Primary Cilium Formation and Ciliary Protein Trafficking Is Regulated by the Atypical MAP Kinase MAPK15 in Caenorhabditis elegans and Human Cells

Abstract

Motile and immotile (or primary) cilia are microtubule-based structures that mediate multiple cellular functions, including the transduction of environmental cues, developmental signaling, cellular motility, and modulation of fluid flow. Although their core architectures are similar, motile and primary cilia exhibit marked structural differences that underlie distinct functional properties. However, the extent to which ciliogenesis mechanisms are shared between these different cilia types is not fully described. Here, we report that the atypical MAP kinase MAPK15 (ERK7/8), implicated in the formation of vertebrate motile cilia, also regulates the formation of primary cilia in Caenorhabditis elegans sensory neurons and human cells. We find that MAPK15 localizes to a basal body subdomain with the ciliopathy protein BBS7 and to cell-cell junctions. MAPK15 also regulates the localization of ciliary proteins involved in cilium structure, transport, and signaling. Our results describe a primary cilia-related role for this poorly studied member of the MAPK family in vivo, and indicate a broad requirement for MAPK15 in the formation of multiple ciliary classes across species.

Keywords: C. elegans; MAPK15; basal body; primary cilia.

Figure 1

MAPK-15 regulates the morphologies of sensory neuron cilia in C. elegans. (A) Genomic structure of C05D10.2 (mapk-15), showing three predicted isoforms (a, b, and c), the TEY activation motif, ATP-binding site, and the mutant alleles (gk1234 and oy112) employed in this study. Exons are denoted by gray rectangles. (B) Representative images of DiI uptake in the amphid (head) and phasmid (tail) neurons of wild-type (WT) and mapk-15(gk1234) worms. Left images from a standard dye-filling assay; right images from a sensitized assay (low concentration DiI). Transgene comprises WT mapk-15 genomic sequence (incorporating a, b, and c isoforms), together with endogenous upstream regulatory sequence. Numbers indicate the percentage of animals exhibiting the phenotype (n > 30 for each strain). Anterior is at left. Bar, 10 μm. (C and D) Representative images and quantification of AWB and ADF cilia defects in the indicated genetic backgrounds. AWB cilia morphologies categorized as: Type I, two normal cilia; Type II, one cilium missing; Type III, one misdirected cilium; Type IV, abnormally branched cilia; Type V, two short cilia; and Type VI, both cilia absent. ADF cilium morphologies categorized as: Type I, two normal cilia; Type II, collapsed cilia; Type III, one misdirected cilium; Type IV, two short cilia; and Type V, one missing cilium. White arrows denote cilia. White arrowheads denote the cilia base. Numbers at top indicate the number of scored neurons. AWB neurons visualized using str-1p::gfp; ADF neurons visualized using srh-142p::rfp. CTD; C-terminal domain. *** P < 0.001 (Generalized Linear Model test and Bonferroni corrections). Anterior is at left. Bar, 5 µm. (E) Box plots of ASH and PHA cilium lengths in WT and mapk-15(gk1234) worms. Horizontal lines denote 25, 50, and 75th percentiles; whiskers denote the full range. Numbers indicate the number of neurons quantified. ASH neurons visualized using sra-6p::gfp; PHA neurons visualized using flp-15p::gfp. *** P < 0.001 (Welch’s t-test vs. WT).

Figure 2

Ultrastructure of amphid channel cilia in WT and mapk-15(gk1234) worms. Representative transmission election microscopy images from serial cross-sections of the amphid pore. Large panels show the entire pore; small panels show individual ciliary axonemes at higher magnifications. Schematics summarize the ultrastructure phenotypes (only 3 of the 10 axonemes are shown for simplicity). Section positions denoted above each image and in schematics. Arrowhead: misdirected ciliary axoneme. Arrows: mispositioned MTs or incomplete B-tubs. Bar, 200 nm (large panels) and 100 nm (small panels). B-tub, B-tubules; DS, distal segment; MS, middle segment; MT, microtubule; PCMC, periciliary membrane component; TZ transition zone.

Figure 3

MAPK-15 is expressed in ciliated sensory neurons and localizes to the ciliary axoneme, basal body, and apical junction. (A) Representative images of worms expressing GFP under the control of mapk-15 upstream regulatory sequences (mapk-15p). DiI costains six pairs of ciliated amphid head neurons and the pair of ciliated PHA/B phasmid tail neurons. Anterior is at left. Bar, 50 μm (left-most panels) and 15 μm (other panels). Lab, inner labial cells; quad, outer labial quadrant cells. (B) Representative images of head sensory neuronal cilia in worms expressing MAPK-15c::GFP (endogenous promoter) and the transition zone marker, TMEM-107::RFP. Anterior is at left. Bar, 2 μm. (C) Representative images of PHA/B and AWB cilia from worms expressing GFP::MAPK-15c (nphp-4 or str-1 promoter) and either mapk-15p::myr-mCherry (PHA/B) or str-1p::mCherry (AWB). Arrows, ciliary axoneme; arrowhead, basal body; asterisk, apical junction. Anterior is at left. Bar, 5 µm. (D) Representative images of phasmid cilia from worms expressing mCherry::MAPK-15c (bbs-8 promoter) and either the basal body marker, GFP::GASR-8 (nphp-4 promoter), or the transition zone marker, NPHP-4::GFP (endogenous promoter). Arrows, ciliary axoneme; arrowhead, basal body; asterisk, apical junction. Anterior is at left. Bar, 5 µm. (E) Representative images of PHA/B and ADF cilia from worms expressing mCherry::MAPK-15c (endogenous promoter for PHA/B; srh-142 promoter for ADF) and AJM-1a::GFP (nphp-4 promoter for PHA/B) or AJM-1::CFP (srh-142 promoter for ADF). Arrows, ciliary axoneme; arrowhead, basal body; asterisk, apical junction. Anterior is at left. Bar, 5 µm. (F) Cartoon representation of MAPK-15 localization (green) to the apical junction, basal body, and middle segments of amphid and phasmid channel cilia. Only one axoneme shown for simplicity. PCMC: periciliary membrane compartment. Socket and sheath cells are glial cells. (G) Representative images of MAPK-15c localization in ADF cilia of wild-type (WT) and mutant animals (n ≥ 16 each). Of hyls-1 mutants, 51% exhibit the shown phenotype. Arrows, ciliary axoneme; arrowhead, basal body; asterisk, apical junction. Anterior is at left. Bar, 5 µm. (H) Representative images of MAPK-15c and AJM-1 localization in ADF cilia of WT and hyls-1 mutant animals (n ≥ 20 each). Arrows, ciliary axoneme; arrowhead, basal body; asterisk, apical junction. Anterior is at left. Bar, 5 µm.

Figure 4

MAPK15 localizes to the ciliary base and regulates ciliogenesis in human cells. (A) Representative images of hTERT-RPE1 cells transiently transfected with MAPK15::GFP, serum starved for 24 hr ,and stained for γ-tubulin (centrosome), ARL13B (cilia), and DNA. Insets show higher magnification images of the centrosomal region. Bar, 5 µm (main images) and 1 µm (magnifications). (B) Representative images of RPTEC/TERT cells transiently transfected with MAPK15::GFP and stained for polyglutamylated tubulin (polyglu. tub, cilia), ZO-1 (tight junctions), and DNA. Blue and yellow dotted lines indicate positions of the XZ and YZ cross‐sections, respectively. Arrows, tight junctions; arrowheads, basal body. Bar, 5 µm. (C) Representative images of serum-starved hTERT-RPE1 stained for endogenous MAPK15, polyglutamylated tubulin (centrioles and cilia), and DNA. Regions within white boxes are shown at higher magnifications in the lower panels. Bar, 5 µm (main images) and 1 µm (magnifications). (D) Representative images of hTERT-RPE1 cells stained for the indicated endogenous proteins. Schematic of centrosome depicts CEP164 and ODF2 localization to distal and subdistal appendages, respectively. Bar, 1 µm. (E–G) Representative images of serum-starved hTERT-RPE1 cells transfected with scrambled siRNA (Scr. siRNA) or an siRNA targeting MAPK15 (MAPK15-si1). Cells stained for ARL13B, polyglutamylated tubulin, and DNA. Regions within white boxes shown at higher magnifications to the right. Bar, 5 µm (main images) and 1 µm (magnifications). ARL13B staining used to mark cilia and determine the percentages of ciliated cells (F) and cilia lengths (G). Data shown are means of five independent experiments. Error bars are ± SEM. For (F), 100–150 cells were assessed per experimental condition. For (G), 30–60 cilia were scored per experimental condition. *** P < 0.001 (ANOVA followed by Dunnett’s post hoc test; vs. Scr. siRNA). (H) hTERT-RPE1 cells stably expressing the indicated rescue constructs were treated as in (E) and percentages of ciliated cells determined using ARL13B as a cilia marker. Data points represented as mean ± SEM of three independent experiments. Per experimental condition, 100–150 cells were scored. *** P < 0.001 (ANOVA followed by Bonferroni post hoc test).

Figure 5

Localization of transition zone (TZ) and basal body (BB) proteins is disrupted in MAPK15-depleted human cells and MAPK-15 mutant worms. (A and B) Representative images and quantification of NPHP-4::GFP, JBTS-14::GFP, DYF-19::GFP, and GFP::GASR-8 localization in the AWB neurons of wild-type (WT) and mapk-15(gk1234) worms. AWB neurons visualized using str-1p::mCherry. GFP-tagged proteins expressed under the str-1 or srd-23 (GASR-8) promoter. Transgene used for rescue is mapk-15p::mapk-15c. Numbers of neurons examined are indicated above each data set. * P < 0.05, ** P < 0.01, and *** P < 0.001 (Generalized Linear Model test and Bonferroni corrections). Arrowheads denote GFP puncta. Arrows denote ciliary axonemes. Anterior is at left. Bar, 5 µm (all images identically scaled). (C and D) Representative images of hTERT-RPE1 cells transfected with nontargeting scrambled siRNA (Scr. siRNA) or an siRNA targeting MAPK15 (MAPK15-si1), serum starved for 48 hr, and stained for NPHP4 (C) or FBF1 (D), polyglutamylated (polyglu.) tubulin, and DNA. Regions within white boxes shown at higher magnification in smaller panels. Bar, 10 µm (large panels) and 2 µm (small panels). Box-and-whisker plots show the relative intensity of NPHP4 at the transition zone (TZ) (C), and FBF1 at the centrosome (D). Horizontal lines denote the 25, 50, and 75th percentiles; whiskers denote the full range. One representative experiment out of three is shown. Per experimental condition, 30–60 cells were scored.* P < 0.05 and *** P < 0.001 (ANOVA followed by Dunnett’s post hoc test; vs. Scr. siRNA). A.U.: arbitrary units.

Figure 6

Localization of ciliary membrane and BBSome proteins in C. elegans and human cells. (A and B) Representative images and quantification of ciliary transmembrane STR-163::GFP (seven transmembrane receptor) and TAX-2::GFP (cyclic nucleotide-gated ion channel subunit) localization in the AWB neurons of wild-type (WT) and mapk-15(gk1234) worms. AWB neurons visualized using an str-1p::mCherry reporter. STR-163 localization categorized as: Type I, signal restricted to cilia; Type II, additional diffuse localization in the PCMC; and Type III, additional localization in the distal dendrite. TAX-2 localization was categorized as: Type I, restricted to ciliary middle segment; Type II, additional localization in the distal dendrite; and Type III, localization throughout the entire ciliary axonemes. N-values indicated above each data set. Only cells retaining both ciliary axonemes were analyzed. Arrowheads denote cilium base. Arrows denote ciliary axoneme (shown only for Type I). Anterior is at left. Bar, 5 µm. (C and D) Representative images and quantification of ciliary transmembrane TAX-4::GFP (cyclic nucleotide-gated ion channel subunit) and membrane-associated ARL-13::tagRFP localization in the AWB neurons of WT and mapk-15(gk1234) worms. Panels show the percentage of worms with indicated pattern of TAX-4 and ARL-13 localization (n ≥ 22 for WT and mapk-15 worms). Only cells retaining both ciliary axonemes were analyzed. Arrowheads denote cilium base. Arrows denote ciliary axonemes (shown only for WT images). Anterior is at left. Bar, 5 µm. (E) Representative images and quantification of OSM-9::GFP (TRPV channel) localization in OLQ neurons. Box plots show the ratio of GFP signal (maximum fluorescence) at the distal ciliary region compared with the proximal ciliary region (and distal dendrite). Horizontal lines denote 25, 50, and 75th percentile; whiskers denote the full range. N-values indicated above each data set. Arrows denote ciliary axonemes (shown only for WT). *** P < 0.001 (Welch’s t-test vs. WT). Anterior is at left. Bar, 5 µm. (F) Representative images and quantification of BBS-7::GFP localization in amphid and PHA/B (phasmid) cilia of WT and mapk-15(gk1234) worms. Schematics show normal and abnormal BBS-7 localizations at the ciliary base. For phasmids, normal and abnormal localization phenotypes also shown in schematics. n > 50 for each data set in graph. Arrowheads denote cilium base. Arrows denote ciliary axonemes (shown only for WT images). Anterior is at top in amphid images and at bottom in phasmid images. Bar, 2 µm. (G and H) Representative images of hTERT-RPE1 cells transfected with nontargeting scrambled siRNA (Scr. siRNA) or an siRNA targeting MAPK15 (MAPK15-si1), serum starved for 48 hr and stained for BBS5 (G) or BBS7 (H), acetylated tubulin (acetyl. tub), and DNA. Regions within white boxes shown at higher magnifications in smaller image panels. Bar, 10 µm (large panels) and 2 µm (small panels). Bar graphs show percentages of cilia positive for BBS5 (G) or BBS7 (H). Data points represented as mean ± SEM of three independent experiments. Per experimental condition, 100–150 cells were scored. * P < 0.05 and ** P < 0.001 (ANOVA followed by Dunnett’s post hoc test; vs. Scr. siRNA).

Figure 7

MAPK15 and BBS7 localize to a distinct subdomain near the distal end of the basal body in human cells. (A) Schematic depicting primary cilium orientation relative to the imaging plane for RPTEC/TERT and hTERT-RPE1 cells. Basal bodies and cilia imaged in cross-section orientation for RPTEC/TERT cells and longitudinal orientation for hTERT-RPE1 cells. (B) Representative single-color stimulated emission depletion (STED) image of endogenous MAPK15 at the basal body in RPTEC/TERT cells. Corresponding confocal images of MAPK15 and polyglutamylated tubulin staining shown to the right. Arrowheads indicate MAPK15 clusters. Bar, 200 nm. (C–L) Representative dual-color STED fluorescence images of RPTEC/TERT (C, E, G, I, and K) and hTERT-RPE1 (D, F, H, J, and L) cells costained with the indicated antibodies. Right-hand panels show merges of both STED images with corresponding confocal image of polyglutamylated tubulin (polyglu. tub) staining. Bar, 200 nm. (M) Scatter plots showing the distribution of ring diameters for ODF2, BBS7, MAPK15, CEP164, and FBF1 signals at the distal end of the basal body. *** P < 0.001 (Kruskel–Wallis followed by Dunnett’s post hoc test). (N) Cartoon summarizing the observed localization patterns. MAPK15/BBS7 are proposed to occupy a more proximal (and narrower) domain than CEP164/FBF1 at the distal appendages.