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BBS4 and BBS5 show functional redundancy in the BBSome to regulate the degradative sorting of ciliary sensory receptors.

Xu Q, Zhang Y, Wei Q, Huang Y, Li Y, Ling K, Hu J - Sci Rep (2015)

Bottom Line: However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive.Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans.Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.

ABSTRACT
Cilia harbor sensory receptors for various signaling cascades critical for vertebrate development. However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive. Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans. BBS-4 directly interacts with BBS-5 and the interaction can be disrupted by a conserved mutation identified in human BBS4. Surprisingly, we found that BBS-4 and BBS-5 act redundantly in the BBSome to regulate the ciliary removal, rather than the ciliary entry or retrograde IFT transport, of various sensory receptors. Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors. Moreover, mammalian BBS4 and BBS5 also interact directly and coordinate the ciliary removal of polycystin 2. Hence, we reveal a novel and highly conserved role for the BBSome in fine-tuning ciliary signaling by regulating the ciliary removal of sensory receptors for lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus

BBS-4 and BBS-5 play redundant roles in the context of cilia.(a) Dye-filling assay was used to examine ciliogenesis in worms. bbs-4 (tm3038) mutant and bbs-5 (gk507) mutant show normal ciliogenesis as the wild type (WT) worms. However, bbs-4 (tm3038); bbs-5 (gk507) double mutants are completely dye-filling minus, which could be fully rescued by introducing a wide copy of BBS-4-GFP or BBS-5-GFP. # denotes complete dye-filling minus. Results were represented as mean ± SD. (b) Schematic diagram of the phasmid cilia in worms. The phasmid has two sensory cilia whose tips bundle together. The axoneme of phasmid cilium contains the middle doublet and the distal singlet segment. In bbs-4; bbs-5 double and bbs-7 single mutants, (c) GFP-tagged IFT-A component CHE-11 is absent in distal segments, (d) GFP-tagged IFT-B component OSM-6 show strong accumulation at cilia tip, and (e) BBS-1 is absent from whole cilia. (f)bbs-4; bbs-5 mutants show defective mating behavior. (g) PKD-2 mislocalizes and accumulates in bbs-4; bbs-5 mutant cilia. Results were represented as mean ± SD. (h) Relative fluorescence intensities for PKD-2-GFP signal in sensory cilia were dot plotted. PKD-2 level increases more than 2 fold in bbs-4; bbs-5 cilia when compared to that in WT animals. Results represented as mean ± SEM. Arrows and arrowheads indicate the base and tip of cilia, respectively. Stars note the junction of middle and distal segment. Brackets indicate the dendrite of polycystin-expressing sensory neuron. Data represent three or more experiments. In each experiment, n > 40 were used in each group. ***p < 0.001; **p < 0.01. Scale bars, 5 μm.
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f1: BBS-4 and BBS-5 play redundant roles in the context of cilia.(a) Dye-filling assay was used to examine ciliogenesis in worms. bbs-4 (tm3038) mutant and bbs-5 (gk507) mutant show normal ciliogenesis as the wild type (WT) worms. However, bbs-4 (tm3038); bbs-5 (gk507) double mutants are completely dye-filling minus, which could be fully rescued by introducing a wide copy of BBS-4-GFP or BBS-5-GFP. # denotes complete dye-filling minus. Results were represented as mean ± SD. (b) Schematic diagram of the phasmid cilia in worms. The phasmid has two sensory cilia whose tips bundle together. The axoneme of phasmid cilium contains the middle doublet and the distal singlet segment. In bbs-4; bbs-5 double and bbs-7 single mutants, (c) GFP-tagged IFT-A component CHE-11 is absent in distal segments, (d) GFP-tagged IFT-B component OSM-6 show strong accumulation at cilia tip, and (e) BBS-1 is absent from whole cilia. (f)bbs-4; bbs-5 mutants show defective mating behavior. (g) PKD-2 mislocalizes and accumulates in bbs-4; bbs-5 mutant cilia. Results were represented as mean ± SD. (h) Relative fluorescence intensities for PKD-2-GFP signal in sensory cilia were dot plotted. PKD-2 level increases more than 2 fold in bbs-4; bbs-5 cilia when compared to that in WT animals. Results represented as mean ± SEM. Arrows and arrowheads indicate the base and tip of cilia, respectively. Stars note the junction of middle and distal segment. Brackets indicate the dendrite of polycystin-expressing sensory neuron. Data represent three or more experiments. In each experiment, n > 40 were used in each group. ***p < 0.001; **p < 0.01. Scale bars, 5 μm.

Mentions: Our previous findings identified the BBSome as an important player in regulating the assembly of IFT machinery in C. elegans25. To study the in vivo roles of BBS proteins, we rigorously tested all available alleles of worm bbs genes. Dye-filling assay is routinely used to examine the biogenesis of worm cilia28. Interestingly, unlike other bbs mutants that show defective ciliogenesis, bbs-4 or bbs-5 single mutants are completely normal in dye-filling assay, indicating that BBS-4 or BBS-5 alone is dispensable for ciliogenesis (Fig. 1a). To further test if BBS-4 and BBS-5 are functionally redundant, we generated bbs-4; bbs-5 double mutants. Remarkably, we found that bbs-4; bbs-5 double mutants show typical cilia defect as observed in other bbs mutants (Fig. 1a). Moreover, introducing a wild-type copy of bbs-4 or bbs-5 gene into bbs-4; bbs-5 could fully restore cilia biogenesis (Fig. 1a). BBS4 is a multiple tetratricopeptide repeats (TPR) containing protein, whereas BBS5 is a pleckstrin homology (PH) domain-containing protein (Fig. S1a and ref.12). It is thus unexpected that two BBSome components that share no similar protein domains (Fig. S1a and S1b) can function redundantly in the context of cilia.


BBS4 and BBS5 show functional redundancy in the BBSome to regulate the degradative sorting of ciliary sensory receptors.

Xu Q, Zhang Y, Wei Q, Huang Y, Li Y, Ling K, Hu J - Sci Rep (2015)

BBS-4 and BBS-5 play redundant roles in the context of cilia.(a) Dye-filling assay was used to examine ciliogenesis in worms. bbs-4 (tm3038) mutant and bbs-5 (gk507) mutant show normal ciliogenesis as the wild type (WT) worms. However, bbs-4 (tm3038); bbs-5 (gk507) double mutants are completely dye-filling minus, which could be fully rescued by introducing a wide copy of BBS-4-GFP or BBS-5-GFP. # denotes complete dye-filling minus. Results were represented as mean ± SD. (b) Schematic diagram of the phasmid cilia in worms. The phasmid has two sensory cilia whose tips bundle together. The axoneme of phasmid cilium contains the middle doublet and the distal singlet segment. In bbs-4; bbs-5 double and bbs-7 single mutants, (c) GFP-tagged IFT-A component CHE-11 is absent in distal segments, (d) GFP-tagged IFT-B component OSM-6 show strong accumulation at cilia tip, and (e) BBS-1 is absent from whole cilia. (f)bbs-4; bbs-5 mutants show defective mating behavior. (g) PKD-2 mislocalizes and accumulates in bbs-4; bbs-5 mutant cilia. Results were represented as mean ± SD. (h) Relative fluorescence intensities for PKD-2-GFP signal in sensory cilia were dot plotted. PKD-2 level increases more than 2 fold in bbs-4; bbs-5 cilia when compared to that in WT animals. Results represented as mean ± SEM. Arrows and arrowheads indicate the base and tip of cilia, respectively. Stars note the junction of middle and distal segment. Brackets indicate the dendrite of polycystin-expressing sensory neuron. Data represent three or more experiments. In each experiment, n > 40 were used in each group. ***p < 0.001; **p < 0.01. Scale bars, 5 μm.
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f1: BBS-4 and BBS-5 play redundant roles in the context of cilia.(a) Dye-filling assay was used to examine ciliogenesis in worms. bbs-4 (tm3038) mutant and bbs-5 (gk507) mutant show normal ciliogenesis as the wild type (WT) worms. However, bbs-4 (tm3038); bbs-5 (gk507) double mutants are completely dye-filling minus, which could be fully rescued by introducing a wide copy of BBS-4-GFP or BBS-5-GFP. # denotes complete dye-filling minus. Results were represented as mean ± SD. (b) Schematic diagram of the phasmid cilia in worms. The phasmid has two sensory cilia whose tips bundle together. The axoneme of phasmid cilium contains the middle doublet and the distal singlet segment. In bbs-4; bbs-5 double and bbs-7 single mutants, (c) GFP-tagged IFT-A component CHE-11 is absent in distal segments, (d) GFP-tagged IFT-B component OSM-6 show strong accumulation at cilia tip, and (e) BBS-1 is absent from whole cilia. (f)bbs-4; bbs-5 mutants show defective mating behavior. (g) PKD-2 mislocalizes and accumulates in bbs-4; bbs-5 mutant cilia. Results were represented as mean ± SD. (h) Relative fluorescence intensities for PKD-2-GFP signal in sensory cilia were dot plotted. PKD-2 level increases more than 2 fold in bbs-4; bbs-5 cilia when compared to that in WT animals. Results represented as mean ± SEM. Arrows and arrowheads indicate the base and tip of cilia, respectively. Stars note the junction of middle and distal segment. Brackets indicate the dendrite of polycystin-expressing sensory neuron. Data represent three or more experiments. In each experiment, n > 40 were used in each group. ***p < 0.001; **p < 0.01. Scale bars, 5 μm.
Mentions: Our previous findings identified the BBSome as an important player in regulating the assembly of IFT machinery in C. elegans25. To study the in vivo roles of BBS proteins, we rigorously tested all available alleles of worm bbs genes. Dye-filling assay is routinely used to examine the biogenesis of worm cilia28. Interestingly, unlike other bbs mutants that show defective ciliogenesis, bbs-4 or bbs-5 single mutants are completely normal in dye-filling assay, indicating that BBS-4 or BBS-5 alone is dispensable for ciliogenesis (Fig. 1a). To further test if BBS-4 and BBS-5 are functionally redundant, we generated bbs-4; bbs-5 double mutants. Remarkably, we found that bbs-4; bbs-5 double mutants show typical cilia defect as observed in other bbs mutants (Fig. 1a). Moreover, introducing a wild-type copy of bbs-4 or bbs-5 gene into bbs-4; bbs-5 could fully restore cilia biogenesis (Fig. 1a). BBS4 is a multiple tetratricopeptide repeats (TPR) containing protein, whereas BBS5 is a pleckstrin homology (PH) domain-containing protein (Fig. S1a and ref.12). It is thus unexpected that two BBSome components that share no similar protein domains (Fig. S1a and S1b) can function redundantly in the context of cilia.

Bottom Line: However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive.Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans.Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.

ABSTRACT
Cilia harbor sensory receptors for various signaling cascades critical for vertebrate development. However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive. Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans. BBS-4 directly interacts with BBS-5 and the interaction can be disrupted by a conserved mutation identified in human BBS4. Surprisingly, we found that BBS-4 and BBS-5 act redundantly in the BBSome to regulate the ciliary removal, rather than the ciliary entry or retrograde IFT transport, of various sensory receptors. Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors. Moreover, mammalian BBS4 and BBS5 also interact directly and coordinate the ciliary removal of polycystin 2. Hence, we reveal a novel and highly conserved role for the BBSome in fine-tuning ciliary signaling by regulating the ciliary removal of sensory receptors for lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus