Biology subjects

Kitaoka, M.

Publications and source records attributed to Kitaoka, M..

6 recordsLinked to original sources

bioRxiv2026

Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism

Infant-associated bifidobacteria utilize lacto-N-biose I (LNB) and galacto-N-biose (GNB), major components of human milk oligosaccharides and intestinal mucin, respectively, through the GNB/LNB metabolic pathway. A UDP-glucose-hexose-1-phosphate uridylyltransferase (GalT) from Bifidobacterium longum JCM 1217 (BlGalT2) catalyzes a key step in this pathway. BlGalT2 belongs to class II GalTs within the histidine triad (HIT) superfamily, a rare protein group distinct from canonical class I GalTs. Here, we determined the crystal structure of BlGalT2 at 2.25 [A] resolution, providing the first three-dimensional structure of a class II GalTs. BlGalT2 is a monomeric enzyme with an architecture distinct from canonical class I GalT, comprising characteristic HIT1 and HIT2 catalytic core subdomains and an extended auxiliary domain that forms a self-contained active site. Kinetic analysis revealed a strong preference for GalNAc-1P over Gal-1P, with a 50-fold lower Km for GalNAc-1P, consistent with a hydrophobic pocket accommodating the N-acetyl group. Metal analysis and activity measurements indicated that Zn2+ is required for full activity, and structural prediction suggested its coordination by residues including the first histidine of the HIT motif. AlphaFold3 prediction and mutational analysis identified residues involved in nucleotide-sugar recognition and supported a Ping-Pong reaction mechanism involving a covalent enzyme-uridine monophosphate intermediate. These findings reveal how class II GalTs have evolved a unique structural framework and substrate specificity for N-acetylated sugars. Moreover, the structure of BlGalT2 fills the final missing gap in bifidobacterial HMO metabolism and provides molecular insight into the adaptation of bifidobacteria to host-derived glycans.

biochemistry↗

bioRxiv2026

A genome quality control program for the haploid stage of spermatogenesis

Genome integrity is fundamental to cellular and organismal health, with the germline genome bearing the most profound impact. During spermatogenesis, post-meiotic germ cells, called spermatids, undergo a series of morphological changes over an extended period (a week in fruit flies, two weeks in humans) to yield mature sperm. Having undergone consecutive meiotic divisions, these spermatids lack homologous chromosomes or sister chromatids, critical templates for accurate DNA repair. How genome integrity of spermatids is ensured during this vulnerable yet prolonged period remains poorly understood. In this study, we show that the process of spermatogenesis is specifically compromised to eliminate damaged nuclei upon {gamma}-irradiation. We further find a non-canonical role of histone variant H2Av in the process of spermatid elimination. Our findings demonstrate the presence of a haploid-specific quality control program that ensures the integrity of the sperm genome transmitted to the next generation.

cell biology↗

bioRxiv2024

Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts

The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we investigate the differences in spindle organization between Xenopus laevis and Xenopus tropicalis, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for Xenopus egg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species. X. laevis spindles overall exhibited a broader range of bundle sizes, while X. tropicalis spindles contained mostly smaller bundles. Moreover, while both species exhibited larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise consisted of very small bundles, and X. laevis spindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring underlying mechanisms. SIGNIFICANCE STATEMENTO_LICorrect spindle morphology is key to its function; however, traditional microscopy methods limit our view of spindle architecture. This study addresses the gap in resolving detailed spindle microtubule organization by using advanced imaging. C_LIO_LIThe research utilizes Expansion Microscopy (ExM) to reveal previously unobservable details of spindle morphology in egg extracts of two Xenopus species (X. laevis and X. tropicalis). This approach provides unprecedented clarity on microtubule arrangement and variations in spindle architecture. C_LIO_LIThis work establishes a new protocol for high-resolution imaging of spindle structures, offering insights into how spindle architecture is adapted in differently-sized spindles to ensure proper function. C_LI

cell biology↗

bioRxiv2022

Molecular conflicts disrupting centromere assembly contribute to Xenopus hybrid inviability

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or over-expressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, whereas the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not, and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA Polymerase I or by preventing collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids. ONE SENTENCE SUMMARYCentromere incompatibilities in inviable Xenopus hybrids are sequence-independent and result from disruption of epigenetic pathways required for centromere maintenance.

cell biology↗

bioRxiv2019

Defective cell death of distinct microglial subsets contributes to ADHD-like behavior in mice

Microglia are resident immune cells in the central nervous system that play essential roles to maintain homeostasis and neuronal function. Microglia are heterogeneous cells but the mechanisms by which they contribute to normal brain development remain unclear. Here,we show that microglia in the developing striatum and thalamus undergo pyroptosis,a type of lytic cell death that occurs as a result of Caspase-1 (CASP1) activation downstream of inflammasomes. We observe that pyroptosis occurs in a spatiotemporally regulated and Casp1-dependent manner during fetal brain development. Mice lacking Casp1 or the inflammasome regulating molecules, NLRP3, IL-1R, and Gasdermin D exhibit behavior changes characterized by hyperactivity, inattention, and impulsivity that are similar to attention-deficit/hyperactivity disorder (ADHD). Furthermore, re-expression of Casp1 in Cx3cr1+ cells including microglia restores normal behavior and cell death. We demonstrate that injection of an NLRP3 inhibitor into pregnant wild-type mice is sufficient to induce ADHD-like behaviors in offspring. These data suggest that microglial inflammasome activation and pyroptosis are essential for normal brain development and that genetic and pharmacological disruptions in this pathway may represent new ADHD risk factors.

neuroscience↗

bioRxiv2018

Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis

Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.

cell biology↗