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Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains.

Tateishi K, Yamazaki Y, Nishida T, Watanabe S, Kunimoto K, Ishikawa H, Tsukita S - J. Cell Biol. (2013)

Bottom Line: We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells.We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type.We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.

ABSTRACT
Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188-806, whereas basal foot formation required aa 1-59 and 188-806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.

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Specific role of centrosomal subdistal appendages (SAs) in stabilizing centriole microtubules (MTs). (A) Immunofluorescence images of MTs in DA+SA+, DA+SA−, and DA−SA− centrioles under nocodazole treatment, an MT-destabilizing condition. WT, (DA+SA+) centriole in WT F9 cells; Δ4/5, (DA+SA−) centriole in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (DA+SA+) centriole in Δ6/7 construct–expressing Odf2-KO F9 cells. (B) Quantification of MT stability. The relative MT stability is shown for the indicated Odf2 construct (*, P = 0.01; **, P = 0.005; n > 200 in more than three independent experiments). (C) Schematic drawing of the specific roles of the appendages of ciliary basal bodies/centrioles. In the proposed model, separate domains in Odf2 serve as the molecular platform on which the appendages are constructed. Note that centriolin associates with ODF2/cenexin at the base of the SA (or where the base would be in the absence of SAs), and recruits vesicles whose cargoes support ciliogenesis. The BF/SAs (red) stabilize MTs, whereas the TFs/DAs (blue) are essential for ciliogenesis. MC, mother centriole; DC, daughter centriole.
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fig5: Specific role of centrosomal subdistal appendages (SAs) in stabilizing centriole microtubules (MTs). (A) Immunofluorescence images of MTs in DA+SA+, DA+SA−, and DA−SA− centrioles under nocodazole treatment, an MT-destabilizing condition. WT, (DA+SA+) centriole in WT F9 cells; Δ4/5, (DA+SA−) centriole in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (DA+SA+) centriole in Δ6/7 construct–expressing Odf2-KO F9 cells. (B) Quantification of MT stability. The relative MT stability is shown for the indicated Odf2 construct (*, P = 0.01; **, P = 0.005; n > 200 in more than three independent experiments). (C) Schematic drawing of the specific roles of the appendages of ciliary basal bodies/centrioles. In the proposed model, separate domains in Odf2 serve as the molecular platform on which the appendages are constructed. Note that centriolin associates with ODF2/cenexin at the base of the SA (or where the base would be in the absence of SAs), and recruits vesicles whose cargoes support ciliogenesis. The BF/SAs (red) stabilize MTs, whereas the TFs/DAs (blue) are essential for ciliogenesis. MC, mother centriole; DC, daughter centriole.

Mentions: We next examined the stabilization of centrosomal microtubules in transfected cells treated with nocodazole, which interferes with microtubule polymerization. We found that under nocodazole treatment, the number of microtubules associated with centrosomes was significantly greater in wild-type (DA+SA+) cells than in Odf2-KO (DA−SA−) cells, based on the number of microtubules in the asters surrounding the centrioles (Fig. 5, A and B). In a previous study, we showed that the cell cycle and centrosomal microtubule-organizing center (MTOC) activity are unaffected by the loss of both appendages (Ishikawa et al., 2005). Consistent with this finding, our results of a microtubule regrowth assay (Brandt and Lee, 1993; Gaglio et al., 1996; Ishikawa et al., 2005) using centrosomes depleted of microtubules by prolonged nocodazole/cold treatment showed that the MTOC activity in the wild-type and Odf2-KO cells was the same (Fig. S3, C and D).


Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains.

Tateishi K, Yamazaki Y, Nishida T, Watanabe S, Kunimoto K, Ishikawa H, Tsukita S - J. Cell Biol. (2013)

Specific role of centrosomal subdistal appendages (SAs) in stabilizing centriole microtubules (MTs). (A) Immunofluorescence images of MTs in DA+SA+, DA+SA−, and DA−SA− centrioles under nocodazole treatment, an MT-destabilizing condition. WT, (DA+SA+) centriole in WT F9 cells; Δ4/5, (DA+SA−) centriole in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (DA+SA+) centriole in Δ6/7 construct–expressing Odf2-KO F9 cells. (B) Quantification of MT stability. The relative MT stability is shown for the indicated Odf2 construct (*, P = 0.01; **, P = 0.005; n > 200 in more than three independent experiments). (C) Schematic drawing of the specific roles of the appendages of ciliary basal bodies/centrioles. In the proposed model, separate domains in Odf2 serve as the molecular platform on which the appendages are constructed. Note that centriolin associates with ODF2/cenexin at the base of the SA (or where the base would be in the absence of SAs), and recruits vesicles whose cargoes support ciliogenesis. The BF/SAs (red) stabilize MTs, whereas the TFs/DAs (blue) are essential for ciliogenesis. MC, mother centriole; DC, daughter centriole.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824012&req=5

fig5: Specific role of centrosomal subdistal appendages (SAs) in stabilizing centriole microtubules (MTs). (A) Immunofluorescence images of MTs in DA+SA+, DA+SA−, and DA−SA− centrioles under nocodazole treatment, an MT-destabilizing condition. WT, (DA+SA+) centriole in WT F9 cells; Δ4/5, (DA+SA−) centriole in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (DA+SA+) centriole in Δ6/7 construct–expressing Odf2-KO F9 cells. (B) Quantification of MT stability. The relative MT stability is shown for the indicated Odf2 construct (*, P = 0.01; **, P = 0.005; n > 200 in more than three independent experiments). (C) Schematic drawing of the specific roles of the appendages of ciliary basal bodies/centrioles. In the proposed model, separate domains in Odf2 serve as the molecular platform on which the appendages are constructed. Note that centriolin associates with ODF2/cenexin at the base of the SA (or where the base would be in the absence of SAs), and recruits vesicles whose cargoes support ciliogenesis. The BF/SAs (red) stabilize MTs, whereas the TFs/DAs (blue) are essential for ciliogenesis. MC, mother centriole; DC, daughter centriole.
Mentions: We next examined the stabilization of centrosomal microtubules in transfected cells treated with nocodazole, which interferes with microtubule polymerization. We found that under nocodazole treatment, the number of microtubules associated with centrosomes was significantly greater in wild-type (DA+SA+) cells than in Odf2-KO (DA−SA−) cells, based on the number of microtubules in the asters surrounding the centrioles (Fig. 5, A and B). In a previous study, we showed that the cell cycle and centrosomal microtubule-organizing center (MTOC) activity are unaffected by the loss of both appendages (Ishikawa et al., 2005). Consistent with this finding, our results of a microtubule regrowth assay (Brandt and Lee, 1993; Gaglio et al., 1996; Ishikawa et al., 2005) using centrosomes depleted of microtubules by prolonged nocodazole/cold treatment showed that the MTOC activity in the wild-type and Odf2-KO cells was the same (Fig. S3, C and D).

Bottom Line: We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells.We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type.We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.

ABSTRACT
Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188-806, whereas basal foot formation required aa 1-59 and 188-806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.

Show MeSH
Related in: MedlinePlus