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Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells

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ABSTRACT

Ciliary transition zone (TZ) assembly is complex and incompletely understood. Vieillard et al. show that Drosophila Cby and Dila cooperate to assemble the TZ and membrane cap, which, together with microtubule remodeling by kinesin-13, is required for axoneme formation in male germ cells.

No MeSH data available.


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Klp59D acts downstream of TZ components to regulate axonemal assembly in spermatogenesis. (A) Live observation of Klp59D::GFP distribution. In spermatocytes, Klp59D is first cytoplasmic and next recruited at centrioles. In elongating spermatids, Klp59D::GFP is present at the tip of the BB and along the axoneme and ciliary cap. (B) Magnification of centrioles and primary cilia in late spermatocytes, showing the localization of Klp59D::GFP in the cilia/TZ extension (labeled with Cby::Tomato) and at the base of the centrioles. (C–E) Confocal imaging of squashed testes. (C) TZ components Dila and Mks1 are recruited normally at centriole tips (Asl antibody) in spermatocytes and spermatids in Klp59D KD compared with control testes. (D) Quantification of Klp59D and Asl relative intensities in control or dila81; cby1 spermatocytes. Significant differences are observed for Klp59D. Scattered plots with mean and SD are shown (control n = 52; dila81; cby1 n = 46). (E) Unc::GFP expression domain is extended in late spermatocytes and spermatids (arrows) in Klp59D KD compared with controls. (F) Cby is still present at the tip of centrioles, but huge axonemal extensions are labeled by CG6652::GFP in Klp59D KD testes. (G) Live cyst imaging using the membrane PLCδPH-mRFP and Unc::GFP reporters. In control cysts, a membrane cap (arrows, straight line indicating the range of the cap) is present at the tip of centrioles and BBs. In Klp59D KD, the membrane cap is absent when aberrant microtubule extensions are observed, as revealed by the extended expression domain of Unc. Bars: (B and D) 1 µm; (A, C, and E–G) 2 µm.
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fig8: Klp59D acts downstream of TZ components to regulate axonemal assembly in spermatogenesis. (A) Live observation of Klp59D::GFP distribution. In spermatocytes, Klp59D is first cytoplasmic and next recruited at centrioles. In elongating spermatids, Klp59D::GFP is present at the tip of the BB and along the axoneme and ciliary cap. (B) Magnification of centrioles and primary cilia in late spermatocytes, showing the localization of Klp59D::GFP in the cilia/TZ extension (labeled with Cby::Tomato) and at the base of the centrioles. (C–E) Confocal imaging of squashed testes. (C) TZ components Dila and Mks1 are recruited normally at centriole tips (Asl antibody) in spermatocytes and spermatids in Klp59D KD compared with control testes. (D) Quantification of Klp59D and Asl relative intensities in control or dila81; cby1 spermatocytes. Significant differences are observed for Klp59D. Scattered plots with mean and SD are shown (control n = 52; dila81; cby1 n = 46). (E) Unc::GFP expression domain is extended in late spermatocytes and spermatids (arrows) in Klp59D KD compared with controls. (F) Cby is still present at the tip of centrioles, but huge axonemal extensions are labeled by CG6652::GFP in Klp59D KD testes. (G) Live cyst imaging using the membrane PLCδPH-mRFP and Unc::GFP reporters. In control cysts, a membrane cap (arrows, straight line indicating the range of the cap) is present at the tip of centrioles and BBs. In Klp59D KD, the membrane cap is absent when aberrant microtubule extensions are observed, as revealed by the extended expression domain of Unc. Bars: (B and D) 1 µm; (A, C, and E–G) 2 µm.

Mentions: We looked for Klp59D distribution using a Klp59D::GFP reporter transgene on live testes cyst preparation. Klp59D::GFP showed a dynamic distribution during germ cell differentiation (Fig. 8 A). In early spermatocytes, Klp59D is present throughout the cytoplasm. During spermatocyte maturation, Klp59D is also enriched at the tips of the centrioles and at their bases in late spermatocytes. Close examination of late spermatocytes show clear enrichment of Klp59D in the ciliary cap protruding from the cell surface (Fig. 8 B). In elongating spermatids, Klp59D is concentrated at the ciliary cap and along the axoneme (Fig. 8 A). We next analyzed the function of Klp59D by expressing shRNA in Drosophila male germ cells. We observed that the distribution of MKS components, Dila, or Cby was apparently normal in Klp59D KD spermatocytes (Fig. 8, C and F), suggesting that Klp59D acts downstream of TZ components. In support of this hypothesis, the amount of Klp59D around centrioles is significantly reduced compared with Asl intensity in the absence of both Dila and Cby (Fig. 8 D). However, Unc distribution was strikingly modified in Klp59D KD cells compared with controls (Fig. 8 E). This phenotype is identical to the one observed in dila81; cby1 double mutants and was confirmed with CG6652::GFP labeling, showing huge axonemal extensions on many centrioles in Klp59D KD cells (Fig. 8 F). Using a cell membrane marker, we observed that the aberrant axoneme elongation was associated with impaired membrane cap formation in male germ cells (Fig. 8 G). Furthermore, EM observations of spermatocytes showed part of the centrioles with aberrant microtubule elongation and disrupted ciliary cap (Fig. S5 D). These observations indicate that membrane cap formation requires strict control of microtubule elongation. Additionally, EM analysis showed a severe disorganization of spermatid axonemes similar to defects observed in dila81; cby1 mutants. Very few axonemes were apparently normal, and most were missing or broken (Fig. S5, B and C). Altogether, our results show that Klp59D is required for timely control of axonemal growth during male germ cell differentiation. Removal of this protein leads to premature microtubule elongation, axonemal structural defects, and defective ciliary cap formation.


Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells
Klp59D acts downstream of TZ components to regulate axonemal assembly in spermatogenesis. (A) Live observation of Klp59D::GFP distribution. In spermatocytes, Klp59D is first cytoplasmic and next recruited at centrioles. In elongating spermatids, Klp59D::GFP is present at the tip of the BB and along the axoneme and ciliary cap. (B) Magnification of centrioles and primary cilia in late spermatocytes, showing the localization of Klp59D::GFP in the cilia/TZ extension (labeled with Cby::Tomato) and at the base of the centrioles. (C–E) Confocal imaging of squashed testes. (C) TZ components Dila and Mks1 are recruited normally at centriole tips (Asl antibody) in spermatocytes and spermatids in Klp59D KD compared with control testes. (D) Quantification of Klp59D and Asl relative intensities in control or dila81; cby1 spermatocytes. Significant differences are observed for Klp59D. Scattered plots with mean and SD are shown (control n = 52; dila81; cby1 n = 46). (E) Unc::GFP expression domain is extended in late spermatocytes and spermatids (arrows) in Klp59D KD compared with controls. (F) Cby is still present at the tip of centrioles, but huge axonemal extensions are labeled by CG6652::GFP in Klp59D KD testes. (G) Live cyst imaging using the membrane PLCδPH-mRFP and Unc::GFP reporters. In control cysts, a membrane cap (arrows, straight line indicating the range of the cap) is present at the tip of centrioles and BBs. In Klp59D KD, the membrane cap is absent when aberrant microtubule extensions are observed, as revealed by the extended expression domain of Unc. Bars: (B and D) 1 µm; (A, C, and E–G) 2 µm.
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fig8: Klp59D acts downstream of TZ components to regulate axonemal assembly in spermatogenesis. (A) Live observation of Klp59D::GFP distribution. In spermatocytes, Klp59D is first cytoplasmic and next recruited at centrioles. In elongating spermatids, Klp59D::GFP is present at the tip of the BB and along the axoneme and ciliary cap. (B) Magnification of centrioles and primary cilia in late spermatocytes, showing the localization of Klp59D::GFP in the cilia/TZ extension (labeled with Cby::Tomato) and at the base of the centrioles. (C–E) Confocal imaging of squashed testes. (C) TZ components Dila and Mks1 are recruited normally at centriole tips (Asl antibody) in spermatocytes and spermatids in Klp59D KD compared with control testes. (D) Quantification of Klp59D and Asl relative intensities in control or dila81; cby1 spermatocytes. Significant differences are observed for Klp59D. Scattered plots with mean and SD are shown (control n = 52; dila81; cby1 n = 46). (E) Unc::GFP expression domain is extended in late spermatocytes and spermatids (arrows) in Klp59D KD compared with controls. (F) Cby is still present at the tip of centrioles, but huge axonemal extensions are labeled by CG6652::GFP in Klp59D KD testes. (G) Live cyst imaging using the membrane PLCδPH-mRFP and Unc::GFP reporters. In control cysts, a membrane cap (arrows, straight line indicating the range of the cap) is present at the tip of centrioles and BBs. In Klp59D KD, the membrane cap is absent when aberrant microtubule extensions are observed, as revealed by the extended expression domain of Unc. Bars: (B and D) 1 µm; (A, C, and E–G) 2 µm.
Mentions: We looked for Klp59D distribution using a Klp59D::GFP reporter transgene on live testes cyst preparation. Klp59D::GFP showed a dynamic distribution during germ cell differentiation (Fig. 8 A). In early spermatocytes, Klp59D is present throughout the cytoplasm. During spermatocyte maturation, Klp59D is also enriched at the tips of the centrioles and at their bases in late spermatocytes. Close examination of late spermatocytes show clear enrichment of Klp59D in the ciliary cap protruding from the cell surface (Fig. 8 B). In elongating spermatids, Klp59D is concentrated at the ciliary cap and along the axoneme (Fig. 8 A). We next analyzed the function of Klp59D by expressing shRNA in Drosophila male germ cells. We observed that the distribution of MKS components, Dila, or Cby was apparently normal in Klp59D KD spermatocytes (Fig. 8, C and F), suggesting that Klp59D acts downstream of TZ components. In support of this hypothesis, the amount of Klp59D around centrioles is significantly reduced compared with Asl intensity in the absence of both Dila and Cby (Fig. 8 D). However, Unc distribution was strikingly modified in Klp59D KD cells compared with controls (Fig. 8 E). This phenotype is identical to the one observed in dila81; cby1 double mutants and was confirmed with CG6652::GFP labeling, showing huge axonemal extensions on many centrioles in Klp59D KD cells (Fig. 8 F). Using a cell membrane marker, we observed that the aberrant axoneme elongation was associated with impaired membrane cap formation in male germ cells (Fig. 8 G). Furthermore, EM observations of spermatocytes showed part of the centrioles with aberrant microtubule elongation and disrupted ciliary cap (Fig. S5 D). These observations indicate that membrane cap formation requires strict control of microtubule elongation. Additionally, EM analysis showed a severe disorganization of spermatid axonemes similar to defects observed in dila81; cby1 mutants. Very few axonemes were apparently normal, and most were missing or broken (Fig. S5, B and C). Altogether, our results show that Klp59D is required for timely control of axonemal growth during male germ cell differentiation. Removal of this protein leads to premature microtubule elongation, axonemal structural defects, and defective ciliary cap formation.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Ciliary transition zone (TZ) assembly is complex and incompletely understood. Vieillard et al. show that Drosophila Cby and Dila cooperate to assemble the TZ and membrane cap, which, together with microtubule remodeling by kinesin-13, is required for axoneme formation in male germ cells.

No MeSH data available.


Related in: MedlinePlus