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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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Aberrant centriolar extension is observed in male germ cells in the absence of the ciliary cap. (A) Transmission EM analysis of spermatocyte cilia. In control late spermatocytes, centrioles (arrow) are docked to the membrane and cilia (arrowhead) extend at the cell surface. In dila81; cby1 mutants, centrioles (arrows) do not dock to the membrane (a3, a4, and a6) and microtubules extend from the centrioles (arrowheads). The centrioles are apparently unaffected and show the central tubule (a2 and a5, arrows). (B) Live imaging of Drosophila spermatocytes labeled with CellMask showing the centrioles (Ana1::GFP) docked to the plasma membrane protruding at the ciliary cap (white arrows) in control cells. The centrioles are present in an inverted orientation under the plasma membrane (dashed line) and not extending a ciliary cap. (C) Squashed spermatocytes and spermatids showing acetylated-tubulin extension (arrows) of the centrioles in double mutants compared with control. Aberrant microtubule extensions are also glutamylated (arrow, GT335 antibody). (D) Confocal imaging of squashed testes showing aberrant extensions of centrioles (arrows) in spermatocytes and spermatids. CG6652::GFP only labels the axoneme and no other microtubules in testes. (E) Scheme summarizing the roles of Cby and Dila in TZ and cilia assembly in Drosophila spermatocytes. Removal of Dila does not affect TZ components (see Fig. S3). The absence of Cby mildly affects MKS protein recruitment. Removal of both completely disorganizes TZ assembly and ciliary cap formation. The absence of ciliary cap is associated with aberrant and premature axonemal extension in spermatocytes. Bars, 2 µm (B–D).
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fig7: Aberrant centriolar extension is observed in male germ cells in the absence of the ciliary cap. (A) Transmission EM analysis of spermatocyte cilia. In control late spermatocytes, centrioles (arrow) are docked to the membrane and cilia (arrowhead) extend at the cell surface. In dila81; cby1 mutants, centrioles (arrows) do not dock to the membrane (a3, a4, and a6) and microtubules extend from the centrioles (arrowheads). The centrioles are apparently unaffected and show the central tubule (a2 and a5, arrows). (B) Live imaging of Drosophila spermatocytes labeled with CellMask showing the centrioles (Ana1::GFP) docked to the plasma membrane protruding at the ciliary cap (white arrows) in control cells. The centrioles are present in an inverted orientation under the plasma membrane (dashed line) and not extending a ciliary cap. (C) Squashed spermatocytes and spermatids showing acetylated-tubulin extension (arrows) of the centrioles in double mutants compared with control. Aberrant microtubule extensions are also glutamylated (arrow, GT335 antibody). (D) Confocal imaging of squashed testes showing aberrant extensions of centrioles (arrows) in spermatocytes and spermatids. CG6652::GFP only labels the axoneme and no other microtubules in testes. (E) Scheme summarizing the roles of Cby and Dila in TZ and cilia assembly in Drosophila spermatocytes. Removal of Dila does not affect TZ components (see Fig. S3). The absence of Cby mildly affects MKS protein recruitment. Removal of both completely disorganizes TZ assembly and ciliary cap formation. The absence of ciliary cap is associated with aberrant and premature axonemal extension in spermatocytes. Bars, 2 µm (B–D).

Mentions: In the absence of both Cby and Dila, we observed that Mks1 and B9d1 (Fig. 6 B) are absent at the tip of BB in Drosophila spermatocytes and spermatids. Cep290 was also almost completely lost (Fig. 6 C; greater than sevenfold reduction), whereas no differences in the overall expression levels of Mks1-GFP or Cep290-GFP could be detected in the testes by Western blot (Fig. S3 B). These observations again reveal strong genetic interactions between dila and cby as MKS proteins and Cep290 are still present in the single cby or dila mutants and their distribution is only slightly modified (Fig. S4). Only Unc was still present in absence of Cby and Dila (Fig. 6 D). However, Unc distribution was altered, with an expansion of its expression domain, suggesting a defective organization of the ring centriole and membrane cap in the dila81; cby1 double mutant. EM observations of the centrioles in Drosophila spermatocytes showed that centrioles did not form a primary cilium-like structure, failed to dock to the plasma membrane, and had an odd orientation inside the cell (Fig. 7 A). No ciliary cap was observed in spermatocytes. Centrioles were apparently normally formed, showing the regular triplet arrangement and the presence of the central tube (Fig. 7 A, a2 and a5, arrow). Strikingly, aberrant microtubule extensions from centrioles were observed (Fig. 7 A, a3, a4, and a6, arrowheads). The absence of docking of the centrioles to the plasma membrane was confirmed by live imaging of centrioles in spermatocytes (Fig. 7 B). In addition, we could confirm the absence of the ciliary cap in spermatids by detecting Rab8, which strongly labels the cap from end of meiosis to late elongation stages (Fig. S3 C). In dila81; cby1 spermatids, Rab8 staining was completely absent from BB distal ends, whereas Rab8 was not affected in dila81 mutants and present at the ciliary cap in a significant fraction (∼35%) of the spermatids in cby1 mutants (Fig. S3 C).


Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells
Aberrant centriolar extension is observed in male germ cells in the absence of the ciliary cap. (A) Transmission EM analysis of spermatocyte cilia. In control late spermatocytes, centrioles (arrow) are docked to the membrane and cilia (arrowhead) extend at the cell surface. In dila81; cby1 mutants, centrioles (arrows) do not dock to the membrane (a3, a4, and a6) and microtubules extend from the centrioles (arrowheads). The centrioles are apparently unaffected and show the central tubule (a2 and a5, arrows). (B) Live imaging of Drosophila spermatocytes labeled with CellMask showing the centrioles (Ana1::GFP) docked to the plasma membrane protruding at the ciliary cap (white arrows) in control cells. The centrioles are present in an inverted orientation under the plasma membrane (dashed line) and not extending a ciliary cap. (C) Squashed spermatocytes and spermatids showing acetylated-tubulin extension (arrows) of the centrioles in double mutants compared with control. Aberrant microtubule extensions are also glutamylated (arrow, GT335 antibody). (D) Confocal imaging of squashed testes showing aberrant extensions of centrioles (arrows) in spermatocytes and spermatids. CG6652::GFP only labels the axoneme and no other microtubules in testes. (E) Scheme summarizing the roles of Cby and Dila in TZ and cilia assembly in Drosophila spermatocytes. Removal of Dila does not affect TZ components (see Fig. S3). The absence of Cby mildly affects MKS protein recruitment. Removal of both completely disorganizes TZ assembly and ciliary cap formation. The absence of ciliary cap is associated with aberrant and premature axonemal extension in spermatocytes. Bars, 2 µm (B–D).
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fig7: Aberrant centriolar extension is observed in male germ cells in the absence of the ciliary cap. (A) Transmission EM analysis of spermatocyte cilia. In control late spermatocytes, centrioles (arrow) are docked to the membrane and cilia (arrowhead) extend at the cell surface. In dila81; cby1 mutants, centrioles (arrows) do not dock to the membrane (a3, a4, and a6) and microtubules extend from the centrioles (arrowheads). The centrioles are apparently unaffected and show the central tubule (a2 and a5, arrows). (B) Live imaging of Drosophila spermatocytes labeled with CellMask showing the centrioles (Ana1::GFP) docked to the plasma membrane protruding at the ciliary cap (white arrows) in control cells. The centrioles are present in an inverted orientation under the plasma membrane (dashed line) and not extending a ciliary cap. (C) Squashed spermatocytes and spermatids showing acetylated-tubulin extension (arrows) of the centrioles in double mutants compared with control. Aberrant microtubule extensions are also glutamylated (arrow, GT335 antibody). (D) Confocal imaging of squashed testes showing aberrant extensions of centrioles (arrows) in spermatocytes and spermatids. CG6652::GFP only labels the axoneme and no other microtubules in testes. (E) Scheme summarizing the roles of Cby and Dila in TZ and cilia assembly in Drosophila spermatocytes. Removal of Dila does not affect TZ components (see Fig. S3). The absence of Cby mildly affects MKS protein recruitment. Removal of both completely disorganizes TZ assembly and ciliary cap formation. The absence of ciliary cap is associated with aberrant and premature axonemal extension in spermatocytes. Bars, 2 µm (B–D).
Mentions: In the absence of both Cby and Dila, we observed that Mks1 and B9d1 (Fig. 6 B) are absent at the tip of BB in Drosophila spermatocytes and spermatids. Cep290 was also almost completely lost (Fig. 6 C; greater than sevenfold reduction), whereas no differences in the overall expression levels of Mks1-GFP or Cep290-GFP could be detected in the testes by Western blot (Fig. S3 B). These observations again reveal strong genetic interactions between dila and cby as MKS proteins and Cep290 are still present in the single cby or dila mutants and their distribution is only slightly modified (Fig. S4). Only Unc was still present in absence of Cby and Dila (Fig. 6 D). However, Unc distribution was altered, with an expansion of its expression domain, suggesting a defective organization of the ring centriole and membrane cap in the dila81; cby1 double mutant. EM observations of the centrioles in Drosophila spermatocytes showed that centrioles did not form a primary cilium-like structure, failed to dock to the plasma membrane, and had an odd orientation inside the cell (Fig. 7 A). No ciliary cap was observed in spermatocytes. Centrioles were apparently normally formed, showing the regular triplet arrangement and the presence of the central tube (Fig. 7 A, a2 and a5, arrow). Strikingly, aberrant microtubule extensions from centrioles were observed (Fig. 7 A, a3, a4, and a6, arrowheads). The absence of docking of the centrioles to the plasma membrane was confirmed by live imaging of centrioles in spermatocytes (Fig. 7 B). In addition, we could confirm the absence of the ciliary cap in spermatids by detecting Rab8, which strongly labels the cap from end of meiosis to late elongation stages (Fig. S3 C). In dila81; cby1 spermatids, Rab8 staining was completely absent from BB distal ends, whereas Rab8 was not affected in dila81 mutants and present at the ciliary cap in a significant fraction (∼35%) of the spermatids in cby1 mutants (Fig. S3 C).

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