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CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base.

Wang WJ, Tay HG, Soni R, Perumal GS, Goll MG, Macaluso FP, Asara JM, Amack JD, Tsou MF - Nat. Cell Biol. (2013)

Bottom Line: The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment.A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear.Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly. CEP162 interacts with core transition zone components, and mediates their association with microtubules. Loss of CEP162 arrests ciliogenesis at the stage of transition zone assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme and ectopically assemble transition zone components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein pre-tethered at centriole distal ends before ciliogenesis to promote and restrict transition zone formation specifically at the cilia base.

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Loss of CEP162 blocks ciliogenesis at the stage of TZ assemblya–f, RPE1 cells transfected with control or CEP162 siRNA followed by serum starvation for 48 hours were stained with antibodies indicated. Only G1 cells that contain two un-duplicated centrioles were scored. In some controls (b, & d-f), both ciliated and non-ciliated G1 cells were shown. Note that the removal of CP110 (b), or the recruitment of IFT88 (d), TCTN1 (e), and cc2d2a (f) occur at wild-type centrioles before cilia formed, as well as at CEP162-deficient centrioles where cilia failed to form. Data were collected from 3 independent experiments. The percentage of cells showing the presence or absence of indicated marker at centrioles was indicated (mean ± SD). No significant differences were detected between control and CEP162 depleted cells. g, TEM images of basal bodies in RPE1 cells transiently transfected with control (left) or CEP162 (middle & right) siRNA, followed by 48hr serum starvation for cilia formation. Serial sections of a basal body (right) in CEP162 knockdown cell were shown in addition to the single-sectioned image (middle). Arrows indicate small membrane vesicles connected to CEP162-deficient centrioles through the distal appendage. Schematic diagrams summarizing the phenotype were shown. Gray links: Membrane-to-MT connections; red: membrane-to-appendage connections. h–k, Control or CEP162-depleted RPE1 cells as described in d–f were examined for the presence of indicated TZ components (red). Data were collected from 3 independent experiments (N=3), and mean% ± SD% was shown. The significance (two-tailed t-test) is indicated, *P < 0.05, **P < 0.001. l, The protein levels of indicated TZ components were examined in control or CEP162-depleted RPE1 cells by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. Total cell counts are shown on the figures as n). More than 40 cells were analyzed for each independent experiment. Table S1 presents the source data for the graphs.
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Figure 3: Loss of CEP162 blocks ciliogenesis at the stage of TZ assemblya–f, RPE1 cells transfected with control or CEP162 siRNA followed by serum starvation for 48 hours were stained with antibodies indicated. Only G1 cells that contain two un-duplicated centrioles were scored. In some controls (b, & d-f), both ciliated and non-ciliated G1 cells were shown. Note that the removal of CP110 (b), or the recruitment of IFT88 (d), TCTN1 (e), and cc2d2a (f) occur at wild-type centrioles before cilia formed, as well as at CEP162-deficient centrioles where cilia failed to form. Data were collected from 3 independent experiments. The percentage of cells showing the presence or absence of indicated marker at centrioles was indicated (mean ± SD). No significant differences were detected between control and CEP162 depleted cells. g, TEM images of basal bodies in RPE1 cells transiently transfected with control (left) or CEP162 (middle & right) siRNA, followed by 48hr serum starvation for cilia formation. Serial sections of a basal body (right) in CEP162 knockdown cell were shown in addition to the single-sectioned image (middle). Arrows indicate small membrane vesicles connected to CEP162-deficient centrioles through the distal appendage. Schematic diagrams summarizing the phenotype were shown. Gray links: Membrane-to-MT connections; red: membrane-to-appendage connections. h–k, Control or CEP162-depleted RPE1 cells as described in d–f were examined for the presence of indicated TZ components (red). Data were collected from 3 independent experiments (N=3), and mean% ± SD% was shown. The significance (two-tailed t-test) is indicated, *P < 0.05, **P < 0.001. l, The protein levels of indicated TZ components were examined in control or CEP162-depleted RPE1 cells by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. Total cell counts are shown on the figures as n). More than 40 cells were analyzed for each independent experiment. Table S1 presents the source data for the graphs.

Mentions: To determine the function of CEP162, the protein was depleted from cells by RNAi (Fig. 2 & 3). Unlike a previous report29, neither mitosis nor centriole duplication were affected by CEP162 depletion (Fig. 2a & b). However, in the absence of CEP162, ciliogenesis was specifically disrupted in retinal pigment epithelial (RPE1) cells (Fig. 2c–e). In vivo studies using zebrafish indicated that the zebrafish CEP162 homolog is maternally supplied and ubiquitously expressed during early embryonic development (Fig. 2f). Partial knockdown of zebrafish Cep162 using antisense morpholino oligonucleotides (MO) results in a series of phenotypes known to associate with cilia defects, including body curvature, hydrocephalus, and left-right asymmetry defects (Fig. 2g–k), which could be partially rescued by expressing RNAi-resistant form of CEP162 (Fig. 2j & k).


CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base.

Wang WJ, Tay HG, Soni R, Perumal GS, Goll MG, Macaluso FP, Asara JM, Amack JD, Tsou MF - Nat. Cell Biol. (2013)

Loss of CEP162 blocks ciliogenesis at the stage of TZ assemblya–f, RPE1 cells transfected with control or CEP162 siRNA followed by serum starvation for 48 hours were stained with antibodies indicated. Only G1 cells that contain two un-duplicated centrioles were scored. In some controls (b, & d-f), both ciliated and non-ciliated G1 cells were shown. Note that the removal of CP110 (b), or the recruitment of IFT88 (d), TCTN1 (e), and cc2d2a (f) occur at wild-type centrioles before cilia formed, as well as at CEP162-deficient centrioles where cilia failed to form. Data were collected from 3 independent experiments. The percentage of cells showing the presence or absence of indicated marker at centrioles was indicated (mean ± SD). No significant differences were detected between control and CEP162 depleted cells. g, TEM images of basal bodies in RPE1 cells transiently transfected with control (left) or CEP162 (middle & right) siRNA, followed by 48hr serum starvation for cilia formation. Serial sections of a basal body (right) in CEP162 knockdown cell were shown in addition to the single-sectioned image (middle). Arrows indicate small membrane vesicles connected to CEP162-deficient centrioles through the distal appendage. Schematic diagrams summarizing the phenotype were shown. Gray links: Membrane-to-MT connections; red: membrane-to-appendage connections. h–k, Control or CEP162-depleted RPE1 cells as described in d–f were examined for the presence of indicated TZ components (red). Data were collected from 3 independent experiments (N=3), and mean% ± SD% was shown. The significance (two-tailed t-test) is indicated, *P < 0.05, **P < 0.001. l, The protein levels of indicated TZ components were examined in control or CEP162-depleted RPE1 cells by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. Total cell counts are shown on the figures as n). More than 40 cells were analyzed for each independent experiment. Table S1 presents the source data for the graphs.
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Figure 3: Loss of CEP162 blocks ciliogenesis at the stage of TZ assemblya–f, RPE1 cells transfected with control or CEP162 siRNA followed by serum starvation for 48 hours were stained with antibodies indicated. Only G1 cells that contain two un-duplicated centrioles were scored. In some controls (b, & d-f), both ciliated and non-ciliated G1 cells were shown. Note that the removal of CP110 (b), or the recruitment of IFT88 (d), TCTN1 (e), and cc2d2a (f) occur at wild-type centrioles before cilia formed, as well as at CEP162-deficient centrioles where cilia failed to form. Data were collected from 3 independent experiments. The percentage of cells showing the presence or absence of indicated marker at centrioles was indicated (mean ± SD). No significant differences were detected between control and CEP162 depleted cells. g, TEM images of basal bodies in RPE1 cells transiently transfected with control (left) or CEP162 (middle & right) siRNA, followed by 48hr serum starvation for cilia formation. Serial sections of a basal body (right) in CEP162 knockdown cell were shown in addition to the single-sectioned image (middle). Arrows indicate small membrane vesicles connected to CEP162-deficient centrioles through the distal appendage. Schematic diagrams summarizing the phenotype were shown. Gray links: Membrane-to-MT connections; red: membrane-to-appendage connections. h–k, Control or CEP162-depleted RPE1 cells as described in d–f were examined for the presence of indicated TZ components (red). Data were collected from 3 independent experiments (N=3), and mean% ± SD% was shown. The significance (two-tailed t-test) is indicated, *P < 0.05, **P < 0.001. l, The protein levels of indicated TZ components were examined in control or CEP162-depleted RPE1 cells by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. Total cell counts are shown on the figures as n). More than 40 cells were analyzed for each independent experiment. Table S1 presents the source data for the graphs.
Mentions: To determine the function of CEP162, the protein was depleted from cells by RNAi (Fig. 2 & 3). Unlike a previous report29, neither mitosis nor centriole duplication were affected by CEP162 depletion (Fig. 2a & b). However, in the absence of CEP162, ciliogenesis was specifically disrupted in retinal pigment epithelial (RPE1) cells (Fig. 2c–e). In vivo studies using zebrafish indicated that the zebrafish CEP162 homolog is maternally supplied and ubiquitously expressed during early embryonic development (Fig. 2f). Partial knockdown of zebrafish Cep162 using antisense morpholino oligonucleotides (MO) results in a series of phenotypes known to associate with cilia defects, including body curvature, hydrocephalus, and left-right asymmetry defects (Fig. 2g–k), which could be partially rescued by expressing RNAi-resistant form of CEP162 (Fig. 2j & k).

Bottom Line: The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment.A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear.Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly. CEP162 interacts with core transition zone components, and mediates their association with microtubules. Loss of CEP162 arrests ciliogenesis at the stage of transition zone assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme and ectopically assemble transition zone components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein pre-tethered at centriole distal ends before ciliogenesis to promote and restrict transition zone formation specifically at the cilia base.

Show MeSH
Related in: MedlinePlus