Identification of a TPX2-Like Microtubule-Associated Protein in Drosophila

Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs). One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38) lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions

Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation.

notes: PMCID: PMC3898610types: Journal Article; Research Support, Non-U.S. Gov'tCopyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. We have tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. Our work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules.Biotechnology and Biological Sciences Research Council PhD studentship (to D.H.)Wellcome Trust Institutional Strategic Support Fund WT097835MF (to J.G.W. and J.M.)

Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis

The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex

Microtubule sliding activity of a kinesin-8 promotes spindle assembly and spindle length control

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by cross-linking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report for the first time on an anti-parallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule destabilizing activity. In conjunction with kinesin-5/Cin8, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a “slide-disassemble” model where Kip3’s sliding and destabilizing activity balance during pre-anaphase. This facilitates normal spindle assembly. However, Kip3’s destabilizing activity dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly

Biophysically Realistic Filament Bending Dynamics in Agent-Based Biological Simulation

An appealing tool for study of the complex biological behaviors that can emerge from networks of simple molecular interactions is an agent-based, computational simulation that explicitly tracks small-scale local interactions – following thousands to millions of states through time. For many critical cell processes (e.g. cytokinetic furrow specification, nuclear centration, cytokinesis), the flexible nature of cytoskeletal filaments is likely to be critical. Any computer model that hopes to explain the complex emergent behaviors in these processes therefore needs to encode filament flexibility in a realistic manner. Here I present a numerically convenient and biophysically realistic method for modeling cytoskeletal filament flexibility in silico. Each cytoskeletal filament is represented by a series of rigid segments linked end-to-end in series with a variable attachment point for the translational elastic element. This connection scheme allows an empirically tuning, for a wide range of segment sizes, viscosities, and time-steps, that endows any filament species with the experimentally observed (or theoretically expected) static force deflection, relaxation time-constant, and thermal writhing motions. I additionally employ a unique pair of elastic elements – one representing the axial and the other the bending rigidity– that formulate the restoring force in terms of single time-step constraint resolution. This method is highly local –adjacent rigid segments of a filament only interact with one another through constraint forces—and is thus well-suited to simulations in which arbitrary additional forces (e.g. those representing interactions of a filament with other bodies or cross-links / entanglements between filaments) may be present. Implementation in code is straightforward; Java source code is available at www.celldynamics.org

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells

Effects of prior osteoporosis treatment on the treatment response of romosozumab followed by denosumab in patients with postmenopausal osteoporosis

Summary: In patients with postmenopausal osteoporosis, prior osteoporosis treatment affected the bone mineral density increase of following treatment with 12 months of romosozumab, although it did not affect that of following treatment with 12 months of denosumab after romosozumab. Purpose: To investigate the effects of prior osteoporosis treatment on the response to treatment with romosozumab (ROMO) followed by denosumab (DMAb) in patients with postmenopausal osteoporosis. Methods: In this prospective, observational, multicenter study, treatment-naïve patients (Naïve; n = 55) or patients previously treated with bisphosphonates (BP; n = 37), DMAb (DMAb; n = 45) or teriparatide (TPTD; n = 17) (mean age, 74.6 years; T-scores of the lumbar spine [LS] − 3.2 and total hip [TH] − 2.6) were switched to ROMO for 12 months, followed by DMAb for 12 months. Bone mineral density (BMD) and serum bone turnover markers were evaluated for 24 months. Results: A BMD increase was observed at 12 and 24 months in the following patients: Naïve (18.2% and 22.0%), BP (10.2% and 12.1%), DMAb (6.6% and 9.7%), and TPTD (10.8% and 15.0%) (P < 0.001 between the groups at both 12 and 24 months) in LS and Naïve (5.5% and 8.3%), BP (2.9% and 4.1%), DMAb (0.6% and 2.2%), and TPTD (4.3% and 5.4%) (P < 0.01 between the groups at 12 months and P < 0.001 at 24 months) in TH, respectively. The BMD increase in LS from 12 to 24 months was negatively associated with the levels of bone resorption marker at 24 months. Incidences of major fragility fractures for the respective groups were as follows: Naïve (5.5%), BP (16.2%), DMAb (11.1%), and TPTD (5.9%). Conclusions: Previous treatment affected the BMD increase of following treatment with ROMO, although it did not affect that of following treatment with DMAb after ROMO.This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/s00198-022-06386-yEbina K., Etani Y., Tsuboi H., et al. Effects of prior osteoporosis treatment on the treatment response of romosozumab followed by denosumab in patients with postmenopausal osteoporosis. Osteoporosis International 33, 1807 (2022