Biology subjects

Fukagawa, T.

Publications and source records attributed to Fukagawa, T..

15 recordsLinked to original sources

bioRxiv2026

Senolytic targeting of CAF-induced KRT17+ colon cancer cells inhibits metastatic invasion

Metastasis remains the leading cause of cancer mortality, yet effective strategies to eliminate metastasis-initiating cells are lacking. Here, we dissected intratumoral heterogeneity in colon cancer using single-cell analyses and identified a KRT17+ slow-cycling cancer cell population that exhibits features of metastasis-initiating cells (L1CAM) and senescent-like cells (CDKN2A, BCL2L1). Spatial transcriptomics and immunostaining revealed that these cells localize at tumor-stroma interfaces, where they are closely associated with TGF-{beta}1-producing subset of cancer-associated fibroblasts (CAFs) and exhibit SMAD3 activation. Mechanistically, TGF-{beta}1 induces KRT17 expression in patient-derived cancer cells, while co-culture with CAFs drives the emergence of KRT17 migratory cells in a KRT17-dependent manner. Functionally, genetic ablation of KRT17 or senolytic targeting of BCL2L1 suppresses peritumoral invasion and liver metastasis in xenograft models. Clinically, KRT17 cells co-localize with TGF-{beta}1 CAFs, and their co-expression with L1CAM correlates with advanced disease stage. These findings support a model in which stromal TGF-{beta} signaling promotes the emergence of a KRT17 invasive cancer cell state with senescence-associated features and suggest that senolytic strategies may represent a potential approach to limit metastatic progression in colon cancer.

cancer biology↗

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↗

bioRxiv2026

Kinetochore Homeostasis is Maintained by Coordinated Chromatin Stabilization and Soluble Buffering

Faithful chromosome segregation requires the coordinated assembly and maintenance of kinetochore complexes. However, the homeostatic mechanisms that maintain these multi-subunit assemblies remain unclear. CENP-T is a chromatin-bound linker that recruits the outer kinetochore modules via the CENP-T-W-S-X complex to form a functional kinetochore. Although CENP-T must be continuously replenished, how its steady-state levels are maintained is unknown. Here we demonstrate that CENP-T homeostasis is actively sustained by two spatially distinct pathways. Using covalent pulse-labeling, we find that CENP-T undergoes cell-cycle-coupled turnover rather than being stably inherited. In situ proximity labeling of chromatin-bound and soluble pools identifies regulators of CENP-T stability, including an unexpected role for the CENP-O complex in stabilizing soluble CENP-T. Mechanistically, CENP-S-X stabilizes chromatin-bound CENP-T through DNA-binding, whereas the CENP-O complex buffers a replenishment-competent CENP-T pool. Together, these conserved, spatially compartmentalized pathways establish an actively maintained homeostatic system that ensures robust kinetochore assembly and faithful chromosome segregation.

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↗

bioRxiv2025

Functional genetics with a hypomorphic CENP-C mutant reveals a regulatory system for chromosome congression

Chromosome congression is a key process that acts to align chromosomes at the spindle equator via kinetochore-microtubule interactions, with defects in chromosome alignment leading to chromosomal instability. However, defining the mechanisms that underlie chromosome congression is limited due to the multiple factors that act in parallel to regulate chromosome movement. Here, we conducted a genome-wide Cas9-based functional genetics screen using a hypomorphic CENP-C mutant that affects its kinetochore interactions. Our analysis identified KIF18A, whose knockout resulted in synthetic lethality with the CENP-C mutant. Further analysis revealed that the synthetic defect was due to a reduction in CENP-E function in the CENP-C mutant. Our work suggests that KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C during early prometaphase. Thus, our analysis enables us to dissect parallel molecular mechanisms for chromosome congression and identify sensitivities and biomarkers that might guide anti-KIF18A chemotherapeutics.

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↗

bioRxiv2024

CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

Establishing the correct kinetochore-microtubule attachment is crucial for faithful chromosome segregation. The kinetochore has various regulatory mechanisms for establishing correct bipolar attachment. However, how the regulations are coupled is not fully understood. Here, we demonstrate a regulatory loop between the kinetochore protein CENP-C and Aurora B kinase, which is critical for the error correction of kinetochore-microtubule attachment. This regulatory loop is mediated through the binding of CENP-C to the outer kinetochore Mis12 complex (Mis12C). Although the Mis12C binding region of CENP-C is dispensable for mouse development and proliferation in human RPE-1 cells, those cells lacking this region display increased mitotic defects. The CENP-C-Mis12C interaction facilitates the centromeric recruitment of Aurora B and the mitotic error correction in human cells. Given that Aurora B reinforces the CENP-C-Mis12C interaction, our findings reveal a positive regulatory loop between Aurora B recruitment and the CENP-C-Mis12C interaction, which ensures chromosome bi-orientation for accurate chromosome segregation.

cell biology↗

bioRxiv2023

Nuclear membrane protein Bqt4 maintains nuclear envelope integrity by recruiting phosphatidic acid

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE.

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

Condensin reorganizes centromeric chromatin during mitotic entry into a bipartite structure stabilized by cohesin

The Structural Maintenance of Chromosomes (SMC) complexes cohesin and condensin establish the 3D organization of mitotic chromosomes1-3. Cohesin is essential to maintain sister chromatid pairing until anaphase onset4, while condensin is important for mitotic centromere structure and elastic resistance to spindle forces5-8. Both complexes are also important to form productive kinetochore-spindle attachments6, 8, 9. How condensin and cohesin work together to shape the mitotic centromere to ensure faithful chromosome segregation remains unclear. Here we show by super-resolution imaging, Capture-C analysis and polymer modeling that vertebrate centromeres are partitioned into two distinct condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres and also in human neocentromeres devoid of satellite repeats, and is therefore a fundamental feature of vertebrate centromere identity. Super-resolution imaging reveals that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to mitotic spindle forces. In its absence, separated bipartite kinetochores frequently engage in merotelic spindle attachments. Consistently, uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells. The two-domain structure of vertebrate regional centromeres described here incorporates architectural roles for both condensin and cohesin and may have implications for avoiding chromosomal instability in cancer 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↗

bioRxiv2020

A super sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair

Auxin-Inducible Degron (AID) technology enables conditional depletion of targeted proteins. However, the applicability of the AID in vertebrate cells has been limited due to cytotoxicity caused by high auxin concentrations. Here, we establish an improved AID system using an engineered orthogonal auxin-TIR1 pair, which exhibits over 1,000 times stronger binding. With ~1,000-fold less auxin concentration, we achieved to generate the AID-based knockout cells in various human and mouse cell lines in a single transfection.

bioengineering↗

bioRxiv2019

Identification of bridgin, an unconventional linker, connects the outer kinetochore to centromeric chromatin

The microtubule-binding outer kinetochore is linked to centromeric chromatin through the inner kinetochore CENP-CMif2, CENP-TCnn1, and CENP-UAme1 pathways. These are the only known kinetochore linker proteins across eukaryotes. Linker proteins are structurally less conserved than their outer kinetochore counterparts. Here, we demonstrate the recurrent loss of most inner kinetochore CCAN, including certain linker proteins during evolution in the fungal phylum of Basidiomycota. By studying the kinetochore interactome, a previously undescribed linker protein, bridgin was identified in the basidiomycete Cryptococcus neoformans, a human fungal pathogen. In vivo and in vitro functional analyses of bridgin reveal that it binds to the outer kinetochore and centromere chromatin simultaneously to ensure accurate kinetochore-microtubule attachments. Unlike known linker proteins, bridgin is recruited by the outer kinetochore. Homologs of bridgin were identified outside fungi. These results showcase a divergent strategy, with a more ancient origin than fungi, to link the outer kinetochore to centromeric chromatin.

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↗