ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis

Abstract

Despite the importance of cilia in cell signaling and motility, the molecular mechanisms regulating cilium formation remain incompletely understood. Herein, we characterize enkurin domain-containing protein 1 (ENKD1) as a novel centrosomal protein that mediates the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole to promote ciliogenesis. We show that Enkd1 knockout mice possess ciliogenesis defects in multiple organs. Super-resolution microscopy reveals that ENKD1 is a stable component of the centrosome throughout the ciliogenesis process. Simultaneous knockdown of ENKD1 and CP110 significantly reverses the ciliogenesis defects induced by ENKD1 depletion. Protein interaction analysis shows that ENKD1 competes with centrosomal protein 97 (CEP97) in binding to CP110. Depletion of ENKD1 enhances the CP110-CEP97 interaction and detains CP110 at the mother centriole. These findings thus identify ENKD1 as a centrosomal protein and uncover a novel mechanism controlling CP110 removal from the mother centriole for the initiation of ciliogenesis.

Keywords: CP110; ENKD1; centrosome; ciliogenesis; cilium.

Figure 1. Enkd1 knockout mice display ciliogenesis defects in multiple organs

1. A

   Strategy for the generation of Enkd1 knockout mice. Deletion of a fragment containing the 173 bp exon 3 and adjacent flanking sequences results in the disruption of ENKD1 protein expression.

2. B

   Identification of wild‐type (WT), heterozygous (HZ), and knockout (KO) mice by PCR and agarose gel electrophoresis.

3. C

   Immunoblotting (IB) of ENKD1 and α‐tubulin in wild‐type and Enkd1 knockout mice.

4. D–F

   Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α‐tubulin (Ace‐α‐tubulin) and γ‐tubulin and DAPI. Scale bar, 2 µm.

5. G, H

   ERG recording images (G) and a‐b wave amplitudes (H, n = 5 mice) show the difference in the ERG a‐b wave amplitude between wild‐type and Enkd1 knockout mice.

6. I, J

   VEP recording images (I) and N2‐P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2‐P2 wave amplitude between wild‐type and Enkd1 knockout mice.

7. K–M

   Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α‐tubulin and DAPI. To quantify the percentage of ciliated cells (L), > 200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm.

8. N–Q

   Images (N) and quantification of the percentage of flagellated sperm (O, n = 9 mice), flagellar length (P, n = 100 sperm from nine mice), and the percentage of sperm with normal motility among flagellated sperm (Q, n = 9 mice) for wild‐type and Enkd1 knockout mice. To quantify the percentage of flagellated sperm (O), > 200 sperm were analyzed for each mouse. To quantify the percentage of sperm with normal motility among flagellated sperm (Q), > 200 flagellated sperm were analyzed for each mouse. In panel N, the arrows indicate two spermatozoa without tails. Scale bar, 50 µm.

9. R, S

   Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), > 200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm.

Data information: Data are presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), ns, not significant.

Figure EV1. Enkd1 knockout mice display ciliogenesis defects in multiple organs

1. A

   Immunoblotting of ENKD1 and GAPDH in various tissues of wild‐type and Enkd1 knockout mice.

2. B–G

   Schematic diagrams (B), ECG recording images (C), and quantifications of R wave duration (D, n = 5 mice), S wave duration (E, n = 5 mice), QRS wave duration (F, n = 5 mice,) and R‐R interval (G, n = 5 mice) show the difference in ECG between wild‐type and Enkd1 knockout mice.

3. H, I

   Immunofluorescence images (H) and quantification of the percentage of ciliated cells (I, n = 3 mice) of mouse trachea stained with antibodies against acetylated α‐tubulin and CEP164 and DAPI. To quantify the percentage of ciliated cells (I), > 150 cells from six images were analyzed for each mouse. Scale bar, 10 µm.

Data information: Data are presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. P < 0.05 (*), P < 0.001 (***), ns, not significant. Source data are available online for this figure.

Figure 2. ENKD1 promotes the initiation of ciliogenesis in cultured cells

1. A

   Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum‐free medium for 48 h.

2. B–D

   Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum‐free medium and stained with antibodies against acetylated α‐tubulin and γ‐tubulin and DAPI. To quantify the percentage of ciliated cells (C), > 100 cells were analyzed for each experiment. Scale bar, 5 µm.

3. E

   Immunoblot analysis of ENKD1 and β‐actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum‐free medium for 48 h.

4. F–H

   Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) and ciliary length (H, n = 100 cilia from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum‐free medium, and stained with antibodies against acetylated α‐tubulin and γ‐tubulin and DAPI. To quantify the percentage of ciliated cells (G), > 150 cells were analyzed for each experiment. Scale bar, 5 µm.

5. I, J

   Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum‐free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), > 80 cells were analyzed for each experiment. Scale bar, 2 µm.

6. K, L

   Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP‐ENKD1, GFP‐ENKD1‐N, GFP‐ENKD1‐M, or GFP‐ENKD1‐C, cultured in serum‐free medium, and stained with the antibody against acetylated α‐tubulin and DAPI. The siRNA‐resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), > 120 cells were analyzed for each experiment. Scale bar, 3 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. P < 0.01 (**), P < 0.001 (***), ns, not significant.

Figure EV2. ENKD1 promotes the initiation of ciliogenesis in cultured cells

1. A

   Immunoblot analysis of ENKD1 and β‐actin in NIH3T3 cells transfected with control or ENKD1 siRNAs and cultured in serum‐free medium for 48 h.

2. B–D

   Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 65 cilia from three independent experiments) for NIH3T3 cells transfected with control or ENKD1 siRNAs, cultured in serum‐free medium and stained with antibodies against acetylated α‐tubulin and γ‐tubulin and DAPI. To quantify the percentage of ciliated cells (C), > 90 cells were analyzed for each experiment. Scale bar, 10 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. P < 0.05 (*), P < 0.01 (**), ns, not significant. Source data are available online for this figure.

Figure EV3. ENKD1 is localized to the centrosome

1. A

   Immunostaining of ENKD1 and Centrin in MEF cells cultured in serum‐free medium. Scale bar, 1 µm.

2. B–D

   Immunofluorescence images (B) and quantifications of fluorescence intensity of ENKD1 (C, n = 50 cells from three independent experiments) and Centrin (D, n = 50 cells from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum‐free medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.

3. E

   Immunofluorescence images of NIH3T3 cells cultured in normal serum or serum‐free medium, and stained with antibodies against ENKD1 and γ‐tubulin and DAPI. Scale bar, 1 µm.

4. F

   Immunofluorescence images of NIH3T3 cells cultured in normal serum or serum‐free medium, and stained with antibodies against ENKD1 and acetylated α‐tubulin and DAPI. Scale bar, 1 µm.

5. G, H

   Immunoblot analysis (G) and immunofluorescence images (H) showing the centrosomal localization domain of ENKD1, in RPE1 cells transfected with GFP‐ENKD1, GFP‐ENKD1‐N, GFP‐ENKD1‐M, or GFP‐ENKD1‐C, cultured in a serum‐starved condition, and stained with the anti‐γ‐tubulin antibody and DAPI. Scale bar, 2 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. P < 0.001 (***), ns, not significant. Source data are available online for this figure.

Figure 3. ENKD1 is a component of the centrosome

1. A

   Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.

2. B

   Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum‐starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 1 µm.

3. C

   Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum‐starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 2 µm.

4. D–F

   3D‐SIM super‐resolution images (D), quantification of the outer toroid diameters (E, n = 20 cells from three independent experiments), and a model illustrating the relative positions of γ‐tubulin, Ninein, ENKD1, CEP164, CEP912, and Centrin (F) within a centriole. RPE1 cells were serum starved and stained with antibodies against the indicated proteins. Scale bar, 1 µm.

5. G

   Immunofluorescence images of RPE1 cells cultured in normal serum or serum‐free medium, and stained with antibodies against ENKD1 and γ‐tubulin and DAPI. Scale bar, 1 µm.

6. H

   Immunofluorescence images of RPE1 cells cultured in serum‐free medium, and stained with antibodies against ENKD1 and acetylated α‐tubulin and DAPI. Scale bar, 1 µm.

7. I

   Immunofluorescence images of RPE1 cells transfected with GFP‐ENKD1 or GFP vector, cultured in a serum‐starved condition, and stained with the antibody against acetylated α‐tubulin and DAPI. Scale bar, 1 µm.

8. J

   Schematic diagrams showing the centrosomal localization domain of ENKD1.

9. K

   3D‐SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum‐free medium (0, 12, 24, or 48 h of serum starvation), and stained with antibodies against ENKD1 and acetylated α‐tubulin. Scale bar, 1 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SD.

Figure 4. ENKD1 functions in the removal of CP110 from the mother centriole

1. A

   A table summarizing the centrosomal localization of cilium‐related proteins in ENKD1‐depleted RPE1 cells.

2. B, C

   Immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum‐free medium, and stained with antibodies against CP110 and acetylated α‐tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), > 120 cells were analyzed for each experiment. Scale bar, 1 µm.

3. D, E

   Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum‐free medium, and stained with antibodies against CEP97 and acetylated α‐tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), > 150 cells were analyzed for each experiment. Scale bar, 1 µm.

4. F–H

   Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum‐free medium for 120 h, and stained with antibodies against CP110 and acetylated α‐tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), > 80 cells were analyzed for each experiment. Scale bar, 1 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. P < 0.01 (**), P < 0.001 (***).

Figure EV4. Loss of ENKD1 does not affect the structural integrity of the centrosome and ciliary vesicle formation

1. A–L

   Immunostaining images of γ‐tubulin and CEP192 (A), PLK4 (C), CEP135 (E), CPAP (G), CEP164 (I), and Rab8a (K) in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum‐free medium. Quantification of the percentage of cells with centrosomal CEP192 (B, n = 3 independent experiments), PLK4 (D, n = 3 independent experiments), CEP135 (F, n = 3 independent experiments), CPAP (H, n = 3 independent experiments), CEP164 (J, n = 3 independent experiments), and Rab8a (L, n = 3 independent experiments). For the quantification, > 150 cells were analyzed for each experiment. Scale bar, 1 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. ns, not significant. Source data are available online for this figure.

Figure 5. ENKD1 regulates the CP110–CEP97 interaction

1. A–C

   Immunoblot analysis (A), immunofluorescence images (B), and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum‐free medium for 48 h, and stained with antibodies against CP110 and acetylated α‐tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), > 180 cells were analyzed for each experiment. Scale bar, 1 µm.

2. D

   Immunoblot analysis of CP110 and β‐actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum‐free medium for 48 h.

3. E, F

   Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum‐starved condition, and stained with antibodies against γ‐tubulin and acetylated α‐tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.

4. G, H

   Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110–CEP97 interaction in the retinal tissues of wild‐type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β‐actin band.

5. I, J

   Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110–CEP97 interaction in serum‐starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β‐actin band.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), ns, not significant.

Figure EV5. Loss of ENKD1 does not affect the length of centrioles

1. A, B

   Immunofluorescence images (A) and quantification of the length of centrioles (B, n = 30 centrioles from three independent experiments) for U2OS cells transfected with the control or ENKD1 siRNAs, cultured in normal serum medium, and stained with antibodies against CEP164 and acetylated α‐tubulin and DAPI. Scale bar, 1 µm.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two‐tailed t‐test was performed. ns, not significant. Source data are available online for this figure.

Figure 6. ENKD1 competes with CEP97 in binding to CP110

1. A

   Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti‐ENKD1 (upper panel) or anti‐CP110 (lower panel) antibody or the corresponding control IgG.

2. B–D

   Immunoprecipitation and immunoblotting showing the interaction of GFP‐ENKD1 with Flag‐CP110 (B and upper panel of D) or HA‐CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D).

3. E

   Identification of the domains of ENKD1 mediating its interaction with CP110.

4. F

   Identification of the domains of CP110 mediating its interaction with ENKD1.

5. G

   Schematic diagrams showing the domains of ENKD1 and CP110 that mediate their interaction.

6. H

   GST pull‐down showing the interaction of purified GST‐CP110‐N with purified His‐ENKD1‐M.

7. I, J

   Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA‐CP110 with Flag‐CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag‐CEP97 band was normalized to that of the β‐actin band.

Data information: Data are from three independent biological repeats and presented as mean ± SEM. Non‐parametric one‐way ANOVA analysis was performed. P < 0.001 (***).