Biology subjects

Mizuno, K.

Publications and source records attributed to Mizuno, K..

17 recordsLinked to original sources

bioRxiv2026

RhoGEF Solo regulates cell density-dependent keratin remodeling and desmosome maturation

Desmosomes (DSMs) are specialized cell-cell adhesion structures that anchor keratin intermediate filaments and play a crucial role in maintaining the mechanical integrity of epithelial tissues. Keratin filaments associate with DSMs in two characteristic patterns: radial spokes extending from DSMs into the cytoplasmic keratin network and circumferential rims connecting neighboring DSMs. Solo is a Rho-guanine nucleotide exchange factor (RhoGEF) involved in cellular responses to mechanical stress and keratin network reorganization. Here, we investigated the role of Solo in regulating keratin-DSM interactions in response to cell density. The number of radial keratin spokes at cell-cell adhesion sites was markedly reduced at high cell density, concomitant with increased Solo localization at these sites. Conversely, Solo knockout (KO) increased the number of radial keratin spokes. Expression of wild-type Solo, but not a GEF-inactive Solo mutant, rescued the increase in radial keratin spokes in confluent Solo-KO cells, indicating that Solo RhoGEF activity is required for this regulation. Inhibition of actin polymerization or ROCK activity also suppressed the reduction in radial spoke density. Furthermore, expansion microscopy and electron microscopy revealed that DSMs in Solo-KO cells remained small and immature even under confluent conditions. Solo was also partially required for the localization of plakoglobin (PG), a core DSM protein, whereas PG-KO markedly increased the number of radial keratin spokes. These findings suggest that increased cell density promotes DSM maturation accompanied by a reduction in radial keratin spokes, and Solo contributes to both processes, potentially through facilitating PG localization at DSMs and regulating Rho-ROCK-dependent actin cytoskeletal remodeling.

cell biology↗

bioRxiv2026

A synNotch-based morphogen detection system reveals sFRP2 enhances Wnt3a signaling

Morphogen gradients provide essential positional information during tissue development, yet the extracellular mechanisms that regulate morphogen transport and presentation remain poorly understood. Here, we introduce a mechanosensitive detection system based on synthetic Notch (synNotch) receptors that selectively detects surface-bound, but not soluble, morphogen complexes. Applying this platform to Wnt signaling, we demonstrate that secreted Frizzled-related protein 2 (sFRP2) promotes the recruitment of Wnt3a to the cell surface via heparan sulfate proteoglycans, enabling coordinated endocytosis and robust activation of canonical Wnt/{beta}-catenin signaling. Notably, sFRP2 extends the effective signaling range of Wnt3a and amplifies Wnt responses under ligand-limiting conditions. In intestinal organoid cultures, sFRP2 enhances Wnt3a-driven growth and induces high-Wnt morphological states with low-level Wnt concentrations. These findings identify sFRP2 as an extracellular carrier that stabilizes surface-bound Wnt3a and regulates both the strength and spatial range of Wnt signaling. More broadly, this work demonstrates the utility of synNotch mechanosensing for dissecting extracellular morphogen dynamics and highlights morphogen carrier proteins as a platform for optimizing organoid culture.

cell biology↗

bioRxiv2025

Actin cytoskeletal remodeling requires the interaction between Solo and LARG in response to substrate stiffness

In response to external mechanical stimuli, cells remodel their actin cytoskeleton. Solo, a Rho guanine nucleotide exchange factor (RhoGEF), is involved in mechanical stress responses. Using BioID, we identified PDZ-RhoGEF (PRG), a member of the RGS-RhoGEF family (regulator of G protein signaling domain-containing RhoGEFs, as a Solo-interacting protein. Moreover, we found that Solo regulates PRG during the mechanical stress response. Furthermore, we identified leukemia-associated RhoGEF (LARG), another RGS-RhoGEF member, as a Solo-interacting protein; however, the functional role of this interaction remains unknown. Therefore, in this study, we investigated the interaction between Solo and LARG and found that LARG localizes to Solo accumulation sites at the basal plane and that LARG is required for Solo-induced actin polymerization. Additionally, Solo is required to maintain LARG activity in cells, and this interaction is related to actin regulation in response to substrate stiffness. We further investigated the relationship between LARG and PRG as a function of Solo. We noted that although they did not competitively localize at Solo accumulation sites, knockdown of either PRG or LARG suppressed Solo-induced actin polymerization to the same extent as double knockdown, indicating that these signaling pathways cooperatively regulate Solo-induced actin polymerization.

cell biology↗

bioRxiv2023

Interaction between Solo and PDZ-RhoGEF is involved in actin cytoskeletal remodeling and response to substrate stiffness

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study was aimed at identifying Solo-interacting proteins using the BioID, a proximal-dependent labeling method and elucidating the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG co-localized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling and RhoA activation. Moreover, overexpression of the binding domains of Solo and PRG had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

cell biology↗

bioRxiv2023

MARK3-mediated Slingshot-1 phosphorylation is essential for polarized lamellipodium formation

Cofilin acts as a key regulator of actin cytoskeletal remodeling via stimulating actin filament disassembly. Cofilin is inactivated by Ser-3 phosphorylation and reactivated by cofilin-phosphatase Slingshot-1 (SSH1). SSH1 is activated upon binding to F-actin, and this activation is inhibited by its phosphorylation at Ser-937 and Ser-978 and the subsequent binding of 14-3-3 proteins. In this study, we identified MARK3 (also named Par-1a and C-TAK1) as a kinase responsible for Ser-937/Ser-978 phosphorylation of SSH1. MARK3-mediated phosphorylation promoted SSH1 binding to 14-3-3 proteins and suppressed its F-actin-assisted cofilin-phosphatase activity. When Jurkat cells were stimulated with SDF-1, actin filaments formed multidirectional F-actin-rich lamellipodia around the cells in the initial stage, and thereafter, they were rearranged as a single polarized lamellipodium to the direction of cell migration. Upon SDF-1 stimulation, SSH1 was translocated into F-actin-rich lamellipodia, but its Ser-937/Ser-978 non-phosphorylatable mutant SSH1(2SA) was retained at the location of the original cortical F-actin. Knockdown of MARK3 or overexpression of SSH1(2SA), similar to SSH1 knockdown, impaired the conversion of multiple lamellipodia to a single polarized lamellipodium. These results indicate that MARK3-mediated Ser-937/Ser-978 phosphorylation is required for SSH1 liberation from F-actin and translocation to lamellipodia, and hence, facilitates the formation of a single polarized lamellipodium for directional cell migration. Our results suggest that the phosphorylation-dephosphorylation cycle of SSH1 is crucial for its localization to lamellipodia via promoting the dissociation-reassociation cycle of SSH1 to F-actin, and thereby the stimulus-induced lamellipodium formation to the direction of cell movement.

cell biology↗

bioRxiv2023

Copy number of the eukaryotic rRNA gene can be counted comprehensively

Gene sequence has been widely used in molecular ecology. For instance, the ribosomal RNA (rRNA) gene has been widely used as a biological marker to understand microbial communities. The variety of the detected rRNA gene sequences reflects the diversity of the microorganisms existing in the analyzed sample. Their biomass also can be estimated by applying quantitative sequencing with the information of rRNA gene copy numbers in genomes, however, the information on rRNA gene copy number is still limited. Especially, the copy number in microbial eukaryotes is much less understood than that of prokaryotes, possibly because of the large and complex structure of eukaryotic genomes. In this communication, we report an alternative approach that is more appropriate from the existing method of quantitative sequencing and demonstrate that the copy number of eukaryotic rRNA can be measured efficiently and comprehensively. By applying this approach widely, information on the eukaryotic rRNA copy number can be determined, and their community structures can be depicted and compared more efficiently.

microbiology↗

bioRxiv2023

Massive RNA editing in ascetosporean mitochondria

Ascetosporeans are parasitic protists of invertebrates. As only two species of Mikrocytida, an ascetosporean subgroup, have ever been sequenced deeply and analyzed using cells isolated from infected organisms, it was shown that their mitochondria are functionally reduced and the organellar genome is lacking. However, molecular studies on other ascetosporeans have not been conducted, and whether reduced mitochondria is common in ascetosporeans remains unclear. In the present study, we established two cultures of Paradinida, another ascetosporean subgroup, and reconstructed their mitochondrial genomes. As they were compared with their RNA-seq data, massive A-to-I and C-to-U types of RNA editing were detected. Many editing sites are shared between two paradinids, but strain-unique sites also exist. As the mitochondrial genes are involved in the electron transfer system, their mitochondria are not functionally reduced, unlike that in Mikrocytida. Furthermore, we detected adenosine deaminase acting on RNA (ADAR), which is a key enzyme of A-to-I substitution, in paradinids as well as several other protists. Immunostaining showed that this ADAR is specifically localized in the mitochondria of paradinids, suggesting that A-to-I substitution in paradinid mitochondria is mediated by ADAR. These findings elucidated the functional diversity and evolutionary process of ascetosporean mitochondria as well as ADAR.

evolutionary biology↗

bioRxiv2022

Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis

The Rab27 effectors play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationship by comparing the exocytic phenotypes in beta cells simultaneously lacking two effectors with those lacking one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy indicate that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. Exophilin-8 also promotes fusion of granules stably docked to the plasma membrane mediated by granuphilin, although it is dispensable for granule residence beneath the plasma membrane. The current study presents the first diagram for multiple intracellular paths of granule exocytosis and for functional hierarchy among different Rab27 effectors in the same cell.

cell biology↗

bioRxiv2022

Immotile cilia of the mouse node sense a fluid flow-induced mechanical force for left-right symmetry breaking

Immotile cilia of crown cells at the node of mouse embryos are required for sensing of a leftward fluid flow1 that gives rise to the breaking of left-right (L-R) symmetry2. The flow-sensing mechanism has long remained elusive, however, with both mechanosensing and chemosensing models having been proposed1, 3-5. Here we show that immotile cilia at the mouse node respond to mechanical force. In the presence of a leftward flow, immotile cilia on the left side of the node bend toward the ventral side whereas those on the right side bend toward the dorsal side. Application of mechanical stimuli to immotile cilia along the dorsoventral axis by optical tweezers induced Ca2+ transients and degradation of Dand5 mRNA--the first known L-R asymmetric molecular events--in the targeted cells. The Pkd2 channel protein was found to be preferentially localized to the dorsal side of immotile cilia on both left and right sides of the node, and the observed induction of Ca2+ transients preferentially by mechanical stimuli directed toward the ventral side could explain the differential response of immotile cilia to the directional flow. Our results thus suggest that immotile cilia at the node sense the direction of fluid flow in a manner dependent on a flow-generated mechanical force.

developmental biology↗

bioRxiv2021

Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration

Skeletal muscle regeneration requires extracellular matrix (ECM) remodeling including an acute and transient breakdown of collagen that produces gelatin. However, the physiological function of such a remodeling process on muscle tissue repair is unclear. Here we elaborate on a bi-phasic effect of gelatin in skeletal muscle regeneration, mediated by hormetic effects of reactive oxygen species (ROS). Low-dose gelatin stimulates ROS production from NADPH oxidase 2 (NOX2) and simultaneously upregulates antioxidant system for cellular defense, reminiscent of the adaptive compensatory process during mild stress. This response triggers the release of myokine IL-6 which stimulates myogenesis and facilitates muscle regeneration. By contrast, high-dose gelatin stimulates ROS overproduction from NOX2 and mitochondrial chain complex, and ROS accumulation by suppressing antioxidant system, triggering release of TNF, which inhibits myogenesis and regeneration. Our findings reveal gelatin-ROS-IL-6/TNF signaling cascades underlying a hormetic response of myogenic cells to gelatin.

physiology↗

bioRxiv2021

Mice lacking the cAMP effector protein POPDC1 show enhanced hippocampal synaptic plasticity

Extensive research has uncovered diverse forms of synaptic plasticity and a wide array of molecular signaling mechanisms that act as positive or negative regulators. Specifically, cAMP-dependent signaling pathways have been crucially implicated in long-lasting synaptic plasticity. In this study, we examine the role of POPDC1 (or BVES), a cAMP effector protein expressed in brain, in modulating hippocampal synaptic plasticity. Unlike other cAMP effectors, such as PKA and EPAC, POPDC1 is membrane-bound and the sequence of the cAMP-binding cassette differs from canonical cAMP-binding domains. These properties suggest that POPDC1 may have a unique role in cAMP-mediated signaling underlying synaptic plasticity. Our results show that POPDC1 is enriched in hippocampal synaptoneurosomes. Acute hippocampal slices from Popdc1 knockout (KO) mice exhibit enhanced long-term potentiation (LTP) induced by a variety of stimulation paradigms, particularly in response to weak stimulation paradigms that in slices from wildtype mice induce only transient LTP. Furthermore, Popdc1 KO mice did not display any further enhancement in forskolin-induced LTP observed following inhibition of phosphodiesterases (PDEs), suggesting a possible modulation of cAMP-PDE signaling by POPDC1. Taken together, these data reveal POPDC1 as a novel player in the regulation of hippocampal synaptic plasticity and as a potential target for cognitive enhancement strategies.

neuroscience↗

bioRxiv2021

Aberrant BCMA Signaling Promotes Tumor Growth by Altering Protein Translation Machinery, a Therapeutic Target for the Treatment of Relapse/Refractory Multiple Myeloma

Recent T Cell therapies have been effective in the treatment of hematological cancers. However, immunotoxicity and treatment relapse pose significant clinical challenges. Here, we revealed distinctive requirement for neutralizing TNF receptor ligands APRIL and BAFF in MM and DLBCL. Furthermore, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as a therapeutic inhibitor of ARPIL and BAFF. While wild-type sBCMA-Fc successfully blocked APRIL signaling with picomolar binding affinity, inhibiting tumor growth in MM models, it lacked efficacy in inhibiting DLBCL progression due to its weak binding for BAFF. To expand the therapeutic utility of sBCMA-Fc, using a protein engineering approach, we generated an affinity enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) with 4-folds and 500-folds enhancement in binding to APRIL and BAFF respectively. The sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, sBCMA-Fc V3 was proven to be a viable clinical candidate by showing adequate toxicity profile and on-target mechanism of action in nonhuman primates. SUMMARYThis study demonstrates the dichotomous function of APRIL and BAFF in MM and DLBCL, that can be safely targeted by an engineered fusion protein designed to trap APRIL and BAFF with ultra-high binding affinity.

cancer biology↗

bioRxiv2021

Double-observer approach with camera traps: Towards an unbiased density estimation of unmarked animal populations

Camera traps are a powerful tool for wildlife surveys. However, camera traps may not always detect animals passing in front. This constraint may create a substantial bias in estimating critical parameters such as the density of unmarked populations. We proposed the double-observer approach with camera traps to counter the constraint, which involves setting up a paired camera trap at a station and correcting imperfect detection with a hierarchal capture-recapture model for stratified populations. We performed simulations to evaluate this approachs reliability and determine how to obtain desirable data for this approach. We then applied it to 12 mammals in Japan and Cameroon. The results showed that the approach could correct imperfect detection as long as paired camera traps detect animals nearly independently (Correlation coefficient < 0.2). Camera traps should be installed to monitor a predefined small focal area from different directions to satisfy this requirement. The field surveys showed that camera trap might miss animals by 3 %-40%, suggesting that current density estimation models relying on perfect detection may underestimate animal density by the same order of magnitude. We hope that our approach will be incorporated into existing density estimation models to improve their accuracy.

ecology↗

bioRxiv2020

Novel multicellular prokaryote discovered next to an underground stream

The emergence of multicellularity is a key event in the evolution of life and is an attractive challenge among researchers, including those investigating the artificial design of cellular behavior1. Multicellular organisms are widely distributed on Earth, and retracing the specific conditions conducive for the initial transition from unicellularity to multicellularity is difficult. However, by examining organisms that inhabit unique (e.g., isolated) environmental niches, we may be able to get a glimpse into primitive multicellularity in the context of a given environment. Here we report the discovery of a new bacterium that displayed multicellular-like characteristics and behavior. The bacterium, which was isolated adjacent to an underground stream in a limestone cave, is to be named Jeongeupia sacculi sp. nov. HS-3. On a solid surface, HS-3 self-organizes its filamentous cells to form an appearance similar to the nematic phase of a liquid crystal2. Mature colonies produce and accommodate clusters of coccobacillus progeny, and release them upon contact with water. HS-3 demonstrated novel, spatiotemporally regulated multicellularity that can resolve the so-called competition-dispersal trade-off problem3. This study illustrates a hypothetical missing link on the emergence of multicellularity.

evolutionary biology↗

bioRxiv2020

Phosphorylation and dephosphorylation of Ser852 and Ser889 control clustering, localization, and function of PAR-3

Cell polarity is essential for various asymmetric cellular events, where the partitioning defective (PAR) protein, PAR3, plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border such as the tight junction in vertebrates and functions as an apical determinant. Although there is much information about the regulators of PAR3 localization, the mechanism involved in PAR3 concentration and localization to the specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2, a stimulator of PAR3 localization, can link PAR3 and protein phosphatase 1 (PP1). The ASPP2-PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 are static in molecular turnover and fail to coordinate rapid reconstruction of the tight junction, supporting that both phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3. Summary statementWe show that phosphorylation and dephosphorylation regulate clustering of PAR-3, a cell polarity-regulating factor, and how the clustering regulation affects localization of PAR-3 and cell-cell junction formation.

cell biology↗

bioRxiv2019

Alzheimers disease-related dysregulation of protein synthesis causes key pathological features with ageing

Alzheimers disease (AD) is characterised by A{beta} and tau pathology as well as synaptic degeneration. Recently, it was suggested that the development of these key disease features may at least in part be due to increased protein synthesis that is regulated by fragile X mental retardation protein (FMRP) and its binding partner CYFIP2. Using an unbiased screen, we show that exposure of primary neurons to A{beta} increases FMRP-regulated protein synthesis and involves a reduction of CYFIP2 levels. Modelling this CYFIP2 reduction in mice, we find A{beta} accumulation, development of pre-tangle tau pathology, gliosis, loss of synapses, and deficits in memory formation with ageing. We conclude that reducing endogenous CYFIP2 expression is sufficient to cause some key features of AD in mice. We therefore propose a central role for CYFIP2 in AD as a modulator of protein synthesis, highlighting a novel direction for therapeutic targeting.

neuroscience↗

bioRxiv2019

Re-evaluation of lysyl hydroxylation in the collagen triple helix: lysyl hydroxylase 1 and prolyl 3-hydroxylase 3 have site-differential and collagen type-dependent roles in lysine hydroxylation.

Collagen is the most abundant protein in humans and is heavily post-translationally modified. Its biosynthesis is very complex and requires three different types of hydroxylation (two for proline and one for lysine) that are generated in the rough endoplasmic reticulum (rER). These processes involve many enzymes and chaperones which were collectively termed the molecular ensemble for collagen biosynthesis. However, the function of some of the proteins in this molecular ensemble is controversial. While prolyl 3-hydroxylase 1 and 2 (P3H1, P3H2) are bona fide collagen prolyl 3-hydroxylases, the function of prolyl 3-hydroxylase 3 (P3H3) is less clear. A recent study of P3H3 null mice demonstrated that this enzyme had no activity as prolyl 3-hydroxylase but may instead act as a chaperone for lysyl hydroxylase 1 (LH1). LH1 is required to generate hydroxylysine for crosslinking within collagen triple helical sequences. If P3H3 is a LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should have similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared lysyl hydroxylation in type I and V collagen from P3H3 and LH1 null mice. Our results indicate LH1 plays a global role for lysyl hydroxylation in triple helical domain of type I collagen while P3H3 is indeed involved in lysyl hydroxylation particularly at crosslink formation sites but is not required for all lysyl hydroxylation sites in type I collagen triple helix. Furthermore, although type V collagen from LH1 null mice surprisingly contained as much hydroxylysine as type V collagen from wild type, the amount of hydroxylysine in type V collagen was clearly suppressed in P3H3 null mice. In summary, our study suggests that P3H3 and LH1 likely have two distinct mechanisms to distinguish crosslink formation sites from other sites in type I collagen and to recognize different collagen types in the rER. Author summaryCollagen is one of the most heavily post-translationally modified proteins in the human body and its post-translational modifications provide biological functions to collagen molecules. In collagen post-translational modifications, crosslink formation on a collagen triple helix adds important biomechanical properties to the collagen fibrils and is mediated by hydroxylation of very specific lysine residues. LH1 and P3H3 show the similar role in lysine hydroxylation for specific residues at crosslink formation sites of type I collagen. Conversely, they have very distinct rules in lysine hydroxylation at other residues in type I collagen triple helix. Furthermore, they demonstrate preferential recognition and modification of different collagen types. Our findings provide a better understanding of the individual functions of LH1 and P3H3 in the rER and also offer new directions for the mechanism of lysyl hydroxylation followed by crosslink formation in different tissues and collagens.

biochemistry↗