Drosophila Klp67A is required for proper chromosome congression and segregation during meiosis I

Drosophila Klp67A belongs to the Kip3 subfamily of Kinesin-type microtubule catastrophe factors. In primary spermatocytes, loss of klp67A leads to defects in karyokinesis and cytokinesis. We show that these cells formed disorganised, bipolar spindles that contained increased numbers of microtubules. The kinetochore fibres were wavy and bent, whereas astral microtubules appeared abnormally robust and formed cortical bundles. Time-lapse studies revealed that during biorientation, the chromosomes in klp67A mutant cells continued to reorient for about twice as long as those in control cells. Metaphase plates were poorly defined in the mutants and often formed at non-equatorial positions. Consistent with the above abnormalities in chromosome congression, we found that in wild-type cells Klp67A associated with prometaphase/metaphase kinetochores before redistributing to the central spindle at anaphase onset. Although the timing of this redistribution of kinetochores argues against a role in anaphase chromosome segregation, dyads in the mutants disjoined but exhibited greatly diminished poleward velocities. They travelled on average at approximately 34% of the velocity of their wild-type counterparts and often decondensed at non-polar locations. Hypomorphic mutations of klp67A may lead to segregation defects

Spindle Formation in the Mouse Embryo Requires Plk4 in the Absence of Centrioles

During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. We show that Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles

Klp67A destabilises pre-anaphase microtubules but subsequently is required to stabilise the central spindle

Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of central spindle microtubules. In mutants anaphase B spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior spindle microtubules then formed diminished bundles of abnormally positioned central spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of central spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior central spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted and regressed as they could not be sustained by the central spindles that became increasing unstable over time and often completely degraded. Consistent with this, actin and anillin failed to form homogenous bands. Thus, the Klp67A microtubule catastrophe factor is required for cytokinesis by regulating both the formation and stability of the central spindle

Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting β-tubulin, suggesting Mgr function is required for tubulin stability. Instability of β-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic β-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not

Wolbachia-Mediated Male Killing Is Associated with Defective Chromatin Remodeling

Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon

Conserved molecular interactions in centriole-to-centrosome conversion.

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila melanogaster and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood.J.F., Z.L., S.S. and N.S.D. are supported from Programme Grant to D.M.G. from Cancer Research UK. H.R. is supported from MRC Programme Grant to D.M.G. J.F. thank the British Academy and the Royal Society for Newton International Fellowship and Z.L. thanks the Federation of European Biochemical Societies for the Long-Term postdoctoral Fellowship. The authors thank Nicola Lawrence and Alex Sossick for assistance with 3D-SIM.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles.

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: first, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies; second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during animal evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.D.M.G. is grateful for a Wellcome Investigator Award, which supported this work. The study was initiated with support from Cancer Research UK

Procentriole elongation and recruitment of pericentriolar material are downregulated in cyst cells as they enter quiescence

The apical region of the Drosophila testis contains a niche with two stem cell populations: germline stem cells (GSCs) and cyst progenitor cells (CPCs). Asymmetrical division of these stem cells leads to gonioblast daughters (which undergo further mitoses) and cyst cell daughters (which withdraw from the cell cycle and become quiescent). Although a considerable body of evidence indicates important roles for centrosomes in spindle orientation and asymmetrical division of GSCs, the behaviour and function of the centrioles in CPCs and their daughters remain unknown. Here, we show that quiescent cyst cells lose centrosome components after two divisions of the spermatogonia they envelop, but keep the centriolar component SAS-6. Cyst cells do have centriole pairs, but they are formed by a mother and a very short daughter that does not elongate or mature. The presence of procentrioles in quiescent cyst cells suggests that the centriole duplication cycle is uncoupled from the G1-S transition and that it might begin even earlier, in mitosis. Failure to enter the cell cycle might result in the improper recruitment of centriolar components at the mother centriole, thus hampering the full elongation of its daughter. Procentriole maturation defects could thus lead to the inability to maintain centrosomal components during development