Biology subjects

Takaki, T.

Publications and source records attributed to Takaki, T..

3 recordsLinked to original sources

bioRxiv2025

Distinct ATRX functions cooperate with 9-1-1 and CST complexesto safeguard replication and telomere integrity

Mutations in the ATRX chromatin remodeller predispose to a developmental genetic disorder and cancer, but how it safeguards genome and telomere stability remains unresolved. Here, we uncover critical dependencies for the CTC1-STN1-TEN1 (CST) complex and RAD9A-HUS1-RAD1 (9-1-1) clamp in ATRX deficient cells. ATRX:CST synthetic lethality manifests following accumulation of telomeric G-rich ssDNA, which results in telomere loss and cell death. Conversely, we attribute ATRX:9-1-1 synthetic lethality to genome-wide ssDNA lesions, which compromise DNA replication. We further show ATRX suppresses DNA damage during replication stress by counteracting the activity of the FAM111A protease. We demonstrate that roles of ATRX in telomere maintenance and replication are genetically separable requiring its ATPase activity and PIP-box, respectively, and independently of its DAXX interaction. Collectively, functions of ATRX in suppressing toxic ssDNA lesions are context-dependent and are key to global DNA replication and telomere integrity.

molecular biology↗

bioRxiv2025

Evaluation of graphene oxide-mediated NET formation using HL-60-derived neutrophil-like cells

Neutrophil extracellular traps (NETs) are chromatin-based structures released by activated neutrophils in response to pathogens or chemical stimuli, contributing to host defense but also implicated in autoimmune disease and inflammation. As NET formation gains attention as an endpoint in in vitro immunotoxicity screening, the lack of reproducible and scalable systems hampers its broader application. Here, we developed an in vitro assay using HL-60-derived neutrophil-like cells (dHL-60) differentiated with all-trans retinoic acid to evaluate NET-inducing activity in a standardized, non-animal model. Graphene oxide (GO), a model nanomaterial known to trigger NETs in primary neutrophils, induced concentration-dependent NET formation in dHL-60 cells, with maximal induction at intermediate doses and attenuation at higher concentrations, likely due to particle aggregation. NET formation was validated by extracellular DNA staining and scanning electron microscopy. GO also induced superoxide-mediated ROS production, as confirmed by electron spin resonance, consistent with canonical NETosis pathways. Furthermore, GO suppressed PMA-induced NET formation, suggesting a dose- and context-dependent immunomodulatory effect. Collectively, our results demonstrate that dHL-60 cells recapitulate key features of NETosis observed in primary neutrophils and provide a practical, reproducible model for investigating immune responses to nanomaterials. This system supports the development of non-animal approaches for assessing immunological effects of chemical substances under controlled in vitro conditions.

pharmacology and toxicology↗

bioRxiv2024

HIV-1 Vpr causes separate cell cycle arrests in G2 and M that activate alternative DNA damage pathways

Vpr is a conserved primate lentiviral accessory protein that induces cell cycle arrest in G2. The precise mechanism of this arrest and its benefit to viral replication is unknown. Here, we show that in addition to G2 arrest, Vpr from HIV-1/SIVcpz and HIV-2 lineages separately induce mitotic arrest through the spindle assembly checkpoint, in contrast to other Vpr proteins that only cause G2 arrest. The G2 arrest was mediated solely by ATR (ataxia telangiectasia and Rad3 related) and this activity caused elevated cellular dNTP levels. The mitotic arrest required ATM (ataxia-telangiectasia mutated) as well as ATR activity and resulted from the formation of HIV-1 Vpr-induced ultra-fine anaphase bridges. Moreover, ectopic expression of the DNA structure-specific endonuclease, MUS81, prevented mitotic but not G2 arrest. Importantly, virion-incorporated Vpr was sufficient to induce cellular changes within 12h post-infection, implying that these events benefit the early stages of HIV infection. Author SummaryVpr is an accessory protein found in primate lentiviruses. Like other retroviral accessory proteins, it is not absolutely required for viral replication but is thought to overcome a cellular factor that negatively regulates infection. The most well-documented effect of Vpr expression is cell cycle arrest in G2. This has been linked to activation of the DNA damage response (DDR) pathway but there are conflicting reports in the literature as to the mechanism behind this. Here, we show that Vpr from some lentiviruses, in fact, cause two separate cell cycle blocks, in G2 and M, that require different DDR pathways. Other Vpr proteins only cause arrest in G2. Furthermore, we show that degradation of one reported target of Vpr, MUS81, is specifically linked to M but not G2 arrest. This indicates that not all Vpr functions are conserved and helps explain contradictory published results. Additionally, we found that virion-incorporated Vpr protein was able to induce cellular changes, including elevated dNTP levels, within 12 hours of infection suggesting that these events enhance early HIV-1 replication events.

microbiology↗