Biology subjects

Kiyomitsu, T.

Publications and source records attributed to Kiyomitsu, T..

10 recordsLinked to original sources

bioRxiv2026

Balanced RCC1 activity organizes the specialized spindle midplane during cleavage divisions

Chromosome-bound RCC1 generates Ran-GTP signals to organize functional spindles for faithful chromosome segregation during mitosis and meiosis. RCC1 is the sole guanine nucleotide exchange factor (GEF) for Ran and is essential for spindle assembly during early, but not late, embryonic divisions. However, how RCC1 organizes the specialized embryonic spindle and when its function changes during early embryogenesis remain unclear. Here, using time-resolved RCC1 depletion and depletion-rescue experiments in medaka embryos, we show that RCC1 GEF activity is specifically required before the blastula stage to organize a specialized metaphase spindle mid-plane that ensures faithful chromosome segregation. Mechanistically, RCC1 promotes the accumulation of the canonical Ran effectors HURP and KIFC1/HSET, and unexpectedly, the microtubule motor dynein at the spindle midplane during early embryonic divisions. Intriguingly, a five-fold increase in RCC1 expression phenocopies RCC1 depletion, disrupting spindle-midplane organization and the accumulation of KIFC1 and dynein in a GEF activity-dependent manner. Together, our findings demonstrate that both insufficient and excessive RCC1 GEF activity compromise embryonic spindle assembly, revealing that balanced Ran activation is required to organize the specialized spindle midplane during vertebrate cleavage divisions. HighlightsRCC1 requirement changes with embryonic spindle remodeling before the blastula stage. RCC1 GEF activity is required to organize the specialized embryonic spindle midplane. RCC1 promotes the accumulation of HURP, KIFC1, and dynein at the spindle midplane. Both insufficient and excessive RCC1 GEF activity disrupt the spindle midplane organization.

cell biology↗

bioRxiv2026

KIFC1 overexpression induces monopolar spindles by preventing centrosome separation during rapid cleavage divisions

Bipolar spindle assembly is essential for accurate chromosome segregation. KIFC1, a conserved Ran- regulated minus-end-directed kinesin-14 motor, accumulates in the nucleus during interphase and promotes chromatin-mediated spindle assembly during mitosis and meiosis. In human oocytes, reduced KIFC1 levels destabilize meiotic spindles, a defect that can be rescued by increasing KIFC1 expression. However, how KIFC1 expression levels affect mitotic spindle stability during cleavage divisions in vertebrates remains unclear. Here, we show that whereas an approximately 50% reduction in KIFC1 causes no detectable defects in spindle assembly, approximately 10-fold overexpression of KIFC1 induces monopolar spindle formation, leading to chromosome mis-segregation and embryonic lethality in medaka early embryos. KIFC1 overexpression results in ectopic centrosomal localization during interphase, impairing the separation of duplicated centrosomes before mitotic entry. Analyses of KIFC1 mutants demonstrated that these centrosome separation defects require KIFC1s microtubule-binding and motor activities and are further enhanced by deletion of KIFC1s nuclear localization sequences. Together, our findings demonstrate that tight regulation of KIFC1 expression and its nuclear sequestration is essential for the proper separation and positioning of duplicated centrosomes before mitotic entry, thereby ensuring efficient bipolar spindle assembly during the rapid cleavage divisions of vertebrate embryos. HighlightsO_LIKIFC1 accumulates in the nucleus and at the embryonic spindle midplane via the Ran pathway. C_LIO_LIPartial KIFC1 depletion does not impair spindle assembly in medaka early embryos. C_LIO_LIKIFC1 overexpression induces monopolar spindles by preventing centrosome separation. C_LIO_LICentrosome separation defects require KIFC1 microtubule-binding and motor activity. C_LI

cell biology↗

bioRxiv2026

Local fluidization of an active cytoplasmic gel partitions large cells

Early animal embryos undergo rapid cleavages that partition cytoplasmic volumes orders of magnitude larger than those of somatic cells1. Each division must reposition nuclei and centrosomes and distribute organelles within minutes, over distances up to hundreds of micrometers2. Cleavage furrows are positioned by microtubule asters3,4, but the mechanical mechanism for long-range transport of cytoplasmic components before cytokinesis was unknown. Here, we show that cytoplasm behaves as a locally switchable active material. Fluidization at the midplane allows bulk actomyosin to convert a local mechanical asymmetry into directed global flows of all components as a composite material. Using an actin-intact cycling Xenopus egg extract together with Xenopus and medaka embryos, we find that F-actin mechanically couples microtubule asters, organelles, nuclei and centrosomes into a gel-like composite that propagates forces over hundreds of micrometers. After mitosis, Aurora B kinase patterns a locally fluidized midplane, from which myosin-II contractility drives coherent cytoplasmic flows. A fluid dynamics model accounts for the observed flow geometry and rates. Our results reveal how local control of the material state of cytoplasm converts mitotic symmetry breaking into long-range intracellular transport and identify bulk actomyosin as the active stress generator that partitions embryonic cytoplasm as a composite gel.

cell biology↗

bioRxiv2026

Spatiotemporal organization of dynein in bulk cytoplasm promotes aster growth and positioning in large embryos

During cleavage in vertebrates, large microtubule asters grow from centrosomes in anaphase. The microtubule motor dynein pulls on these asters in bulk cytoplasm and positions centrosomes to determine cleavage geometry. However, how aster growth and pulling are coordinated remains unclear. We discovered that small metaphase asters generate a halo-like enrichment of cytoplasmic dynein at the aster periphery in medaka early embryos. In anaphase, dynein relocates from the halo to growing asters and neighboring membranous organelles, coincident with aster expansion and centrosome movement. Dynein inhibition or localized halo disruption prevents centrosome movement. Unexpectedly, dynein inhibition also suppresses aster growth and causes ectopic cytoplasmic microtubule nucleation, which leads to ectopic furrows. We propose that inactive dynein that accumulated at the metaphase aster periphery is activated in anaphase and incorporates both microtubule nucleators and organelles into growing asters to coordinately promote aster growth and pulling for efficient centrosome positioning in rapidly-dividing, large embryos.

cell biology↗

bioRxiv2025

CDK1-dependent N-terminal NuMA phosphorylation promotes dynein-dynactin-NuMA assembly for accurate chromosome segregation

The microtubule-based motor dynein and its cofactor dynactin fulfil essential functions throughout the cell cycle, including organelle transport and mitotic spindle assembly. To achieve these diverse functions, dynein-dynactin associates with different activating adaptors. Nuclear Mitotic Apparatus (NuMA) is a mitosis-specific adaptor that connects dynein-dynactin with microtubules to focus mitotic spindle poles. NuMAs C-terminal microtubule-binding activity is promoted by mitotic phosphorylation, but regulation of NuMAs N-terminal interaction with dynein-dynactin remains unclear. Here, we combine a membrane-tethering assay, quantitative proteomics, and live functional analyses in human cells to show that the interaction between NuMAs N-terminus and dynein-dynactin is cell cycle-regulated and driven by mitotic phosphorylation. We identify highly conserved CDK1 consensus sites proximal to NuMAs dynein heavy chain-binding site, which are phosphorylated by CDK1-Cyclin B1 in cells and in vitro. This CDK1-dependent phosphorylation, together with NuMAs Spindly-like motif, is crucial for stable dynein-dynactin-NuMA (DDN) complex formation. Replacement of endogenous NuMA with phosphorylation-deficient NuMA mutants leads to aberrant dynein distribution on mitotic spindles, resulting in chromosome mis-segregation and micronucleus formation. Together, our results highlight CDK1-dependent N-terminal NuMA phosphorylation as a crucial mitotic switch that constitutes a regulatable core of multivalent interactions with dynein-dynactin to assemble stable DDN complexes for accurate chromosome segregation.

cell biology↗

bioRxiv2023

Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos

Despite drastic cellular changes during cleavage divisions, a mitotic spindle is assembled in each blastomere to accurately segregate duplicated chromosomes. Recent studies indicate that early embryonic divisions are highly error-prone in bovines and humans. However, processes and mechanisms of embryonic spindle assembly remain little understood in vertebrates. Here, we established live functional assay systems in medaka fish (Oryzias latipes) embryos by combining CRISPR knock-in with an auxin-inducible degron technology. In contrast to mammals, mitoses during cleavage divisions are very rapid (<12 min), but segregation errors are rarely observed. Importantly, we found that the Ran-GTP gradient assembles a specialized, dense microtubule network at the spindle midplane during metaphase, which is essential for faithful chromosome segregation in early embryos. In contrast, Ran-GTP becomes dispensable for chromosome segregation in later stages, where spindles are morphologically remodeled into short, somatic-like spindles lacking the dense microtubule network. We propose that the specialized Ran-based spindle structure ensures high fidelity of chromosome segregation in large, vertebrate early embryos.

developmental biology↗

bioRxiv2022

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles

Micronuclei resulting from improper chromosome segregation foster chromosomal instability in somatic cell division cycles. To prevent micronuclei formation, bundled kinetochore-microtubules called k-fibers must be properly connected to all sister kinetochores on chromosomes via their plus-ends, whereas k-fiber minus-ends must be clustered at the two opposing spindle poles throughout mitosis. The bipolar attachment between sister kinetochores and k-fiber plus-ends is carefully monitored by the spindle assembly checkpoint and further promoted by error-correction mechanisms. However, how k-fiber minus-end clustering is maintained and monitored remains poorly understood. Here, we show that degradation of the Nuclear Mitotic Apparatus (NuMA) protein by auxin-inducible degron technologies in human cells results in micronuclei formation through k-fiber minus-end detachment from focused spindle poles during metaphase. Importantly, this k-fiber minus-end detachment creates misaligned chromosomes that maintain chromosome biorientation and do not activate the mitotic checkpoint, resulting in lagging chromosomes in anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA-mediated minus-end clustering of k-fibers and spindle microtubules is critical for faithful chromosome segregation. Similar to erroneous merotelic kinetochore attachments, detachment of k-fiber minus-ends from metaphase spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

cell biology↗

bioRxiv2018

Importin-β targets HURP to kinetochore-fibers in coordination with Ran-GTP in human mitotic cells

During mitosis, a bipolar spindle is assembled around chromosomes to efficiently capture chromosomes. Previous work proposed that a chromosome-derived Ran-GTP gradient promotes spindle assembly around chromosomes by liberating spindle assembly factors (SAFs) from inhibitory importins. However, Rans dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic degradation assays to dissect Rans mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle pole focusing or for targeting TPX2. In contrast, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions. We demonstrated that Ran-GTP and importin-{beta} coordinately promote HURPs dynamic microtubule binding-dissociation cycle near chromosomes, which results in stable kinetochore-fiber formation. Intriguingly, this pathway acts to establish proper spindle length preferentially during prometaphase, rather than metaphase. Together, we propose that the Ran pathway is required to activate SAFs specifically near chromosomes, but not generally during human mitotic spindle assembly. Ran-dependent spindle assembly is likely coupled with parallel pathways to activate SAFs, including NuMA, for spindle pole focusing away from chromosomes. HighlightsO_LIUsing auxin-inducible degron technology, we developed mitotic degradation assays for the Ran pathway in human cells. C_LIO_LIThe Ran pathway is non-essential to activate NuMA for spindle pole focusing. C_LIO_LIThe Ran pathway dynamically polarizes HURP and defines mitotic spindle length preferentially during prometaphase. C_LIO_LIRan-GTP is required to activate SAFs specifically near chromosomes, but not generally, in human mitotic cells. C_LI

cell biology↗

bioRxiv2018

Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment/activation by NuMAs N-terminal long arm, and NuMAs direct microtubule-binding activities to achieve a multiplicity of microtubule interactions. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

cell biology↗

bioRxiv2017

Human microcephaly ASPM protein is a spindle pole-focusing factor

Nonsense mutations in the ASPM gene have been most frequently identified among familial microcephaly patients. Depletion of the Drosophila orthologue causes spindle pole unfocusing during mitosis in multiple cell types. However, it remains unknown whether human ASPM has a similar function. Here, using CRISPR-based gene knockout (KO) and RNA interference combined with auxin-inducible degron, we show that ASPM functions in spindle pole organisation during mitotic metaphase redundantly with another microcephaly protein CDK5RAP2 (also called CEP215) in human tissue culture cells. Deletion of the ASPM gene alone did not affect spindle morphology or mitotic progression. However, when the pericentriolar material protein CDK5RAP2 was depleted in ASPM KO cells, spindle poles were unfocused during prometaphase and anaphase onset was significantly delayed. The phenotypic analysis of CDK5RAP2-depleted cells suggested that the pole-focusing function of CDK5RAP2 is independent of its known function to localise the kinesin-14 motor HSET or activate the{gamma} -tubulin complex. Finally, a hypomorphic mutation identified in ASPM microcephaly patients similarly caused spindle pole unfocusing in the absence of CDK5RAP2, suggesting a possible link between spindle pole disorganisation and microcephaly.

cell biology↗