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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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Cby and Dila cooperate to build the Drosophila TZ in sensory cilia. (A) Whole-mount staining of chordotonal organs of the second antennal segment showing neuronal cell bodies and dendrites (Futsch) and cilia (GT335 antibody). Cilia are missing in dila81; cby1 double mutants. (B) EM analysis of cross section (top) reveals an almost complete absence of cilia, the few remaining ones being severely disorganized compared with control (arrows). On longitudinal sections (bottom), the two BBs (DBB, distal basal body; PBB, proximal basal body) are visible at the tip of the dendrites, but no cilium and TZ are built (white arrow). In some cases, the two BBs are not aligned properly (right), and accumulation of vesicles is seen (asterisk). (C) EM analysis of cross sections at the base of the cilia. Transition fibers (arrows) can be seen connecting the BB to the membrane in control or cby1 flies. In dila81; cby1, the BB showed possible distal extensions, but these did not connect the membrane. (D and E) Whole-mount staining of pupae antennae. (D) Centrioles (Ana1::GFP) are still present at the tip of the dendrites (HRP staining) in dila81; cby1 mutants. (E) Cep290 and Mks1 are not recruited at TZ in dila81; cby1 chordotonal organs. (F) Scheme of the defects observed after removing Dila (*, adapted from Ma and Jarman, 2011), Cby (**, adapted from Enjolras et al., 2012), or both (this study). In dila81 mutant antennae, the TZ is apparently normal by EM. In the cby1 mutant, the TZ shows incomplete ultrastructural defects. In the double mutant, no TZ is built. Bars, 10 µm (A, D, and E).
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fig5: Cby and Dila cooperate to build the Drosophila TZ in sensory cilia. (A) Whole-mount staining of chordotonal organs of the second antennal segment showing neuronal cell bodies and dendrites (Futsch) and cilia (GT335 antibody). Cilia are missing in dila81; cby1 double mutants. (B) EM analysis of cross section (top) reveals an almost complete absence of cilia, the few remaining ones being severely disorganized compared with control (arrows). On longitudinal sections (bottom), the two BBs (DBB, distal basal body; PBB, proximal basal body) are visible at the tip of the dendrites, but no cilium and TZ are built (white arrow). In some cases, the two BBs are not aligned properly (right), and accumulation of vesicles is seen (asterisk). (C) EM analysis of cross sections at the base of the cilia. Transition fibers (arrows) can be seen connecting the BB to the membrane in control or cby1 flies. In dila81; cby1, the BB showed possible distal extensions, but these did not connect the membrane. (D and E) Whole-mount staining of pupae antennae. (D) Centrioles (Ana1::GFP) are still present at the tip of the dendrites (HRP staining) in dila81; cby1 mutants. (E) Cep290 and Mks1 are not recruited at TZ in dila81; cby1 chordotonal organs. (F) Scheme of the defects observed after removing Dila (*, adapted from Ma and Jarman, 2011), Cby (**, adapted from Enjolras et al., 2012), or both (this study). In dila81 mutant antennae, the TZ is apparently normal by EM. In the cby1 mutant, the TZ shows incomplete ultrastructural defects. In the double mutant, no TZ is built. Bars, 10 µm (A, D, and E).

Mentions: Strikingly, when we combined cby and dila mutations, we observed severe consequences on the function and architecture of sensory neurons. Flies are completely uncoordinated and cilia are absent in chordotonal neurons as observed by immunofluorescence (IF) and EM analyzes (Fig. 5, A and B). Centrioles fail to build a TZ (Fig. 5, B and C), and Cep290 and Mks1 are absent at dendritic tips (Fig. 5 E). In dila81; cby1 double mutants, we did not manage to observe typical features of docked BB such as transition fibers, present both in control or cby1 mutant flies (Fig. 5 C). However, centrioles are present at the tip of the dendrites and apparently show distal projections but are not connected to the membrane (Fig. 5, C and D). This phenotype differs from observations of mutants of genes encoding centriolar proteins, such as plp, for which all BBs fail to reach the distal part of the dendrite (Galletta et al., 2014).


Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells
Cby and Dila cooperate to build the Drosophila TZ in sensory cilia. (A) Whole-mount staining of chordotonal organs of the second antennal segment showing neuronal cell bodies and dendrites (Futsch) and cilia (GT335 antibody). Cilia are missing in dila81; cby1 double mutants. (B) EM analysis of cross section (top) reveals an almost complete absence of cilia, the few remaining ones being severely disorganized compared with control (arrows). On longitudinal sections (bottom), the two BBs (DBB, distal basal body; PBB, proximal basal body) are visible at the tip of the dendrites, but no cilium and TZ are built (white arrow). In some cases, the two BBs are not aligned properly (right), and accumulation of vesicles is seen (asterisk). (C) EM analysis of cross sections at the base of the cilia. Transition fibers (arrows) can be seen connecting the BB to the membrane in control or cby1 flies. In dila81; cby1, the BB showed possible distal extensions, but these did not connect the membrane. (D and E) Whole-mount staining of pupae antennae. (D) Centrioles (Ana1::GFP) are still present at the tip of the dendrites (HRP staining) in dila81; cby1 mutants. (E) Cep290 and Mks1 are not recruited at TZ in dila81; cby1 chordotonal organs. (F) Scheme of the defects observed after removing Dila (*, adapted from Ma and Jarman, 2011), Cby (**, adapted from Enjolras et al., 2012), or both (this study). In dila81 mutant antennae, the TZ is apparently normal by EM. In the cby1 mutant, the TZ shows incomplete ultrastructural defects. In the double mutant, no TZ is built. Bars, 10 µm (A, D, and E).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Cby and Dila cooperate to build the Drosophila TZ in sensory cilia. (A) Whole-mount staining of chordotonal organs of the second antennal segment showing neuronal cell bodies and dendrites (Futsch) and cilia (GT335 antibody). Cilia are missing in dila81; cby1 double mutants. (B) EM analysis of cross section (top) reveals an almost complete absence of cilia, the few remaining ones being severely disorganized compared with control (arrows). On longitudinal sections (bottom), the two BBs (DBB, distal basal body; PBB, proximal basal body) are visible at the tip of the dendrites, but no cilium and TZ are built (white arrow). In some cases, the two BBs are not aligned properly (right), and accumulation of vesicles is seen (asterisk). (C) EM analysis of cross sections at the base of the cilia. Transition fibers (arrows) can be seen connecting the BB to the membrane in control or cby1 flies. In dila81; cby1, the BB showed possible distal extensions, but these did not connect the membrane. (D and E) Whole-mount staining of pupae antennae. (D) Centrioles (Ana1::GFP) are still present at the tip of the dendrites (HRP staining) in dila81; cby1 mutants. (E) Cep290 and Mks1 are not recruited at TZ in dila81; cby1 chordotonal organs. (F) Scheme of the defects observed after removing Dila (*, adapted from Ma and Jarman, 2011), Cby (**, adapted from Enjolras et al., 2012), or both (this study). In dila81 mutant antennae, the TZ is apparently normal by EM. In the cby1 mutant, the TZ shows incomplete ultrastructural defects. In the double mutant, no TZ is built. Bars, 10 µm (A, D, and E).
Mentions: Strikingly, when we combined cby and dila mutations, we observed severe consequences on the function and architecture of sensory neurons. Flies are completely uncoordinated and cilia are absent in chordotonal neurons as observed by immunofluorescence (IF) and EM analyzes (Fig. 5, A and B). Centrioles fail to build a TZ (Fig. 5, B and C), and Cep290 and Mks1 are absent at dendritic tips (Fig. 5 E). In dila81; cby1 double mutants, we did not manage to observe typical features of docked BB such as transition fibers, present both in control or cby1 mutant flies (Fig. 5 C). However, centrioles are present at the tip of the dendrites and apparently show distal projections but are not connected to the membrane (Fig. 5, C and D). This phenotype differs from observations of mutants of genes encoding centriolar proteins, such as plp, for which all BBs fail to reach the distal part of the dendrite (Galletta et al., 2014).

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