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Research Article
Tubulin-binding cofactor E-like (TBCEL), the protein product of the mulet gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization
James J. Fabrizio, Janet Rollins, Christopher W. Bazinet, Stephanie Wegener, Iryna Koziy, Rachel Daniel, Vincent Lombardo, Dwaine Pryce, Kavita Bharrat, Elissa Innabi, Marielle Villanobos, Gabriela Mendoza, Elisa Ferrara, Stephanie Rodway, Matthew Vicioso, Victoria Siracusa, Erin Dailey, Justin Pronovost, Simon Innabi, Vrutant Patel, Nicole DeSouza, Danielle Quaranto, Amir Niknejad
Biology Open 2020 9: bio049080 doi: 10.1242/bio.049080 Published 26 February 2020

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Figures

  • Fig. 1.
    Fig. 1.

    Mitochondrial dynamics in mulet mutant testes as revealed by don juan-GFP (dj-GFP). Positive control testes [A,B, ms(2)4210/CyO; dj-GFP] depict the coordination between the organized bundle of F-actin cones and a wave front of mitochondrial whorls as revealed by dj-GFP (arrowheads). Optical sections through ms(2)4210/ ms(2)4210; dj-GFP mutant testes (C,D) reveal asynchronous arrangement of the investment cones that maintain association with mitochondria (white arrowheads). Other mitochondria in both control and mutant testes appear behind the investment cones (yellow arrowheads). Scale bars: 10 µm.

  • Fig. 2.
    Fig. 2.

    Cytoplasmic microtubules are in excess prior to spermatid individualization and persist after individualization failure in mulet mutant cysts as observed by transmission electron microscopy. Cross-sections through wild-type (A–D) and mlt1/mlt1 (E–H) spermatid cysts are shown at low (12,000×, A,B,E.F), medium (20,000×, C,G) and high (50,000×, D,H) magnifications. Prior to individualization, wild-type cysts exhibit loosely packed sperm tails in a syncytium (A,B). Small, barely distinct cytoplasmic microtubules are observed around the axonemes in one cyst (B, inset, green arrowheads). Cross-sections through wild-type cysts after individualization reveal tightly packed individualized spermatids with little cytoplasm between the flagella (C,D). Cross sections through mlt1 mutant cysts prior to individualization are comparable to wild type (E), but upon close inspection, they reveal an excess of cytoplasmic microtubules around the axonemes (E, inset, green arrowheads). After the passage of the IC, mlt cysts reveal large deposits of excess cytoplasm (F, white arrows) as well as the occasional cross-section through an investment cone (F, white arrowheads) and individualized sections of flagella (F, red arrowheads). Axonemes in mlt mutant testes are consistently surrounded by an array of cytoplasmic microtubules (G,H arrowheads). Scale bar: 1 µm.

  • Fig. 3.
    Fig. 3.

    Hypomorphic and amorphic mulet mutant males exhibit counterintuitive individualization phenotypes. Wild-type testes exhibit intact ICs that form waste-bags at the apical end of the testis (arrowheads in A). While testes from hypomorphic mlt[EP-CG12214]/Df mutant males exhibit severely disrupted ICs that fail to form waste bags (arrowheads in B), testes from homozygous null mlt[G18151] mutant males exhibit mildly disrupted ICs that appear to form waste-bag-like structures at the apical end of the testes (arrowheads in C,D). Scale bars: 30 µm.

  • Fig. 4.
    Fig. 4.

    Rescue of the mulet mutant phenotype using tub-Gal4. Positive control testes [A, EP-CG12214 or Df(2R)BSC281/CyO] reveal intact ICs (arrowheads in A), while investment cones in the negative control [B, EP-CG12214/Df(2R)BSC281] are disorganized (arrowheads in B). Expression of TBCEL under tub-Gal4 control in hemizygous mutant testes most often resulted in partial rescue of the phenotype [C,D, EP-CG12214/Df(2R)BSC281; tub-Gal4 UAS-mCD8-GFP] as characterized by improved organization of the IC (arrowheads in C,D). Males with two copies of the EP-CG12214 (E,F; EP-CG12214/EP-CG12214;tub-Gal4 UAS-mCD8-GFP) very often exhibited full rescue, as characterized by ICs that were indistinguishable from wild type (arrowheads in E,F). Scale bar: 20 µm.

  • Table 1.
  • Fig. 5.
    Fig. 5.

    Germline-specific knockdown of TBCEL using bam-Gal4-VP16 phenocopies mulet. (A,B) Low magnification (100×) images of testes dissected from negative control UAS-CG12214 RNAi males. (C–H) Images on the right are higher-magnification (200×) close-ups of images on the left (100×). Control testes exhibit the presence of waste-bags and intact ICs (arrowheads in A and B, respectively). UAS-CG12214RNAi; bam-Gal4-VP16 testes exhibited IC defects comparable to mulet. Specifically, at 25°C there was mild disruption of the ICs (white arrowheads in C,D). (E,F) More severe disruptions were observed at 28°C (white arrowheads in E,F), consistent with a higher level of RNAi at the elevated temperature. Milder defects, comparable to the null mutant phenotype, were observed in testes dissected from UAS-CG12214RNAi/UAS-dicer; bam-Gal4-VP16 males (white arrowheads in G,H). Green arrowheads in C and H indicate F-actin sleeves, which were regularly observed in testes from knockdown males. Scale bars: (A–C,E,G) 40 µm; (D,F,H) 20 µm.

  • Table 2.
  • Table 3.
  • Fig. 6.
    Fig. 6.

    Model for IC disruptions by cytoplasmic microtubules. (A) Normal ICs in wild-type cysts, absent cytoplasmic microtubules. (B) Mild IC disruptions caused by persistence of a few microtubule fragments when TBCEL is moderately reduced. (C) Severe IC disruptions caused by persistence of many microtubule fragments when TBCEL is heavily reduced. (D) Almost normal ICs permitted by alternate microtubule tracks when TBCEL is absent.

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Research Article
Tubulin-binding cofactor E-like (TBCEL), the protein product of the mulet gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization
James J. Fabrizio, Janet Rollins, Christopher W. Bazinet, Stephanie Wegener, Iryna Koziy, Rachel Daniel, Vincent Lombardo, Dwaine Pryce, Kavita Bharrat, Elissa Innabi, Marielle Villanobos, Gabriela Mendoza, Elisa Ferrara, Stephanie Rodway, Matthew Vicioso, Victoria Siracusa, Erin Dailey, Justin Pronovost, Simon Innabi, Vrutant Patel, Nicole DeSouza, Danielle Quaranto, Amir Niknejad
Biology Open 2020 9: bio049080 doi: 10.1242/bio.049080 Published 26 February 2020
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