Isolation of Cytokinesis Mutants
To isolate mutations affecting cytokinesis during Drosophila male meiosis, we screened a collection of 1955 lines carrying ethyl methonesulfonate-induced mutations that cause male sterility. These sterile lines were isolated from ~12,000 viable lines, generated in C. Zuker's laboratory (see MATERIALS AND METHODS). Analysis by phase-contrast microscopy revealed that 64 of these male sterile lines produced spermatids with an abnormally large nebenkern and multiple normal-sized nuclei, indicating defects in cytokinesis but not in chromosome segregation. Twenty-six of these 64 lines showed <20% abnormal spermatids, most of which had a large nebenkern associated with only two nuclei. Although potentially interesting, these mutant lines were not studied further because their meiotic abnormalities were too rare to allow cytological analysis of the cytokinetic defect (see below).
We next performed inter se complementation tests among the remaining 11 second chromosome lines and the remaining 27 third chromosome mutant lines. The analysis of spermatid morphology in each heteroallelic combination led to identification of 23 complementation groups, seven on the second and 16 on the third chromosome. Representative mutant alleles from these groups were then tested for complementation with several known Drosophila
cytokinesis mutations. Mutations in one of the complementation groups on chromosome 2 were allelic to diaphanous
(Castrillon and Wasserman, 1994
). Two of the complementation groups on chromosome 3 proved to be pebble
(Hime and Saint, 1992
; Lehner, 1992
) and four wheel drive
(Brill et al., 2000
). No new alleles of chickadee
(Cooley et al., 1992
(Adams et al., 1998
(Sunkel and Glover, 1988
), or twinstar
(Gunsalus et al., 1995
) were identified.
Representative strong alleles of the remaining 20 complementation groups were mapped by meiotic recombination and tested for failure to complement a set of deficiencies that, together, deleted more than two-thirds of the appropriate chromosome. Both the recombination and deficiency mapping experiments were scored on the basis of the spermatid phenotype. In four of the third chromosome lines, the lesions responsible for the cytokinesis phenotype did not map to a single locus in recombination analysis, suggesting additive or synergistic effects of more than one mutation. In each case, the line was the only representative of its complementation group and was not studied further. For the remaining 16 complementation groups, the representative mutant allele analyzed behaved simply in recombination mapping and could be mapped to a single interval. In some cases, the interval could be narrowed down to a discrete chromosomal region defined by a deficiency that failed to complement the mutation for the spermatid phenotype ().
Map positions of genes identified by cytokinesis-defective mutants
Each of the 19 complementation groups identified in our screen (the 16 new genes, dia, fwd, and pbl) contained mutant alleles that displayed high frequencies of abnormal spermatids (). Mutant males commonly had spermatids with a large nebenkern and two or four normal-sized nuclei ( and ), indicating failure of cytokinesis during one or both the meiotic divisions, respectively. Occasionally, spermatids with three nuclei were observed, suggesting failure of cytokinesis during the first meiotic division, followed by budding off of only one daughter cell during the second meiotic division (). A few mutants produced spermatids with eight or more nuclei, most likely due to defects in the mitotic divisions preceding meiosis ().
Abnormal spermatids in cytokinesis-defective mutants
Cytological Characterization of Cytokinesis Mutants
To define the primary defects in cytokinesis leading to the formation of spermatids with multiple nuclei, we examined meiotic division in representative mutant males for each of the 19 genes identified in our screen. Testis preparations were stained with anti-tubulin antibodies to visualize spindle microtubules and with rhodamine-phalloidin to visualize the F-actin–based contractile ring. Testis preparations of all mutants were also immunostained for two additional proteins involved in Drosophila
cytokinesis: anillin and Klp3A. Anillin is an actin-binding protein that accumulates at the equatorial cortex of ana-telophase cells (Field and Alberts, 1995
; Hime et al., 1996
; Giansanti et al., 1999
), and Klp3A is a kinesin-like protein that concentrates at the central spindle midzone (Williams et al., 1995
In wild-type spermatocytes, anillin accumulates at the cell cortex during late anaphase, forming a circumferential band at the cell equator (). At this time, the central spindle has just begun to assemble and contains many antiparallel microtubules that overlap at the center of the cell (); the appearance of the anillin ring coincides with the first detectable accumulation of Klp3A at the spindle midzone (Giansanti et al., 1999
; Giansanti, Bonaccorsi and Gatti, unpublished observations). The F-actin contractile ring forms in a subsequent stage of ana-telophase and precisely colocalizes with the anillin band. Anillin and F-actin continue to colocalize throughout ring constriction, whereas the central spindle midzone continues to accumulate Klp3A and becomes increasingly pinched (Figures , , and ). At the end of cytokinesis, the F-actin ring disassembles, whereas anillin becomes incorporated into a stable ring canal (Hime et al., 1996
; Giansanti et al., 1999
Anillin localization in wild-type primary spermatocytes. Cells are stained for tubulin, anillin, and DNA. (A) Early telophase. (B) Mid-telophase. (C) Late telophase showing aster separation (arrows) at the spindle poles. Bar, 10 μm.
Visualization of the actin ring in wild-type primary spermatocytes. Cells are stained for tubulin, actin, and DNA. (A) Early telophase. (B) Mid-telophase. (C) Late telophase showing aster separation (arrows) at the spindle poles. Bar, 10 μm.
Klp3A localization in wild-type primary spermatocytes. Cells are stained for tubulin, Klp3A, and DNA. (A) Early telophase. (B) Late telophase showing aster separation at the spindle poles. Bar, 10 μm.
Progression through telophase I involves morphological transformations of the spindle poles as well as of the central spindle and contractile ring. In early and mid-telophase I, each spindle pole displays a single centrosome that nucleates an astral array of microtubules. As telophase I progresses, the centrioles comprising each centrosome separate, giving rise to two separate asters that rotate toward the opposite sides of the nucleus, in preparation for the second meiotic division (Figures and ). The contractile ring and the central spindle are usually nearly fully constricted by the time the asters looked separated (Figures and ). Thus, aster separation provides a useful marker for identification of late telophase cells. We have used this marker to characterize the mutant phenotypes of primary spermatocytes undergoing the first meiotic division. However, we also examined the second meiotic division in all mutants. In all cases, the defects observed in telophase II cells were fully consistent with those seen in telophase I figures (our unpublished data).
An examination of preparations from mutant testes revealed that in one mutant, fermata (fer), most spermatocytes were very large and contained multiple dividing nuclei, consistent with the fer spermatid phenotype (). The presence of multiple, overlapping meiotic spindles within the same large cells (our unpublished data) precluded a reliable definition of the cytokinesis defect of this mutant. The other 18 mutants, however, displayed discrete defects in components of the cytokinetic machinery (). Based on our phenotypic analyses, we divided the genes identified by these mutations into the four classes described below.
Cytological characterization of telophases of cytokinesis-defective mutants
Genes Required for Anillin Localization, Central Spindle Formation, and Contractile Ring Assembly
Double staining of pbl spermatocytes for either anillin and tubulin or F-actin and tubulin revealed that wild-type function of pbl is required for the assembly of all the major structures present at the equator of spermatocytes in anatelophase. Spermatocytes from pbl homozygotes or hemizygotes lacked the anillin cortical band throughout anaphase and telophase (n = 150; ). In addition, ana-telophase spermatocytes from pbl mutants failed to form both the central spindle and the F-actin ring ( and ). Instead of a robust and well-organized central spindle (Figures and ), these mutant ana-telophases displayed only sparse microtubules between the daughter nuclei and did not show any Klp3A accumulation at the center of the cell ().
Figure 5. Cytological phenotype of pbl mutant spermatocytes. Cells are stained for tubulin, DNA, and either anillin, actin, or Klp3A. (A and D) Early telophases. (B and C) Late telophases. Note the absence of an organized central spindle with Klp3A at its midzone, (more ...)
The formation of a normal anillin ring also requires wild-type function of dia
. A continuous anillin ring did not form in ana-telophases from dia
mutants; instead, only patches of anillin were detected at the equator of these cells (). These anillin patches were observed in both early and late telophase figures, suggesting that dia
mutations affect anillin band formation rather than its stability. Spermatocytes carrying the new dia
allele identified in our screen also lacked an F-actin ring and failed to form a well-organized central spindle with Klp3A in the middle, consistent with previous studies of other dia
alleles ( and ; Giansanti et al., 1998
Figure 6. Cytological phenotype of dia mutant spermatocytes. Cells are stained for tubulin, DNA and either anillin or actin. (A) Early telophase. (B) Late telophase. (C) Mid-telophase. Note the discontinuity of the anillin band and the absence of both the contractile (more ...)
Genes Required for Both Central Spindle and F-Actin Ring Assembly
Mutations in five genes, scapolo
), james bond
), and smeagol
), affected the formation of both the central spindle and the F-actin ring but did not prevent anillin accumulation at the equator of dividing spermatocytes. In we present images illustrating the phenotype of scpo
; images from the other mutants in this class are shown in the supplemental material section (Figures and ). Spermatocytes from males mutant for any of these five genes lacked both an organized central spindle and an F-actin ring throughout ana-telophase ( and ; Figures 1 and 2 in Supplemental Material), just as those from pbl
mutants (Figures and and ). However, in scpo, cbe, bond, sau
, and sgo
mutants, anillin accumulated normally at the equator of late anaphase cells, forming a circumferential band indistinguishable from that seen in wild type (, and ; Figure 2 in Supplemental Material). This anillin band, unlike that of wild type (), failed to constrict properly during telophase (Figure 7 and Figure 2 in Supplemental Material). Most late telophases from these five mutants displayed anillin rings that were either unconstricted or only poorly constricted. In these rings, anillin was usually more diffuse than in wild type (Figure 7 and Figure 2 in Supplemental Material), suggesting that proper morphology of a late telophase anillin ring depends on the presence of a normal actin ring. These observations confirm that anillin concentration at the equatorial cortex occurs independently of either central spindle or actin ring assembly (Giansanti et al., 1999
) and suggest that constriction of the anillin ring may normally be driven by constriction of the F-actin ring.
Figure 7. Cytological phenotype of scpo mutant spermatocytes. Cells are stained for tubulin, DNA, and either anillin, actin, or Klp3A. (A) Late telophase lacking both an organized central spindle and the actin ring. (B and C) Late telophases with severely defective (more ...)
Genes Required for F-Actin Ring Constriction and Central Spindle Stability
Ten genes, four score
), four way stop
), funnel cakes
), onion rings
), four wheel drive
), and giotto
) are primarily required for constriction of the F-actin ring (Figures and and ). Spermatocytes from bru, cia, fro, fsco, fun, fws, omt
, and onr
displayed similar abnormalities in their cytokinetic structures. We present images from fsco
spermatocytes to illustrate phenotypes typical for this class of mutants (Figures , , ); images from bru, cia, fro, fun, fws, omt
, and onr
mutants are shown in the Supplemental Material (Figures and ) and in Farkas et al.
). A normallooking actin ring assembled in fsco
spermatocytes during early telophase ( and ). However, in most mid- and late telophase cells of this mutant, the actin ring was either as large as in early telophase or was only slightly constricted ( and ). In addition, in many late telophase figures the poorly constricted actin ring either seemed broken into several pieces or consisted of what seemed to be remnants of the early telophase ring ( and ). fsco
early telophases also displayed a regular anillin band that precisely colocalized with the actin ring (; our unpublished data); anillin continued to colocalize with the unconstricted/broken actin ring of fsco
mutants throughout mid- and late telophase (; our unpublished data).
Figure 8. Cytological phenotype of fsco mutant cells. Cells are stained for tubulin, DNA, and actin. (A) fsco mid-telophase with a normal central spindle and a regular actin ring. (B and C) Late telophases displaying unconstricted (B) and broken (C) actin rings (more ...)
Figure 9. Anillin localization in fsco mutant spermatocytes. Cells are stained for tubulin, DNA and anillin. (A) Early fsco telophase with a normal anillin band. (B) Late telophase with a poorly constricted anillin band and a severely defective central spindle. (more ...)
Figure 10. Klp3A localization in fsco mutant spermatocytes. Cells are stained for tubulin, DNA, and Klp3A. (A) Early fsco telophase with a normal Klp3A signal at the central spindle midzone. (B) Late telophase with a severely defective central spindle and no Klp3A (more ...)
Spermatocytes from fsco mutants also exhibited progressive defects in the central spindle. In most fsco early telophases, the central spindle seemed normal and showed normal accumulation of KLP3A at its midzone ( and ). However, in mid- and late telophase figures, the central spindle was consistently either less dense than in wild type or completely disorganized (Figures , , and ). Central spindles less robust than their wild-type counterparts showed a reduction in KLP3A concentration at the midzone, whereas disorganized central spindles did not exhibit a detectable KLP3A signal (). In general, in both fsco and the other mutants of its class, the farther a cell had advanced through telophase, the more the central spindle and the actin ring seemed disrupted (Figures , , and ; Figures 3 and 4 in Supplemental Material). In addition, the degree of central spindle abnormality correlated with the degree of actin ring defect. Telophase cells with an intact central spindle showed a complete actin ring, cells with a less robust central spindle tended to have a broken actin ring, and cells without a central spindle displayed only remnants of the contractile ring. Together, our observations indicate that fsco, bru, cia, fro, fun, fws, omt, and onr spermatocytes have the ability to form both the central spindle and the contractile ring during early telophase. However, as mutant cells progress through telophase, the actin ring fails to constrict properly and the central spindle disassembles.
Examination of living spermatocytes from fsco, bru, cia, fro, fun, fws, omt, and onr males revealed defective cleavage furrow ingression (), as predicted from observation of fixed material. Typical results are presented in , showing cytokinesis in fun and fsco primary spermatocyte over time. In the wild-type ana-telophase cell shown in , the furrow advanced over a period of 15 min, leading to the formation of two daughter cells connected by a thin cytoplasmic bridge. In the fun and fsco cells, after a limited ingression, the furrow regressed, resulting into a clear failure of cytokinesis ().
Figure 11. In vivo time course of cleavage furrow in living spermatocytes under oil. Panels in each set are consecutive images taken at 5-min intervals. In wild type (wt), a furrow is initiated ~10 min after anaphase onset (left panel of the set) and continues (more ...)
Wild-type functions of gio
also seem to be required for furrow ingression, but at a later stage than the genes in the fsco
class. In early telophases and most midtelophases from fwd
mutants, the central spindle and the contractile ring both seemed normal. However, most fwd
late telophases displayed actin rings that were not fully constricted ( and ). In ~10% of these telophases, the unconstricted actin rings were broken and central spindles seemed less dense than in their wild-type counterparts (). In some late fwd
telophases displaying a high degree of ring constriction, the F-actin rings seemed thicker than in wild-type cells at the same stage (), as described previously for fwd
mutants (Brill et al., 2000
). However, thick actin rings were also seen in some of the rare telophases with constricted rings we occasionally observed in fsco, bru, cia, fro, fun, fws, omt
, and onr
mutants. In both fwd
spermatocytes, the anillin band formed normally and constricted to the same extent as the actin ring, as observed in the other mutants of the fsco
class (our unpublished data).
Figure 12. Defects in ring morphology in telophases from fwd mutants. Cells are stained for tubulin (green), actin (orange), and DNA (blue). (A) Early mutant telophase showing a regular actin ring. (B) Late telophase with an incompletely constricted actin ring. (more ...)
Genes Required for Actin Ring Disassembly
In spermatocytes from bird nest soup (bns) mutants, the central spindle had a normal morphology throughout telophase. Both the F-actin and the anillin ring assembled normally and exhibited a normal degree of constriction in midtelophase cells. However, in late telophase cells, the actin and anillin rings were both abnormal. The actin rings were commonly misshapen, containing excess F-actin compared with wild-type cells at the same stage, and seemed to lack an internal lumen (). We did not detect a clear lumen in 28 actin rings from bns late telophases, whereas approximately one-half the wild-type cells at the same stage displayed hollow actin rings. Most anillin rings of bns late telophases had a lumen but were more constricted than in wild-type late telophases (). Consistent with the latter observation, ring canals in bns spermatids were often smaller than in wild-type (our unpublished data). These results suggest that wild-type function of bns may be required for normal disassembly of the actin ring during late telophase.
Figure 13. Cytological phenotype of bns mutants. The cells shown in A and B are stained for tubulin (green), actin (orange), and DNA (blue); the telophases shown in C and D are stained for anillin (green), actin (red), and DNA (blue). (A) Mid-telophase with a normal (more ...)