Biology subjects

Shiba, K.

Publications and source records attributed to Shiba, K..

11 recordsLinked to original sources

bioRxiv2026

KK-LC-1 reveals evolutionary divergence in the regulation of sperm motility between humans and mice

CT83 (KK-LC-1) is a cancer-testis antigen originally identified in human lung cancer cells and has recently attracted attention as a potential target for cancer therapy. Although KK-LC-1 orthologs have been identified in up to 160 animal species, a murine homolog had not previously been identified, hindering in vivo analysis of its physiological function. In this study, we identified the mouse homolog of KK-LC-1 and performed a comparative analysis of its properties in humans and mice, together with an investigation of its biological function using gene knockout (KO) mice. The murine Kk-lc-1 gene is located on the X chromosome and, like its human counterpart, contains an N-terminal transmembrane domain. In both humans and mice, KK-LC-1 is expressed specifically in the testis and localizes to the head and tail regions of sperm. Analysis of Kk-lc-1-deficient mice revealed normal spermatogenesis, and both male and female KO mice were fertile. However, sperm from Kk-lc-1-deficient males exhibited reduced motility caused by decreased flexibility of the midpiece and failed to penetrate the oocyte zona pellucida in vitro. This defect was rescued by artificial insemination using epididymal sperm, suggesting that maternal factors in vivo may compensate for reduced sperm motility. Although impaired sperm motility during in vitro fertilization (IVF) was rescued by murine Kk-lc-1, functional rescue by human KK-LC-1 was not observed. These findings indicate that KK-LC-1 contributes to sperm motility but is not essential for fertility. Moreover, species-specific differences in KK-LC-1-mediated regulation of sperm motility suggest functional divergence during evolution. The role of KK-LC-1 in sperm motility should therefore be considered in the clinical development of cancer therapies targeting KK-LC-1.

developmental biology↗

bioRxiv2026

Establishing genetically controlled, closed 1 colonies of an ascidian

Recent technological advances have made many "non-model" organisms accessible for experimental studies. However, reference inbred strains were not necessarily available, especially in marine invertebrates, and genetic background of organisms used for experiments are often non-uniform. This situation potentially affects experimental reproducibility. Although ascidians, Ciona intestinalis (type A, or C. robusta), are a widely used marine animal for many areas of experimental biology including developmental studies, no reference strains have been obtained despite extensive efforts. As an alternative way to improve reproducibility, we have established and maintained ascidian colonies through intra-population breeding every year from 2016 to 2020, and monitored genomic variants of these colonies. This method does not reduce genetic variations but instead manages and monitors genetic variations in the colonies, providing an easy and cost-effective way of increasing experimental reproducibility. Furthermore, we recently upgraded these genetically isolated, closed colonies that were re-established every year, and have maintained them for more than three years only through intra-population breeding and occasional back-cross using cryopreserved sperm. Genetic variants that we revealed using 3.7 tera-bases of sequence data will help to design future experiments in this species. Our data also show that two wild-populations, which were used to establish the colonies, have maintained distinct genetic backgrounds, although their habitats are directly linked to the Pacific Ocean and only 170 km apart. More importantly, genetic information regarding these colonies will undoubtedly improve experimental reproducibility and traceability, and our method will provide a realistic solution for performing reproducible experiments using non-model organisms.

developmental biology↗

bioRxiv2025

Spiral-Sensing and Fold-Change Detection Direct Ascidian Sperm to the Egg

From microorganisms to marine sperm, successful navigation toward chemical cues relies on the integration of sensory inputs with locomotion. Ascidian sperms exhibit a robust chemotactic strategy by swimming along circular paths whose centres form large-scale spirals, guiding them toward attractant-releasing eggs. Although their intracellular Ca2+ dynamics exhibit rhythmic bursts aligned with flagellar modulation, the underlying computational principles remain unclear. Here, we propose a minimal model that captures ascidian sperm chemotaxis through a combination of swimming trajectory control and an internal signalling model with fold-change detection (FCD). This model responds to relative rather than absolute changes in the stimulus concentration, achieving scale-invariant signal processing. Concurrently, the spiral swimming path enables spatial sampling of the concentration field, allowing the cell to infer gradient direction. Our findings demonstrate that the integration of a resilient signalling module and physically structured sampling trajectory enables effective chemotaxis in complex environments.

cell biology↗

bioRxiv2025

A cellular entity retaining only its replicative core: Hidden archaeal lineage with an ultra-reduced genome

Defining the minimal genetic requirements for cellular life remains a fundamental question in biology. Genomic exploration continually reveals novel microbial lineages, often exhibiting extreme genome reduction, particularly within symbiotic relationships. Here, we report the discovery of Candidatus Sukunaarchaeum mirabile, a novel archaeon with an unprecedentedly small genome of only 238 kbp --less than half the size of the smallest previously known archaeal genome-- from a dinoflagellate-associated microbial community. Phylogenetic analyses place Sukunaarchaeum as a deeply branching lineage within the tree of Archaea, representing a novel major branch distinct from established phyla. Environmental sequence data indicate that sequences closely related to Sukunaarchaeum form a diverse and previously overlooked clade in microbial surveys. Its genome is profoundly stripped-down, lacking virtually all recognizable metabolic pathways, and primarily encoding the machinery for its replicative core: DNA replication, transcription, and translation. This suggests an unprecedented level of metabolic dependence on a host, a condition that challenges the functional distinctions between minimal cellular life and viruses. The discovery of Sukunaarchaeum pushes the conventional boundaries of cellular life and highlights the vast unexplored biological novelty within microbial interactions, suggesting that further exploration of symbiotic systems may reveal even more extraordinary life forms, reshaping our understanding of cellular evolution. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/651781v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@f5fbd6org.highwire.dtl.DTLVardef@168bee5org.highwire.dtl.DTLVardef@1ccb02forg.highwire.dtl.DTLVardef@14315fa_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2025

Drastic genome reduction driven by parasitic lifestyle: Two complete genomes of endosymbiotic bacteria possibly hosted by a dinoflagellate

Bacteria with endosymbiotic lifestyles often exhibit dramatic genome reduction. While the reduction of genomes in intracellular symbionts of animals, including parasitic bacteria, has been extensively studied, less is known about such bacteria associated with single-celled eukaryotes. Here, we report the genomes of two novel gammaproteobacterial lineages, RS3 and XS4, identified as putative parasitic endosymbionts of the dinoflagellate Citharistes regius. Phylogenetic analyses suggest that RS3 and XS4 belong to the family Fastidiosibacteraceae within the order Beggiatoales, forming independent lineages therein. The genomes of RS3 and XS4 are 529 Kbp and 436 Kbp in size, respectively, revealing drastic genome reduction compared to related bacterial genomes. XS4, which has a particularly reduced genome with low GC content, uses an alternative genetic code, in which UGA assigned tryptophan. The small genomes of RS3 and XS4 encode a limited number of proteins, retaining only approximately 20% of the predicted ancestral proteome. Metabolic reconstruction suggests that RS3 and XS4 are energy parasites, heavily dependent on their host for essential metabolites. Furthermore, we found that the ancestor of both genomes likely acquired an ADP:ATP antiporter gene via horizontal gene transfer, an event that may have enabled their evolution as energy parasites by facilitating the acquisition of ATP from their host. These observations on novel bacteria with highly reduced genomes expand our understanding of the phylogenetic and genomic diversity of endosymbiotic bacteria in protists.

evolutionary biology↗

bioRxiv2024

Apusomonad photophobic behavior highlights cytoskeletal responses to blue light in early eukaryotes

Light response is a fundamental characteristic of eukaryotic organisms, both unicellular and multicellular. However, no photoresponse has previously been reported in Apusomonadida, a group of small, free-living biflagellates phylogenetically positioned as a sister group to Opisthokonta (animals, fungi, and their unicellular relatives). Apusomonads are thus crucial for understanding the evolution of opisthokonts. Here, we report for the first time an avoidance response to blue light in the apusomonad Podomonas kaiyoae. This avoidance response is accompanied by an increase in gliding velocity, transient changes in flagellar waveforms, and alterations in cell shape. Dynamic cell contraction is induced by an increase in intracellular free Ca2+ and is inhibited by either a dynein inhibitor or an actin-disrupting drug. These findings suggest that the photophobic behavior of Podomonas kaiyoae relies on cytoskeletal responses mediated by both the dynein/tubulin and myosin/actin systems, which were acquired early in eukaryotic evolution. The dominance of the posterior flagellum in cilia-driven directional changes further supports the phylogenetic placement of apusomonads as a sister group to opisthokonts rather than other eukaryotic lineages.

evolutionary biology↗

bioRxiv2024

Convergent reductive evolution of cyanobacteria in symbiosis with Dinophysiales dinoflagellates

The diversity of marine cyanobacteria has been extensively studied due to their vital roles in ocean primary production. However, little is understood about the diversity of cyanobacterial species involved in symbiotic relationships. In this study, we successfully sequenced the complete genome of a cyanobacterium in symbiosis with Citharistes regius, a dinoflagellate species thriving in the open ocean. A phylogenomic analysis revealed that the cyanobacterium (CregCyn) belongs to the marine picocyanobacterial lineage, akin to another cyanobacterial symbiont (OmCyn) of a different dinoflagellate closely related to Citharistes. Nevertheless, these two symbionts are distinct lineages, suggesting independent origins of their symbiotic lifestyles. Despite the distinct origins, the genome analyses of CregCyn revealed shared characteristics with OmCyn, including an obligate symbiotic relationship with the host dinoflagellates and a degree of genome reduction. In contrast, a detailed analysis of genome subregions unveiled that the CregCyn genome carries genomic islands that are not found in the OmCyn genome. The presence of the genomic islands implies that exogenous genes have been integrated into the CregCyn genome at some point in its evolution. This study contributes to our understanding of the complex history of the symbiosis between dinoflagellates and cyanobacteria, as well as the genomic diversity of marine picocyanobacteria.

evolutionary biology↗

bioRxiv2023

Differentiation of Large Extracellular Vesicles in Oral Fluid: Combined Protocol of Small Force Centrifugation and Pattern Analysis

Extracellular vesicles (EVs) in biofluids are highly heterogeneous entities in terms of their origins and physicochemical properties. Considering the application of EVs in diagnostic and therapeutic fields, it is of extreme importance to establish differentiating methods by which focused EV subclasses are operationally defined. Several differentiation protocols have been proposed; however, they have mainly focused on smaller types of EVs, and the heterogeneous nature of large EVs has not yet been fully explored. In this report, to classify large EVs into subgroups based on their physicochemical properties, we have developed a protocol, named EV differentiation by sedimentation patterns (ESP), in which entities in the crude large EV fraction are first moved through a density gradient of iodixanol with small centrifugation forces, and then the migration patterns of molecules through the gradients are analyzed using a non-hierarchical data clustering algorithm. Based on this method, proteins in the large EV fractions of oral fluids clustered into three groups: proteins shared with small EV cargos and enriched in immuno-related proteins (Group 1), proteins involved in energy metabolism and protein synthesis (Group 2), and proteins required for vesicle trafficking (Group 3). These observations indicate that the physiochemical properties of EVs, which are defined through low-speed gradient centrifugation, are well associated with their functions within cells. This protocol enables the detailed subclassification of EV populations that are difficult to differentiate using conventional separation methods.

molecular biology↗

bioRxiv2022

"Two-story building" of a ctenophore comb plate provides structural and functional integrity for motility of giant multicilia

Comb plates (CPs) are large compound cilia uniquely seen in a basal metazoan group of ctenophores.1-3 Tens of thousands of cilia are bundled in a CP via structures connecting adjacent cilia, called compartmenting lamella (CL), which are the basis for the structural iridescent color and the coordination of ciliary movement of the CP.4-6 We previously reported the first component of CL, CTENO64, and found that it was convergently acquired in ctenophores and was essential for determination of ciliary orientation.3 However, CTENO64 is localized only in the proximal region of the CL; therefore, the molecular architecture of CL over the entire length has not been elucidated. Here, we identified a second CL component, CTENO189. This ctenophore-specific protein was present in the distal region of the CL, with a localization clearly segregated from CTENO64. Knockdown of the CTENO189 gene with morpholino antisense oligonucleotides resulted in complete loss of CLs in the distal region, but did not affect either the formation of CP or the orientation of each cilium. However, the hexagonal distribution of cilia was disarranged, and the metachronal coordination of CP along a comb row was lost in the CTENO189 morphants. The morphant CP showed asymmetric ciliary-type movement in normal seawater, and in a high-viscosity solution, it could not maintain the normal waveforms, becoming a symmetric flagellar-type. Our findings demonstrate a "two-story building" of CP, comprising the proximal CL, as the building foundation that rigidly fixes the ciliary orientation. The distal CL would reinforce the elastic connection among cilia to overcome the hydrodynamic drag of giant multiciliary plates.

cell biology↗

bioRxiv2020

Calaxin is essential for the transmission of Ca2+-dependent asymmetric waves in sperm flagella

Regulation of waveform asymmetry in sperm flagella is critical for changes in sperm swimming trajectory as seen during sperm chemotaxis towards eggs. Ca2+ is known as an important regulator of asymmetry in flagellar waveforms. A calcium sensor protein, calaxin, which is associated with the outer arm dynein, plays a key role in the sperm waveform regulation in a Ca2+-dependent manner. However, the molecular mechanism underlying the regulation of asymmetric waves by Ca2+ and calaxin remains unclear. We performed experiments using caged ATP to elucidate the formation and propagation of asymmetric flagellar waves in the sperm of the ascidian Ciona intestinalis. Demembranated sperm cells were suspended in a solution containing caged ATP and reactivated using UV flash photolysis. Initial bends were formed at the base and propagated towards the tip of flagella; however, the bend direction was different between asymmetric and symmetric waves. A calaxin inhibitor, repaglinide, had no effect on initial bend formation, but significantly inhibited the generation of the second flagellar bend in the reverse direction, resulting in the failure of asymmetric wave formation and propagation. These results suggest that calaxin plays a critical role in Ca2+-dependent transmission of flagellar asymmetric waveforms.

cell biology↗

bioRxiv2018

Microtubule dynamics is required for rapid coiling of haptonemata in haptophyte algae

A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is \"coiling\", which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. The persistence lengths calculated from the curvature of the haptonematal microtubules indicated their unusual flexibility. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca2+, suggesting changes in the microtubule surface lattice. Addition of Ca2+ caused bend propagation toward the proximal region. These results indicate that switching microtubule conformation with the aid of Ca2+-binding microtubule-associated proteins is responsible for rapid haptonematal coiling.\n\nSummary StatementMicroscopy observations and pharmacological experiments revealed that the rapid coiling of a non-motor microtubule-based motile organelle, the haptonema, is explained by conformational changes of microtubules, including twisting and writhing.

cell biology↗