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. 2016 Jul 1;27(13):2133-44.
doi: 10.1091/mbc.E15-07-0490. Epub 2016 May 18.

PACRG, a protein linked to ciliary motility, mediates cellular signaling

Catrina M Loucks  1 , Nathan J Bialas  1 , Martijn P J Dekkers  2 , Denise S Walker  3 , Laura J Grundy  3 , Chunmei Li  1 , P Nick Inglis  1 , Katarzyna Kida  4 , William R Schafer  3 , Oliver E Blacque  4 , Gert Jansen  2 , Michel R Leroux  5
Affiliations

PACRG, a protein linked to ciliary motility, mediates cellular signaling

Catrina M Loucks et al. Mol Biol Cell. .

Abstract

Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon-associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan.

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Figures

FIGURE 1:
FIGURE 1:
PCRG-1::GFP fusion protein localization and pcrg-1 mutant alleles. (A) Gene model for E04F6.2 (pcrg-1) in the nematode. Positions and relative sizes of the two deletion alleles examined in this study (gk480, tm2597), as well as the position of the X-box sequence. (B) Localization of PCRG-1::GFP constructs A (991–base pair promoter) and B (3182–base pair promoter) to the four OLQ and the two ASE cilia of C. elegans. Left, schematic diagram outlining fusion protein localization in the nematode. Bases of cilia (basal bodies) are indicated by orange ovals, cilia by green lines, and dendrites by gray lines. Scale bar, 5 μm.
FIGURE 2:
FIGURE 2:
pcrg-1 animals show a defect in behavioral plasticity. (A) Naive responses to NaCl are not affected in pcrg-1 animals. Chemotaxis responses of wild-type (black line) and pcrg-1(tm2597) (green line) animals to various concentrations of NaCl. (B) Mutation of pcrg-1 affects gustatory plasticity. Worms are washed for 15 min in a low-salt buffer (black bars) or a buffer containing 100 mM NaCl (gray bars) and tested for chemotaxis to 25 mM NaCl. Wild-type animals show avoidance of NaCl after preexposure, whereas the two pcrg-1 mutants and the two transgenic strains carrying extra copies of pcrg-1 remain attracted to NaCl. **Statistically significantly different compared with wild type (p < 0.001). (C) pcrg-1 interacts with G-protein signal transduction in gustatory plasticity. Worms are washed for 15 min in a low-salt buffer (black bars) or a buffer containing 100 mM NaCl (gray bars) and tested for chemotaxis to 25 mM NaCl. *, Statistically significantly different compared to wild type (p < 0.05). **, Statistically significantly different compared to wild type (p < 0.001). Comparisons were also performed between double mutants and their corresponding single mutants. The only statistically significantly differences (p < 0.001) were between pcrg-1 and odr-3, pcrg-1 and pcrg-1;odr-3, pcrg-1, and gpa-11, and pcrg-1 and pcrg-1;gpa-11. Means + SEM; n > 100 for all assays.
FIGURE 3:
FIGURE 3:
C. elegans pcrg-1 animals show a significant decrease in lifespan compared with wild type. (A) Both alleles of pcrg-1 (gk480, tm2597) show a significant decrease in lifespan compared with the wild-type N2 strain. (B) Expression of PCRG-1::GFP (construct A with 991– base pair promoter) rescues the lifespan defect shown in the gk480 allele. (C) Expression of PCRG-1::GFP (construct B with 3182–base pair promoter) rescues the lifespan defect shown in the tm2597 allele. (D) Expression of PCRG-1::GFP (construct B) in the wild-type (N2) background does not affect the lifespan of the worm. For number of animals and p values comparing differences between strains in lifespan experiments, see Supplemental Table S1.
FIGURE 4:
FIGURE 4:
PCRG-1 does not appear to interact genetically with G-proteins to affect lifespan. (A) The lifespan of a pcrg-1;gpc-1 double mutant is not statistically different from that of gpc-1. (B) The lifespan of a pcrg-1;gpa-1 double mutant is not statistically different from that of gpa-1. (C) The lifespan of a pcrg-1;gpa-11 double mutant is significantly different from that of either single mutant. (D) The lifespan of a pcrg-1;gpa-11XS mutant strain is significantly different from that of gpa-11XS and pcrg-1 mutants. (E) The lifespan of a pcrg-1;odr-3 double mutant is significantly different from that of either single mutant. (F) The lifespan of a pcrg-1;odr-3; gpa-11XS mutant strain is indistinguishable from that of odr-3;gpa-11XS. For number of animals and p values comparing differences between strains in lifespan experiments, see Supplemental Table S1.
FIGURE 5:
FIGURE 5:
PCRG-1 functions in a parallel pathway to ciliary proteins to regulate longevity. (A) The lifespan of a pcrg-1;osm-5 double mutant is significantly different from that of either single mutant. (B) The lifespan of a pcrg-1;che-11 double mutant is significantly different from that of either single mutant. (C) The lifespan of a pcrg-1;nphp-1 double mutant is significantly different from that of either single mutant. (D) The lifespan of a pcrg-1;nphp-4 double mutant is significantly different from that of either single mutant. For number of animals and p values comparing differences between strains in lifespan experiments, see Supplemental Table S1.
FIGURE 6:
FIGURE 6:
PCRG-1 functions upstream of DAF-16 and likely the insulin signaling pathway to regulate longevity. (A) The lifespan of a pcrg-1(tm2597);daf-16 double mutant is indistinguishable from that of either single mutant. (B) The lifespan of a pcrg-1(tm2597);daf-2 double mutant is indistinguishable from that of daf-2. (C) The lifespan of a pcrg-1;eat-2 double mutant is significantly different from that of either single mutant. (D) The lifespan of a pcrg-1;clk-1 double mutant is significantly different from that of either single mutant. For number of animals and p values comparing differences between strains in lifespan experiments, see Supplemental Table S1.
FIGURE 7:
FIGURE 7:
efhc-1 mutant alleles, localization of EFHC-1::GFP, and a reduction in lifespan in efhc-1 mutants. (A) Gene model for the Y49A10A.1 (efhc-1) gene in the nematode. Positions and size/nature of the two alleles examined in this study (gk424336 and tm6235). (B) Localization of EFHC-1::GFP to the four OLQ and the four CEP cilia of C. elegans. Basal body regions (BB) are indicated by orange ovals, cilia by green lines, and dendrites by gray lines. Scale bar, 5 μm. (C) Both efhc-1(gk424336) and efhc-1(tm6235) worms show significantly decreased lifespans compared with wild type (N2). (D) The reduced lifespan in efhc-1 mutants is similar to the lifespan of pcrg-1 mutants, as well as of double mutants, suggesting that the two genes work in the same genetic pathway to regulate lifespan. For number of animals and p values comparing differences between strains in lifespan experiments, see Supplemental Table S1.
FIGURE 8:
FIGURE 8:
OLQ neurons and/or cilia appear to play a role in longevity. (A) The ultrastructure of OLQ cilia in pcrg-1 mutants shown by representative TEM images from serial cross sections. Bottom, striated rootlet at the base of OLQ cilia that is unaltered in pcrg-1 mutants. Middle, proximal region of OLQ cilia. In wild-type cilia, four outer doublets are connected in a square by thick cross-bridges and to the center with fine radial arms surrounded by additional microtubule doublets. In pcrg-1 mutants, microtubule doublets no longer form a square but frequently form a triangle instead, and radial arms are missing. Top, distal region of OLQ cilia where wild-type animals retain microtubule doublets connected in a square but lose additional doublets. pcrg-1 mutants completely lose connections between doublets and retain a small number of doublets. Scale bars, 200 nm. (B) pcrg-1 animals with PCRG-1 fused to GFP and driven by the ocr-4 promoter specifically in OLQ neurons show increased longevity compared with nontransgenic, pcrg-1 siblings. (C) Genetic ablations of OLQ neurons by expression of egl-1(d) (two lines) show increased longevity compared with nontransgenic, wild-type siblings.
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