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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.

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B9d2 and Tctn are required to assemble the MKS complex and organize the TZ. (A, B, and D–F). IF analysis of squashed testes. (A and B) Cby and Cep290 localization domains are reduced in length at the TZ of the B9d2, tctnΔ mutant spermatocytes or spermatids. (C) Quantification of Cep290, Cby, and centriole lengths in meiosis stage in control or B9d2, tctnΔ mutant germ cells. Scattered plots with mean and SD are shown. Cep290 and Asl: control n = 39 and B9d2, tctnΔ n = 45; Cby: control n = 54 and B9d2, tctnΔ n = 51. ****, P < 0.0001; **, P < 0.01; ***, P < 0.001. (D and E) Mks1 and B9d1 are absent from the TZ in B9d2, tctnΔ mutant germ cells. (F) B9d1 localization (red) is only rescued when introducing both B9d2 and Tctn, the latter being fused to GFP. No rescue is observed when introducing B9d2 alone. (G) Scheme recapitulating the consequences of B9d2, tctnΔ deletion. In the absence of B9d2 and Tctn, the TZ is shorter, as revealed by Cep290 or Cby staining, and all other MKS components are missing. WT, wild type. Bars, 2 µm.
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fig4: B9d2 and Tctn are required to assemble the MKS complex and organize the TZ. (A, B, and D–F). IF analysis of squashed testes. (A and B) Cby and Cep290 localization domains are reduced in length at the TZ of the B9d2, tctnΔ mutant spermatocytes or spermatids. (C) Quantification of Cep290, Cby, and centriole lengths in meiosis stage in control or B9d2, tctnΔ mutant germ cells. Scattered plots with mean and SD are shown. Cep290 and Asl: control n = 39 and B9d2, tctnΔ n = 45; Cby: control n = 54 and B9d2, tctnΔ n = 51. ****, P < 0.0001; **, P < 0.01; ***, P < 0.001. (D and E) Mks1 and B9d1 are absent from the TZ in B9d2, tctnΔ mutant germ cells. (F) B9d1 localization (red) is only rescued when introducing both B9d2 and Tctn, the latter being fused to GFP. No rescue is observed when introducing B9d2 alone. (G) Scheme recapitulating the consequences of B9d2, tctnΔ deletion. In the absence of B9d2 and Tctn, the TZ is shorter, as revealed by Cep290 or Cby staining, and all other MKS components are missing. WT, wild type. Bars, 2 µm.

Mentions: We next analyzed the molecular organization of the TZ in absence of B9d2 and Tctn (Figs. 4 and S1). Cby and Cep290 are still recruited at the TZ (Fig. 4, A and B). We measured both the length of the centriole, labeled for Asl, and the length of the TZ labeled with Cep290 or Cby in absence of B9d2 and Tctn (Fig. 4 C), and we observed a small but significant decrease of TZ length that was correlated with an increase in centriole size. Strikingly, we observed a complete disruption of the MKS complex, as Mks1 and B9d1 are not recruited to the TZ in B9d2, tctnΔ spermatocytes (Fig. 4, D and E). B9d1 localization was rescued by reintroducing both tctn and B9d2 (Fig. 4 F). This shows that B9d2 and Tctn proteins are required to organize the MKS complex and that the absence of several proteins of the MKS complex at the TZ is largely dispensable for cilia assembly (Fig. 4 G). Based on this observation, we conclude that the MKS complex alone has only a subtle role in the organization of the TZ and ciliary cap in Drosophila sperm cells.


Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells
B9d2 and Tctn are required to assemble the MKS complex and organize the TZ. (A, B, and D–F). IF analysis of squashed testes. (A and B) Cby and Cep290 localization domains are reduced in length at the TZ of the B9d2, tctnΔ mutant spermatocytes or spermatids. (C) Quantification of Cep290, Cby, and centriole lengths in meiosis stage in control or B9d2, tctnΔ mutant germ cells. Scattered plots with mean and SD are shown. Cep290 and Asl: control n = 39 and B9d2, tctnΔ n = 45; Cby: control n = 54 and B9d2, tctnΔ n = 51. ****, P < 0.0001; **, P < 0.01; ***, P < 0.001. (D and E) Mks1 and B9d1 are absent from the TZ in B9d2, tctnΔ mutant germ cells. (F) B9d1 localization (red) is only rescued when introducing both B9d2 and Tctn, the latter being fused to GFP. No rescue is observed when introducing B9d2 alone. (G) Scheme recapitulating the consequences of B9d2, tctnΔ deletion. In the absence of B9d2 and Tctn, the TZ is shorter, as revealed by Cep290 or Cby staining, and all other MKS components are missing. WT, wild type. Bars, 2 µm.
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fig4: B9d2 and Tctn are required to assemble the MKS complex and organize the TZ. (A, B, and D–F). IF analysis of squashed testes. (A and B) Cby and Cep290 localization domains are reduced in length at the TZ of the B9d2, tctnΔ mutant spermatocytes or spermatids. (C) Quantification of Cep290, Cby, and centriole lengths in meiosis stage in control or B9d2, tctnΔ mutant germ cells. Scattered plots with mean and SD are shown. Cep290 and Asl: control n = 39 and B9d2, tctnΔ n = 45; Cby: control n = 54 and B9d2, tctnΔ n = 51. ****, P < 0.0001; **, P < 0.01; ***, P < 0.001. (D and E) Mks1 and B9d1 are absent from the TZ in B9d2, tctnΔ mutant germ cells. (F) B9d1 localization (red) is only rescued when introducing both B9d2 and Tctn, the latter being fused to GFP. No rescue is observed when introducing B9d2 alone. (G) Scheme recapitulating the consequences of B9d2, tctnΔ deletion. In the absence of B9d2 and Tctn, the TZ is shorter, as revealed by Cep290 or Cby staining, and all other MKS components are missing. WT, wild type. Bars, 2 µm.
Mentions: We next analyzed the molecular organization of the TZ in absence of B9d2 and Tctn (Figs. 4 and S1). Cby and Cep290 are still recruited at the TZ (Fig. 4, A and B). We measured both the length of the centriole, labeled for Asl, and the length of the TZ labeled with Cep290 or Cby in absence of B9d2 and Tctn (Fig. 4 C), and we observed a small but significant decrease of TZ length that was correlated with an increase in centriole size. Strikingly, we observed a complete disruption of the MKS complex, as Mks1 and B9d1 are not recruited to the TZ in B9d2, tctnΔ spermatocytes (Fig. 4, D and E). B9d1 localization was rescued by reintroducing both tctn and B9d2 (Fig. 4 F). This shows that B9d2 and Tctn proteins are required to organize the MKS complex and that the absence of several proteins of the MKS complex at the TZ is largely dispensable for cilia assembly (Fig. 4 G). Based on this observation, we conclude that the MKS complex alone has only a subtle role in the organization of the TZ and ciliary cap in Drosophila sperm cells.

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