FY2022 Annual Report

Cell Division Dynamics Unit

Assistant Professor
Tomomi Kiyomitsu

cddu FY2022 Annual Report 01

Abstract

Cell division is a fundamental process in all organisms to create two daughter cells from a mother cell. During mitosis in eukaryotes, a microtubule-based structure called the mitotic spindle is assembled to segregate duplicated chromosomes into daughter cells. The bipolar nature of the spindle is critical for the accurate inheritance of genomic information. In addition, spindle positioning is critical to control daughter cell fates and tissue morphogenesis during the development of multi-cellular organisms since spindle positioning controls the distribution modes of polarized cell-fate determinants as well as the size and location of daughter cells.

In the Cell Division Dynamics unit, we are studying the mechanisms of bipolar spindle assembly, positioning, and their coordination in vertebrate mitosis using advanced cell-biological and biochemical approaches. We are currently focusing on the following two projects:

  1. Mechanisms of bipolar spindle maintenance in mitotic human cells
  2. Mechanisms of spindle assembly and positioning in symmetrically-dividing Medaka early embryonic cells

In FY2022 (April 2022- March 2023), we continued both human cell and Medaka projects with the following members (see below). Regarding the human cell projects, we published a research paper in Current Biology (van Toorn et al., 2023 Feb 6; 33(3):572-580.e2. doi: 10.1016/j.cub.2022.12.017.), and a Method paper to Methods in Molecular Biology (Kiyomitsu, 2023; 2623:73-85. doi: 10.1007/978-1-0716-2958-1_5.). In addition, we are preparing a research paper (Boerner et al., in preparation) related to the above project 1.

Regarding the Medaka projects, we succeeded in establishing four transgenic Medaka strains and in performing two-color live cell imaging in the Medaka early embryos. We are preparing a research paper (Ai Kiyomitsu et al., in preparation). We also started a collaboration with Dr. Ansai (Tohoku Univ.) using an internal SHINKA grant.

1. Staff

Human cell projects

  • Marvin van Toorn, Technician (April 2022- )

Medaka projects

  • Ai Kiyomitsu, Science and Technology Associate
  • Shiang Jyi Hwang, Technician
  • Ayaka Mori, Technician (September 2022- )

Research Unit Administrator

  •  Tomomi Teruya, (August 2019-)

2. Students

  • Yang Ming, a rotation student (Sep -Dec 2022)
  • Koya Shimabukuro, an intern student (Oct 2022-March 2023).

3. Collaborations

3.1 Mechanisms of spindle positioning in Medaka early embryos

  • Minoru Tanaka, Nagoya University
  • Toshiya Nishimura, Nagoya University (currently Hokkaido University)

3.2 Establishment of functional analyses in Medaka early embryos

  • Satoshi Ansai, Tohoku University (currently Kyoto University)
  • Masato Kanemaki, National Institute of Genetics

4. Activities and Findings

4.1 A new pathway that causes micronuclei in mitosis

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements. To prevent micronuclei formation in mitosis, the dynamic plus-ends of bundled kinetochore-microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes, whereas k-fiber minus-ends must be clustered at the two opposing spindle poles which are normally connected with centrosomes. The establishment of chromosome biorientation via k-fiber plus-ends is carefully monitored by the spindle assembly checkpoint (SAC). However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. In this fiscal year, we showed that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole created misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also caused minus-end clustering defects in non-transformed Rpe1 cells, but additionally induced centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we found that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells generally by maintaining k-fiber minus-end clustering, but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell-types or contexts. Similar to erroneous merotelic kinetochore attachments, detachment of k-fiber minus-ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

4.2 Mechanisms of bipolar spindle maintenance in mitotic human cells

Cytoplasmic dynein and nuclear mitotic apparatus (NuMA) protein are well-conserved spindle-pole localizing proteins in vertebrates. Accumulating evidence indicates that NuMA targets and activates dynein at the minus-end of spindle microtubules to provide robust clustering of microtubules into a focused, bipolar spindle. However, it remains unclear how dynein-NuMA complexes function to maintain microtubule minus-end focusing at the spindle poles in human metaphase cells. In this fiscal year, we systematically depleted dynein and its binding partners, NuMA, dynactin and LIS1, during metaphase using the recently developed AID-mediated metaphase depletion assay in HCT116 cells (Tsuchiya et al., Current Biology 2021). Metaphase depletion of dynein, dynactin and LIS1 disrupted bipolar spindle structure, whereas NuMA depletion, unexpectedly, caused minor defects in spindle-pole focusing. Our results indicate that two functionally distinct populations of dynein maintain spindle-pole focusing; one works with NuMA using dynein’s motile activity, but the other functions independently of NuMA at the proximity of the centrosomes (Boerner et al., in preparation).

4.3 Mechanisms of Ran-based spindle assembly in Medaka early embryos

A gradient of GTP-bound form of Ran (Ran-GTP) has been recognized as one of chromosome-derived signals that facilitate spindle assembly around chromosomes. Prior studies demonstrated that the Ran-GTP gradient is critical for acentrosomal spindle assembly in female meiosis, but dispensable for bipolar spindle formation in somatic human cells. Although multiple pathways including Ran-GTP and centrosomes coordinately assemble mitotic spindles in somatic cells, it remains unclear how these pathways contribute to organizing large embryonic spindles in vertebrates. In this fiscal year, we analyzed requirement of Ran-GTP for spindle assembly in Medaka embryos using dual-color live imaging, a dominant negative Ran mutant, and an auxin inducible degron2 (AID2)-based protein knockdown system. In contrast to typical somatic spindles, we found that embryonic spindles have two unique features: a dense microtubule network around chromosomes and precocious centrosome separation from spindle poles during mitosis. Importantly, depletion of RCC1, a GEF for Ran, diminished the dense microtubule network around chromosomes and caused severe chromosome segregation defects specifically in early embryonic divisions. Together, we propose that, in contrast to smaller somatic cells, Ran-GTP plays essential roles in embryonic spindle assembly for accurate chromosome segregation in vertebrates.

5. Publications

5.1 Journals

  • van Toorn M, Gooch A, Boerner S, Kiyomitsu T*.NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles. Curr Biol. (2023) Feb 6;33(3):572-580.e2.
  • Kiyomitsu T*.Using Optogenetics to Spatially Control Cortical Dynein Activity in Mitotic Human Cells.Methods Mol Biol. (2023) 2623:73-85.s

5.2 Oral Presentations

Tomomi Kiyomitsu, How does universal chromosome segregation machinery adapt to specialized cleavage divisions in vertebrate embryos? The annual meeting of Molecular Biology Society of Japan, November 30th, 2022

6 Intellectual Property Rights and Other Specific Achievements

External grants received

  • The JST FOREST program

7 Meetings and Events

  • Co-organized a scientific symposium, Cell Biology across Boundaries, with Dr. Iain Cheeseman (Whitehead Institute/MIT) at the annual meeting of Molecular Biology Society of Japan, November 30th, 2022.
  • Co-organized MBSJ-ASCB-EMBO joint workshop Part 1&2 and MBSJ special program, with Dr. Iain Cheeseman (Whitehead Institute/MIT) at the annual meeting of Molecular Biology Society of Japan, November 31st – December 2nd 2022.