Biology subjects

Kanai, Y.

Publications and source records attributed to Kanai, Y..

14 recordsLinked to original sources

bioRxiv2026

Genome reduction increases endosymbiont sensitivity and promotesmutualism evolution

Endosymbiosis is a major source of biological innovation, enabling hosts to explore new ecological niches. While genome reduction in endosymbionts is well documented, its impact on the evolutionary transition from parasitism to mutualism remains unclear. Regulatory genes are lost disproportionately as bacterial genomes shrink, as we confirm in endosymbiont-containing lineages. We therefore model metabolic competition between hosts and endosymbionts, representing endosymbiont genome size by the number of independently regulated sets of reactions. In our simulations, hosts that grow poorly alone adapt best when harboring moderately genome-reduced endosymbionts rather than large-genome ones. This advantage goes beyond the lower resource use of smaller endosymbionts. By decomposing endosymbiont metabolic responses to changes in host-provided metabolites into "adaptability" and "sensitivity," we demonstrate that moderate genome reduction increases the sensitivity of endosymbionts to host metabolic conditions, rendering them more exploitable through host evolution. This suggests that genome reduction plays a more constructive role in mutualism evolution than previously appreciated, facilitating host-level selection for exploitable endosymbionts rather than being merely a passive consequence of endosymbiosis. Our systems-level approach, focusing on regulatory capacity rather than specific gene functions, provides a general framework for understanding how genome reduction affects the evolutionary trajectory from parasitic to mutualistic endosymbionts, and may help explain the frequent replacement of highly reduced endosymbionts in nature.

evolutionary biology↗

bioRxiv2026

Selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility in female mice

In mammals, ovarian follicle development is a highly coordinated process that underlies female fertility. Granulosa cells expressing anti-Mullerian hormone (AMH) are widely used as a marker of growing follicles. However, the in vivo roles of granulosa cells in follicular development and female fertility remain unclear. Here, we analyzed AMH-toxin receptor-mediated cell knockout (AMH-TRECK) transgenic (Tg) mice on a NOG background, in which AMH-expressing granulosa cells are specifically depleted by diphtheria toxin (DT). We first found that, after a single DT injection into postnatal AMH-TRECK Tg females, AMH-expressing granulosa cells in primary and secondary follicles exhibited cleaved caspase-3 signals 1 day later and were depleted 4 days later. Second, after repeated DT injections weekly from 1 to 7 weeks of age in AMH-TRECK Tg females, antral follicles and corpora lutea were rarely observed, and the numbers of primordial, primary, and secondary follicles were decreased. Following PMSG and hCG stimulation, repeated DT-injected Tg females exhibited a reduced number of ovulated oocytes with a low proportion of mature oocytes, resulting in reduced IVF rates and fertility. Further, after a cessation of repeated DT treatment, ovarian weight and follicular development recovered: the numbers of primary, secondary, and antral follicles were recovered, whereas the primordial follicle pool remains reduced. We conclude that selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility. Our model enables assessment of the in vivo effects of granulosa cell depletion and may provide a useful platform for future transplantation-based studies to understand complex follicular dynamics.

developmental biology↗

bioRxiv2026

Ectopic hAMH-driven SOX17 expression induces hyperplastic Sertoli valve formation in mouse testes

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

developmental biology↗

bioRxiv2024

Laboratory evolution of the bacterial genome structure through insertion sequence activation

The genome structure critically impacts bacterial physiology, ecology, and evolution. However, its evolution, driven by transposons called insertion sequences (IS), has been challenging to track in laboratories due to its slow pace. Here, we accelerated this process by introducing multiple copies of a high-activity IS into Escherichia coli. Mimicking bursts of IS copies in host-restricted endosymbionts and pathogens, we evolved the bacteria under relaxed neutral conditions. Within ten weeks, we observed a median of 24.5 IS insertions per genome, comparable to a decade of wild-type evolution. Long-read sequencing revealed extensive IS-mediated genome rearrangements, resulting in novel IS variants and genome size changes exceeding {+/-}5 %. By achieving such drastic genome evolution under relaxed selection, our study establishes a baseline for assessing the fitness effects of IS insertions, genome size changes, and rearrangements. This work paves the way for experimentally studying bacterial genome structure evolution, complementing analyses of genome structures in nature.

microbiology↗

bioRxiv2023

Structural basis of anticancer drug recognition and amino acid transport by LAT1

LAT1 (SLC7A5) transports large neutral amino acids and their derivatives across the plasma membrane and plays pivotal roles in cancer cell proliferation, immune response and drug delivery across the blood-brain barrier. Despite recent advances in structural understanding of LAT1, how it discriminates substrates and inhibitors including the clinically relevant anticancer drugs remains elusive. Here we report six structures of LAT1, captured in three different conformations and bound with diverse bioactive ligands, elucidating its substrate transport and inhibitory mechanisms. JPH203, also known as nanvuranlat or KYT-0353 and currently in clinical trials as an anticancer drug, binds to the wide-open substrate-binding pocket of LAT1. It adopts a U-shaped conformer, with its amino-phenylbenzoxazol moiety pushing against transmembrane helix 3 (TM3), bending TM10 and arresting the transporter in the outward-facing conformation. In contrast, the physiological substrate L-Phe does not exhibit such inhibitory interactions, whereas melphalan, a slow substrate, poses steric hindrance in the pocket, explaining its inhibitory activity. Unexpectedly, the "classical" system L inhibitor BCH induces an occluded state, a key structural intermediate required for substrate transport. Trans stimulation assays show that BCH facilitates transporter turnover and is therefore a transportable substrate. These findings provide a structural framework for the intricate mechanisms of substrate recognition and inhibition of LAT1, paving the way for developing more specific and effective drugs against it.

biochemistry↗

bioRxiv2023

Morc1 re-establishes heterochromatin on activated transposons and shapes the host transcriptome in gonocytes

Following reprogramming of DNA methylation, numerous transposable elements (TEs) are transiently activated in male prenatal gonocytes. Persistent expression of such TEs to adulthood leads to arrest of germ cell development. However, how these TEs are re-silenced has been unexplored. Here, we found that DNA-binding protein Morc1 re-established H3K9me3-marked heterochromatin on activated TEs, which involved methyltransferase SetDB1. Although Morc1 also triggered DNA methylation, the types of Morc1-targeted TEs for each epigenetic modification were different, suggesting that these two mechanisms were largely independent of each other. Significant overlap between TEs targeted by Morc1 and those by Miwi2, a nuclear PIWI protein, indicated that piRNA-loaded Miwi2 conferred target specificity to Morc1. Activated TEs drove transcription of adjacent genes by acting as ectopic cis-regulatory elements. Such a disrupting effect of TEs on the transcriptome was compensated by Morc1. Thus, Morc1 ensures proper interactions of TEs with host genes through re-establishment of heterochromatin in gonocytes.

bioinformatics↗

bioRxiv2022

Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

BackgroundDuring mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aortas intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17, is expressed in IAHCs, and that, among them, CD45lowc-Kithigh cells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity. MethodsRNA-sequencing (RNA-seq) analysis was done to identify genes that are up- regulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study and firstly the whole-mount immunostaining was done. We conducted a luciferase reporter assay to identify the Sox17 binding site for Rasip1 gene induction. We also analyzed the cluster formation and the multi-lineage colony forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells. ResultsThe increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity, while Rasip1 knock-down in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability. ConclusionsRasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multi-lineage colony forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multi-lineage colony forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.

developmental biology↗

bioRxiv2021

Operon formation by insertion sequence IS3 in Escherichia coli

Operons are a hallmark of the genomic and regulatory architecture of prokaryotes. However, the mechanism by which two genes placed far apart gradually come close and form operons remains to be elucidated. Here, we propose a new model of the origin of operons: Mobile genetic elements called insertion sequences can facilitate the formation of operons by consecutive insertion-deletion-excision reactions. This mechanism barely leaves traces of insertion sequences and is difficult to detect in evolution in nature. We performed, to the best of our knowledge, the first experimental demonstration of operon formation, as a proof of concept. The insertion sequence IS3 and the insertion sequence excision enhancer are genes found in a broad range of bacterial species. We introduced these genes into insertion sequence-less Escherichia coli and found that, supporting our hypothesis, the activity of the two genes altered the expression of genes surrounding IS3, closed a 2.7 kilobase pair gap between a pair of genes, and formed new operons. This study shows how insertion sequences can facilitate the rapid formation of operons through locally increasing the structural mutation rates and highlights how coevolution with mobile elements may shape the organization of prokaryotic genomes and gene regulation.

genomics↗

bioRxiv2021

Structural insights into human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity, that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. This is the first characterization of molecular features of EAAT2 and the selective inhibition mechanism, underlying structure-based drug design for EAAT2.

molecular biology↗

bioRxiv2020

Lab-on-a-graphene-FET detection of key molecular events underpinning influenza virus infection and effect of antiviral drugs

Small solid-state devices are candidates for accelerating biomedical assays/drug discovery, however their potential remains unfulfilled. Here, we demonstrate that graphene-field effect transistors (FET) can be used to successfully detect the key molecular events underlying viral infections and the effect of antiviral drugs. Our device success is achieved by bio-mimicking the host-cell surface during an influenza infection at the graphene channel. In-situ AFM confirms the biological interactions at the sialic acid-functionalized graphene: viral hemagglutinin (HA) binds to sialic acid, and neuraminidase (NA) reacts with the sialic acid-HA complex. The graphene-FET detects HA binding to sialic acid, and NA cleavage of sialic acid. The inhibitory effect of the drug "zanamivir" on NA-sialic acid interactions is monitored in real-time; the reaction rate constant of NA-sialic acid reaction was successfully determined. We demonstrate that graphene-FETs are powerful platforms for measurement of biomolecular interactions and contribute to future deployment of solid-state devices in drug discovery/biosensing.

bioengineering↗

bioRxiv2019

Periosteum-derived Osteocrin regulates bone growth through both endochondral ossification and intramembranous ossification

During development of long bones, two mechanistically distinct processes contribute to long- and short-axis growth. Endochondral ossification in the growth plate leads to the long-axis growth, while intramembranous ossification including apposition in the periosteum regulates the short axis growth. Here, we show that periosteal osteoblast-derived secretory peptide, Osteocrin (OSTN), promotes both types of long bone growth through potentiation of signaling by C-type natriuretic peptide (CNP), because OSTN inhibits the clearance of CNP by binding to natriuretic peptide receptor 3 (NPR3). The mice lacking OSTN showed less bone mass in trabecular and cortical regions than the control mice, suggesting the dual functions of OSTN in long bone growth. We found that OSTN regulated trabecular bone formation by inducing proliferation and maturation of chondrocytes possibly through enhancing CNP-dependent signaling. Besides the contribution of OSTN to long axis growth, we demonstrated that OSTN together with CNP induced osteoblast differentiation of periosteum-derived multipotent progenitor cells expressing NPR3. These data suggest that OSTN induces long bone growth through endochondral ossification and osteoblast specification of multipotent progenitor cells in the periosteum.

developmental biology↗

bioRxiv2019

Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc

The L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 [A] resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.

biochemistry↗

bioRxiv2019

Sox17 expression in endocardium precursor cells regulates heart development in mice

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Cardiac progenitor cells (CPCs) that express the homeobox gene Nkx2-5 give rise to the endocardium. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ CPCs that express the Sry-type HMG box gene Sox17 specifically differentiate into the endocardium in mouse embryos. Approximately 20% to 30% of Nkx2-5+CPCs transiently express Sox17 from embryonic day (E) 7.5 to E8.5.Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, resulting in anomalous morphology of the heart, likely as a consequence of down-regulation of NOTCH signaling. Changes in gene expression profile in both the endocardium and myocardium preceded the reduction in NOTCH-related gene expression in the mutant embryos, suggesting that Sox17 expression in the endocardium regulates an unknown signal required for nurturing of the myocardium. Our results thus provide insight into differentiation of the endocardium and its role in heart development. SignificanceThe endocardium is vital for vertebrate heart development; however, the molecular mechanisms regulating fate determination and differentiation remain largely unknown. Here, we show that a part of the earliest cardiac progenitor cells (CPCs) transiently and exclusively express Sry-type HMG box gene Sox17 in the mouse embryo. Sox17-expressing CPCs specifically differentiate to the endocardium. Sox17 biases the fate of CPCs toward the endocardium, and regulates proliferation and cellular behavior cell autonomously. Conversely, Sox17 in the endocardium regulates the myocardium non-cell autonomously. Notably, Sox17 is required for the ventricular trabeculation via the NOTCH signal that is not directly induced but maintained by Sox17. This study, thus, sheds light on endocardium development.

developmental biology↗

bioRxiv2018

Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

The nuclear receptor NR5A1 (also known as Ad4BP, or SF1) is essential for the initial steps of mammalian gonadal development. The Nr5a1 gene is equally expressed in XX and XY gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in XX gonads. We recently reported a case of 46, XX disorder of sex development (DSD) in which ectopically expressed NR5A1 in XX gonads led to an ovo-testicular phenotype, suggesting that excess NR5A1 can direct the development of immature XX gonads towards testicular formation. However, a direct causal relationship has not been demonstrated in an animal model. Here, using a Wt1-BAC (bacterial artificial chromosome) transgene system, we generated two lines of mice overexpressing Nr5a1 in the fetal gonads at different levels. One of these lines (Tg-S), highly expressing Nr5a1, revealed that enforced Nr5a1 expression alone is insufficient to switch the fate of the 46,XX gonads toward testicular formation in mice. In the other line (Tg-A) expressing Nr5a1 at lower level, ovarian development was compromised, with multi-oocyte follicles, reduced number of matured follicles, and impaired expression of Wnt4, resulting in late onset infertility at 20 weeks after birth. The phenotype was similar to that of genetically modified mice with impaired Notch signaling. Indeed, the expression level of Notch2 and 3 was significantly reduced in Tg-A mice, and the ovarian phenotype in Tg-A mice was almost completely rescued by in utero treatment with a Notch2 agonist HMN2-29. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine tuning of Notch signaling levels.\n\nAUTHOR SUMMARYSexual development is a process of differentiation from undifferentiated bipotential gonads, and insight into sexual differentiation will bring important new knowledge to our understanding of organogenesis. The nuclear receptor NR5A1 which is essential for mammalian gonadal development, is equally expressed in both gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in XX gonads. We have recently demonstrated that this down-regulation is mediated by ovarian transcription factor, Forkhead box L2 (FOXL2). This finding raised two key questions, whether Nr5a1 can function as a male sex-determining factor, and whether the repression is essential for appropriate ovarian development. By generating two lines Tg mice in XX gonads with different enforced expression levels of Nr5a1, our present study revealed that alterations in Nr5a1 dosage, either reduced or excessive, result in pathological effects in ovarian development and female fertility, indicating that the precise control of Nr5a1 at the transcriptional level is essential for optimal ovarian development. We envisage that the improved understanding of how this pathway regulates ovarian development and female fertility would aid the development of artificial somatic ovarian cells, which in turn may provide a valuable treatment option in reproductive medicine.\n\nABBREVIATIONS

developmental biology↗