The conserved kinase NHK-1 is essential for mitotic progression and unifying acentrosomal meiotic spindles in Drosophila melanogaster

Conventional centrosomes are absent from the spindle in female meiosis in many species, but it is not clear how multiple chromosomes form one shared bipolar spindle without centrosomes. We identified a female sterile mutant in which each bivalent chromosome often forms a separate bipolar metaphase I spindle. Unlike wild type, prophase I chromosomes fail to form a single compact structure within the oocyte nucleus, although the integrity of metaphase I chromosomes appears to be normal. Molecular analysis indicates that the mutant is defective in the conserved kinase nucleosomal histone kinase-1 (NHK-1). Isolation of further alleles and RNA interference in S2 cells demonstrated that NHK-1 is also required for mitotic progression. NHK-1 itself is phosphorylated in mitosis and female meiosis, suggesting that this kinase is part of the regulatory system coordinating progression of mitosis and meiosis

Planar cell polarity: the Dachsous/Fat system contributes differently to the embryonic and larval stages of Drosophila.

The epidermal patterns of all three larval instars (L1-L3) ofDrosophilaare made by one unchanging set of cells. The seven rows of cuticular denticles of all larval stages are consistently planar polarised, some pointing forwards, others backwards. In L1 all the predenticles originate at the back of the cells but, in L2 and L3, they form at the front or the back of the cell depending on the polarity of the forthcoming denticles. We find that, to polarise all rows, the Dachsous/Fat system is differentially utilised; in L1 it is active in the placement of the actin-based predenticles but is not crucial for the final orientation of the cuticular denticles, in L2 and L3 it is needed for placement and polarity. We find Four-jointed to be strongly expressed in the tendon cells and show how this might explain the orientation of all seven rows. Unexpectedly, we find that L3 that lack Dachsous differ from larvae lacking Fat and we present evidence that this is due to differently mislocalised Dachs. We make some progress in understanding how Dachs contributes to phenotypes of wildtype and mutant larvae and adults.This work was generously supported by the Wellcome Trust: a Project Grant [086986] and, later, two successive Investigator Awards, [096645 and 107060] awarded to P.A.L., as well as [100986] to D.S. P.S. thanks Fundaçã o para a Ciência e a Tecnologia and the Cambridge Philosophical Society for research studentships.This is the final version of the article. It first appeared from The Company of Biologists via https://doi.org/10.1242/bio.01715

Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis

Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently from centrosomes, and the molecular requirements of this pathway remain to be understood. Here we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. Firstly, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates, for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation

Concerted action of Aurora B, Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation

The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. Here we report that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in Drosophila mitosis. We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation

Planar Polarity Specification through Asymmetric Subcellular Localization of Fat and Dachsous

Two pathways regulate planar polarity: the core proteins and the Fat-Dachsous-Four-jointed (Ft-Ds-Fj) system. Morphogens specify complementary expression patterns of Ds and Fj that potentially act as polarizing cues. It has been suggested that Ft-Ds-Fj-mediated cues are weak and that the core proteins amplify them [1, 2]. Another view is that the two pathways act independently to generate and propagate polarity [3, 4]: if correct, this raises the question of how gradients of Ft and Ds expression or activity might be interpreted to provide strong cellular polarizing cues and how such cues are propagated from cell to cell. Here, we demonstrate that the complementary expression of Ds and Fj results in biased Ft and Ds protein distribution across cells, with Ft and Ds accumulating on opposite edges. Furthermore, boundaries of Ft and Ds expression result in subcellular asymmetries in protein distribution that are transmitted to neighboring cells, and asymmetric Ds localization results in a corresponding asymmetric distribution of the myosin Dachs. We show that the generation of subcellular asymmetries of Ft and Ds and the core proteins is largely independent in the wing disc and additionally that ommatidial polarity in the eye can be determined without input from the Ft-Ds-Fj system, consistent with the two pathways acting in parallel

Four-Jointed Modulates Growth and Planar Polarity by Reducing the Affinity of Dachsous for Fat

The Drosophila genes fat (ft) and dachsous (ds) encode large atypical cadherins that collaborate to coordinately polarize cells in the plane of the epithelium (planar cell polarity) and to affect growth via the Warts/Hippo pathway [1–3]. Ft and Ds form heterodimeric bridges that convey polarity information from cell to cell [4–7]. four-jointed (fj) is a modulator of Ft/Ds activity that acts in a graded fashion in the abdomen, eye, and wing [8–11]. Genetic evidence indicates that Fj acts via Ds and/or Ft [4, 6–9, 12], and here we demonstrate that Fj can act independently on Ds and on Ft. It has been reported that Fj has kinase activity and can phosphorylate a subset of cadherin domains of both Ft and Ds in vitro [13]. We have used both cell and in vitro assays to measure binding between Ft and Ds. We find that phosphorylation of Ds reduces its affinity for Ft in both of these assays. By expressing forms of Ds that lack the defined phosphorylation sites or have phosphomimetic amino acids at these positions, we demonstrate that effects of Fj on wing size and planar polarity can be explained by Fj phosphorylating these sites

Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

Microtubule-associated proteins regulate microtubule (MT) dynamics spatially and temporally, which is essential for proper formation of the bipolar mitotic spindle. The XMAP215 family is comprised of conserved microtubule-associated proteins that use an array of tubulin-binding tumor overexpressed gene (TOG) domains, consisting of six (A–F) Huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin (HEAT) repeats, to robustly increase MT plus-end polymerization rates. Recent work showed that TOG domains have differentially conserved architectures across the array, with implications for position-dependent TOG domain tubulin binding activities and function within the XMAP215 MT polymerization mechanism. Although TOG domains 1, 2, and 4 are well described, structural and mechanistic information characterizing TOG domains 3 and 5 is outstanding. Here, we present the structure and characterization of Drosophila melanogaster Mini spindles (Msps) TOG3. Msps TOG3 has two unique features as follows: the first is a C-terminal tail that stabilizes the ultimate four HEAT repeats (HRs), and the second is a unique architecture in HR B. Structural alignments of TOG3 with other TOG domain structures show that the architecture of TOG3 is most similar to TOG domains 1 and 2 and diverges from TOG4. Docking TOG3 onto recently solved Stu2 TOG1· and TOG2·tubulin complex structures suggests that TOG3 uses similarly conserved tubulin-binding intra-HEAT loop residues to engage α- and β-tubulin. This indicates that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence. The similarity of TOG domains 1–3 and the divergence of TOG4 suggest that a TOG domain array with polarized structural diversity may play a key mechanistic role in XMAP215-dependent MT polymerization activity