Biology subjects

Kuwana, S.

Publications and source records attributed to Kuwana, S..

4 recordsLinked to original sources

bioRxiv2026

Surface Tension and Stalk Elongation Drive Dictyostelium Morphogenesis

We investigate the mechanical principles underlying fruiting body morphogenesis in Dictyostelium discoideum. Quantitative shape analysis based on the Young--Laplace law, together with AFM indentation measurements, indicate surface tension as the dominant tissue-scale force acting on the culminating fruiting body. Based on this observation, we construct a hydrodynamic phase-field model with tunable surface and interfacial tensions, and analyze its behavior numerically. Our results show that, once a stalk begins to form, the elevation of the cell mass arises naturally through a dewetting process. Through quantitative comparisons with experimental measurements, we identify the mechanical conditions required for detachment from the substrate and for establishment of the characteristic morphology of the culminating fruiting body. Together, our model analysis highlights the importance of stalk-tip elongation and tissue-scale surface and interfacial tensions in the construction of large-scale three-dimensional tissues.

biophysics↗

bioRxiv2026

Adhesion-mediated transition to a mesenchymal-like, fan-shaped migration mode in Dictyostelium discoideum

Cells migrate with varying degrees of polarization and directional persistence as exemplified by epithelial, mesenchymal and amoeboid cell types. Depending on the physiological and developmental context, these states are often interchangeable, reflecting the plastic and adaptive nature of the cytoskeleton. However, general principles governing such motility-mode transitions remain poorly established, and it is unclear whether they apply to non-metazoan cells. Here, we report previously overlooked features of the amoebozoan Dictyostelium discoideum, demonstrating that it undergoes pronounced adhesion-dependent changes in both motility and morphology. Unlike the well-known pseudopodia-rich forms observed on weakly adhesive surfaces, cells on highly adhesive substrates adopt fan-shaped morphologies reminiscent of cultured mesenchymal cells. These cells are characterized by lamellipodia-like protrusions enriched in the SCAR/WAVE complex, large focal adhesion-like plaques, F-actin-independent front-rear gradients of Ras/Rap activity. Furthermore, they exhibit a marked increase in cortical stiffness dependent on F-actin, talins, and the RhoA homolog RacE. Their high directional persistence depends on the persistent localization of the SCAR/WAVE complex, talin-mediated substrate anchoring, and RacE-dependent stabilization of the cell rear. We propose that adhesion-engaged remodeling of cell polarity and cortical mechanics is an evolutionarily ancient feature that predates the specialization of adhesion receptors.

cell biology↗

bioRxiv2025

A nanobody-based degron system for targeted protein knockdown in Dictyostelium discoideum

BackgroundsThe cellular slime mold Dictyostelium discoideum is a widely used model system for studying basic processes in cell and developmental biology. While genetic tools, such as targeted gene disruption by homologous recombination and genome editing using CRISPR/Cas9, are well-established in D. discoideum, efficient methods for conditional loss-of-function studies are limited. Here, we developed a nanobody-based degron system for D. discoideum based on ALFA-tagged protein recruitment to the Skp1-Cullin-F-box (SCF) complex. ResultsALFA-tagged Histone H1 was efficiently degraded by expressing anti-ALFA nanobody (NbALFA) fused to the D. discoideum FbxD F-box domain ( dictyGrad-ALFA). Cell type-specific targeting was achieved by expressing dictyGrad-ALFA under prestalk- and prespore-specific gene promoters. Furthermore, targeting of adenylyl cyclase A (ACA) resulted in the expected aggregation-deficient phenotype, validating the efficacy of dictyGrad-ALFA-mediated protein depletion. Cell type-specific ACA degradation delayed development but eventually resulted in normal fruiting bodies. Our ALFA-tag approach was further used for conditional knockdown in combination with the auxin-inducible degron 2 (AID2) system, which relies on indole-3-acetic acid (IAA)-dependent binding between NbALFA-mAID and a OsTIR-F-box-Skp1A fusion protein. We obtained efficient IAA-induced degradation in prestalk cells; however, efficiency was low in other cell types. ConclusionsTogether, these systems pave the way for conditional and cell type-specific protein degradation in D. discoideum, enabling functional analyses of essential genes for development and survival.

cell biology↗

bioRxiv2024

Multi-color fluorescence live-cell imaging in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imaging of D. discoideum has greatly facilitated studies on fundamental topics, including cytokinesis, phagocytosis, and cell migration. Additionally, its unique life cycle places Dictyostelium at the forefront of understanding aggregative multicellularity, a recurring evolutionary trait found across the Opisthokonta and Amoebozoa clades. The use of multiple fluorescent proteins (FP) and labels with separable spectral properties is critical for tracking cells in aggregates and identifying co-occurring biomolecular events and factors that underlie the dynamics of the cytoskeleton, membrane lipids, second messengers, and gene expression. However, in D. discoideum, the number of frequently used FP species is limited to two or three. In this study, we explored the use of new-generation FP for practical 4- to 5-color fluorescence imaging of D. discoideum. We showed that the yellow fluorescent protein Achilles and the red fluorescent protein mScarlet-I both yield high signals and allow sensitive detection of rapid gene induction. The color palette was further expanded to include blue (mTagBFP2 and mTurquosie2), large Stoke-shift LSSmGFP, and near-infrared (miRFP670nano3) FPs, in addition to the HaloTag ligand SaraFluor 650T. Thus, we demonstrated the feasibility of deploying 4- and 5- color imaging of D. discoideum using conventional confocal microscopy.

cell biology↗