N/A | ||
| RAB-28T49N | ND | ND | N/A | ||
| bbs-8 | Enlarged | Yes, surrounding all segments | This work | ||
| arl-6 | ND | ND | N/A | ||
| Other phenotypes | |||||
| Phenotype | rab-28(tm2636) | rab-28(gk1040) | arl-6 | bbs-8 | |
| PKD-2 localization in CEMs | Mislocalized | Normal (Jensen et al., 2016) | ND | ND | |
| CIL-7 localization in CEMs | Mislocalized | Mislocalized | Mislocalized | Mislocalized | |
| Environmental release of EVs from heads | Normal | Normal | Normal | Normal | |
Additional files
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Supplementary file 1
Key resources table.
- https://cdn.elifesciences.org/articles/50580/elife-50580-supp1-v1.docx
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Supplementary file 2
Primers used in this study.
- https://cdn.elifesciences.org/articles/50580/elife-50580-supp2-v1.xlsx
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Supplementary file 3
Means and SEM for all figures.
- https://cdn.elifesciences.org/articles/50580/elife-50580-supp3-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50580/elife-50580-transrepform-v1.docx
