SummaryCentrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer [1]. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms [2–4]. Although Plk4’s centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent.
Centrioles play a key role in the development of the fly. They are needed for the correct formation of centrosomes, the organelles at the poles of the spindle that can persist as microtubule organizing centers (MTOCs) into interphase. The ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome but also as the basal body, the structure that templates the formation of cilia and flagellae. Thus the structure and functions of the centriole, the centrosome, and the basal body have an impact upon many aspects of development and physiology that can readily be modeled in Drosophila. Centrosomes are essential to give organization to the rapidly increasing numbers of nuclei in the syncytial embryo and for the spatially precise execution of cell division in numerous tissues, particularly during male meiosis. Although mitotic cell cycles can take place in the absence of centrosomes, this is an error-prone process that opens up the fly to developmental defects and the potential of tumor formation. Here, we review the structure and functions of the centriole, the centrosome, and the basal body in different tissues and cultured cells of Drosophila melanogaster, highlighting their contributions to different aspects of development and cell division.
Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.
One of the great advantages of Drosophila melanogaster as model organism is the availability of balancer chromosomes. These chromosomes suppress recombination with their homologues, allowing the maintenance of lethal and sterile mutants as balanced heterozygotes. All balancers carry dominant markers that are visible in adult flies, but only a subset have markers that unambiguously distinguish homozygous mutant larvae from their heterozygous siblings. The latter balancers include those that express high levels of the GFP protein under the indirect control, via the UAS/GAL4 system, of either the Kruppel (Kr), 1 or hsp70, 2 promoter. In addition, there are direct-drive balancers that express GFP under the control of the actin A5c promoter 3 or YFP under the control of Deformed (Dfd HZ2.7rev) or glass (GMR) enhancer elements. 4 Each of the GFP-or YFP-expressing balancers has specific advantages, but all share a common drawback: These balancers require the use of a dissecting microscope equipped with an UV light source, which for reliable fluorescence detection is preferably used in the dark.The TM6B balancer 5 carries the Tb 1 dominant mutation, which results in squat larvae and pupae. We have been using this balancer for many years to unambiguously distinguish homozygous mitotic mutants dying at late larval stages from their heterozygous siblings (reviewed in ref. 6). This balancer proved particularly useful when mutant larvae are rare and one has to examine several vials (or bottles) to find third instar larvae suitable for dissection and cytological analysis. To generate new tools for easy detection of larvae homozygous for lethal mutations on the X or the second chromosome, we decided to generate males to w/w females (TMS is a Δ2-3-bearing third chromosome balancer expressing the P-element transposase described in ref. 10); from approximately 1,000 progeny we recovered two FM7 chromosomes that co-segregated with Tb (we propose to name these balancers FM7-TbA and FM7-TbB). To generate a second chromosome balancer with a P{Tb 1 } insertion we used a CyO balancer bearing the additional markers S and bw 1 (designated as CyO-Sbw in FlyBase). We crossed w/w; P{Tb 1 }/CyOSbw; TMS/+ females to w; CyO/Sco males and recovered three CyO-Sbw-P{Tb 1 } chromosomes from approximately 1,000 progeny (we propose to name these balancers CyO-TbA, CyO-TbB and CyO-TbC). To assess the utility of these FM7a and CyO Tb-bearing balancers, we compared their Tb phenotype with the Tb 1 mutant phenotype associated with TM6B. 5 To quantify the squat phenotype elicited by the balancers we measured the axial ratio 11 of larvae and pupae (AR, length/width) heterozygous for each balancer. It has been previously shown that the AR does not depend on larval and pupal size and provides a reliable measure of the Tb phenotype.9 As shown in figure 1, the ARs observed in FM7-TbA, FM7-TbB, CyO-TbA, CyO-TbB, CyOTbC and TM6B heterozygotes are fully comparable and significantly different from those of wild-type or non-Tb-bearing larvae and pupae. We thus co...
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