The role of anillin during meiotic cytokinesis of Drosophila males

Anillin is a 190 kDa actin-binding protein that concentrates in the leading edges of furrow canals during Drosophila cellularization and in the cleavage furrow of both somatic and meiotic cells. We analyzed anillin behavior during D. melanogaster spermatogenesis, and focused on the relationships between this protein and the F-actin enriched structures. In meiotic anaphases anillin concentrates in a narrow band around the cell equator. Cytological analysis of wild-type meiosis and examination of mutants defective in contractile ring assembly (chickadee and KLP3A), revealed that the formation of the anillin cortical band occurs before, and does not require the assembly of the F-actin based contractile ring. However, once the acto-myosin ring is assembled, the anillin band precisely colocalizes with this cytokinetic structure, accompanying its contraction throughout anaphase and telophase. In chickadee and KLP3A mutant ana-telophases the cortical anillin band fails to constrict, indicating that its contraction is normally driven by the cytokinetic ring. These findings, coupled with the analysis of anillin behavior in twinstar mutants, suggested a model on the role of anillin during cytokinesis. During anaphase anillin would concentrate in the cleavage furrow before the assembly of the contractile ring, binding the equatorial cortex, perhaps through its carboxy-terminal pleckstrin homology (PH) domain. Anillin would then interact with the actin filaments of the acto-myosin ring through its actin-binding domain, anchoring the contractile ring to the plasma membrane throughout cytokinesis

Chromatin staining of Drosophila testes

This protocol describes chromatin staining of Drosophila testes. To visualize DNA, preparations fixed using methanol-acetone, paraformaldehyde, or formaldehyde can be stained with several DNAbinding dyes. If the slides are to be examined with a fluorescence microscope equipped with filters that permit ultraviolet (UV) excitation, suitable dyes for DNA staining are Hoechst 33258 or 4′,6- diamidino-2-phenylindole (DAPI). If the slides are to be analyzed with a confocal microscope not equipped with a UV laser, DNA can be stained with either propidium iodide or TOTO-3 iodide. © 2012 Cold Spring Harbor Laboratory Press

Drosophila male meiosis as a model system for the study of cytokinesis in animal cells

Drosophila male meiosis offers unique opportunities for mutational dissection of cytokinesis. This system allows easy and unambiguos identification of mutants defective in cytokinesis through the examination of spermatid morphology. Moreover, cytokinesis defects and protein immunostaining can be analyzed with exquisite cytological resolution because of the large size of meiotic spindles. In the past few years several mutations have been isolated that disrupt meiotic cytokinesis in Drosophila males. These mutations specify genes required for the assembly, proper constriction or disassembly of the contractile ring. Molecular characterization of these genes has identified essential components of the cytokinetic machinery, highlighting the role of the central spindle during cytokinesis. This structure appears to be both necessary and sufficient for signaling cytokinesis. In addition, many data indicate that the central spindle microtubules cooperatively interact with elements of the actomyosin contractile ring, so that impairment of either of these structures prevents the formation of the other

F-actin staining of Drosophila testes.

Preparations of Drosophila testes fixed with paraformaldehyde can be stained for F-actin according to the protocol described here. This staining procedure is particularly suitable for staining the male fusome and the cytokinetic contractile ring

Methanol-acetone fixation of Drosophila testes

This protocol describes two techniques for methanol-acetone fixation of Drosophila melanogaster testis squashes. The first method results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics show most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation method results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is poor preservation of chromosome structure. In most instances, the chromosomes do not show a distinct morphology and tend to coalesce into one or more masses of chromatin. The second technique for methanol-acetone fixation described here has proved to be particularly suitable for γ-tubulin and centrosomin immunostaining. It results in preparations having the same characteristics as those obtained with the first method. © 2011 Cold Spring Harbor Laboratory Press

Immunostaining of Drosophila testes

Fixed Drosophila testis preparations can be immunostained with a variety of antibodies. This article describes a general procedure for immunostaining. The concentration of the primary antibody will vary with both the type of antibody and the type of incubation and should be determined empirically each time. © 2011 Cold Spring Harbor Laboratory Press

Genetic dissection of meiotic cytokinesis in Drosphila males

mutants defective in meiotic cytokinesis can be easily identified by their multinucleate spermatids. Moreover, the large size of meiotic spindles allows characterization of mutant phenotypes with exquisite cytological resolution. We have screened a collection of 1955 homozygous mutant male sterile lines for those with multinucleate spermatids, and thereby identified mutations in 19 genes required for cytokinesis. These include 16 novel loci and three genes, diaphanous, four wheel drive, and pebble, already known to be involved in Drosophila cytokinesis. To define the primary defects leading to failure of cytokinesis, we analyzed meiotic divisions in male mutants for each of these 19 genes. Examination of preparations stained for tubulin, anillin, KLP3A, and F-actin revealed discrete defects in the components of the cytokinetic apparatus, suggesting that these genes act at four major points in a stepwise pathway for cytokinesis. Our results also indicated that the central spindle and the contractile ring are interdependent structures that interact throughout cytokinesis. Moreover, our genetic and cytological analyses provide further evidence for a cell type-specific control of Drosophila cytokinesis, suggesting that several genes required for meiotic cytokinesis in males are not required for mitotic cytokinesis