Biology subjects

Ariyoshi, M.

Publications and source records attributed to Ariyoshi, M..

8 recordsLinked to original sources

bioRxiv2026

CCAN forms the spherical shell-like structure in interphase kinetochores

The kinetochore is an essential structure for the faithful chromosome segregation during mitosis. The 16-protein complex known as the constitutive centromere-associated network (CCAN) localizes the kinetochore throughout the cell cycle and forms the basis of the kinetochore. Although a structural model of an individual CCAN unit has been proposed through cryo-electron microscopy analysis, little is known about how multiple CCAN units are organized within cells. Here, using in silico and in vivo analyses, we demonstrate that multiple CCAN units form a spherical, shell-like structure in interphase DT40 kinetochores. CENP-T and CENP-N localize to the CCAN shell, and the CENP-C cupin domain clusters at the center. Molecular dynamics simulations of CCAN assembly suggest that CENP-C contributes to the orientation of CCAN units, leading to the formation of a spherical shell-like structure via CENP-C oligomerization. Super-resolution imaging reveals the spherical shell-like structure at DT40 kinetochores, and knocking out CENP-C abolishes the structure, supporting the reliability of the predicted models. These findings provide important insights into the spatial organization of functional kinetochores within cells.

molecular biology↗

bioRxiv2025

Dual pathways via CENP-C and Mis18C recruit HJURP for CENP-A deposition into vertebrate centromeres

Centromere position is specified and maintained by sequence-independent epigenetic mechanisms in vertebrate cells, with the incorporation of the centromere-specific histone H3 variant CENP-A into chromatin being a key event for centromere specification. Although many models for CENP-A incorporation have been proposed, much remains unknown. In this study, we reveal that the CENP-A chaperone HJURP directly binds to the C-terminal domain of chicken CENP-C in vitro and that this interaction is essential for new CENP-A incorporation in chicken DT40 cells. While existing models have suggested that HJURP is recruited by the Mis18 complex (Mis18C), here, we propose that CENP-C and Mis18C provide dual recruitment pathways for HJURP localization to centromeres in DT40 cells. We demonstrate that both HJURP localization and new CENP-A incorporation are completely abolished in Mis18C knockout cells expressing an HJURP mutant lacking CENP-C binding ability. Furthermore, co-immunoprecipitation experiments reveal that CENP-C, HJURP and Mis18C form a tight association in the chromatin fraction. These two pathways are critical for robust CENP-A incorporation to maintain centromere position in vertebrate cells.

cell biology↗

bioRxiv2024

Structural Basis of betaKNL2 Centromeric Targeting Mechanism and Its Role in Plant-Specific Kinetochore Assembly

The kinetochore is an essential protein complex that ensures proper chromosome segregation during cell division. Kinetochore assembly is initiated by the incorporation of CENP-A/CENH3. This process depends on KNL2/M18BP1 and CENP-C proteins. In plants, two variants of KNL2, KNL2 and {beta}KNL2, are present. Both possess the conserved SANTA domain, while KNL2 additionally has the centromere-targeting CENPC-k motif. Despite lacking the CENPC-like motif, the plant-specific {beta}KNL2 localizes to centromeres and aids in CENP-A/CENH3 loading. We found that efficient centromeric targeting of {beta}KNL2 requires the SANTA domain and the C-terminal part, while nuclear targeting depends on a conserved C-terminal motif-III. Structural predictions and experimental validations reveal that {beta}KNL2 forms homodimers and interacts with centromeric DNA and KNL2. We confirm that centromeric targeting of {beta}KNL2 depends on KNL2 in a tissue-dependent manner. Our findings provide crucial insights into the unique mechanisms of plant-specific kinetochore assembly, highlighting {beta}KNL2s essential role in this process. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/605747v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@30b331org.highwire.dtl.DTLVardef@14494a3org.highwire.dtl.DTLVardef@17a6336org.highwire.dtl.DTLVardef@7ad8f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Molecular details and phospho-regulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells

To establish bi-polar attachments of microtubules to sister chromatids, an inner kinetochore subcomplex, the constitutive centromere-associated network (CCAN), is assembled on centromeric chromatin and recruits the microtubule-binding subcomplex called the KMN network. Although it is important to characterize the interaction between CCAN and KMN, it is difficult to evaluate the significance of each interaction in cells, since CCAN proteins CENP-C and CENP-T independently bind to the Mis12 complex (Mis12C) of KMN. Here, we demonstrate molecular details of the CENP-T-Mis12C interaction using chicken DT40 cells lacking CENP-C-Mis12C interaction. Based on AlphaFold2 predictions, we identified two binding surfaces for the CENP-T-Mis12C interaction, demonstrating that each surface is critical for Mis12C recruitment and cell viability. This interaction via two interaction surfaces is cooperatively regulated by dual phosphorylation of Dsn1 (a Mis12C component) and CENP-T, ensuring robust CENP-T-Mis12C interaction and proper mitotic progression. These findings deepen our understanding of kinetochores assembly in cells.

cell biology↗

bioRxiv2022

The centromere/kinetochore is assembled through CENP-C oligomerization

The kinetochore is an essential protein complex for accurate chromosome segregation. The constitutive centromere-associated network (CCAN), a subcomplex of the kinetochore, associates with centromeric chromatin providing a platform for the kinetochore assembly. A CCAN protein, CENP-C, is thought to be a central hub for the centromere/kinetochore organization. However, the crucial role of CENP-C in centromeres remains to be elucidated. Here, we demonstrated that both the CCAN-binding domain and C-terminal Cupin domain of CENP-C are necessary and sufficient for chicken CENP-C function. Our structural and biochemical analyses revealed that the Cupin domain of chicken and human CENP-C is self-oligomerization domain, which is crucial for centromeric chromatin organization. CENP-C mutants lacking the oligomerization interface cause mislocalization of CCAN and cell death. Based on these results, we conclude that the CENP-C oligomerization plays a crucial role in centromere function via providing the robust centromeric chromatin in vertebrate cells.

cell biology↗

bioRxiv2022

The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2

Centromere protein A (CENP-A) nucleosome is an epigenetic marker that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP) on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNK2 remains unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing a CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for CENP-A deposition. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 alters its binding partner at the centromere during chicken cell cycle progression.

biochemistry↗

bioRxiv2022

Transcription factors that specify cell fate activate cell cycle regulator genes to determine cell numbers in ascidian larval tissues

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programs. Although cell cycle control in animal development has been studied extensively, such links are not well understood. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. In the present study, we first showed that maternal factors promote approximately 10 rounds of cell division without zygotic gene expression. Notochord and muscle cells stop dividing after fewer than 10 rounds of cell division, and we show that a Cdk inhibitor (Cdkn1.b) is responsible. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells divided more than 10 times, and Myc, which encodes a proto-oncogenic transcription factor, is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by the same developmental programs that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm was under control of the same developmental programs that specify the developmental fate of mesenchyme and endoderm. Because these transcription factors that regulate Cdkn1.b and Myc are essential factors for fate specification of these tissues, cell fate specification and cell cycle control are linked by those transcription factors. In other words, ectodermal, mesodermal, and endodermal tissues in ascidian embryos control the cell cycle through Cdkn1.b and Myc, which are under the control of transcription factors that specify cell fate.

developmental biology↗

bioRxiv2020

Cystal structure of inhibitor-bound human MSPL/TMPRSS13 that can activate high pathogenic avian influenza

Infection of certain influenza viruses is triggered when its hemagglutinin (HA) is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, while the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R{downarrow} sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal -helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

biophysics↗