The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I, but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule crosslinker Ase1 as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.
The outer kinetochore serves as a platform for initiating the spindle assembly checkpoint and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S/CENP-X is involved in regulating the spindle assembly checkpoint and kinetochore-microtubule attachments has not been well characterized. Employing live-cell microscopy and yeast genetics, we found that the fission yeast CENP-S/CENP-X counterpart Mhf1/Mhf2 plays crucial roles in promoting the spindle assembly checkpoint and regulating chromosome segregation. The absence of Mhf2 attenuates the spindle assembly checkpoint, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Aurora-B/Ark1 to the kinetochore. Hence, our findings constitute a model in which Mhf1/Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex that is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments.
<p><strong>Objective</strong>: Quantitative analysis of spindle dynamics in mitosis through fluorescence microscopy requires tracking spindles elongation in noisy image sequences. Deterministic methods, which use typical microtubule detection and tracking methods, perform poorly in the case of the sophisticated background of spindles. <strong>Methods</strong>: In this paper, we present SpindlesTracker, a fully automatic and extensible workflow that can efficiently analyze time-lapse images’ dynamic spindle mechanism. First, we design a novel deep neural network: YOLOX-SP (YOLOX for spindle). It consists of double branches responsible for spindle bounding boxes and endpoints detection. Then an improved SORT algorithm is used to link the same identity in different frames. Subsequently, we pair endpoints that fall into the same spindle bounding box as the spindle poles. Finally, we introduce the minimal cost path (MCP) algorithm to extract the continuous, single-pixel spindle skeleton. <strong>Result</strong>: SpindlesTracker is evaluated in all aspects of detection, tracking, and skeleton extraction through a fission yeast dataset. It achieves 84.1% mAP in bounding box detection and over 90% accuracy in endpoint detection. And for tracking, the comparison results show that the improved SORT algorithm increases by 1.3% in multiple object tracking accuracy (MOTA) and by 6.5% in multiple object tracking precision (MOTP). In addition, the statistical result shows that the mean error of spindle length is within 1 ?m. <strong>Conclude</strong>: SpindlesTracker provides a new baseline for multiple spindles analysis. <strong>Significance</strong>: This workflow could be easily extended to other microtubule or filamentous structures. The code is released on GitHub.</p>
42 43 The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented 44 homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms 45 operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy 46 to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the 47 conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during 48 meiosis I, but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis 49 I requires the synergism between Klp2 and the conserved microtubule crosslinker Ase1 as the absence of 50 both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for 51 regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying 52 meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and 53 mitotic events. 54 55
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