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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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Cilia tips modified by CEP162 swell exceedingly and discharge ciliary contentsa, RPE1 cells expressing GFP-tagged CEP162tNC1C2 were traced by time-lapse fluorescence microscopy. Images were taken at 2-min time interval. Existing cilia tips were marked by arrows, and newly formed tips were marked with arrowheads. b–e, HA- or GFP-tagged CEP162ΔCC3 or CEP162tNC1C2 was conditionally expressed in RPE1 cells followed by serum starvation for 48hr. Cells were stained with antibodies against CEP290 (b), RPGRIP1L (c), IFT88 (d), Arl13b (e) in red, acetylated-tubulin (green) and HA (blue). DNA was stained with DAPI. Arrows indicate the CEP162-positive structures released from cilia tips. f, Correlative light-scanning electron microscopy images of control cells (left), or RPE1 cells expressing GFP-tagged CEP162ΔCC3 (middle & right). GFP-CEP162ΔCC3 (green) marked cilia tip (middle & right). Primary cilia in control cells (left) were labeled with acetylated-tubulin (green). Arrows indicate cilia tips. Merged images were shown under low SEM magnification. g, Correlate LM-SEM images of the swollen structure released from cilia tips in RPE1 cells expressing GFP-tagged CEP162ΔCC3. Released structures from cilia tip were marked with GFP (green). Merged images were shown under low SEM magnification.
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Figure 7: Cilia tips modified by CEP162 swell exceedingly and discharge ciliary contentsa, RPE1 cells expressing GFP-tagged CEP162tNC1C2 were traced by time-lapse fluorescence microscopy. Images were taken at 2-min time interval. Existing cilia tips were marked by arrows, and newly formed tips were marked with arrowheads. b–e, HA- or GFP-tagged CEP162ΔCC3 or CEP162tNC1C2 was conditionally expressed in RPE1 cells followed by serum starvation for 48hr. Cells were stained with antibodies against CEP290 (b), RPGRIP1L (c), IFT88 (d), Arl13b (e) in red, acetylated-tubulin (green) and HA (blue). DNA was stained with DAPI. Arrows indicate the CEP162-positive structures released from cilia tips. f, Correlative light-scanning electron microscopy images of control cells (left), or RPE1 cells expressing GFP-tagged CEP162ΔCC3 (middle & right). GFP-CEP162ΔCC3 (green) marked cilia tip (middle & right). Primary cilia in control cells (left) were labeled with acetylated-tubulin (green). Arrows indicate cilia tips. Merged images were shown under low SEM magnification. g, Correlate LM-SEM images of the swollen structure released from cilia tips in RPE1 cells expressing GFP-tagged CEP162ΔCC3. Released structures from cilia tip were marked with GFP (green). Merged images were shown under low SEM magnification.

Mentions: The consequence of having a TZ-like structure at cilia tips was further examined. RPE1 cells expressing GFP-tagged CEP162tNC1C2 were imaged by time-lapse fluorescence microscopy. The GFP-CEP162 signal was seen first at the cilia tip as expected, but surprisingly, it became detached from the tip, and released into the extracellular environment (Fig. 7a and Supplementary movie 1). In a few cases, violent bursts of cilia tips were observed (Supplementary movie 2). Similar phenotypes were also seen in cells expressing CEP162ΔCC3. The detached, CEP162-positive structures, scattering amongst cells, were also positive for the TZ marker CEP290 (Fig. 7b) or RPGRIP1l (Fig. 7c), IFT marker IFT88 (Fig. 7d), and ciliary membrane marker Arl13b (Fig. 7e), indicating that CEP162-modified cilia are actively discharging ciliary contents from their tips. Correlative light-scanning electron microscopy revealed that CEP162-modified tips are severely swollen (Fig. 7f), forming a blister-like configuration. The swelling is likely a consequence of excess accumulation of proteins at tips, including CEP162, and other ciliary materials (e.g. TZ components, IFT, and Arl13b). We don’t understand how these ciliary materials are excessively trapped there, although it is consistent with the idea that some form of a barrier is built, separating the tip from the rest of the ciliary compartment. The bulging structures released from cilia tips differed in size, and were irregularly shaped (Fig. 7g), suggesting that they are membranous structures randomly breaking away from the exceedingly enlarged tip. However, the GFP signal was retained over time in some of these detached structures (Supplementary Movie 1), rather than being diluted or dispersed, an indication of intact membrane-coated structures. Note that secretion-like activities have been reported to occur at the flagella tip in Chlamydomonas32, 33. Together, these observations demonstrate that the spatial restriction of TZ assembly to the cilia base is critical for proper cilia structure and function, and that one important role of centriole-distal-end proteins is to provide such spatial cues.


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)

Cilia tips modified by CEP162 swell exceedingly and discharge ciliary contentsa, RPE1 cells expressing GFP-tagged CEP162tNC1C2 were traced by time-lapse fluorescence microscopy. Images were taken at 2-min time interval. Existing cilia tips were marked by arrows, and newly formed tips were marked with arrowheads. b–e, HA- or GFP-tagged CEP162ΔCC3 or CEP162tNC1C2 was conditionally expressed in RPE1 cells followed by serum starvation for 48hr. Cells were stained with antibodies against CEP290 (b), RPGRIP1L (c), IFT88 (d), Arl13b (e) in red, acetylated-tubulin (green) and HA (blue). DNA was stained with DAPI. Arrows indicate the CEP162-positive structures released from cilia tips. f, Correlative light-scanning electron microscopy images of control cells (left), or RPE1 cells expressing GFP-tagged CEP162ΔCC3 (middle & right). GFP-CEP162ΔCC3 (green) marked cilia tip (middle & right). Primary cilia in control cells (left) were labeled with acetylated-tubulin (green). Arrows indicate cilia tips. Merged images were shown under low SEM magnification. g, Correlate LM-SEM images of the swollen structure released from cilia tips in RPE1 cells expressing GFP-tagged CEP162ΔCC3. Released structures from cilia tip were marked with GFP (green). Merged images were shown under low SEM magnification.
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Figure 7: Cilia tips modified by CEP162 swell exceedingly and discharge ciliary contentsa, RPE1 cells expressing GFP-tagged CEP162tNC1C2 were traced by time-lapse fluorescence microscopy. Images were taken at 2-min time interval. Existing cilia tips were marked by arrows, and newly formed tips were marked with arrowheads. b–e, HA- or GFP-tagged CEP162ΔCC3 or CEP162tNC1C2 was conditionally expressed in RPE1 cells followed by serum starvation for 48hr. Cells were stained with antibodies against CEP290 (b), RPGRIP1L (c), IFT88 (d), Arl13b (e) in red, acetylated-tubulin (green) and HA (blue). DNA was stained with DAPI. Arrows indicate the CEP162-positive structures released from cilia tips. f, Correlative light-scanning electron microscopy images of control cells (left), or RPE1 cells expressing GFP-tagged CEP162ΔCC3 (middle & right). GFP-CEP162ΔCC3 (green) marked cilia tip (middle & right). Primary cilia in control cells (left) were labeled with acetylated-tubulin (green). Arrows indicate cilia tips. Merged images were shown under low SEM magnification. g, Correlate LM-SEM images of the swollen structure released from cilia tips in RPE1 cells expressing GFP-tagged CEP162ΔCC3. Released structures from cilia tip were marked with GFP (green). Merged images were shown under low SEM magnification.
Mentions: The consequence of having a TZ-like structure at cilia tips was further examined. RPE1 cells expressing GFP-tagged CEP162tNC1C2 were imaged by time-lapse fluorescence microscopy. The GFP-CEP162 signal was seen first at the cilia tip as expected, but surprisingly, it became detached from the tip, and released into the extracellular environment (Fig. 7a and Supplementary movie 1). In a few cases, violent bursts of cilia tips were observed (Supplementary movie 2). Similar phenotypes were also seen in cells expressing CEP162ΔCC3. The detached, CEP162-positive structures, scattering amongst cells, were also positive for the TZ marker CEP290 (Fig. 7b) or RPGRIP1l (Fig. 7c), IFT marker IFT88 (Fig. 7d), and ciliary membrane marker Arl13b (Fig. 7e), indicating that CEP162-modified cilia are actively discharging ciliary contents from their tips. Correlative light-scanning electron microscopy revealed that CEP162-modified tips are severely swollen (Fig. 7f), forming a blister-like configuration. The swelling is likely a consequence of excess accumulation of proteins at tips, including CEP162, and other ciliary materials (e.g. TZ components, IFT, and Arl13b). We don’t understand how these ciliary materials are excessively trapped there, although it is consistent with the idea that some form of a barrier is built, separating the tip from the rest of the ciliary compartment. The bulging structures released from cilia tips differed in size, and were irregularly shaped (Fig. 7g), suggesting that they are membranous structures randomly breaking away from the exceedingly enlarged tip. However, the GFP signal was retained over time in some of these detached structures (Supplementary Movie 1), rather than being diluted or dispersed, an indication of intact membrane-coated structures. Note that secretion-like activities have been reported to occur at the flagella tip in Chlamydomonas32, 33. Together, these observations demonstrate that the spatial restriction of TZ assembly to the cilia base is critical for proper cilia structure and function, and that one important role of centriole-distal-end proteins is to provide such spatial cues.

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