Biology subjects

Sawai, S.

Publications and source records attributed to Sawai, S..

14 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↗

bioRxiv2025

An antiparallel cell circulation driven by self-alignment induces phase separation

Active tissues exhibit diverse collective dynamics, yet the cell-cell interactions that generate ordered microscopic flows remain poorly understood. Here, we show that antiparallel cell circulation can emerge from self-aligned, polarity-dependent tension gradients. Using a minimal vertex model of confluent tissues, we studied polar cells that align their polarity with their own velocity and impose polarity-dependent tension gradients along cell-cell contacts, without relying on substrate traction. This behavior can be generalized as a minimal interaction in which forces transmitted between cells act with opposite signs, reminiscent of action-reaction forces, organizing cells into stable interlocking antiparallel lanes. In mixtures of motile and nonmotile cells, this circulation drives phase separation, in which motile cells spontaneously form persistent domains. Accordingly, we identified similar antiparallel circulation patterns in two-dimensional aggregates of Dictyostelium discoideum, supporting the biological relevance of the mechanism. Together, these results demonstrate that self-aligned tension gradients provide a robust and underappreciated route to dynamic microscopic pattern formation in multicellular systems.

biophysics↗

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↗

bioRxiv2024

Slow diffusion and signal amplification on membranes regulated by phospholipase D

Control of molecular diffusion is pivotal for highly fluidic membranes to serve as substrates for biochemical reactions and the self-assembly of molecular machinery driving membrane protrusions. Lateral diffusion in membranes depends on lipid composition, which is highly diverse and homeostatically controlled in living cells. Due to the complexity of the underlying processes, its impact on molecular diffusion remain largely unclear. In this study, we show that lipid diffusion in model membranes is markedly decreased in cytosolic extracts. The reduction in lipid diffusivity could be pharmacologically inhibited by targeting phospholipase D (PLD), and addition of PLD to membranes mimicked the reduction in diffusion. Phosphatidic acid, a direct product of PLD, diffused slowly in model membranes and reduced the diffusivity of surrounding lipids. Furthermore, we demonstrated that PLD specifically controls the lateral diffusion of a myristoylated protein in cells, possibly due to auxiliary electrostatic interactions between cationic residues located near the lipidated tail and anionic phospholipids. PLD controlled the size and lifetime of localized patches of phosphatidylinositol (3,4,5) triphosphates that specify regions of membrane protrusions. Overall, the results of this study suggest that PLD controls the lateral diffusion of certain membrane proteins, which play key roles in phosphoinositide signaling. Significance StatementIn living cells, many biochemical reactions occur in confined regions on the membranes, facilitating the local occurrence of specific events, such as membrane protrusion. This is puzzling from a physical perspective because the membrane is a two-dimensional fluidic structure that should allow molecules to spread freely. Herein, we found that the fluidity of artificial membranes was markedly reduced by adding extracts from the cell cytoplasm. A lipid-modifying protein phospholipase D (PLD) was found to be responsible for this and it regulates the diffusion of membrane proteins in cells. This study suggests the novel role of PLD as a regulator of molecular diffusion and its impact on phosphoinositide production that serves as an important signal for cell deformation.

biophysics↗

bioRxiv2024

Traveling wave chemotaxis of neutrophil-like HL-60 cells

The question of how changes in chemoattractant concentration translate into the chemotactic response of immune cells serves as a paradigm for the quantitative understanding of how cells perceive and process temporal and spatial information. Here, using a microfluidic approach, we analyzed the migration of neutrophil-like HL-60 cells to a traveling wave of the chemoattractants fMLP and leukotriene B4 (LTB4). We found that under a pulsatile wave that travels at a speed of 95 and 170 {micro}m/min, cells move forward in the front of the wave but slow down and randomly orient at the back due to temporal decrease in the attractant concentration. Under a slower wave, cells re-orient and migrate at the back of the wave; thus, cell displacement is canceled out or even becomes negative as cells chase the receding wave. FRET-based analysis indicated that these patterns of movement correlated well with spatiotemporal changes in Cdc42 activity. Furthermore, pharmacological perturbations suggested that migration in front of the wave depends on Cdc42, whereas that in the back of the wave depends more on PI3K/Rac and ROCK. These results suggest that pulsatile attractant waves may recruit or disperse neutrophils, depending on their speed and degree of cell polarization. Significance statementO_LIThe way neutrophil chemotaxis is directed by attractants is thought to depend on temporal and spatial concentration changes, however the response to a transient pulsatile stimulus has not been well explored. C_LIO_LIA moderately fast traveling wave of fMLP and leukotrieneB4 (LTB4) directs unidirectional cell migration. Under slow waves, cells respond to the back of the wave; thus, cell displacement is canceled out. Cdc42, PI3K, Rac, and ROCK contributed differently to responses at the front and back of the wave. C_LIO_LIThese findings suggest that traveling waves of attractant can guide immune cell recruitment and dispersal. C_LI

cell biology↗

bioRxiv2023

Random walk and cell morphology dynamics in Naegleria gruberi

Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systems Dictyostelium and neutrophils over the years have uncovered generality in their overall cell movement pattern. Under no directional cues, the centroid movement can be quantitatively characterized by their persistence to move in a straight line and the frequency of re-orientation. Mathematically, the cells essentially behave as a persistent random walker with memory of two characteristic time-scale. Such quantitative characterization is important from a cellular-level ethology point of view as it has direct connotation to their exploratory and foraging strategies. Interestingly, outside the amoebozoa and metazoa, there are largely uncharacterized species in the excavate taxon Heterolobosea including amoeboflagellate Naegleria. While classical works have shown that these cells indeed show typical amoeboid locomotion on an attached surface, their quantitative features are so far unexplored. Here, we analyzed the cell movement of Naegleria gruberi by employing long-time phase contrast imaging to automatically track individual cells. We show that the cells move as a persistent random walker with two time-scales that are close to those known in Dictyostelium and neutrophils. Similarities were also found in the shape dynamics which is characterized by the appearance, splitting and annihilation of the curvature waves along the cell edge. Our analysis based on the Fourier descriptor and a neural network classifier point to importance of morphology features unique to Naegleria including complex protrusions and the transient bipolar dumbbell morphologies.

cell biology↗

bioRxiv2022

Evolutionary stability of social commitment

Conflict resolution between individual cells and a group is essential for multicellularity. The social amoeba Dictyostelium discoideum switches between solitary growth and social fruitification depending on nutrient availability. Under starvation, cells form fruiting bodies consisting of spores and non-viable altruistic stalk cells. Once cells socially committed, they complete fruitification even with a renewed source of nutrients. This social commitment is puzzling because it deprives individual cells of benefits of quickly resuming solitary growth. One idea posits that traits that facilitate premature de-commitment are somehow hindered from being selected. We studied outcomes of premature de-commitment by forced refeeding. We show that when refed cells resume sociality together with non-refed cells, besides some becoming solitary outside of fruiting bodies, a large fraction was redirected to a sub-region of altruistic stalk regardless of their original fate. The refed cells exhibited reduced cohesivity and were sorted out to the altruistic positions in morphogenesis. Furthermore, a theoretical model considering evolution of cell-cell association revealed a valley in the fitness landscape that prevents invasion of de-committing mutants. Our results provide a general scheme that naturally penalizes withdrawal from a society by evolving a specific division of labor that less cohesive individuals become altruists. Significance StatementEvolution of unicellular to multicellular organisms must resolve conflicts of reproductive interests between individual cells and the group. In the social amoeba Dictyostelium, a transition from a solitary to multicellular group occurs under starvation. Once cells commit themselves to multicellular organization, the process continues even when shifting to an environment that favors solitary growth. Our study revealed that cells forced to partially revert to a de-committed state take an altruistic role through interaction with socially committed cells. The de-committed cells exhibited reduced cohesivity and were sorted out to altruistic positions in morphogenesis. This inevitably penalizes selfish cells that revert to solitary growth too quickly. Our results explain group-level behavior that is apparently difficult to understand from an individual-level fitness.

evolutionary biology↗

bioRxiv2021

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, it is still unknown how actomyosin contractility generates force and maintains cellular morphology. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility. The system, named OptoMYPT, combines a catalytic subunit of the type I phosphatase-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination was sufficient to induce dephosphorylation of myosin regulatory light chains and decrease in traction force at the subcellular level. The OptoMYPT system was further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We found that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system will provide new opportunities to understand cellular and tissue mechanics.

cell biology↗

bioRxiv2020

Comparative mapping of crawling-cell morphodynamics in deep learning-based feature space

Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.

cell biology↗

bioRxiv2020

Micro-topographical guidance of macropinocytic signaling patches

In fast moving cells such as amoeba and immune cells, spatio-temporal regulation of dendritic actin filaments shapes large-scale plasma membrane protrusions. Despite the importance in migration as well as in particle and liquid ingestion, how these processes are affected by the micrometer-scale surface features is poorly understood. Here, through quantitative imaging analysis of Dictyostelium on micro-fabricated surfaces, we show that there is a distinct mode of topographically guided cell migration phagotaxis directed by the macropinocytic Ras/PI3K signaling patches. The topography guidance was PI3K-dependent and involved nucleation of a patch at the convex curved surface and confinement at the concave surface. Due to the topography-dependence, constitutive cup formation for liquid uptake in the axenic strain is also destined to trace large surface features. Given the fact that PI3K-dependency of phagocytosis are restricted to large particles in both Dictyostelium and immune cells, topography-dependency and the dual-use of membrane cups may be wide-spread.

biophysics↗

bioRxiv2020

Three-dimensional morphodynamics simulations of macropinocytic cups

*Macropinocytosis is non-specific uptake of the extracellular fluid playing ubiquitous roles in cell growth, immune-surveillance as well as virus entry. Despite its widespread occurrence, it remains unclear how its initial cup-shaped plasma membrane extensions forms without external physical support as in phagocytosis or curvature inducing proteins as in clathrin-mediated endocytosis. Here, by developing a novel computational framework that describes the coupling between bistable reaction-diffusion processes of active signaling patches and membrane deformation, we demonstrate that protrusive force localized to the edge of the patches can give rise to the self-enclosing cup structure without further assumption of local bending or contraction. Efficient uptake requires an appropriate balance between the patch size and the magnitude of protrusive force relative to the cortical tension. Furthermore, our model exhibits a variety of known morphology dynamics including cyclic cup formation, coexistence and competition between multiple cups and cup splitting indicating that these complex morphologies self-organize through mutually dependent dynamics between the reaction-diffusion process and membrane deformation.

biophysics↗

bioRxiv2018

Tissue self-organization based on collective cell migration by contact activation of locomotion and chemotaxis

Despite their central role in multicellular organization, navigation rules that dictate cell rearrangement remain much to be elucidated. Contact between neighboring cells and diffusive attractant molecules are two of the major determinants of tissue-level patterning, however in most cases, molecular and developmental complexity hinders one from decoding the exact governing rules of individual cell movement. A primordial example of tissue patterning by cell rearrangement is found in the social amoeba Dictyostelium discoideum where the organizing center or the tip self-organize as a result of sorting of differentiating prestalk and prespore cells. Due to its relatively simple and conditional multicellularity, the system provides a rare case where the process can be fully dissected into individual cell behavior. By employing microfluidics and microsphere-based manipulation of navigational cues at the single-cell level, here we uncovered a previously overlooked mode of Dictyostelium cell migration that is strictly directed by cell-cell contact. The cell-cell contact signal is mediated by E-set Ig-like domain containing heterophilic adhesion molecules TgrB1/TgrC1 that act in trans to induce plasma membrane recruitment of SCAR complex and formation of dendritic actin networks, and the resulting cell protrusion competes with those induced by chemoattractant cAMP. Furthermore, we demonstrate that both prestalk and prespore cells can protrude towards the contact signal as well as to chemotax towards cAMP, however when given both signals, prestalk cells orient towards the chemoattractant whereas prespore cells choose the contact signal. These data suggest a new model of cell sorting by competing juxtacrine and diffusive cues each with potential to drive its own mode of collective cell migration. The present findings not only resolve the long standing question of how cells sort in Dictyostelium but also cast light on the remarkable parallels in collective cell migration that evolved independently in metazoa and amoebozoa.

cell biology↗