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Shibuya, H.

Publications and source records attributed to Shibuya, H..

7 recordsLinked to original sources

A patient-derived LMX1B variant causes tissue-specific manifestations of nail-patella syndrome in mice

Nail-patella syndrome (NPS) is a multisystem disorder caused by pathogenic variants in LMX1B and is characterized by dysplasia of the nails and patellae as well as extraskeletal complications such as progressive nephropathy and glaucoma. We generated a CRISPR/Cas9 knock-in mouse carrying the R252Q substitution, corresponding to a human LMX1B variant associated with renal-predominant disease. Phenotypic analysis revealed that homozygous mice were viable, but they displayed marked growth retardation and severe bilateral ocular opacity. Interestingly, while this model exhibited clear skeletal and ocular defects, the renal phenotype was relatively mild, although increased urinary albumin excretion, focal glomerular basement membrane abnormalities, and subtle changes in renal gene expression were detected. Beyond the classical NPS hallmarks, mutant mice also displayed midbrain morphological abnormalities, suggesting broader developmental consequences of this LMX1B variant. This patient-derived variant model not only recapitulates the pleiotropic features of NPS but also demonstrates organ-specific susceptibility to the R252Q substitution, providing a foundation for elucidating the complex molecular mechanisms underlying multisystem disease.

genetics↗

A Data-Analysis Pipeline for High-Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT-SELEX) in the Characterization of Telomeric Proteins

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that safeguard them from triggering inappropriate DNA damage signaling. POT1, a member of the mammalian shelterin complex, binds single-stranded (ss) telomeric DNA and blocks the activation of the ATR kinase-mediated DNA damage response at telomeres. Yet until recently, it was poorly understood how the double-stranded (ds)-ss telomeric junction was protected from DNA damage response factors. An initial study of the DNA-binding activity of human POT1 (hPOT1) using systematic evolution of ligands by exponential enrichment (SELEX) and subsequent investigation revealed that POT1 contains a binding pocket, known as the POT-hole, that binds the 5 phosphorylated dC of the telomeric ds-ss junction. The amino acid residues composing the POT-hole show full sequence identity with telomeric proteins from diverse eukaryotes, including Caenorhabditis elegans POT-1. The current study builds on this SELEX method, developing an extensive analysis pipeline for SELEX datasets sequenced by next-generation sequencing and achieving a deeper analysis of the resulting sequences. We validated our approach by applying it to the DNA-binding domain of hPOT1, yielding results consistent with a previous SELEX study. Furthermore, we employ our pipeline to characterize the DNA-binding activity of C. elegans proteins that are considered homologs of hPOT1: POT-1, POT-2, POT-3, and MRT-1. Our analysis suggests that all four proteins show a binding preference for G-enriched DNA sequences, with POT-1 additionally binding secondary structural elements. Overall, we present a bioinformatics pipeline that is accessible and applicable for determining the nucleic acid-binding properties of a variety of proteins.

biochemistry↗

Non-viral derivation of induced pluripotent stem cells from the canine umbilical cord

In our previous study, canine induced pluripotent stem cells (iPSCs) were successfully generated from skin-derived fibroblasts, without the use of viral vectors. However, for clinical application of canine iPSCs in veterinary regenerative medicine, iPSCs generated from less invasive cell sources would be desirable. Therefore, the purpose of this study was to generate iPSCs from canine umbilical cords discarded at fetal birth. Canine umbilical cords were cut into small pieces and cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum. Episomal vectors carrying 10 reprogramming gene sets were introduced into fibroblasts obtained from the umbilical cord using electroporation. When putative iPSCs colonies emerged, constitutive cell characterization was performed to evaluate cell morphology, proliferative potential, alkaline phosphatase staining, expression of stem cell markers, and the ability to differentiate into a trilineage following embryoid body and teratoma formation. Multiple putative iPSC colonies formed when reprogramming gene sets were introduced into fibroblasts obtained from the canine umbilical cord. The resulting colony cells stained positive for alkaline phosphatase, and showed expression of OCT4, SOX2, NANOG, SSEA1, and SSEA3 on fluorescent immunostaining for stem cell markers. Furthermore, the mRNA expression of canine endogenous OCT4, SOX2, and NANOG significantly increased, confirming the multi-differentiation potential of cells after embryoid and teratoma formation. In this study, iPSCs were successfully generated from canine umbilical cord without the use of a viral vector. Furthermore, canine umbilical cord-derived iPSCs were successfully cultured in a feeder-free manner. This study contributes to the development of veterinary regenerative medicine by using canine iPSCs.

cell biology↗

MEILB2-BRME1 forms a V-shaped DNA clamp upon BRCA2-binding in meiotic recombination

DNA double-strand break repair by homologous recombination has a specialised role in meiosis by generating crossovers that enable the formation of haploid germ cells. This requires meiosis-specific MEILB2-BRME1, which interacts with BRCA2 to facilitate loading of recombinases onto resected DNA ends. Here, we report the crystal structure of the MEILB2-BRME1 2:2 core complex, revealing a parallel four-helical assembly that recruits BRME1 to meiotic double-strand breaks in vivo. It forms an N-terminal {beta}-cap that binds to DNA, and a MEILB2 coiled-coil that bridges to C-terminal ARM domains. Upon BRCA2-binding, MEILB2-BRME1 2:2 complexes dimerize into a V-shaped 2:4:4 complex, with rod-like MEILB2-BRME1 components arranged at right-angles. The {beta}-caps located at the tips of the MEILB2-BRME1 limbs are separated by 25 nm, allowing them to bridge between DNA molecules. Thus, we propose that BRCA2 induces MEILB2-BRME1 to function as a DNA clamp, connecting resected DNA ends or homologous chromosomes to facilitate meiotic recombination.

biochemistry↗

1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm-egg fusion

Sperm acrosomal membrane proteins, such as IZUMO1 and SPACA6, play an essential role in mammalian sperm-egg fusion. How their biosynthesis is regulated during spermiogenesis has largely remained unknown. Here, we show that the 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik encodes a type-II transmembrane protein that is expressed in early spermatids but not in mature spermatozoa. 1700029I15Rik is associated with proteins involved in N-glycosylation, disulfide isomerisation, and ER- Golgi trafficking, suggesting its involvement in nascent protein processing. 1700029I15Rik knockout testis has a normal level of sperm plasma membrane proteins, but decreased expression of multiple acrosomal membrane proteins. The knockout sperm exhibit elevated ubiquitinated proteins and upregulated ER-associated degradation; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway in spermiogenesis for the assembly of acrosomal membrane proteins. Significance StatementIn sexually reproducing species, life begins with the fusion between a sperm and an egg. Multiple sperm acrosomal membrane proteins have been reported indispensable for sperm-egg fusion in mammals, yet the mechanism underlying their biosynthesis remains unknown. The present study demonstrates the existence of a 1700029I15Rik-mediated pathway specifically coordinating the processing and assembly of acrosomal membrane proteins. It represents an intriguing paradigm where the biosynthesis of proteins destined for various subcellular compartments might be orchestrated in a spatiotemporal manner. Given 1700029I15Rik is highly conserved in human, our findings provide potential insights into the aetiology of idiopathic male infertility and the development of a novel contraceptive approach involving molecular interventions in the maturation of gamete fusion-required acrosomal proteins.

developmental biology↗

The KASH5 protein involved in meiotic chromosomal movements is a novel dynein activating adaptor

Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.

cell biology↗

Intergenerational hormesis is regulated by heritable 18S rRNA methylation

Heritable non-genetic information can regulate a variety of complex phenotypes. However, what specific non-genetic cues are transmitted from parents to their descendants are poorly understood. Here, we perform metabolic methyl-labelling experiments to track the heritable transmission of methylation from ancestors to their descendants in the nematode Caenorhabditis elegans. We find that methylation is transmitted to descendants in proteins, RNA, DNA and lipids. We further find that in response to parental starvation, fed naive progeny display reduced fertility, increased heat stress resistance, and extended longevity. This intergenerational hormesis is accompanied by a heritable increase in N6-dimethyl adenosine (m6,2A) on the 18S ribosomal RNA at adenosines 1735 and 1736. We identified the conserved DIMT-1 as the m6,2A methyltransferase in C. elegans and find that dimt-1 is required for the intergenerational hormesis phenotypes. This study provides the first labeling and tracking of heritable non-genetic material across generations and demonstrates the importance of rRNA methylation for regulating the heritable response to starvation.

genetics↗