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. 2022 Sep 5;11:e78383. doi: 10.7554/eLife.78383

Figure 4. CLS1-4 are conserved ciliary localization signals affecting INPP5E function.

(a) CLS1-4 are highly evolutionarily conserved in vertebrates, including human (NP_063945.2), mouse (AAH80295.1), python (XP_007441606.1), crow (XP_039417670.1), toad (XP_002935265.1), and zebrafish (NP_001096089.2). Consensus sequences are shown below. (b) AlphaFold model of INPP5E 3D structure (AF-Q9NRR6-F1) depicting predicted locations of CLS1 (red), CLS2 (green), CLS3 (pink) and CLS4 (yellow). Active site in cyan. Beta-strands and alpha-helices in yellow and brown, respectively. Proline-rich N-terminal region (aa 1–200), predicted to be highly flexible, is not shown. CLS1 is probably also part of a flexible region, and its position in the AlphaFold model has a low confidence score (pLDDT). See Uniprot entry Q9NRR6 for more details. (c) Rescue assay assessing the ability of INPP5E or its mutants to lower the abnormally high TULP3 levels characteristic of INPP5E-KO cilia. The indicated constructs were transfected into INPP5E-KO RPE1 cells, generated via CRISPR-Cas9 (Figure 4—figure supplement 2). Cells were fixed and stained for EGFP, acetylated tubulin (AcTub), TULP3, and DNA (DAPI), as indicated. Scale bar, 10 µm. Note how untransfected INPP5E-KO cells have high ciliary TULP3 levels, as previously described. Transfected cell cilia are labeled with asterisks in the merge panels: yellow asterisks for rescued TULP3-negative cilia, and red asterisks for non-rescued TULP3-positive cilia. (d) Quantitation of the rescue experiment shown in (c). For each construct, the percentage of TULP3-negative transfected-cell cilia was counted. Data come from five independent experiments. Each point in the graph indicates an independent transfection. Between 12 and 39 transfected-cell cilia were counted per transfection (with exception of the highest data point in ΔCLS2, where only 9 cilia could be counted). Experiments were performed in parallel with two different INPP5E-KO clones (clones 3 and 12). Graph shows individual data points, color-coded by clone as indicated, and the overall median is indicated with a line. Two-way ANOVA revealed significant differences between constructs (p<0.0001) but no significant differences between the clones. All data were then analyzed by one-way ANOVA followed by Tukey tests. Significance is shown relative to EGFP unless otherwise indicated. p<0.0001 (****); p<0.05 (*); not significant (n.s.).

Figure 4—source data 1. Source data for Figure 4d.
Data from n=5 independent experiments.

Figure 4.

Figure 4—figure supplement 1. Generation of puromycin-sensitive hTERT-RPE1 cells by CRISPR/Cas9.

Figure 4—figure supplement 1.

(a) hTERT-RPE1 cells were originally immortalized with pGRN145, a plasmid expressing both human telomerase (hTERT) and puromycin N-acetyltransferase (PAC) from Streptomyces alboniger, a 199 aa enzyme that confers puromycin resistance (top). Three single guide RNAs were selected in the region encoding aa 25–62 of PAC (bottom). A pool of these gRNAs was then used for CRISPR/Cas9-mediated knockout of PAC gene in hTERT-RPE1 cells. (b) Phase contrast images of WT and PAC-KO hTERT-RPE1 cells right before (0 hr) or after treatment for 60 hr with 2 µg/ml puromycin. This treatment was sufficient to completely kill PAC-KO cells but did not noticeably affect WT cells. (c) Dose-response curves showing percentage confluence of WT or PAC-KO cells after 60 hr treatment with the indicated puromycin doses. Approximate IC50 values are 18 µg/ml for WT and 0.6 µg/ml for PAC-KO cells, a 30-fold difference.
Figure 4—figure supplement 2. Generation of INPP5E-KO hTERT-RPE1 cells by CRISPR/Cas9.

Figure 4—figure supplement 2.

(a) Top: diagram of human INPP5E gene depicting its 10 exons as boxes, with coding sequence in orange. Middle: the three indicated guide RNAs (arrows) were used to target INPP5E exon-1 in RPE-PS cells using CRISPR. Bottom: the genomic regions of interest from clones 3 and 12 were PCR-amplified, ligated into pJET1.2/blunt vector, transformed, and DNA from bacterial colonies sequenced (6 and 4 colonies for clones 3 and 12, respectively). All sequenced clones contained the same truncating mutation (P31Rfs101), which causes frameshift and only leaves intact the first 30 residues of INPP5E. (b) Immunofluorescence staining of clone-3 (INPP5E-KO) and its parental RPE-PS cells (WT) with the indicated antibodies. Ciliary staining of INPP5E is seen in WT but not INPP5E-KO cells, which only show non-specific staining outside cilia. Same results were obtained for clone-12 (not shown). (c) Immunofluorescence staining of clone-3 (INPP5E-KO) and its parental RPE-PS cells (WT) with the indicated antibodies. As previously reported, TULP3 strongly accumulates in INPP5E-KO cilia. Same results were obtained for clone-12 (not shown).
Figure 4—figure supplement 3. CLS1 and CLS4 mutants are only seen at the transition zone in methanol-fixed cells.

Figure 4—figure supplement 3.

(a) The indicated EGFP-INPP5E constructs were expressed in hTERT-RPE1 cells, which were fixed with methanol as described in Materials and methods. Immunofluorescence was carried out with antibodies against EGFP, polyglutamylated alpha-tubulin (polyE), and gamma-tubulin (γTub), as indicated. Scale bar, 5 mm. (b) Percentage of cilia with the specified EGFP-INPP5E localization was quantified for each of the constructs in (a). Data are from an individual experiment. The number of transfected cell cilia counted for each construct was, from left to right: n=31,13,15,10,12,10. (c) Pixel intensity profiles of the images in (a). A line was drawn along the cilium using Fiji/ImageJ, and the Plot Profile function of this program was used to obtain the densitometric data for each channel. Note how EGFP-INPP5E WT, ΔCLS1, ΔCLS4 and ΔCLS1 +4 accumulate at the transition zone, distal from the basal body (γTub, blue) and proximal to the axoneme (polyE, red).