Spindle assembly and cytokinesis in the absence of chromosomes during Drosophila male meiosis

Abstract

A large body of work indicates that chromosomes play a key role in the assembly of both a centrosomal and centrosome-containing spindles. In animal systems, the absence of chromosomes either prevents spindle formation or allows the assembly of a metaphase-like spindle that fails to evolve into an ana-telophase spindle. Here, we show that Drosophila secondary spermatocytes can assemble morphologically normal spindles in the absence of chromosomes. The Drosophila mutants fusolo and solofuso are severely defective in chromosome segregation and produce secondary spermatocytes that are devoid of chromosomes. The centrosomes of these anucleated cells form robust asters that give rise to bipolar spindles that undergo the same ana-telophase morphological transformations that characterize normal spindles. The cells containing chromosome-free spindles are also able to assemble regular cytokinetic structures and cleave normally. In addition, chromosome-free spindles normally accumulate the Aurora B kinase at their midzones. This suggests that the association of Aurora B with chromosomes is not a prerequisite for its accumulation at the central spindle, or for its function during cytokinesis.

Figure 1.

First meiotic division in fsl mutant males. Cells were stained for tubulin (green), centrin (orange), and DNA (blue). (a and b) Meiotic division in wild-type males. (a) Metaphase I; (b) Late telophase I; (c–e) Meiotic division in fsl males. (c) Metaphase I; (d) Late telophase I with nonsegregating chromosomes at the center of the cell; (e) Late telophase I with all chromosomes segregating to only one of the two presumptive daughter cells. Bar, 10 μm.

Figure 2.

Spindle formation and dynamics in chromosome-free fsl secondary spermatocytes. Cells were stained for tubulin (green), centrin (orange), and DNA (blue). (a–e) Second meiotic division in wild type males. (a) Metaphase; (b) Early anaphase, (c) Late anaphase; (d) Early telophase; (e) Late telophase. (a′–e′) Spindles of chromosome-free fsl secondary spermatocytes. (a′) Metaphase-like; (b′) Early anaphase-like; (c′) Late anaphase-like; (d′) Early telophase-like; (e′) Late telophase-like. Bar, 10 μm.

Figure 3.

Pav accumulates at the central spindle midzone of chromosome-free telophases. Cells were stained for tubulin (green), Pav (orange), and DNA (blue). (a) Telophase II from wild-type males. (b) Chromosome-free telophase II from fsl mutants. Bar, 10 μm.

Figure 4.

Aurora B distribution in wild-type and fsl spermatocytes. Cells were stained for tubulin (green), Aurora B (orange), and DNA (blue). (a) Wild-type metaphase I; (b) fsl metaphase I; (c) Wild-type telophase I; (d) fsl telophase I; (e) Chromosome-free fsl early telophase II; (f) Chromosome-free fsl late telophase II. Note that Aurora B concentrates in the central spindle midzone in the absence of chromosomes. Bar, 10 μm.

Figure 5.

Cytokinesis in the absence of chromosomes in fsl mutants. (a–c) Telophase II figures stained for tubulin (green), DNA (blue), and either actin (a, orange), myosin II (b, orange), or anillin (c, orange). Note that the cytokinetic structures of chromosome-free cells are comparable to those of chromosome-containing cells. (d and e) Live spermatids from wild-type (d) and fsl (e) males. Note that in fsl mutants, some nebenkern (arrowheads) are not associated with nuclei. Bars, 10 μm.