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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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CEP162 interacts with CEP290 and mediates its association with MTsa, Endogenous CEP162 or CEP290 in HEK293T cell extracts were immunoprecipitated, and co-precipitated proteins were probed with indicated antibodies by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. b, Mapping the CEP290 binding domain of CEP162. 293T cells expressing various Flag-tagged fragments of CEP162 were subjected to immunoprecipitations and western blot with antibodies as indicated. Uncropped images of immunoblots are presented in Supplementary Fig. S1. c, RPE1 cells at different cell cycle stages were processed for immunofluorescence with anti-CEP290 (red), anti-CEP162 (green), and anti-centrin (blue) antibodies. DNA (DAPI, blue). d, Spindle association of CEP290 depends on CEP162. RPE1 cells transfected with control, CEP162, or CEP290 siRNA for 72 hours were stained with indicated antibodies against acetylated-tubulin (green), CEP162 (red), and CEP290 (blue). DNA (DAPI, blue). e, Overexpression of CEP162 induced MT bundles recruiting endogenous CEP290. U2OS cells transiently expressing HA-tagged CEP162FL or HA-tagged CEP162tNC1C2 were stained with antibodies against CEP162 (green), CEP290 (red) and DAPI (DNA, blue). f, Overexpression of CEP162 but not CEP290 induced MT bundles. NIH3T3 cells transiently expressing HA-tagged human CEP162FL or GFP-tagged mouse CEP290FL were stained with antibodies against α-tubulin (Red), CEP162 (HA, green) or CEP290 (GFP, green). g, Centriolar localizations of CEP290 and CEP162 are independent of each other. RPE1 cells were transfected with control, CEP162, or CEP290 siRNA for 96 hours. Cells were stained with anti-centrin (green), anti-CEP162 (red), and anti-CEP290 (blue) antibodies.
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Figure 5: CEP162 interacts with CEP290 and mediates its association with MTsa, Endogenous CEP162 or CEP290 in HEK293T cell extracts were immunoprecipitated, and co-precipitated proteins were probed with indicated antibodies by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. b, Mapping the CEP290 binding domain of CEP162. 293T cells expressing various Flag-tagged fragments of CEP162 were subjected to immunoprecipitations and western blot with antibodies as indicated. Uncropped images of immunoblots are presented in Supplementary Fig. S1. c, RPE1 cells at different cell cycle stages were processed for immunofluorescence with anti-CEP290 (red), anti-CEP162 (green), and anti-centrin (blue) antibodies. DNA (DAPI, blue). d, Spindle association of CEP290 depends on CEP162. RPE1 cells transfected with control, CEP162, or CEP290 siRNA for 72 hours were stained with indicated antibodies against acetylated-tubulin (green), CEP162 (red), and CEP290 (blue). DNA (DAPI, blue). e, Overexpression of CEP162 induced MT bundles recruiting endogenous CEP290. U2OS cells transiently expressing HA-tagged CEP162FL or HA-tagged CEP162tNC1C2 were stained with antibodies against CEP162 (green), CEP290 (red) and DAPI (DNA, blue). f, Overexpression of CEP162 but not CEP290 induced MT bundles. NIH3T3 cells transiently expressing HA-tagged human CEP162FL or GFP-tagged mouse CEP290FL were stained with antibodies against α-tubulin (Red), CEP162 (HA, green) or CEP290 (GFP, green). g, Centriolar localizations of CEP290 and CEP162 are independent of each other. RPE1 cells were transfected with control, CEP162, or CEP290 siRNA for 96 hours. Cells were stained with anti-centrin (green), anti-CEP162 (red), and anti-CEP290 (blue) antibodies.

Mentions: To determine whether CEP162 bridges TZ components to the axoneme, we examined its relationship with CEP290. CEP290 forms a complex with multiple ciliopathy molecules, including NPHP515, Cc2d2a, Tctn1, Mks1, and more17, and mediates the membrane-to-MT connection in the TZ20. However, whether CEP290 associates directly or indirectly with ciliary MTs is unclear. Immunoprecipitation of endogenous CEP162 pulled down endogenous CEP290 (Fig. 5a), and vice versa, indicating that CEP162 and CEP290 are present in the same protein complex. Further analyses revealed that the N-terminal region of CEP162 that contains CC1 and CC2 is required to pull down endogenous CEP290 (Fig. 5b), the same region where the axoneme-binding activity resides. Endogenous CEP290 localized to the distal ends of centrioles throughout the cell cycle (Fig. 5c), and to mitotic spindles during mitosis (Fig. 5d, top), similar to CEP162. Intriguingly, CEP162 localizes to spindle MTs in the absence of CEP290 (Fig. 5d, middle), but CEP290 requires CEP162 to associate with MTs (Fig. 5d, bottom). Furthermore, overexpression of CEP162 induced MT bundles that recruited endogenous CEP290 (Fig. 5e), whereas overexpression of CEP290 failed to decorate MTs or induce MT bundles (Fig. 5f), demonstrating that CEP290 is not a MT-binding protein but associates with MTs through CEP162. Interestingly, however, the localization of CEP162 and CEP290 at centriole distal ends is independent of each other (Fig. 5g), explaining the recruitment of Ccdc2a and Tctn1 to centrioles in the absence of CEP162 (Fig. 3e & f), and further suggesting that CEP290 may serve as an anchor that recruit other TZ components to the vicinity of the TZ, whereas CEP162 is to act as a bridge to seal the gap, linking nearby TZ components to ciliary MTs.


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)

CEP162 interacts with CEP290 and mediates its association with MTsa, Endogenous CEP162 or CEP290 in HEK293T cell extracts were immunoprecipitated, and co-precipitated proteins were probed with indicated antibodies by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. b, Mapping the CEP290 binding domain of CEP162. 293T cells expressing various Flag-tagged fragments of CEP162 were subjected to immunoprecipitations and western blot with antibodies as indicated. Uncropped images of immunoblots are presented in Supplementary Fig. S1. c, RPE1 cells at different cell cycle stages were processed for immunofluorescence with anti-CEP290 (red), anti-CEP162 (green), and anti-centrin (blue) antibodies. DNA (DAPI, blue). d, Spindle association of CEP290 depends on CEP162. RPE1 cells transfected with control, CEP162, or CEP290 siRNA for 72 hours were stained with indicated antibodies against acetylated-tubulin (green), CEP162 (red), and CEP290 (blue). DNA (DAPI, blue). e, Overexpression of CEP162 induced MT bundles recruiting endogenous CEP290. U2OS cells transiently expressing HA-tagged CEP162FL or HA-tagged CEP162tNC1C2 were stained with antibodies against CEP162 (green), CEP290 (red) and DAPI (DNA, blue). f, Overexpression of CEP162 but not CEP290 induced MT bundles. NIH3T3 cells transiently expressing HA-tagged human CEP162FL or GFP-tagged mouse CEP290FL were stained with antibodies against α-tubulin (Red), CEP162 (HA, green) or CEP290 (GFP, green). g, Centriolar localizations of CEP290 and CEP162 are independent of each other. RPE1 cells were transfected with control, CEP162, or CEP290 siRNA for 96 hours. Cells were stained with anti-centrin (green), anti-CEP162 (red), and anti-CEP290 (blue) antibodies.
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Figure 5: CEP162 interacts with CEP290 and mediates its association with MTsa, Endogenous CEP162 or CEP290 in HEK293T cell extracts were immunoprecipitated, and co-precipitated proteins were probed with indicated antibodies by western blot. Uncropped images of immunoblots are presented in Supplementary Fig. S1. b, Mapping the CEP290 binding domain of CEP162. 293T cells expressing various Flag-tagged fragments of CEP162 were subjected to immunoprecipitations and western blot with antibodies as indicated. Uncropped images of immunoblots are presented in Supplementary Fig. S1. c, RPE1 cells at different cell cycle stages were processed for immunofluorescence with anti-CEP290 (red), anti-CEP162 (green), and anti-centrin (blue) antibodies. DNA (DAPI, blue). d, Spindle association of CEP290 depends on CEP162. RPE1 cells transfected with control, CEP162, or CEP290 siRNA for 72 hours were stained with indicated antibodies against acetylated-tubulin (green), CEP162 (red), and CEP290 (blue). DNA (DAPI, blue). e, Overexpression of CEP162 induced MT bundles recruiting endogenous CEP290. U2OS cells transiently expressing HA-tagged CEP162FL or HA-tagged CEP162tNC1C2 were stained with antibodies against CEP162 (green), CEP290 (red) and DAPI (DNA, blue). f, Overexpression of CEP162 but not CEP290 induced MT bundles. NIH3T3 cells transiently expressing HA-tagged human CEP162FL or GFP-tagged mouse CEP290FL were stained with antibodies against α-tubulin (Red), CEP162 (HA, green) or CEP290 (GFP, green). g, Centriolar localizations of CEP290 and CEP162 are independent of each other. RPE1 cells were transfected with control, CEP162, or CEP290 siRNA for 96 hours. Cells were stained with anti-centrin (green), anti-CEP162 (red), and anti-CEP290 (blue) antibodies.
Mentions: To determine whether CEP162 bridges TZ components to the axoneme, we examined its relationship with CEP290. CEP290 forms a complex with multiple ciliopathy molecules, including NPHP515, Cc2d2a, Tctn1, Mks1, and more17, and mediates the membrane-to-MT connection in the TZ20. However, whether CEP290 associates directly or indirectly with ciliary MTs is unclear. Immunoprecipitation of endogenous CEP162 pulled down endogenous CEP290 (Fig. 5a), and vice versa, indicating that CEP162 and CEP290 are present in the same protein complex. Further analyses revealed that the N-terminal region of CEP162 that contains CC1 and CC2 is required to pull down endogenous CEP290 (Fig. 5b), the same region where the axoneme-binding activity resides. Endogenous CEP290 localized to the distal ends of centrioles throughout the cell cycle (Fig. 5c), and to mitotic spindles during mitosis (Fig. 5d, top), similar to CEP162. Intriguingly, CEP162 localizes to spindle MTs in the absence of CEP290 (Fig. 5d, middle), but CEP290 requires CEP162 to associate with MTs (Fig. 5d, bottom). Furthermore, overexpression of CEP162 induced MT bundles that recruited endogenous CEP290 (Fig. 5e), whereas overexpression of CEP290 failed to decorate MTs or induce MT bundles (Fig. 5f), demonstrating that CEP290 is not a MT-binding protein but associates with MTs through CEP162. Interestingly, however, the localization of CEP162 and CEP290 at centriole distal ends is independent of each other (Fig. 5g), explaining the recruitment of Ccdc2a and Tctn1 to centrioles in the absence of CEP162 (Fig. 3e & f), and further suggesting that CEP290 may serve as an anchor that recruit other TZ components to the vicinity of the TZ, whereas CEP162 is to act as a bridge to seal the gap, linking nearby TZ components to ciliary MTs.

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