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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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Odf2 gene sequences required to generate cilia and reconstitute the appendages of ciliary basal bodies. (A) Schematic representation of the Odf2 deletion constructs, which were transfected into Odf2-KO F9 cells. LZ, leucine zipper motif. (B) Immunofluorescence for GFP (Odf2), γ-tubulin (centrioles/basal bodies), and acetylated tubulin (primary cilia) to examine the generation of cilia. Bars, 1 µm. (C) Percentage of cilia on centrioles in cells expressing the indicated construct (n > 100 in more than three independent experiments). (D) Electron micrographs showing transition fibers (TFs) and/or basal feet (BF) on ciliary basal bodies. Thin-section electron microscopic images show cross sections (Cross) and longitudinal sections (Longitudinal) of primary cilia. Tomography, UHVEMT images of basal bodies; TFs, blue arrows; BF, red arrowheads; WT, (TF+BF+) basal bodies in WT F9 cells; Δ4/5, (TF+BF−) basal bodies in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (TF+BF+) basal bodies in Δ6/7 construct–expressing Odf2-KO F9 cells. Bars, 0.2 µm. More than five samples were analyzed in each case. The cross section electron micrographs in the top two rows are the same images as in the top two rows of Fig. S3 A, where they are annotated for TFs and BF. Insets, schematic drawings of electron microscopic images of basal bodies.
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fig1: Odf2 gene sequences required to generate cilia and reconstitute the appendages of ciliary basal bodies. (A) Schematic representation of the Odf2 deletion constructs, which were transfected into Odf2-KO F9 cells. LZ, leucine zipper motif. (B) Immunofluorescence for GFP (Odf2), γ-tubulin (centrioles/basal bodies), and acetylated tubulin (primary cilia) to examine the generation of cilia. Bars, 1 µm. (C) Percentage of cilia on centrioles in cells expressing the indicated construct (n > 100 in more than three independent experiments). (D) Electron micrographs showing transition fibers (TFs) and/or basal feet (BF) on ciliary basal bodies. Thin-section electron microscopic images show cross sections (Cross) and longitudinal sections (Longitudinal) of primary cilia. Tomography, UHVEMT images of basal bodies; TFs, blue arrows; BF, red arrowheads; WT, (TF+BF+) basal bodies in WT F9 cells; Δ4/5, (TF+BF−) basal bodies in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (TF+BF+) basal bodies in Δ6/7 construct–expressing Odf2-KO F9 cells. Bars, 0.2 µm. More than five samples were analyzed in each case. The cross section electron micrographs in the top two rows are the same images as in the top two rows of Fig. S3 A, where they are annotated for TFs and BF. Insets, schematic drawings of electron microscopic images of basal bodies.

Mentions: We first transfected full-length GFP-tagged Odf2 into Odf2-KO F9 cells, and immunostained for GFP (to detect GFP-ODF2/cenexin), acetylated α-tubulin (to detect primary cilia), and γ-tubulin (to detect basal bodies and centrioles; Fig. 1, A and B). The full-length Odf2 gene product was recruited to centrioles and ciliary basal bodies, and ciliogenesis was restored in Odf2-KO F9 cells by the full-length GFP-tagged Odf2 construct (Fig. 1, B and C).


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)

Odf2 gene sequences required to generate cilia and reconstitute the appendages of ciliary basal bodies. (A) Schematic representation of the Odf2 deletion constructs, which were transfected into Odf2-KO F9 cells. LZ, leucine zipper motif. (B) Immunofluorescence for GFP (Odf2), γ-tubulin (centrioles/basal bodies), and acetylated tubulin (primary cilia) to examine the generation of cilia. Bars, 1 µm. (C) Percentage of cilia on centrioles in cells expressing the indicated construct (n > 100 in more than three independent experiments). (D) Electron micrographs showing transition fibers (TFs) and/or basal feet (BF) on ciliary basal bodies. Thin-section electron microscopic images show cross sections (Cross) and longitudinal sections (Longitudinal) of primary cilia. Tomography, UHVEMT images of basal bodies; TFs, blue arrows; BF, red arrowheads; WT, (TF+BF+) basal bodies in WT F9 cells; Δ4/5, (TF+BF−) basal bodies in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (TF+BF+) basal bodies in Δ6/7 construct–expressing Odf2-KO F9 cells. Bars, 0.2 µm. More than five samples were analyzed in each case. The cross section electron micrographs in the top two rows are the same images as in the top two rows of Fig. S3 A, where they are annotated for TFs and BF. Insets, schematic drawings of electron microscopic images of basal bodies.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Odf2 gene sequences required to generate cilia and reconstitute the appendages of ciliary basal bodies. (A) Schematic representation of the Odf2 deletion constructs, which were transfected into Odf2-KO F9 cells. LZ, leucine zipper motif. (B) Immunofluorescence for GFP (Odf2), γ-tubulin (centrioles/basal bodies), and acetylated tubulin (primary cilia) to examine the generation of cilia. Bars, 1 µm. (C) Percentage of cilia on centrioles in cells expressing the indicated construct (n > 100 in more than three independent experiments). (D) Electron micrographs showing transition fibers (TFs) and/or basal feet (BF) on ciliary basal bodies. Thin-section electron microscopic images show cross sections (Cross) and longitudinal sections (Longitudinal) of primary cilia. Tomography, UHVEMT images of basal bodies; TFs, blue arrows; BF, red arrowheads; WT, (TF+BF+) basal bodies in WT F9 cells; Δ4/5, (TF+BF−) basal bodies in Δ4/5 construct–expressing Odf2-KO F9 cells; Δ6/7, (TF+BF+) basal bodies in Δ6/7 construct–expressing Odf2-KO F9 cells. Bars, 0.2 µm. More than five samples were analyzed in each case. The cross section electron micrographs in the top two rows are the same images as in the top two rows of Fig. S3 A, where they are annotated for TFs and BF. Insets, schematic drawings of electron microscopic images of basal bodies.
Mentions: We first transfected full-length GFP-tagged Odf2 into Odf2-KO F9 cells, and immunostained for GFP (to detect GFP-ODF2/cenexin), acetylated α-tubulin (to detect primary cilia), and γ-tubulin (to detect basal bodies and centrioles; Fig. 1, A and B). The full-length Odf2 gene product was recruited to centrioles and ciliary basal bodies, and ciliogenesis was restored in Odf2-KO F9 cells by the full-length GFP-tagged Odf2 construct (Fig. 1, B and C).

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