Biology subjects

Kitagawa, D.

Publications and source records attributed to Kitagawa, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Gap junctions in the alimentary tract regulate reproductive span in C. elegans

Aging does not occur uniformly throughout an organism but is instead differentially regulated across distinct physiological systems. In particular, reproductive aging is often temporally distinct from somatic aging and exhibits species-specific trajectories, suggesting that different physiological functions may age independently. Here we show that mutation of inx-20, an innexin family gene encoding a gap junction component, markedly extends reproductive span, with only a minor increase in overall lifespan. Furthermore, this extension of reproductive span persists in a feminized genetic background, thereby precluding the possibility that it is driven by altered sperm dynamics. inx-20 is expressed in a specific subset of cells within the alimentary tract, and its expression is selectively repressed in a fraction of these cells during dauer diapause, suggesting a role in nutrient responses. Genetic analyses suggest that inx-20 operates via a distinct mechanism that does not intersect with the TGF-{beta} and IIS-FOXO pathways, which are established regulators of reproductive span. Collectively, our results suggest that gap junctions in the alimentary tract are a selective determinant of reproductive span, capable of extending it substantially without a commensurate effect on lifespan.

genetics↗

bioRxiv2026

AKIP1 is an inner scaffold component required for centriole integrity

Centrioles are microtubule-based organelles that play crucial roles in various biological processes, including cell division, ciliogenesis and flagellar assembly. These functions require the preservation of centriole structural integrity, yet the molecular mechanisms that maintain this integrity remain incompletely understood. In this study, through large-scale gene co-dependency analysis and ultrastructure expansion microscopy (U-ExM), we identified A-kinase interacting protein 1 (AKIP1) as a novel component of the centriole inner scaffold, a luminal structural framework that supports centriole integrity. Analysis of large-scale functional genomic data from DepMap placed AKIP1 in a functional cluster with the inner scaffold components POC1A, POC1B, and POC5. Consistently, AKIP1 colocalizes with these clustered inner scaffold components in the central core region of centrioles, forming a multicomponent interaction network. We further show that AKIP1 is recruited to centrioles during G2 phase, subsequent to the recruitment of other inner scaffold components, and that its centriolar localization depends on these components. AKIP1 depletion leads to defects in centriolar microtubules and abnormal centriole morphology. Together, our findings establish AKIP1 as a downstream component of the inner scaffold that contributes to the maintenance of centriole integrity.

cell biology↗

bioRxiv2026

Pathway-Centric Integration of CRISPR Fitness with Molecular Features Draws Cancer State Maps

Cancer cells display heterogeneous pathway activity that shapes therapeutic vulnerability, but mapping it remains challenging. Transcriptomic scores do not directly measure functional activity, and CRISPR knockout data alone lack molecular interpretability. We introduce StateMap, a pathway-centric framework integrating gene expression and genome-wide CRISPR knockout fitness data from the Cancer Dependency Map. For a given pathway, StateMap selects features by co-dependency and mutual information, then projects cell lines into a low-dimensional space reflecting pathway activity and molecular state. Applied to the Hippo pathway, it resolved five functional states refining the YAP-on/YAP-off dichotomy. Notably, the Hippo-strong state showed selective dependence on integrin V{beta}5; ITGAV depletion triggered Hippo-dependent cell aggregation and G1 arrest via enhanced cell-cell adhesion. Machine learning transfer to TCGA identified a matching subtype with poor prognosis, nominated NNMT as a biomarker, and predicted sensitivity to the V inhibitor Cilengitide. StateMap enables pathway-specific state mapping and discovery of state-selective therapeutic vulnerabilities.

cancer biology↗

bioRxiv2024

Latent mitotic vulnerability of AML cells induced by therapeutic agents

Acute myeloid leukemia (AML) is a hematopoietic malignancy with a poor prognosis. Understanding the unidentified properties of AML cells is beneficial for the identification of novel therapeutic strategies for AML. In this study, we uncover the vulnerabilities of AML cells in mitosis when exposed to therapeutic agents. Through comparative analysis of large-scale data quantifying drug effects on cancer cell proliferation, the drug targeting the cell cycle and mitosis are predicted to possess high cytotoxicity against AML cell lines. Consistently, live-cell imaging with microwell devices demonstrates that clinical drugs targeting the cell cycle processes, such as idarubicin, pevonedistat and vincristine, potently induce mitotic cell death in AML cells. While these therapeutic agents also induce cell death through S/G2 phase arrest, the cytotoxic effects during mitosis are notably more pronounced. Furthermore, by employing additional inhibition of Chk1 to override the G2/M checkpoint, the AML cells stalled in the S/G2 phase prematurely enter mitosis, resulting in a significant increase in cell death. Collectively, these results unveiled the latent mitotic vulnerabilities of AML cells, providing a basis for developing novel therapeutic interventions.

cancer biology↗

bioRxiv2024

Comprehensive analysis of end-modified long dsDNA donors in CRISPR-mediated endogenous tagging

CRISPR-mediated endogenous tagging is a powerful gene editing technique for studying protein dynamics and function in their native cellular environment. While the use of 5 modified DNA donors has emerged as a promising strategy to improve the typically low efficiency of knock-in gene editing, the underlying mechanisms remain poorly understood. In this study, we conducted a comprehensive analysis of end-modified long linear dsDNA donors in CRISPR-mediated endogenous tagging in human non-transformed cells. In-depth analysis of repair patterns reveals that 5 biotinylation of dsDNA donors significantly reduces imprecise insertions, thereby enhancing homology-directed repair (HDR)-mediated precise insertion efficiency. Notably, the impact of biotinylation on repair patterns resembles that of non-homologous end joining (NHEJ) pathway inhibition, suggesting its role in preventing NHEJ-mediated mis-integration. Moreover, combining biotin modification with NHEJ inhibitor treatment further improves bi-allelic knock-in efficiency. Overall, this study provides novel insights into the mechanisms by which 5 modifications enhance precise knock-ins and demonstrates their potential for achieving high-efficient, prercise endogenous tagging in human cells.

cell biology↗

bioRxiv2024

Multimodal mechanisms of human centriole engagement and disengagement

The DNA and the centrioles are the only cellular structures that uniquely replicate to produce identical copies, which is crucial for proper chromosome segregation in mitosis. A new centriole termed "daughter" is progressively assembled adjacent to a pre-existing, "mother" centriole. Only after the daughter centriole is structurally completed as an identical copy, it disengages from its mother to become the core of a new functional centrosome. The mechanisms preventing precocious disengagement of the immature copy have been previously unknown. Here, we identify three key centriole-associated mechanisms that maintain the mother-daughter engagement: the cartwheel, the torus, and the pericentriolar material pathways. Among these, the torus is critical for establishing the characteristic orthogonal engagement between the mother and daughter centrioles. Furthermore, we show that the engagement mediated by the cartwheel and the torus pathways is released stepwise through structural changes in the daughter centriole, known as centriole blooming and centriole distancing, respectively. Disruption at any stage of these structural transitions leads to the failure of all subsequent steps, ultimately blocking centriole disengagement and centrosome conversion at the end of mitosis. Overall, this study provides a comprehensive understanding of how the maturing daughter centriole progressively disengages from its mother through multiple maturation steps, to ensure its complete structure and conversion into an independent centrosome.

cell biology↗

bioRxiv2023

Comparable analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging

CRISPR-mediated endogenous tagging, utilizing the homology-directed repair (HDR) of DNA double-strand breaks (DSBs) with exogenously incorporated donor DNA, is a powerful tool in biological research. Inhibition of the non-homologous end joining (NHEJ) pathway has been proposed as a promising strategy for improving the low efficiency of accurate knock-in via the HDR pathway. However, the influence of alternative DSB repair pathways on gene knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR/Cas-mediated knock-in, even under conditions where NHEJ is inhibited. Suppression of the microhomology-mediated end joining (MMEJ) or the single strand annealing (SSA) repair mechanisms leads to a reduction in distinct patterns of imprecise repair, thereby elevating the efficiency of accurate knock-in. Furthermore, a novel reporter system shows that the SSA pathway contributes to a specific pattern of imprecise repair, known as asymmetric HDR. Collectively, our study uncovers the involvement of multiple DSB repair pathways in CRISPR/Cas-mediated gene knock-in and proposes alternative approaches to enhance the efficiency of precise gene knock-in.

genomics↗

bioRxiv2023

Group comparison based on genetic information reveals lineage-specific therapeutic vulnerabilities in acute myeloid leukemia

Cancer is a genetic disease with specific mutations or fusions. Therapies targeting cancer cell-specific essential genes are expected to have efficient anticancer effects with fewer side effects. To explore such cancer cell-specific vulnerabilities, we established a two-group comparison system to predict essential genes in each cancer subtype using the data from the Cancer Dependency Map (DepMap). We applied this analytical method to acute myeloid leukemia (AML) and identified PCYT1A and BCL2L1 as a specific vulnerability in MLL-rearranged AML and TP53-mutated AML, respectively. Interestingly, further investigation revealed that PCYT1A is in fact a critical regulator in monocytic AML including those with MLL-rearrangements, and BCL2L1 is essential in acute erythroid leukemia in which TP53 is frequently mutated. These results highlighted the importance of cell of origin, rather than the genetic aberrations alone, to identify subtype-specific vulnerabilities in AML. The DepMap-based two-group comparison approach could accelerate the discovery of subtype-specific therapeutic targets in diverse cancers.

cancer biology↗

bioRxiv2022

An antioxidant screen identifies ascorbic acid for prevention of light-induced mitotic prolongation in live cell imaging

Phototoxicity is an important issue in fluorescence live imaging of light-sensitive cellular processes such as mitosis, especially in high spatiotemporal resolution microscopy that often requires high-intensity illumination. Among several approaches to reduce phototoxicity, the addition of antioxidants to the imaging media has been used as a simple and effective method. However, it remains unknown what are the optimal antioxidants that could prevent phototoxicity-induced defects during mitosis in fluorescence live cell microscopy. In this study, we analyzed the impact of phototoxicity on the mitotic progression in fluorescence live imaging of human diploid cells and performed a screen to identify the most efficient antioxidative agents that reduce it. Quantitative analysis shows that high amounts of light illumination cause various mitotic defects such as prolonged mitosis and delays of chromosome alignment and centrosome separation. Among several antioxidants known to reduce cellular phototoxicity, our screen reveals that ascorbic acid significantly alleviates these phototoxic effects in mitosis. Furthermore, we demonstrate that the addition of ascorbic acid to the imaging media enables fluorescence imaging of mitotic events at very high temporal resolution without obvious photodamage. Thus, this study provides a simple and practical method to effectively reduce the phototoxic effects on mitotic processes in fluorescence live cell imaging.

cell biology↗

bioRxiv2022

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, for insertion of long transgenes such as fluorescent reporters in human cells, the optimal type of HDR donors remains unclear. In this study, we established a simple and efficient CRISPR-mediated knock-in method for long transgenes using linear dsDNA and ssDNA donors, and systematically compared the performance of these two donors for endogenous gene tagging in human non-transformed diploid cells. Quantification using flow cytometry revealed higher efficiency of fluorescent tagging with dsDNA donors than with ssDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was higher with dsDNA donors than with ssDNA. Moreover, in off-target integration analyses, dsDNA and ssDNA were comparably prone to non-homologous integration. These results indicate that ssDNA is not superior to dsDNA as long HDR donors for gene knock-in in human cells.

cell biology↗

bioRxiv2021

LEXAS: a web application for life science experiment search and suggestion

MotivationIn cellular biology, researchers design wet experiments by reading the relevant articles and considering the described experiments and results. Today, researchers spend a long time exploring the literature in order to plan experiments. ResultsTo accelerate experiment planning, we have developed a web application named LEXAS (Life-science EXperiment seArch and Suggestion). LEXAS curates the description of biomedical experiments and suggests the experiments on genes that could be performed next. To develop LEXAS, we first retrieved the descriptions of experiments from full-text biomedical articles archived in PubMed Central. Using these retrieved experiments and biomedical knowledgebases and databases, we trained a machine learning model that suggests the next experiments. This model can suggest not only reasonable genes but also novel genes as targets for the next experiment as long as they share some critical features with the gene of interest. Availability and implementationLEXAS is available at https://lexas.f.u-tokyo.ac.jp/ and provides users with two interfaces: search and suggestion. The search interface allows users to find a comprehensive list of experiment descriptions, and the suggestion interface allows users to find a list of genes that could be analyzed along with possible experiment methods. The source code is available at https://github.com/lexas-f-utokyo/lexas. Contactito-delightfully-kei@g.ecc.u-tokyo.ac.jp Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2021

Stress-dependent dynamic and reversible formation of cytoskeleton-like filaments and gel-transition by tardigrade tolerance proteins.

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "Desolvataion-induced ReversiblY condensing Proteins (DRYPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRYPs, 3 of which were major components of DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. CAHS filamentation increases cell stiffness to resist deformation and improves resistance to dehydration-like stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and also contribute to the exceptional physical stability in a dehydrated state.

biochemistry↗

bioRxiv2021

Quantitative analysis of CRISPR-del for complete gene knockout in human diploid cells

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion/deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked-out by the indel-based gene disruption. In this study, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform "complete" gene knockout efficiently in non-transformed human diploid RPE1 cells. By optimizing several procedures, the CRISPR-del pipeline allowed us to generate knockout cell lines harboring bi-allelic large chromosomal deletions in a high-throughput manner. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enables the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is a high-throughput approach for performing "complete" gene knockout in RPE1 cells.

cell biology↗

bioRxiv2020

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. While centrosomes facilitate bipolar spindle formation, the individual functions of the centriole and PCM in mitosis remain elusive. Herein, we show the redundant roles of the centriole and PCM in bipolar spindle formation in human cells. Upon depletion of the PCM scaffold components, pericentrin and CDK5RAP2, centrioles remained able to recruit CEP192 onto their walls, which was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the chemical perturbation of polo-like kinase 1, a critical kinase for PCM assembly, efficiently suppressed the proliferation of various cancer cell lines from which centrioles were removed. Overall, these data suggest that the centriole and PCM cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation in human cells.

cell biology↗

bioRxiv2020

Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase to ensure proper centriole duplication cycle

Centrioles duplicate in the interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Co-depletion of Cep57 and Cep57L1 induces precocious centriole disengagement in the interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation.

cell biology↗

bioRxiv2020

DONSON, a gene responsible for microcephalic primordial dwarfism, ensures proper centriole duplication cycle by maintaining centriole engagement during interphase.

Microcephalic primordial dwarfism (MPD) is a genetic disorder characterized by short stature and microcephaly. MPD-related genes are known to regulate centrosome biogenesis, DNA replication or the DNA damage response. Although some of the MPD-related proteins that are implicated in DNA replication localize to centrosomes, how these proteins affect centrosome biogenesis remains mostly elusive. Here, we revisit the potential function of these DNA replication mediators in human centrosome biogenesis. Among these proteins, depletion of DONSON leads to excessive number of centrosomes in interphase, caused by precocious centriole disengagement. Such disengaged centrioles are converted to centrosomes, followed by centriole reduplication during interphase. These extra centrosomes lead to abnormal spindle formation and chromosome segregation errors. Importantly, similar defects are observed in MPD patients cells with DONSON mutations, suggesting a possible cause of the disease. Overall, these results indicate that DONSON is involved in regulating the centriole duplication cycle by ensuring the maintenance of centriole engagement during interphase.

cell biology↗

bioRxiv2019

Feedback loops in the Plk4-STIL-HsSAS6 network coordinate site selection for procentriole formation

Centrioles are duplicated once in every cell cycle, ensuring the bipolarity of the mitotic spindle. Although the core components have been identified, how they cooperate to achieve high fidelity in centriole duplication remains poorly understood. Here, we show that in centriole duplication the accumulation of STIL and HsSAS6, components of the cartwheel structure, provides negative feedback in the centriolar dynamics of Plk4. By live-cell imaging of endogenously tagged proteins in human cells throughout the entire cell cycle, we quantitatively tracked the dynamics of the critical duplication factors: Plk4, STIL, and HsSAS6. Centriolar Plk4 peaks and then starts decreasing during the late G1 phase, which coincides with the accumulation of STIL at centrioles. Shortly thereafter, the HsSAS6 level increases steeply at the procentriole assembly site. We also show that both STIL and HsSAS6 are necessary for attenuating Plk4 levels. Furthermore, our mathematical modeling and simulation convincingly reproduce the dynamics of these three proteins at centrioles, and suggest that the STIL-HsSAS6 complex in the cartwheel has a negative feedback effect on centriolar Plk4. Combined, these findings illustrate how the dynamic behavior of and interactions between the critical duplication factors coordinate the centriole-duplication process.

cell biology↗

bioRxiv2018

A theory of centriole duplication based on self-organized spatial pattern formation

In each cell cycle, centrioles are duplicated to produce a single copy of each pre-existing centriole. At the onset of centriole duplication, the master regulator Polo-like kinase 4 (Plk4) undergoes a dynamic change in its spatial pattern on the periphery of the pre-existing centriole, forming a single duplication site. However, the significance and mechanisms of this pattern transition remain largely unknown. Using super-resolution imaging, we found that centriolar Plk4 exhibits periodic discrete patterns resembling pearl necklaces, frequently with single prominent foci. We constructed mathematical models that simulated the pattern formation of Plk4 to gain insight into the discrete ring patterns. The simulations incorporating the self-organization properties of Plk4 successfully generated the experimentally observed patterns. We therefore propose that the self-patterning of Plk4 is crucial for the regulation of centriole duplication. These results, defining the mechanisms of self-organized regulation, provide a fundamental principle for understanding centriole duplication.

cell biology↗