Biology subjects

Tanaka, T. U.

Publications and source records attributed to Tanaka, T. U..

7 recordsLinked to original sources

bioRxiv2025

Contractile peri-nuclear actomyosin network PANEM repositions chromosomes from unfavorable locations to facilitate their interaction with the mitotic spindle

Chromosomes must efficiently and properly interact with the mitotic spindle during prometaphase for correct segregation in anaphase. Chromosomes at the nuclear periphery or behind the spindle poles interact less efficiently with the mitotic spindle, increasing the risk of missegregation. The mechanisms that mitigate such risks in unperturbed cells are unknown. An actomyosin network (PANEM) forms around the nucleus during prophase. While the myosin-II-dependent PANEM contraction immediately after nuclear envelope breakdown (NEBD) facilitates chromosome interaction with the mitotic spindle, the mechanism by which it does so remains unclear. Here, using human cell lines, we show that immediately after NEBD, PANEM contraction directly pushes chromosomes at the nuclear periphery or behind spindle poles toward the center of cells. Detailed tracking of kinetochore movements following light-induced activation of a myosin-II inhibitor reveals that this inward movement of chromosomes facilitates kinetochores initial interaction with spindle microtubules. It also promotes the onset of kinetochores congression towards the spindle mid-plane, but not congression itself once it starts. Thus, PANEM contraction ensures high-fidelity chromosome segregation by relocating chromosomes from unfavorable locations. Since some chromosomally unstable cancer cells fail to establish PANEM during early mitosis, the absence of PANEM may contribute to numerical chromosomal instability in these cells. Impact statementAn actomyocin network (PANEM) forms around the nucleus in prophase, and its contraction repositions peripheral and polar chromosomes to facilitate their interaction with the mitotic spindle, ensuring their correct segregation.

cell biology↗

bioRxiv2024

Exclusion of condensin I from the nucleus during prophase coordinates mitotic chromosome reorganization to complete sister chromatid resolution

AbstractDuring early mitosis, chromosomes are reorganized from their relatively decompacted interphase state into characteristic mitotic rod-shaped structures. This process is important to allow correct segregation of newly replicated sister chromatids to the opposite spindle poles during anaphase. To facilitate mitotic chromosome organization, two protein complexes named condensin I and condensin II play crucial roles. Condensin II is particularly important for achieving sister chromatid separation (resolution) whilst condensin I is required for chromosome condensation (compaction). Although sister chromatid resolution occurs 15-20 min earlier before chromosome compaction, it is not yet clear how these events are temporally coordinated or whether this temporal coordination is important to ensure chromosome segregation later in mitosis. One hypothesis is that the temporal coordination is achieved through different subcellular localisation of two condensin complexes; whilst condensin II localizes in the nucleus, condensin I is restricted to the cytoplasm, during interphase and prophase. In this study we tested this hypothesis by engineering the localization of condensin I to the nucleus. We monitored sister chromatid resolution and chromosome compaction by real-time imaging that visualized selected neighboring chromosome loci. We found that localization of condensin I to the nucleus led to precocious chromosome compaction during prophase with a similar timing to sister chromatid resolution. We also monitored later stages of mitosis and found that cells expressing nuclear condensin I subsequently exhibited frequent chromosome mis-segregation in anaphase. Therein, the majority of mis-segregated chromosomes consisted of lagging chromosomes involving both sister chromatids. This suggests that the temporal control of mitotic chromosome reorganization is crucial for high-fidelity chromosome segregation. In conclusion, the exclusion of condensin I from the nucleus during prophase delays chromosome compaction and allows condensin II to complete sister chromatid resolution, which ensures correct chromosome segregation later in mitosis.

cell biology↗

bioRxiv2023

Aurora B spatial separation from its outer kinetochore substrates stabilizes chromosome biorientation when tension is applied

For correct chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (biorientation). For this, aberrant kinetochore- microtubule interaction must be resolved (error correction) by Aurora B kinase. Once biorientation is formed, tension is applied across sister kinetochores, stabilizing kinetochore- microtubule interactions. The mechanism for this tension-dependent process has been debated. Here we study how localizations of Aurora B at different kinetochore sites affect the establishment and maintenance of biorientation in budding yeast. In the absence of the physiological Aurora B-INCENP recruitment mechanisms, engineered recruitment of Aurora B-INCENP to the inner kinetochore (Mif2) prior to biorientation supports the subsequent establishment of biorientation. By contrast, an engineered Aurora B-INCENP recruitment to the outer kinetochore (Ndc80) fails to support biorientation establishment. Furthermore, when the physiological Aurora B-INCENP recruitment mechanisms are present, an engineered Aurora B-INCENP recruitment to Mif2 during metaphase (after biorientation establishment) does not affect biorientation maintenance. By contrast, an engineered Aurora B-INCENP recruitment to Ndc80 during metaphase leads to disruption of biorientation, which is dependent on the kinase activity of Aurora B. Taken together, our results suggest that spatial separation of Aurora B from its outer kinetochore substrates is required to stabilize kinetochore-microtubule interaction when biorientation is formed and tension is applied on this interaction. Meanwhile, Aurora B shows dynamic turnover (or exchange) on the centromere and kinetochore during early mitosis. It has been thought that this turnover is crucial for error correction and biorientation, as it may help Aurora B reach its substrates in distance and/or may facilitate the Aurora B activation on the mitotic spindle. However, using the engineered Aurora B-INCENP recruitment to the inner kinetochore, we demonstrate that, even without such a turnover, Aurora B-INCENP can efficiently support biorientation. Altogether, our study provides important insights into how Aurora B promotes error correction and biorientation in a tension-dependent manner.

cell biology↗

bioRxiv2019

Aurora B-INCENP localization at centromeres/inner kinetochores is essential for chromosome bi-orientation in budding yeast

To promote chromosome bi-orientation, Aurora B kinase weakens and disrupts aberrant kinetochore-MT interaction. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. Pertinent to this debate, it was shown that Bir1 (yeast Survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, is dispensable for bi-orientation, raising the possibility that Aurora B localization at centromeres is not required for bi-orientation. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres/inner kinetochores is essential for bi-orientation, refuting the above possibility. Our results give important insight into how Aurora B disrupts kinetochore-MT interaction in a tension-dependent manner, to promote chromosome bi-orientation.

cell biology↗

bioRxiv2018

Contractile actomyosin network on nuclear envelope remnants positions chromosomes for mitosis

To ensure proper segregation during mitosis, chromosomes must be efficiently captured by kinetochore microtubules and subsequently aligned on the mitotic spindle. The efficacy of chromosome capture by the mitotic spindle can be influenced by how widely chromosomes are scattered in space. Here, we quantify chromosome-scattering volume (CSV) and find that it is reduced immediately after nuclear envelope breakdown (NEBD) in human cells. The reduction of CSV occurs independently of microtubules and is therefore not an outcome of interactions between chromosomes and the spindle. We find that, prior to NEBD, an acto-myosin network is assembled in a LINC complex-dependent manner on the cytoplasmic surface of the nuclear envelope. This acto-myosin network remains around chromosomes soon after NEBD, and its myosin-II-mediated contraction reduces CSV and facilitates chromosome interaction with spindle microtubules.

cell biology↗

bioRxiv2018

In vitro reconstitution of kinetochore-microtubule interface reveals a fundamental error correction mechanism

For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles; this is called bi-orientation. To establish bi-orientation prior to chromosome segregation, any aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase that phosphorylates outer kinetochore components. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still not fully understood how kinetochore-microtubule interactions are exchanged during error correction. Here we reconstituted the kinetochore-microtubule interface of budding yeast in vitro by attaching the Ndc80 complexes (Ndc80C) to nanobeads. These Ndc80C-nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores, on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. We suggest that the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment and promotes the exchange to a new lateral attachment, leading to error correction. Our study reveals a fundamental mechanism of error correction for establishment of bi-orientation.

cell biology↗

bioRxiv2018

Live-cell imaging of marked chromosome regions reveals dynamics of mitotic chromosome resolution and compaction

When human cells enter mitosis, chromosomes undergo substantial changes in their organisation to resolve sister chromatids and compact chromosomes. Despite the fundamental importance of this phenomenon to genome stability, we still do not fully comprehend the timing and coordination of these events. To address these questions, we need to evaluate the progression of both sister chromatid resolution and chromosome compaction in one assay. We achieved this by analysing changes in configuration of marked chromosome regions over time, with high spatial and temporal resolution. This assay showed that sister chromatid resolution is an iterative process that begins in late G2 phase and completes in prophase. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase whilst local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, our assay allowed quantitative evaluation of the timing and efficiency of condensin II and I activities in promoting sister chromatid resolution and chromosome compaction, respectively. Thus, our real-time assay sheds new light on the dynamics of mitotic chromosome resolution and compaction.

cell biology↗