Biology subjects

Ito, K. K.

Publications and source records attributed to Ito, K. K..

8 recordsLinked to original sources

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

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↗

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↗

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↗