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First published online 30 August 2006
doi: 10.1242/dev.02555

Development 133, 3859-3870 (2006)
Published by The Company of Biologists 2006
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General and cell-type specific mechanisms target TRPP2/PKD-2 to cilia

Young-Kyung Bae1,2, Hongmin Qin3,*, Karla M. Knobel2, Jinghua Hu2, Joel L. Rosenbaum3 and Maureen M. Barr2,{dagger}

1 Laboratory of Genetics,, Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI 53705-2222, USA.
2 School of Pharmacy, Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI 53705-2222, USA.
3 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA.


Figure 1
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  		Fig. 1. PKD-2 localizes to cilia and ER in male-specific sensory neurons. (A-G) Images are projected z-series obtained by laser-scanning confocal microscopy unless indicated otherwise. (A) A lateral view of an adult male expressing Ppkd-2::GFP (pkd-2 promoter driving GFP expression). GFP is expressed in four CEMs in the head, 16 ray B neurons, and the HOB neuron in the tail. Scale bar: 100 µm. (B,C) PKD-2::GFP localizes in the cell bodies (arrows), small dendritic and axonal puncta, and ciliary endings (arrowheads). Scale bars: 10 µm. (B) CEMs, (C) RnB ray (three arrows) and HOB (one arrow) neuronal cell bodies, and ciliary regions (ciliary membrane and base, arrowheads). (D,E) GFP-tagged TRAM expressed under (Ppkd-2::TRAM::GFP) localizes in cell bodies (arrows), dendritic puncta and ciliary bases (open arrowheads), but not the cilium proper. Scale bars: 10 µm. (D) CEMs, (E) HOB and RnB neurons. (F,G) GFP-KDEL ER marker driven by the pkd-2 promoter shows reticular structure in cell bodies and is excluded from ciliary and dendritic compartment of the cells. Open arrowheads indicate the absence of GFP in cilia. (H) Epifluorescence images showing overlapping expression of PKD-2::GFP and Ppkd-2::TRAM::DsRed2 in RnB cell bodies.

 

Figure 2
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  		Fig. 2. PKD-2::GFP moves rapidly and bidirectionally in dendrites. (A) A montage of PKD-2::GFP particles shows both anterograde (black arrows, from the cell body to cilium) and retrograde (white arrowheads) movement. The broken white line indicates stationary particles. Scale bar: 5 µm. (B) A kymograph depicting PKD-2::GFP particle motility in the dendrite. The horizontal and vertical axes represent time and distance, respectively. Vertical scale bar: 5 µm. The left arrowhead illustrates the changes from anterograde to retrograde and the right arrowhead reflects retrograde to anterograde changes. Saltatory movement is also detected (arrows). (C) Distribution of PKD-2 particle velocities in dendrites. The particles have distinct velocities for retrograde (light boxes) and anterograde (dark boxes) directions. The velocities are shown as the average ± s.e.m. Time lapse movies are available upon request.

 

Figure 3
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  		Fig. 3. PKD-2::GFP localization is altered in unc-101, lov-1 and IFT mutants. (A,D,G,J) Cartoons of a sensory neuron expressing PKD-2::GFP (shown in green) for each genotype. (B,E,H,K) CEMs, (C,F,I,L) RnBs and HOB neurons. (A-C) Wild-type localization of PKD-2::GFP. PKD-2 in ciliary endings (ciliary membrane and ciliary base, arrowheads) in CEMs and RnBs. (D-F) In unc-101(m1) mutants, PKD-2::GFP is uniformly distributed throughout neurons, including axons, cell bodies, dendrites (thick arrows) and cilia (arrowhead). (G-I) In lov-1(sy582) mutants, PKD-2::GFP aggregates within the cell bodies (arrows) and is expressed in a lower level in ray cilia (arrowhead in 3I). The boxed inset in I shows a closer view of a ray cell body containing PKD-2::GFP aggregates. (J-L) In daf-10(p821) mutants, PKD-2::GFP accumulates in the ciliary base (arrowheads). In the RnBs, PKD-2::GFP also mislocalizes to dendrites (thick arrow). This mutant phenotype is consistent in other IFT single and double mutant backgrounds. Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. PKD-2::GFP abundance in cilia is altered in lov-1 and IFT mutants. (A,B) Fluorescence intensity in CEM cilia (Fcilia) shows that PKD-2::GFP accumulates in IFT mutants. (A) Fcilia of PKD-2::GFP in CEMs in wild-type, lov-1, daf-10 and osm-5 backgrounds. Fcilia in the IFT mutants (daf-10 and osm-5) is approximately three times greater than in wild type. (B) Ratio (Fcilia/Fcell body) shows that PKD-2::GFP abundance in CEM cilia is increased in comparison with the cell bodies in IFT mutants (daf-10 and osm-5). (C-E) Fluorescence intensity measurement in RnB ray cilia of wild type and lov-1 mutants reveals reduced ciliary localization in lov-1 background. (C) PKD-2::GFP localization in RnB ray cilia is often below detectable levels in lov-1 mutants. R1B to R5B are selected because they are easily distinguished from other rays. The percentage reflects the number of rays exhibiting detectable ciliary localization divided by total number of ray pairs scored. Those cilia with detectable GFP were used for ciliary measurement in D and E. (D) Fcilia in RnB cilia showed that PKD-2::GFP levels in lov-1 mutants are significantly reduced. (E) Fcilia/Fcell body ratio in rays reveals decreased PKD-2::GFP ciliary abundance in lov-1 mutants. Error bars indicate s.e.m. Non-parametric Mann-Whitney tests with two-tailed P-value were performed between wild type and each genotype. ns, not significant (P>0.05); ***P<0.001; n, number of cilia measured.

 

Figure 5
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  		Fig. 5. Ectopic PKD-2 expression (Punc-119::PKD-2::GFP) reveals cell-type specific factors required for PKD-2 localization. (A,B) A wild-type hermaphrodite expressing Punc-119::PKD-2::GFP. (A) PKD-2::GFP localizes to neuronal cell bodies in the head, but GFP is excluded from ciliary endings. Small arrowheads indicate the anatomical location of OLQ (outer labial quadrant) cilia in the left panels. PKD-2::GFP is absent from cilia in the head sensillae of the adult hermaphrodite. (B) In the hermaphrodite tail, PKD-2::GFP is retained in cell bodies of many neurons including the ciliated phasmid sensory neurons. Phasmid cell bodies were identified by their location (arrows); small arrowhead indicates approximate anatomical location of phasmid cilia. PKD-2::GFP is absent from cilia in the phasmids of the adult hermaphrodite. (C,D) A wild-type male expressing PKD-2::GFP in every neuron. (C) In the head, PKD-2::GFP is localized to the ciliary endings of CEMs (large arrowheads in the left panels). (D) In the male tail, PKD-2::GFP is detected in ciliated endings (large arrowhead, R3B cilium) of all B-type ray neurons, except R6B (asterisk), which does not normally express the polycystins. (E,F)Punc-119::PKD-2::GFP expression in the unc-101(m1) mutant. Arrows indicate PKD-2::GFP dendritic expression in polycystin-expressing neurons. (E) In the head, PKD-2::GFP is distributed throughout male-specific CEM neurons. In all other neurons, PKD-2::GFP remains in cell bodies. (F) In the tail, PKD-2::GFP is uniformly distributed only in all B-type ray neurons (arrow) except R6B (asterisks). unc-101 males are Mab (male abnormal) and often fail to develop all ray process. (G) In the lov-1 mutant, PKD-2::GFP aggregates in CEM cell bodies (arrows) but not in other neurons, and PKD-2::GFP is detectable in CEM cilia (large arrowheads), which are identified by location and cell body morphology. The inset shows one CEM cell body with PKD-2::GFP aggregates (compare with the Fig. 3I inset). A subset of z-series confocal sections is projected to generate the inset. (H) PKD-2::GFP in lov-1 RnB ray neurons forms aggregates in cell bodies (arrow), and the level of ciliary staining is reduced. Large arrowhead points at R3B cilia with reduced PKD-2 expression. Scale bars: 10 µm. The anterior outlines of the worm are drawn by hand according to the transmission images. (I) The percentage of ciliary localization of Punc-119::PKD-2::GFP in non-native and native cells. The reason that a small fraction (3-7%) of native cells do not localize PKD-2::GFP in native cilia may be due to mosaicism of the extrachromosomal array. When PKD-2::GFP is detected in R6B cilia, PKD-2::GFP staining is reduced and mislocalized to the distal dendrite. In the hermaphrodite, there is no detectable ciliary PKD-2::GFP expression in the head. Specifically, OLQ cilia were scored because of their distinct shape and location. One-hundred and eight pairs of RnBs were scored. For hermaphrodites (OLQs and phasmids), a total of 112 animals were scored. There are four OLQ and phasmid neurons in each animal. WT, wild type

 

Figure 6
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  		Fig. 6. PKD-2::GFP CEM ciliary distribution in wild type and IFT mutants. The first column depicts a cartoon showing two out of four CEM cilia in the male nose. Column 2 shows PKD-2::GFP ciliary distribution patterns. Column 4 shows Ppkd-2::TBB-4::DsRed2 labeling CEM ciliary axonemes of wild type and IFT mutants. Column 3 is the merged image of Columns 2 and 4. Scare bars: 5 µm. (A) In wild type, PKD-2::GFP is enriched in the ciliary base (transition zone and distal-most dendrite, bracket) and along the ciliary membrane (dashed bracket). (B-E) PKD-2::GFP accumulates in the ciliary bases (bracket) and abnormal cilia (dashed bracket) of IFT mutants (B,C) In osm-3 and osm-5 mutants, PKD-2::GFP accumulates at the bases of stunted cilia. (D) In the che-3 cytoplasmic dynein mutant, cilia form and PKD-2 accumulates at the ciliary bases and along the ciliary membrane. (E) In the klp-11 kinesin II mutant (and kap-1, not shown), CEM cilia morphology is abnormal and PKD-2 accumulates at ciliary bases and along the ciliary membrane. (F) Kinesin II is expressed in polycystin-expressing neurons. The polycystin-expressing neurons are labeled with a pkd-2 promoter driven DsRed2 (left panel). KAP-1::GFP (right panel) is enriched in the ciliary compartment, but also found in dendrites (arrow) and axons (arrowhead). HOB, hook B neuron. (A) Reproduced, with permission, from Qin et al. (Qin et al., 2005Go).

 

Figure 7
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  		Fig. 7. Male mating behavior is abnormal in IFT mutants. Response and location of vulva efficiency is scored for each genotype. Two independent integrated PKD-2::GFP transgenic lines (myIs1 and myIs4) rescue Response and Lov defects of the pkd-2 mutant (only myIs4 is shown). Three IFT kinesin mutants (osm-3, kap-1, klp-11) exhibit Response and Lov defects. Non-null osm-3(e1806) exhibits wild-type male mating behavior (Barr and Sternberg, 1999Go). Error bars indicate s.e.m. between multiple assays, and each assay consisted of at least 20 animals. An asterisk marks wherever the data are significantly different from wild type (*P<0.05, **P<0.01, ***P<0.001). n, number of males examined.

 

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© The Company of Biologists Ltd 2006