Biology subjects

Yoshida, S.

Publications and source records attributed to Yoshida, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Transient interdomain interactions shape the conformational ensemble governing RNA recognition by the tandem RRMs of Sex-lethal

RNA recognition motif (RRM) proteins frequently contain multiple RNA-binding domains connected by flexible linkers, yet the contribution of transient interdomain interactions to RNA recognition remains incompletely understood. Here, we investigated the structural organization of the tandem RRMs of the Drosophila melanogaster splicing regulator Sex-lethal (Sxl) using solution NMR spectroscopy in combination with rational protein engineering, restrained docking and RNA-binding studies. Progressive extension of the native interdomain linker resulted in a gradual decrease in rotational coupling between the two RRMs and continuous chemical shift changes, demonstrating that the RNA-free protein samples a dynamic conformational ensemble rather than behaving as two independently tumbling domains. NMR-guided docking identified a compact arrangement compatible with the experimental data and suggested a transient interface partially overlapping the RNA-binding surfaces. Surprisingly, a mutant designed to weaken this interface produced the opposite effect: instead of increasing interdomain mobility, it exhibited enhanced rotational coupling while remaining natively folded, indicating a redistribution of the conformational ensemble rather than disruption of the domain architecture. Both linker extension and the mutant reduced RNA-binding affinity, and the mutant additionally diminished sequence discrimination, demonstrating that perturbations shifting the conformational equilibrium in either direction compromise RNA recognition. Together, our results demonstrate that RNA recognition by Sxl is governed not by a single apo structure but by a finely balanced conformational ensemble, and that perturbing this equilibrium in either direction compromises high-affinity and sequence-selective RNA binding.

biophysics↗

bioRxiv2026

Three-dimensional vascular microenvironments uncover endothelial plasticity during TGF-β2-driven vascular remodeling

The tumor microenvironment plays a pivotal role in tumor development, harboring elements such as endothelial cells, immune cells, fibroblasts, and soluble factors such as transforming growth factor-{beta} (TGF-{beta}) family. TGF-{beta} family regulates cell development and promotes tumor invasion, metastasis, angiogenesis, and endothelial-to-mesenchymal transition (EndoMT). Here, we investigate the effects of TGF-{beta} signaling on vascular remodeling using a three-dimensional (3D) vascular network in a microfluidic device. Using both a co-culture (3D-Co) and simplified endothelial monoculture (3D-CM), we demonstrate that TGF-{beta} signaling reduces the quality and functionality of the vasculature by regressing them. In addition, we observed the upregulation of EndoMT-related markers in mRNA and protein expressions, suggesting the induction of EndoMT in 3D vascular networks. The increased vascular permeability stimulated by TGF-{beta}2 also supports the loss of endothelial identity in the 3D-Co. Transcriptomic analysis revealed the coordinated activation of pathways associated with cell migration and EndoMT, along with the suppression of cell cycle progression. A comparative analysis of two-dimensional (2D) and 3D cultures revealed a fundamentally distinct endothelial response to TGF-{beta}2 in the 3D context, including metabolic reprogramming. These findings demonstrate that the 3D microenvironment critically modulates endothelial responses to TGF-{beta} and enables the emergence of vascular phenotypes not captured in 2D systems. This study provides a more physiologically relevant platform to investigate endothelial dysfunction and vascular remodeling.

bioengineering↗

bioRxiv2025

Bats create a silent frequency band to detect prey through Doppler shift compensation

Acquiring information efficiently through sensory inputs is essential for animal survival. Animals with active sensory systems that emit their own signals often optimize the design and use of these signals according to context and purpose1-6. In this study, we reveal a previously unrecognized function of Doppler shift compensation (DSC) in bats. During flight, bats actively lower the frequency of their echolocation calls so that echoes remain stable at a reference frequency (fref) despite Doppler shifts caused by movement7-12. We demonstrate that DSC does not simply serve, as previously thought, to align echoes with the acoustic fovea--a narrow band of maximal auditory sensitivity13,14--but in addition, suppresses background noise for detecting prey-derived signals. Using phantom echo playbacks and on-board microphone recordings, we show that bats selectively compensate for the highest-frequency echoes rather than the most intense ones. This process shifts all clutter echoes below fref, leaving the spectral band above fref free of stationary-object echoes and secures a "quiet frequency band". Recordings during prey capture and noise playback experiments revealed that spectral glints from fluttering moth wings appear in this "quiet frequency band" and are exploited for prey detection. This mechanism enhances the high-fidelity detection of prey echoes even in cluttered environments. Such findings reveal a sensory strategy in which animals actively create silence in a critical frequency range. It represents a conceptual advance in active sensing and auditory scene analysis, highlighting how evolution shapes sensory systems to extract reliable information under noisy natural conditions.

animal behavior and cognition↗

bioRxiv2025

Clonal Dynamics in the Male Germline from Development to Next Generation

Germ cells secure the continuity and evolutionary potential of species. Yet how reproductive success is allocated among the founder cells of the mammalian male germline, the primordial germ cells (PGCs), remains unknown. Here, we quantitatively traced individual PGCs, non-invasively barcoded in mouse embryos, across their establishment, lifelong maintenance within the testis, spermatogenesis, and transmission to the next generation. We found that highly skewed clonal contributions arise from a very early stochastic bottleneck during migration to the developing testes and remain stable throughout adult reproductive life. Reproductive success during adulthood was proportional to the embryonically established PGC clone size. Mathematical modeling shows that the patchy compartmentalization of PGC clones within the extended thin seminiferous tubules maintain clonal diversity and safeguard against the expansion of clones that gain a selective advantage. These findings uncover fundamental principles governing the development, stability, and evolutionary transmission of the mammalian germline.

developmental biology↗

bioRxiv2025

Maternal care during early development is necessary for the acquisition of a calming response to back stroking

In many mammals, early postnatal interactions between caregivers and offspring involve rich physical contact, including stroking, holding, and grooming. Offspring typically remain calm and close to the caregiver during such stimulation. Although these behaviors are thought to support emotional regulation and bonding during infancy, the underlying mechanisms and the role of prior experience remain unclear. Here, we demonstrate that back stroking induces a conserved calming response in both human infants and mouse pups, characterized by reduced movement and heart rate. In mouse pups, stroking further facilitated sleep onset, increased EEG delta power, and attenuated stress-induced corticosterone elevations. These sleep-promoting and stress-buffering effects were absent in artificially reared pups deprived of postnatal maternal contact, underscoring the importance of early tactile experience. Transcriptomic analysis revealed downregulation of the voltage-gated calcium channel subunit gene Cacna1b in the hypothalamus of artificially reared pups. Moreover, knockdown of hypothalamic Cacna1b in maternally reared pups abolished stroking-induced calming and sleep-promoting effects. This study identifies a conserved, experience-dependent calming response to affiliative tactile input, biologically embedded through plasticity, that supports physiological regulation and stress resilience during early development. Our results highlight that affiliative tactile sensation, like discriminative tactile sensation, depends on early experience to organize neural mechanisms regulating internal states. SignificanceEarly-life physical contact with caregivers is essential for healthy emotional and physiological development in mammals, but its underlying mechanisms remain unclear. We show that back stroking induces a conserved calming response in both human infants and mouse pups, reducing movement, heart rate, and promoting sleep. These effects depend on early tactile experience and are mediated by Cacna1b expression in the hypothalamus. Our findings identify a plastic, experience-dependent pathway through which affiliative tactile input modulates internal states, revealing how parental care supports stress resilience and sleep physiology during early development.

neuroscience↗

bioRxiv2025

CAKUT variants in PRPF8, DYRK2, and CEP78: implications for splicing and ciliogenesis

IntroductionCongenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of chronic kidney disease in children and young adults. Although over 50 monogenic causes have been identified, many remain unresolved. PRPF8 is a core spliceosome component, essential for pre-mRNA splicing, and further localizes to the distal mother centriole to promote ciliogenesis. MethodsWe performed trio exome sequencing in 208 CAKUT families and identified strong variants in PRPF8 and the EDD-DYRK2-DDB1VprBP complex. Functional validation included splicing assays in yeast (Saccharomyces cerevisiae), Sonic hedgehog (Shh) signaling in RPE-1 cells, co-immunoprecipitation for protein complex assembly, and in situ hybridization in mouse embryos. Protein interactions were modeled using AlphaFold. ResultsWe identified heterozygous de novo or inherited variants in PRPF8, DYRK2, DDB1, EDD and CEP78. Yeast assays revealed that while most PRPF8 variants preserved growth and splicing at consensus splice sites, the de novo PRPF8R1681W variant impaired splicing of non-consensus splice sites and was inviable at elevated temperature. CAKUT variants failed to rescue prp28-1 and U4-cs1 alleles but showed variant-specific synthetic interactions with brr2-1, including weak suppression or synthetic sickness at elevated temperatures. Shh signaling was reduced in [~]50% of PRPF8 variants expressed in RPE-1 cells. CEP78 truncating variants abrogated binding to CEP350 and VPRBP. Two DYRK2 variants disrupted EDD-DYRK2-DDB1VprBP complex formation without affecting kinase activity. In situ hybridization revealed strong Prpf8 expression in the developing collecting duct and urothelium. ConclusionVariants in PRPF8 and components of the EDD-DYRK2-DDB1VprBP complex may contribute to CAKUT through impaired pre-mRNA splicing and defective ciliogenesis. These findings uncover an entirely new functional network of candidate genes for CAKUT and ciliopathies, significantly broadening our understanding of disease mechanisms and offering novel entry points for mechanistic studies. Translational StatementOur study identifies a previously unrecognized molecular network involving PRPF8 and the EDD-DYRK2-DDB1VprBP complex, revealing a novel pathogenic mechanism in CAKUT. These results introduce a new class of candidate genes and pathways essential for kidney development. As the genetic etiology of CAKUT remains unknown in most patients, our findings underscore the need for targeted genetic testing and functional studies to enhance diagnosis, advance mechanistic insight, and enable more personalized clinical management.

genetics↗

bioRxiv2025

Boundary-guided cell alignment drives mouse epiblast maturation

Symmetry breaking and pattern formation are critical events that occur throughout embryonic development. In early mouse development, a mass of non-polarized epiblast (EPI) cells in the blastocyst forms the egg-cylinder, while cells become apico-basally polarized and build a radial configuration. Yet, what drives the formation of this tissue architecture remains unclear. Here, we demonstrate that orientational patterning of EPI cells is dictated by heterogeneous tissue boundaries, which then defines central lumen positioning. We show that EPI cells progressively orient perpendicular to the visceral endoderm (VE) boundary enriched with laminin and active integrin {beta}1, but parallel to the extraembryonic ectoderm interface. These orientation dynamics are consistent with general boundary-induced alignment effects in polar materials, with a topological defect predicting the position where the pro-amniotic cavity nucleates. Knockout of laminin {gamma}1 and integrin {beta}1 confirms the essential role of adhesion at the EPI-VE-boundary. The established EPI pattern, in turn, facilitates ERK activation to ensure proper EPI maturation. Together, these findings present the mechanistic basis and functional significance of EPI tissue patterning.

developmental biology↗

bioRxiv2025

HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis

mTORC1 is a central regulator of cell growth that is directly activated by the small GTPase Rheb on the lysosomal membrane in response to growth factors. We recently reported that polyubiquitinated Rheb facilitates its interaction with mTORC1, leading to mTORC1 activation. However, the molecular mechanisms underlying the ubiquitination of Rheb and its interaction with mTORC1 for activation are not fully understood. In this study, we demonstrate that HUWE1, an E3 ubiquitin ligase, preferentially interacts with ubiquitinated Rheb and is essential for the interaction between Rheb and mTOR, as well as for mTORC1 activation. Additionally, HUWE1 is necessary for Rheb to interact with CAD, a crucial enzyme involved in de novo pyrimidine biosynthesis, and for its activation through the mTORC1-S6K1 pathway. Knocking down HUWE1 in cultured cells or in the liver tissues of mice inhibits mTORC1 activity without affecting the phosphorylation of Akt or TSC2, nor does it affect the lysosomal localization of TSC2 or mTORC1. Furthermore, HUWE1 specifically enhances the expression of CAD without influencing other enzymes involved in de novo pyrimidine synthesis and maintains UMP levels in the hepatocytes of liver tissues. These findings indicate that HUWE1 serves as a key organizer of the Rheb ubiquitin complex, amplifying mTORC1 activity and playing a vital role in stimulating de novo pyrimidine synthesis by enhancing CAD expression and activity. HighlightsO_LIHUWE1 and CAD preferentially interact with ubiquitinated Rheb. C_LIO_LIHUWE1 further ubiquitinates Rheb and supports Rheb interaction with mTORC1 and CAD. C_LIO_LIHUWE1 stimulates mTORC1 activity through its E3 ligase activity. C_LIO_LIHUWE1 also supports pyrimidine synthesis by enhancing CAD expression. C_LI

biochemistry↗

bioRxiv2025

Glucosylation of Endogenous Haustorium-inducing factors Underpins Kin Avoidance in Parasitic Plants

Parasitic plants rarely attack themselves, suggesting the activity of a kin-avoidance mechanism. In the root parasitic plant Phtheirospermum japonicum, prehaustorium formation is triggered by host-secreted haustorium-inducing factors (HIFs), but it is unresponsive to its own root exudates. Here we report the identification of the spontaneous prehaustorium 1 (spoh1) mutant, which forms prehaustoria without external HIFs. spoh1 harbors a point mutation in the gene encoding UDP-glycosyltransferase GT72B1, an enzyme that glucosylates and thereby inactivates phenolic HIFs. Notably, PjGT72B1 possesses a different substrate specificity from its ortholog of the host Arabidopsis. Introduction of PjGT72B1 in Arabidopsis reduced HIF activity, indicating that HIF glucosylation regulates haustorium induction by hosts. Our findings suggest that parasitic plants have evolved kin-avoidance mechanisms through the glucosylation of endogenous HIFs.

plant biology↗

bioRxiv2025

AMPK is dispensable for physiological podocyte and glomerular functions but prevents glomerular fibrosis in experimental diabetes

AMP-activated protein kinase (AMPK) has been postulated to be crucial in regulating various renal physiology and pathophysiology processes, including energy metabolism, ion and water transport, inflammation, and hypertrophy. However, the specific roles of AMPK in the podocyte, a cell critical for maintaining glomerular filtration, have not been fully explored using genetic model animals. In this study, we generated mice lacking both AMPK 1 and 2 catalytic subunits in glomerular podocytes (pmut). Our findings revealed that, surprisingly, AMPK is dispensable for normal podocyte function. These knockout mice could live as long as their wild-type littermates without showing any pathological alterations in their glomeruli or glomerular function at two years of age. However, under type 1 diabetic conditions, the diabetic pmut mice exhibited increased lipid and collagen accumulation and an elevated expression of mesenchymal proteins in their glomeruli. They also showed more significant albuminuria compared to control diabetic mice. Under high glucose culture conditions, glomeruli isolated from pmut mice demonstrated a reduced expression of mitochondrial genes (e.g., Ndufv2) and increased leakage of mitochondrial components. Additionally, there was heightened expression of genes associated with nucleotide sensing and pro-inflammatory pathways (including mb21d2, IL-1 beta, and NF-kB). These observations suggest that while AMPK is not necessary for podocyte function in healthy kidneys, it is crucial for preventing glomerular fibrosis resulting from lipotoxicity and inflammation under diabetic conditions.

cell biology↗

bioRxiv2025

Neofunctionalized RGF pathways drive haustorial organogenesis in parasitic plants

Parasitic plants initiate rapid de novo organogenesis of a specialized feeding structure called a haustorium upon contact with their hosts. Currently, little is known about the internal signals regulating haustorium development. Here, we identify root meristem growth factor (RGF) peptides in Phtheirospermum japonicum as endogenous inducers of prehaustorium formation. Treatment with specific RGF peptides in the absence of hosts triggered prehaustoria and induced expression of PjYUC3, a gene required for auxin biosynthesis and prehaustorium formation. CRISPR-mediated knockouts showed that PjRGFR1 and PjRGFR3, receptors activated by the haustorium specific RGF peptides PjRGF2 and PjRGF5, are essential for prehaustorium formation, revealing functional redundancy. Phylogenic analyses indicate that PjRGF2 is broadly conserved among Orobanchaceae, whereas PjRGF5 appears to have recently evolved through tandem multiplication and neofunctionalization. Our findings establish RGF peptides and their corresponding receptors as critical components of haustorium developmental signaling and provide insights into the evolutionary trajectories that shape plant parasitism. TeaserPlant peptide hormones regulate and induce the parasitic plant specialized organ for connecting to and feeding from the host.

plant biology↗

bioRxiv2024

Self-organization of spermatogenic wave coordinates sustained sperm production in the mouse testis

Spermatogenesis takes place in the testis, relying on the ordered turnover of differentiating cells supplied from stem cells. Classic histological analyses have revealed that this process shows hierarchical spatiotemporal patterning known as the spermatogenic cycle, wave, and descent of segmental order, indicative of currently underexplored mechanisms of tissue- and organ-scale homeostasis. Here, using mice, we conducted high-resolution, wide-field, and ultra long-term live imaging studies in vivo and ex vivo, combined with whole-organ mapping of differentiation stages. Such trans-scale measures demonstrate how stereotypic local cell turnover is coordinated into characteristic phase waves propagating along the seminiferous tubules, further organized into organ-scale patterning over the tubule loops. Minimal mathematical modeling shows that such higher-order dynamics can emerge from the local coupling of autonomous oscillators, which are rooted in delayed feedback interplay between stem and differentiating cells via retinoic acid signaling. These findings highlight a self-organization mechanism underpinning organ-scale homeostasis and constant sperm production.

developmental biology↗

bioRxiv2024

Spatial Transcriptomics Reveals the Temporal Architecture of the Seminiferous Epithelial Cycle and Precise Sertoli-Germ Synchronization

Spermatogenesis is characterized by the seminiferous epithelial cycle, a periodic pattern of germ cell differentiation with a wave-like progression along the length of seminiferous tubules. While key signaling and metabolic components of the cycle are known, the transcriptional changes across the cycle and the correlations between germ cell and somatic lineages remain undefined. Here, we use spatial transcriptomics via RNA SeqFISH+ to profile 2,638 genes in 216,090 cells in mouse testis and identify a periodic transcriptional pattern across tubules that precisely recapitulates the seminiferous epithelial cycle, enabling us to map cells to specific timepoints along the developmental cycle. Analyzing gene expression in somatic cells reveals that Sertoli cells exhibit a cyclic transcriptional profile closely synchronized with germ cell development while other somatic cells do not demonstrate such synchronization. Remarkably, in mouse testis with drug-induced ablation of germ cells, Sertoli cells independently maintain their cyclic transcriptional dynamics. By analyzing expression data, we identify an innate retinoic acid cycle, a network of transcription factors with cyclic activation, and signaling from germ cells that could interact with this network. Together, this work leverages spatial geometries for mapping the temporal dynamics and reveals a regulatory mechanism in spermatogenesis where Sertoli cells oscillate and coordinate with the cyclical progression of germ cell development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/620681v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@10a78c7org.highwire.dtl.DTLVardef@7f5a95org.highwire.dtl.DTLVardef@113f6b6org.highwire.dtl.DTLVardef@1aa41f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

Kinetochores promote timely spindle bipolarization to prevent microtubule attachment errors in oocytes

Incorrect kinetochore-microtubule attachment leads to chromosome segregation errors. The risk of incorrect attachments is particularly high in acentrosomal oocytes, where kinetochores are surrounded by randomly oriented microtubules until spindle bipolarization. How the timing of acentrosomal spindle bipolarization is coordinated with kinetochore-microtubule attachment is unknown. Here, we show that in mouse oocytes, kinetochores promote spindle bipolarization when microtubule attachment is unstable in early prometaphase. The kinetochore kinase MPS1 allows this function by regulating multiple pathways, including those mediated by the kinetochore proteins NDC80-NUF2 at their C-terminal domains and the antiparallel microtubule crosslinker PRC1. Inhibition of MPS1 delays spindle bipolarization to late prometaphase, thereby increasing incorrect kinetochore-microtubule attachments. Artificially accelerating spindle bipolarization by overexpressing KIFC1 prevents incorrect kinetochore-microtubule attachment in MPS1-inhibited oocytes. Thus, kinetochores autonomously promote timely acentrosomal spindle bipolarization prior to stabilizing microtubule attachment, thereby reducing the risk of egg aneuploidy.

cell biology↗

bioRxiv2024

Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction

Chronic kidney disease (CKD) and atherosclerotic heart disease, frequently associated with dyslipidemia and hypertension, represent significant health concerns. We investigated the interplay among these conditions, focusing on the role of oxidized low-density lipoprotein (oxLDL) and angiotensin II (Ang II) in renal injury via G protein q subunit (Gq) signaling. We hypothesized that oxLDL enhances Ang II-induced Gq signaling via the AT1 (Ang II type 1 receptor)-LOX1 (lectin-like oxLDL receptor) complex. Based on CHO and renal cell model experiments, oxLDL alone did not activate Gq signaling. However, when combined with Ang II, it significantly potentiated Gq-mediated inositol phosphate 1 production and calcium influx in cells expressing both LOX-1 and AT1 but not in AT1-expressing cells. This suggests a critical synergistic interaction between oxLDL and Ang II in the AT1-LOX1 complex. Conformational studies using AT1 biosensors have indicated a unique receptor conformational change due to the oxLDL-Ang II combination. In vivo, wild-type mice fed a high-fat diet with Ang II infusion presented exacerbated renal dysfunction, whereas LOX-1 knockout mice did not, underscoring the pathophysiological relevance of the AT1-LOX1 interaction in renal damage. These findings highlight a novel mechanism of renal dysfunction in CKD driven by dyslipidemia and hypertension and suggest the therapeutic potential of AT1-LOX1 receptor complex in patients with these comorbidities.

molecular biology↗

bioRxiv2024

Neural signatures of human psychological resilience driven by acute stress

Neurophysiological mechanisms underlying psychological resilience, the ability to overcome adversity1,2, have been extensively studied in animals. However, in comparison with animals, human resilience is unique in that it is underpinned by higher cognitive functions, such as self-confidence and a positive attitude to challenges3,4. Given these discrepancies, the neurophysiological mechanisms underlying human resilience remain unclear. To address this issue, we recorded multimodal responses after acute stress exposure over 1.5 hours using functional brain imaging and peripheral physiological measurements. Here, we showed that the degree of individual resilience is indexed by multiple changes in neural dynamics 1 hour after acute stress. Both functional magnetic resonance imaging and electroencephalography show that activity in the cortical salience network and power in high-beta and gamma oscillations increase in less resilient individuals. Contrastingly, activity in the cortical default mode network and spontaneous activity in the posterior hippocampus increase in more resilient individuals. Machine learning analysis confirmed that, 1 hour after stress exposure, the functional connectivity in the salience network was the most influential, followed by that in the default mode network, gamma power, high-beta power, and hippocampal activity. The neurophysiological dynamics for resilience do not occur as previously thought, but rather in a time-lagged manner against stress exposure. Our findings Shed light on a new approach to recovery from stress-induced deficits such as delayed neuromodulation after a stressful event.

neuroscience↗

bioRxiv2024

Human spermatogonial stem cells retain states with a foetal-like signature

Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling it has been proposed that undifferentiated human spermatogonia can be sub-classified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc)RNA-seq libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate state 0B with a suppressive transcriptomic program that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells - which are mitotically arrested - can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mouse (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive life while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes.

cell biology↗

bioRxiv2024

A cellular assay system revealed the deamidase-mediated regulation of the Arg-mediated N-end rule protein degradation pathway

Regulated protein production and degradation are essential for maintaining proteostasis in eukaryotic cells. The N-end rule, or N-degron pathway, is a protein degradation machinery in which the N-terminal amino acid is the mark of the protein degradation via the proteasome. The N-end rule is a conserved protein disposal machinery in eukaryotic cells. However, the precise cellular mechanisms and their physiological roles are not fully understood. Herein, we report the development of an Arg-mediated N-end rule assay system using artificial substrates expressed in cultured cell lines. We demonstrated that the N-end rule degradation is significantly influenced by the expression levels of N-terminal amino acid-modifying enzymes, including NTAN1, NTAQ1, and ATE1. In the N-terminal Asn protein pathway, an increase in NTAN1 or ATE1 expression promotes its disposal via the N-end rule degradation pathway. Interestingly, overexpression of NTAQ1 decreased the degradation of the protein bearing Gln at the N-terminus. Computational prediction of NTAQ1 and ATE1 complex formation suggests that the outer loop region of NTAQ1 is involved in its interaction with ATE1 and that the NTAQ1 overexpression may negatively affect this interaction. Our findings suggest that the degradation activity of the Arg/N-end rule pathway is positively or negatively regulated by deamidase expression levels and propose a higher degree of control of protein degradation by the Arg/N-end rule within cells.

cell biology↗