Biology subjects

Saitoh, S.

Publications and source records attributed to Saitoh, S..

10 recordsLinked to original sources

bioRxiv2025

Compromised ESCRT signalling is sufficient for resistance to the Target of Rapamycin Complex inhibitor Torin1 in fission yeast

BackgroundFission yeast cells defective in Golgi-endosomal sorting display high resistance to Torin1, a pan-Target of Rapamycin (TOR) inhibitor. TOR complexes regulate the ESCRT system to integrate nutrient availability with cell division. TOR activity is frequently deregulated in cancer, making it an attractive therapeutic target. Deregulated ESCRT activity has also been associated with cancer, but its role in mediating drug resistance is not fully understood. Herein, we have investigated the role of the ESCRT system in regulating sensitivity to Torin1. MethodsGrowth assays were used to monitor the growth of yeast cells. The effect of Torin1 on protein expression was monitored by immunoblotting. Fluorescence microscopy was used to investigate the action of Torin1 on protein localization. ResultsThe ESCRT system mediates Torin1-induced degradation of amino acid and glucose transporters. The expression of these transporters at the plasma membrane is not abolished in ESCRT mutants. Mutants unable to effectively ubiquitylate these transporters are also resistant to Torin1. Impaired ESCRT-mediated protein degradation is associated with strong resistance to Torin1. ConclusionsMutations in genes encoding ESCRT components have been reported in cancer. We present evidence that compromised ESCRT signalling is sufficient for resistance to Torin1. Cells defective in ESCRT signalling or ubiquitin homeostasis are highly resistant to Torin1. Our studies demonstrate that defective ESCRT-mediated proteolysis can suppress sensitivity to Torin1.

cell biology↗

bioRxiv2025

A Priori recognition for biological phenomena prior to empirical understanding

Biological phenomena include unrecognized events. These unrecognized events often unknowingly increase observer bias, which inhibits open and reproducible science. In this study, we focused on the recognition procedures underlying primary data and modeled cell population behavior. Using agent-based modeling (ABM), we demonstrated that cellular behaviors can be categorized into 11 distinct types, a framework we defined as the Behavioral Eleven Cell Class (BECC). We further validated BECC by describing the differential STAT3 phosphorylation patterns in leukocyte subsets and polarized T cell differentiation in OT-II transgenic mice. BECC serves as a novel descriptive method with three defining features: (i) it functions as a symbolic system independent of numerical or linguistic constraints, (ii) it represents the minimal unit of recognition, and (iii) it enables the expression of inherently unrecognizable phenomena. BECC allows for observer subjectivity and the relativity of results while enforcing rigorous recognition. This unique approach provides a practical and conceptual basis for advancing open and reproducible science.

systems biology↗

bioRxiv2024

In quiescent G0 phase, Schizosaccharomyces pombe Mis4 ensures full nuclear separation during the subsequent M phase

Evolutionarily conserved Mis4 establishes cohesion between replicated sister chromatids in vegetatively proliferating cells. In the fission yeast, Schizosaccharomyces pombe, defects in Mis4 lead to premature separation of sister chromatids, resulting in fatal chromosome mis-segregation during mitosis. In humans, NIPBL, an ortholog of Mis4, is responsible for a severe multisystem disorder called Cornelia de Lange syndrome. We previously reported that Mis4 is also essential in non-proliferating quiescent cells. Whereas wild-type fission yeast cells can maintain high viability for long periods without cell division in the quiescent G0 phase, mis4-450 mutant cells cannot. In this report, we show that Mis4 is not required for cells to enter G0 phase, but is essential to exit from it. When resuming mitosis after passage of G0, mis4 mutant cells segregated sister chromatid successfully, but insufficiently separated daughter nuclei consequently formed dikaryon-like cells. These findings suggest a novel role of Mis4/NIPBL in non-dividing quiescent cells, which is prerequisite for full nuclear separation upon resumed mitosis. As most human cells are in the quiescent state, this study may facilitate development of novel therapies for human diseases caused by Mis4/NIPBL deficiency.

cell biology↗

bioRxiv2024

Phosphorylation of Aly3 C-terminus impedes aberrant endocytosis of S. pombe hexose transporter Ght5

In fission yeast, Schizosaccharomyces pombe, transcriptional upregulation and cell-surface localization of the hexose transporter, Ght5, are required for cell proliferation in low glucose. As the target of rapamycin complex 2 (TORC2) signaling pathway inhibits -arrestin Aly3-dependent endocytosis of Ght5, we hypothesized that this endocytosis was inhibited by phosphorylation. To identify phosphorylation sites required for cell proliferation in low glucose, serine and threonine residues of Aly3 and Ght5 reportedly phosphorylated were replaced with alanine. We found that C-terminal serine residues of Aly3, but not Ght5, are necessary for proliferation in low glucose. Expression of Aly3 protein unphosphorylated at the C-terminus led to increased ubiquitination and vacuolar accumulation of Ght5 in low glucose, but reversion of one of the alanine residues to serine reduced ubiquitination and vacuolar accumulation of Ght5. Also, Aly3 physically interacted with the HECT-type ubiquitin ligases Pub1 and Pub3, and these interactions were required for surface localization of Ght5 and proliferation in low glucose. This study reveals mechanisms by which Aly3 is regulated so that fission yeast can adapt to nutritional stress.

cell biology↗

bioRxiv2024

Arrayed CRISPRi library to suppress genes required for Schizosaccharomyces pombe viability

The fission yeast, Schizosaccharomyces pombe, is an excellent eukaryote model organism to study essential biological processes. Its genome contains [~]1200 genes essential for cell viability, most of which are evolutionarily conserved. To study these essential genes, resources enabling conditional perturbation of target genes are required. Here, we constructed comprehensive arrayed libraries of plasmids and strains to knock down essential genes in S. pombe, using a dCas9-mediated CRISPRi. These libraries cover [~]98% of all essential genes in fission yeast. We estimate that in [~]60% of the strains in the library, transcription of a target gene was repressed so efficiently that cell proliferation was significantly inhibited. To demonstrate usefulness of these libraries, we performed metabolic analyses with knockdown strains and revealed a flexible interaction among metabolic pathways. Libraries established in this study enable comprehensive functional analyses of essential genes in S. pombe and will facilitate understanding of essential biological processes in eukaryotes.

cell biology↗

bioRxiv2024

High Shear Stress Reduces ERG Causing Endothelial-Mesenchymal Transition and Pulmonary Arterial Hypertension

Pathological high shear stress (HSS, 100 dyn/cm2) is generated in distal pulmonary arteries (PA) (100-500 m) in congenital heart defects and in progressive PA hypertension (PAH) with inward remodeling and luminal narrowing. Human PA endothelial cells (PAEC) were subjected to HSS versus physiologic laminar shear stress (LSS, 15 dyn/cm2). Endothelial-mesenchymal transition (EndMT), a feature of PAH not previously attributed to HSS, was observed. H3K27ac peaks containing motifs for an ETS-family transcription factor (ERG) were reduced, as was ERG-Kruppel-like factors (KLF)2/4 interaction and ERG expression. Reducing ERG by siRNA in PAEC during LSS caused EndMT; transfection of ERG in PAEC under HSS prevented EndMT. An aorto-caval shunt was preformed in mice to induce HSS and progressive PAH. Elevated PA pressure, EndMT and vascular remodeling were reduced by an adeno-associated vector that selectively replenished ERG in PAEC. Agents maintaining ERG in PAEC should overcome the adverse effect of HSS on progressive PAH.

cell biology↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗

bioRxiv2020

Five patterns of cell signaling pathways associated with cell behavior

Cell signaling pathway is complex systems. Here, we present a concept for a new approach to analyze cell signaling pathway associated with cell behavior. In theoretically, cell behavior is recognized by energy and fluctuation. In this study, we measured phosphorylation level of signal transducers in a cell and fluctuation of the phosphorylation level in the cell population using flow cytometry. Flow cytometric data of mean fluorescence intensity (MFI) and coefficient variation (CV) were considered to the energy and the fluctuation, respectively. Topologically, the changes of MFI and CV were categorized into five patterns (we tentatively named as attractive, subsequent, passive, counter, and negative arbiter). In this study, we clarified the relationship between the cell behavior and the five patterns. Furthermore, combining the five patterns can define the signaling pathways, such as simple activated signal, oscillating signal, regulatory signal, robust signal, or homeostatic signal. These observations provide a proof of concept for general strategy to use the five patterns for connection between cell signaling pathway and cell behavior.

systems biology↗

bioRxiv2020

Selective and controlled myelin formation by individual interfascicular oligodendrocytes in the mouse corpus callosum

Single oligodendrocytes produce myelin sheath around multiple axons in the central nervous system. Interfascicular oligodendrocytes (IOs) in the white matter are aligned in rows and facilitate nerve conduction, but their detailed morphologies remain largely unknown. In the present study, we three-dimensionally reconstructed seven IOs in a row in the murine corpus callosum using serial block face-scanning electron microscopy (SBF-SEM). These morphologically polarized IOs extended a thick process with numerous branches from the cytoplasm-rich part of the cell and formed myelin sheaths preferentially around distant axons. The multiple branched processes of each IO myelinated multiple axons having similar diameters with restricted myelin thicknesses, indicating that individual IOs have their own myelination profiles even on distinct target neurons. Consistent with the finding, the IOs transduced and visualized with the rabies viral vector expressing GFP showed statistically significant variations in the myelination patterns. We further reconstructed the sheath immediately adjacent to those derived from the seven IOs; the thicknesses of both sheaths were significantly correlated despite emanating from different IOs. These results proposed a rule that myelination by individual IOs, regulated by interaction with ensheathed axons, is selective in specific axons and at the same time orchestrated at the whole cell level. Main pointsCallosal oligodendrocytes form myelin sheaths preferentially around distant axons. Each oligodendrocyte myelinate multiple axons having similar diameters with restricted myelin thicknesses. The thicknesses of adjacent myelin sheaths are similar.

neuroscience↗

bioRxiv2019

Identification of 15 new bypassable essential genes of fission yeast

Every organism has a different set of genes essential for its viability. This indicates that an organism can become tolerant to the loss of an essential gene under certain circumstances during evolution, via the manifestation of masked alternative mechanisms. In our quest to systematically uncover masked mechanisms in eukaryotic cells, we developed an extragenic suppressor screening method using haploid spores deleted of an essential gene in the fission yeast Schizosaccharomyces pombe. We screened for the bypass suppressors of lethality of 92 randomly selected genes that are essential for viability in standard laboratory culture conditions. Remarkably, extragenic mutations bypassed the essentiality of as many as 20 genes (22%), 15 of which have not been previously reported. Half of the bypass-suppressible genes were involved in mitochondria function; we also identified multiple genes regulating RNA processing. 18 suppressible genes were conserved in the budding yeast Saccharomyces cerevisiae, but 13 of them were non-essential in that species. These trends are consistent with a recent independent bypass-of-essentiality (BOE) screening of 142 fission yeast genes conducted with more elaborate methodology (Li et al., 2019). Thus, our study reinforces the emerging view that BOE is not a rare event and that each organism may be endowed with secondary or backup mechanisms that can substitute for primary mechanisms in various biological processes. Furthermore, the robustness of our simple spore-based methodology paves the way for genome-scale BOE screening.

genetics↗