bims-micesi 2023-06-04 papers

Issue of 2023‒06‒04
five papers selected by
Valentina Piano
Uniklinik Köln

J Cell Biol. 2023 Aug 07. pii: e202208018. [Epub ahead of print]222(8):

The Ndc80-Cdt1-Ska1 complex is a central processive kinetochore-microtubule coupling unit.

Amit Rahi, Manas Chakraborty, Shivangi Agarwal, Kristen M Vosberg, Shivani Agarwal, Annie Y Wang, Richard J McKenney, Dileep Varma.

It is known that microtubule-binding proteins including the Ska1 complex and the DNA replication licensing factor, Cdt1, enable the kinetochore-localized Ndc80 complex to form robust kinetochore-microtubule attachments. However, it is not clear how the Ndc80 complex is stably coupled to dynamic spindle microtubule plus-ends. Here, we have developed a conditional auxin-inducible degron approach to reveal a function for Cdt1 in chromosome segregation and kinetochore-microtubule interactions that is separable from its role in DNA replication licensing. Further, we demonstrate that a direct interaction between Cdt1 and Ska1 is required for recruiting Cdt1 to kinetochores and spindle microtubules. Cdt1 phosphorylation by Cdk1 kinase is critical for Ska1 binding, kinetochore-microtubule attachments, and mitotic progression. Furthermore, we show that Cdt1 synergizes with Ndc80 and Ska1 for microtubule binding, including forming a diffusive, tripartite Ndc80-Cdt1-Ska1 complex that can processively track dynamic microtubule plus-ends in vitro. Taken together, our data identify the Ndc80-Cdt1-Ska1 complex as a central molecular unit that can promote processive bidirectional tip-tracking of microtubules by kinetochores.

DOI: https://doi.org/10.1083/jcb.202208018

Cell Rep. 2023 May 25. pii: S2211-1247(23)00579-X. [Epub ahead of print]42(6): 112568

EWSR1 maintains centromere identity.

Risa Kitagawa, Yohei Niikura, Argentina Becker, Peter J Houghton, Katsumi Kitagawa.

The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif in vitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.

Keywords: CENP-A; CENP-A maintenance; CP: Molecular biology; EWSR1; EWSR1-FLI1; Ewing sarcoma; Ewing sarcoma breakpoint region 1; Ewing sarcoma oncogenic fusion protein; centromere; centromere identity; kinetochore; phase separation

DOI: https://doi.org/10.1016/j.celrep.2023.112568

IEEE J Biomed Health Inform. 2023 May 30. PP

SpindlesTracker: An Automatic and Low-cost Labeled Workflow for Spindle Analysis.

Zhongzhong Li, Yanze Jian, Jiatong Hu, Chuanmin Zhang, Xuechun Meng, Ji Liu.

Quantitative analysis of spindle dynamics in mitosis through fluorescence microscopy requires tracking spindle elongation in noisy image sequences. Deterministic methods, which use typical microtubule detection and tracking methods, perform poorly in the sophisticated background of spindles. In addition, the expensive data labeling cost also limits the application of machine learning in this field. Here we present a fully automatic and low-cost labeled workflow that efficiently analyzes the dynamic spindle mechanism of time-lapse images, called SpindlesTracker. In this workflow, we design a network named YOLOX-SP which can accurately detect the location and endpoint of each spindle under box-level data supervision. We then optimize the algorithm SORT and MCP for spindle's tracking and skeletonization. As there was no publicly available dataset, we annotated a S.pombe dataset that was entirely acquired from the real world for both training and evaluation. Extensive experiments demonstrate that SpindlesTracker achieves excellent performance in all aspects, while reducing label costs by 60%. Specifically, it achieves 84.1% mAP in spindle detection and over 90% accuracy in endpoint detection. Furthermore, the improved algorithm enhances tracking accuracy by 1.3% and tracking precision by 6.5%. Statistical results also indicate that the mean error of spindle length is within 1 μm. In summary, SpindlesTracker holds significant implications for the study of mitotic dynamic mechanisms and can be readily extended to the analysis of other filamentous objects. The code and the dataset are both released on https://github.com/lizhogn/SpindlesTrackerGitHub.

DOI: https://doi.org/10.1109/JBHI.2023.3281454

Nat Commun. 2023 Jun 01. 14(1): 3172

Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Ksenia Smurova, Michela Damizia, Carmela Irene, Stefania Stancari, Giovanna Berto, Giulia Perticari, Maria Giuseppina Iacovella, Ilaria D'Ambrosio, Maria Giubettini, Réginald Philippe, Chiara Baggio, Elisabetta Callegaro, Andrea Casagranda, Alessandro Corsini, Vincenzo Gentile Polese, Anna Ricci, Erik Dassi, Peter De Wulf.

Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae, we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5'-3'exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.

DOI: https://doi.org/10.1038/s41467-023-38920-9

Nat Commun. 2023 Jun 02. 14(1): 3209

Two RhoGEF isoforms with distinct localisation control furrow position during asymmetric cell division.

Emilie Montembault, Irène Deduyer, Marie-Charlotte Claverie, Lou Bouit, Nicolas J Tourasse, Denis Dupuy, Derek McCusker, Anne Royou.

Cytokinesis partitions cellular content between daughter cells. It relies on the formation of an acto-myosin contractile ring, whose constriction induces the ingression of the cleavage furrow between the segregated chromatids. Rho1 GTPase and its RhoGEF (Pbl) are essential for this process. However, how Rho1 is regulated to sustain furrow ingression while maintaining correct furrow position remains poorly defined. Here, we show that during asymmetric division of Drosophila neuroblasts, Rho1 is controlled by two Pbl isoforms with distinct localisation. Spindle midzone- and furrow-enriched Pbl-A focuses Rho1 at the furrow to sustain efficient ingression, while Pbl-B pan-plasma membrane localization promotes the broadening of Rho1 activity and the subsequent enrichment of myosin on the entire cortex. This enlarged zone of Rho1 activity is critical to adjust furrow position, thereby preserving correct daughter cell size asymmetry. Our work highlights how the use of isoforms with distinct localisation makes an essential process more robust.

DOI: https://doi.org/10.1038/s41467-023-38912-9