Biology subjects

Fukuyama, M.

Publications and source records attributed to Fukuyama, M..

13 recordsLinked to original sources

bioRxiv2026

N-terminal intrinsically disordered region mediates self-catalytic interfacial nucleation of Aspergillus oryzae hydrophobin RolA

Hydrophobins are biosurfactant proteins that coat the cell surfaces of filamentous fungi. On the conidial surface, hydrophobins self-assemble into rodlets, forming a dense hydrophobic film that promotes air-dispersibility. Although rodlet formation is closely associated with the physiology of filamentous fungi, its underlying molecular mechanisms remain largely unknown. Previously, we revealed that RolA, a hydrophobin derived from Aspergillus oryzae, forms rodlets at the air-water interface. In this study, we focused on the flexible N-terminal region of RolA, which lacks a well-defined tertiary structure, and hypothesized that this intrinsically disordered region regulates rodlet formation. To investigate its role, we used RolA mutants with reduced charges in the N-terminal region and analyzed the rodlet formation process on the surface of a water-in-air sessile droplet using atomic force microscopy. In addition, we quantitatively characterized rodlet formation at the air-water interface by applying a kinetic perspective to the interfacial tension change profiles obtained from dynamic surface tension measurements. The results suggested that RolA first forms a monolayer at the air-water interface, then rodlet formation proceeds through the continuous supply of free RolA monomers from the bulk phase to the interfacial RolA film. Our molecular dynamics simulations of RolA at the interface supported a model in which RolA molecules within the interfacial film interact with free monomers in the bulk phase through their N-terminal regions. These results reveal a previously unidentified role of the N-terminal region in rodlet formation and provide a more comprehensive framework for understanding the molecular mechanism underlying RolA rodlet formation.

biophysics↗

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↗

bioRxiv2026

Local aromatic interactions define temperature sensitivity of phase separation in an intrinsically disordered protein.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins is highly sensitive to environmental conditions, yet the molecular basis of sharp temperature responsiveness remains poorly understood. Here, we investigate a sequence-encoded mechanism underlying the pronounced temperature sensitivity of phase separation using Sup35NM, the intrinsically disordered domain of the yeast prion Sup35. We show that a tyrosine-rich local structural region within this domain encodes strong temperature responsiveness of droplet formation. Mutational analyses reveal that tyrosine residues mediate both intramolecular interactions that stabilize local structure and intermolecular interactions required for LLPS. Substitution of the tyrosine residues with alanine disrupts local structure and weakens intermolecular interactions, thereby diminishing temperature sensitivity. In contrast, substitution with phenylalanine promotes rapid droplet gelation, abolishes internal fluidity, suppresses amyloid formation, and confers resistance to temperature-induced dissolution. Based on these findings, we propose a molecular model in which finely tuned, moderately weak aromatic interactions among tyrosines enable reversible local compaction that is sensitive to temperature, thereby generating a sharp phase transition. These results suggest that amino acid sequences encode not only phase separation propensity but also the sensitivity of condensates to environmental perturbations, providing a framework for understanding how intrinsically disordered proteins act as molecular sensors of cellular conditions.

biochemistry↗

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↗

bioRxiv2022

Reconstitution of ribosome self-replication outside a living cell

Ribosome biogenesis, a recursive process of pre-existing ribosomes self-replicating nascent ones, is pivotal in the self-replication of life. In Escherichia coli, three ribosomal RNAs (rRNAs) are transcribed, and 54 ribosomal proteins (r-proteins) are synthesized by pre-existing ribosomes as structural components1, 2. They are cotranscriptionally assembled in a cooperative hierarchy under the support of [~]100 accessory factors1-3. The reconstitution of ribosome biogenesis outside a living cell is an essential goal to understand the self-replication of life. However, this goal could not have been achieved so far due to its complexity. Here, we report the successful in vitro reconstitution of the entire ribosome biogenesis process. We hypothesized that mimicking in vivo ribosome biogenesis1-6 could result in in vitro ribosome biogenesis. Specifically, we found that coactivating the transcription of an rRNA operon, as well as the transcription and translation of 54 r-protein genes encoding r-proteins, and the coordinated ribosomal assembly in a cytoplasm-mimicking reaction solution, resulted in highly efficient in vitro reconstitution of ribosome biogenesis. Our achievement represents a critical step toward revealing fundamental principles underlying the self-replication of life and creating self-replicating artificial cells7. We also succeeded in engineering rRNA and r-proteins by only adding mutant ribosomal genes in the reaction, enabling high-throughput and unconstrained creation of artificial ribosomes with altered or enhanced functionality8-12.

synthetic 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↗

bioRxiv2019

Patched regulates lipid homeostasis by controlling cellular cholesterol levels

AbstractHedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat storage and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPAR homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a novel role for PTCH in organelle structure maintenance and fat metabolism.

cell biology↗