Binocular stereo-microscopy for deforming intact amoeba.

A powerful and convenient method for measuring three-dimensional (3D) deformation of moving amoeboid cells will assist the progress of environmental and cytological studies as protists amoebae play a role in the fundamental environmental ecosystem. Here we develop an inexpensive and useful method for measuring 3D deformation of single protists amoeba through binocular microscopy and a newly proposed algorithm of stereo-scopy. From the movies taken from the left and right optical tubes of the binocular microscope, we detect the 3D positions of many intrinsic intracellular vesicles and reconstruct cellular surfaces of amoeboid cells in 3D space. Some observations of sampled behaviors are shown in a single-celled organism of Amoeba proteus. The resultant surface time series is then analyzed to obtain surface velocity, curvature and volume increasing rates of pseudo-pods for characterizing the movements of amoeboid cells. The limitations and errors of this method are also discussed.

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists.

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site.

Simple mechanosense and response of cilia motion reveal the intrinsic habits of ciliates.

An important habit of ciliates, namely, their behavioral preference for walls, is revealed through experiments and hydrodynamic simulations. A simple mechanical response of individual ciliary beating (i.e., the beating is stalled by the cilium contacting a wall) can solely determine the sliding motion of the ciliate along the wall and result in a wall-preferring behavior. Considering ciliate ethology, this mechanosensing system is likely an advantage in the single cell's ability to locate nutrition. In other words, ciliates can skillfully use both the sliding motion to feed on a surface and the traveling motion in bulk water to locate new surfaces according to the single "swimming" mission.

Protein import complexes in the mitochondrial outer membrane of Amoebozoa representatives.

BACKGROUND: An ancestral trait of eukaryotic cells is the presence of mitochondria as an essential element for function and survival. Proper functioning of mitochondria depends on the import of nearly all proteins that is performed by complexes located in both mitochondrial membranes. The complexes have been proposed to contain subunits formed by proteins common to all eukaryotes and additional subunits regarded as lineage specific. Since Amoebozoa is poorly sampled for the complexes we investigated the outer membrane complexes, namely TOM, TOB/SAM and ERMES complexes, using available genome and transcriptome sequences, including transcriptomes assembled by us. RESULTS: The results indicate differences in the organization of the Amoebozoa TOM, TOB/SAM and ERMES complexes, with the TOM complex appearing to be the most diverse. This is reflected by differences in the number of involved subunits and in similarities to the cognate proteins of representatives from different supergroups of eukaryotes. CONCLUSIONS: The obtained results clearly demonstrate structural variability/diversity of these complexes in the Amoebozoa lineage and the reduction of their complexity as compared with the same complexes of model organisms.

Caenorhabditis elegans transfers across a gap under an electric field as dispersal behavior.

Interactions between different animal species are a critical determinant of each species' evolution and range expansion. Chemical, visual, and mechanical interactions have been abundantly reported, but the importance of electric interactions is not well understood. Here, we report the discovery that the nematode Caenorhabditis elegans transfers across electric fields to achieve phoretic attachment to insects. First, we found that dauer larvae of C. elegans nictating on a substrate in a Petri dish moved directly to the lid through the air due to the electrostatic force from the lid. To more systematically investigate the transfer behavior, we constructed an assay system with well-controlled electric fields: the worms flew up regardless of whether a positive or negative electric field was applied, suggesting that an induced charge within the worm is related to this transfer. The mean take-off speed is 0.86 m/s, and the worm flies up under an electric field exceeding 200 kV/m. This worm transfer occurs even when the worms form a nictation column composed of up to 100 worms; we term this behavior "multiworm transfer." These observations led us to conclude that C. elegans can transfer and attach to the bumblebee Bombus terrestris, which was charged by rubbing with flower pollen in the lab. The charge on the bumblebee was measured with a coulomb-meter to be 806 pC, which was within the range of bumblebee charges and of the same order of flying insect charges observed in nature, suggesting that electrical interactions occur among different species.

Three-dimensional architecture and assembly mechanism of the egg-shaped shell in testate amoeba Paulinella micropora.

Unicellular euglyphid testate amoeba Paulinella micropora with filose pseudopodia secrete approximately 50 siliceous scales into the extracellular template-free space to construct a shell isomorphic to that of its mother cell. This shell-constructing behavior is analogous to building a house with bricks, and a complex mechanism is expected to be involved for a single-celled amoeba to achieve such a phenomenon; however, the three-dimensional (3D) structure of the shell and its assembly in P. micropora are still unknown. In this study, we aimed to clarify the positional relationship between the cytoplasmic and extracellular scales and the structure of the egg-shaped shell in P. micropora during shell construction using focused ion beam scanning electron microscopy (FIB-SEM). 3D reconstruction revealed an extensive invasion of the electron-dense cytoplasm between the long sides of the positioned and stacked scales, which was predicted to be mediated by actin filament extension. To investigate the architecture of the shell of P. micropora, each scale was individually segmented, and the position of its centroid was plotted. The scales were arranged in a left-handed, single-circular ellipse in a twisted arrangement. In addition, we 3D printed individual scales and assembled them, revealing new features of the shell assembly mechanism of P. micropora. Our results indicate that the shell of P. micropora forms an egg shape by the regular stacking of precisely designed scales, and that the cytoskeleton is involved in the construction process.

Light-sheet microscopy reveals dorsoventral asymmetric membrane dynamics of Amoeba proteus during pressure-driven locomotion.

Amoebae are found all around the world and play an essential role in the carbon cycle in the environment. Therefore, the behavior of amoebae is a crucial factor when considering the global environment. Amoebae change their distribution through amoeboid locomotion, which are classified into several modes. In the pressure-driven mode, intracellular hydrostatic pressure generated by the contraction of cellular cortex actomyosin causes the pseudopod to extend. During amoeboid locomotion, the cellular surface exhibits dynamic deformation. Therefore, to understand the mechanism of amoeboid locomotion, it is important to characterize cellular membrane dynamics. Here, to clarify membrane dynamics during pressure-driven amoeboid locomotion, we developed a polkadot membrane staining method and performed light-sheet microscopy in Amoeba proteus, which exhibits typical pressure-driven amoeboid locomotion. It was observed that the whole cell membrane moved in the direction of movement, and the dorsal cell membrane in the posterior part of the cell moved more slowly than the other membrane. In addition, membrane complexity varied depending on the focused characteristic size of the membrane structure, and in general, the dorsal side was more complex than the ventral side. In summary, the membrane dynamics of Amoeba proteus during pressure-driven locomotion are asymmetric between the dorsal and ventral sides. This article has an associated interview with the co-first authors of the paper.

Simple dynamics underlying the survival behaviors of ciliates.

Ciliates are swimming microorganisms in aquatic environments. Habitats where ciliates accumulate include nutrient-rich solid-liquid interfaces such as pond bottom walls and waterweed surfaces. The ciliates stay near the walls to survive. We investigated the dynamics of the near-wall behavior of ciliates. In experiments, the ciliates were made to slide on a flat wall of glass substrate. When encountering the wall, the wall-side cilia of the cells stop their motion and lose their propelling activity, which indicates that the ciliates have a mechano-sensing system for cilia beating. Based on the experimental results, we hypothesized that the ciliary thrust force that propels the cell body becomes asymmetric, and the asymmetry of the thrust force generates a head-down torque to keep the cell sliding on the wall. To prove this hypothesis, we performed numerical simulations by using a developed hydrodynamic model for swimming ciliates. The model revealed that the loss of cilia activity on the wall side physically induces a sliding motion, and the aspect ratio of the cell body and effective cilium area are critical functions for the sliding behavior on a wall. In addition, we investigated the stability of the sliding motion against an external flow. We found that ciliates slide upstream on a wall. Interestingly, the dynamics of this upstream sliding, called rheotaxis, were also explained by the identical physical conditions for no-flow sliding. Only two simple physical conditions are required to explain the dynamics of ciliate survival behavior. This review article is an extended version of the Japanese article, Fluid Dynamic Model Reveals a Mechano-sensing System Underlying the Behavior of Ciliates, published in SEIBUTSU BUTSURI Vol. 61, p. 16-19 (2021).

Switching of behavioral modes and their modulation by a geometrical cue in the ciliate Stentor coeruleus.

Protists ubiquitously live in nature and play key roles in the food web chain. Their habitats consist of various geometrical structures, such as porous media and rigid surfaces, affecting their motilities. A kind of protist, Stentor coeruleus, exhibits free swimming and adhering for feeding. Under environmental and culture conditions, these organisms are often found in sediments with complex geometries. The determination of anchoring location is essential for their lives. However, the factors that induce the behavioral transition from swimming to adhering are still unknown. In this study, we quantitatively characterized the behavioral transitions in S. coeruleus and observed the behavior in a chamber with dead ends made by a simple structure mimicking the environmental structures. As a result, the cell adheres and feeds in narrow spaces between the structure and the chamber wall. It may be reasonable for the organism to hide itself from predators and capture prey in these spaces. The behavioral strategy for the exploration and exploitation of spaces with a wide variety of geometries in their habitats is discussed.

Accumulation of Tetrahymena pyriformis on Interfaces.

The behavior of ciliates has been studied for many years through environmental biology and the ethology of microorganisms, and recent hydrodynamic studies of microswimmers have greatly advanced our understanding of the behavioral dynamics at the single-cell level. However, the association between single-cell dynamics captured by microscopic observation and pattern dynamics obtained by macroscopic observation is not always obvious. Hence, to bridge the gap between the two, there is a need for experimental results on swarming dynamics at the mesoscopic scale. In this study, we investigated the spatial population dynamics of the ciliate, Tetrahymena pyriformis, based on quantitative data analysis. We combined the image processing of 3D micrographs and machine learning to obtain the positional data of individual cells of T. pyriformis and examined their statistical properties based on spatio-temporal data. According to the 3D spatial distribution of cells and their temporal evolution, cells accumulated both on the solid wall at the bottom surface and underneath the air-liquid interface at the top. Furthermore, we quantitatively clarified the difference in accumulation levels between the bulk and the interface by creating a simple behavioral model that incorporated quantitative accumulation coefficients in its solution. The accumulation coefficients can be compared under different conditions and between different species.

Near-wall rheotaxis of the ciliate Tetrahymena induced by the kinesthetic sensing of cilia.

To survive in harsh environments, single-celled microorganisms autonomously respond to external stimuli, such as light, heat, and flow. Here, we elucidate the flow response of Tetrahymena, a well-known single-celled freshwater microorganism. Tetrahymena moves upstream against an external flow via a behavior called rheotaxis. While micrometer-sized particles are swept away downstream in a viscous flow, what dynamics underlie the rheotaxis of the ciliate? Our experiments reveal that Tetrahymena slides along walls during upstream movement, which indicates that the cells receive rotational torque from shear flow to control cell orientation. To evaluate the effects of the shear torque and propelling speed, we perform a numerical simulation with a hydrodynamic model swimmer adopting cilia dynamics in a shear flow. The swimmer orientations converge to an upstream alignment, and the swimmer slides upstream along a boundary wall. The results suggest that Tetrahymena automatically responds to shear flow by performing rheotaxis using cilia-stalling mechanics.

Uni-cellular integration of complex spatial information in slime moulds and ciliates.

Single-celled organisms show a fascinating faculty for integrating spatial information and adapting their behaviour accordingly. As such they are of potential interest for elucidating fundamental mechanisms of developmental biology. In this mini-review we highlight current research on two organisms, the true slime mould Physarum polycephalum and the ciliates Paramecium and Tetrahymena. For each of these, we present a case study how applying physical principles to explain behaviour can lead to the understanding of general principles possibly relevant to developmental biology.

Influence of cellular shape on sliding behavior of ciliates.

Some types of ciliates accumulate on solid/fluid interfaces. This behavior is advantageous to survival in nature due to the presence of sufficient nutrition and stable environments. Recently, the accumulating mechanisms of Tetrahymena pyriformis at the interface were investigated. The synergy of the ellipsoidal shape of the cell body and the mechanosensing feature of the cilia allow for cells to slide on interfaces, and the sliding behavior leads to cell accumulation on the interfaces. Here, to examine the generality of the sliding behavior of ciliates, we characterized the behavior of Paramecium caudatum, which is a commonly studied ciliate. Our experimental and numerical results confirmed that P. caudatum also slid on the solid/fluid interface by using the same mechanism as T. pyriformis. In addition, we evaluated the effects of cellular ellipticity on their behaviors near the wall with a phase diagram produced via numerical simulation.

Non-periodic oscillatory deformation of an actomyosin microdroplet encapsulated within a lipid interface.

Active force generation in living organisms, which is mainly involved in actin cytoskeleton and myosin molecular motors, plays a crucial role in various biological processes. Although the contractile properties of actomyosin have been extensively investigated, their dynamic contribution to a deformable membrane remains unclear because of the cellular complexities and the difficulties associated with in vitro reconstitution. Here, by overcoming these experimental difficulties, we demonstrate the dynamic deformation of a reconstituted lipid interface coupled with self-organized structure of contractile actomyosin. Therein, the lipid interface repeatedly oscillates without any remarkable periods. The oscillatory deformation of the interface is caused by the aster-like three-dimensional hierarchical structure of actomyosin inside the droplet, which is revealed that the oscillation occurs stochastically as a Poisson process.

Wrinkling of a spherical lipid interface induced by actomyosin cortex.

Actomyosin actively generates contractile forces that provide the plasma membrane with the deformation stresses essential to carry out biological processes. Although the contractile property of purified actomyosin has been extensively studied, to understand the physical contribution of the actomyosin contractile force on a deformable membrane is still a challenging problem and of great interest in the field of biophysics. Here, we reconstitute a model system with a cell-sized deformable interface that exhibits anomalous curvature-dependent wrinkling caused by the actomyosin cortex underneath the spherical closed interface. Through a shape analysis of the wrinkling deformation, we find that the dominant contributor to the wrinkled shape changes from bending elasticity to stretching elasticity of the reconstituted cortex upon increasing the droplet curvature radius of the order of the cell size, i.e., tens of micrometers. The observed curvature dependence is explained by the theoretical description of the cortex elasticity and contractility. Our present results provide a fundamental insight into the deformation of a curved membrane induced by the actomyosin cortex.

Reconstruction of active regular motion in amoeba extract: dynamic cooperation between sol and gel states.

Amoeboid locomotion is one of the typical modes of biological cell migration. Cytoplasmic sol-gel conversion of an actomyosin system is thought to play an important role in locomotion. However, the mechanisms underlying sol-gel conversion, including trigger, signal, and regulating factors, remain unclear. We developed a novel model system in which an actomyosin fraction moves like an amoeba in a cytoplasmic extract. Rheological study of this model system revealed that the actomyosin fraction exhibits shear banding: the sol-gel state of actomyosin can be regulated by shear rate or mechanical force. Furthermore, study of the living cell indicated that the shear-banding property also causes sol-gel conversion with the same order of magnitude as that of shear rate. Our results suggest that the inherent sol-gel transition property plays an essential role in the self-regulation of autonomous translational motion in amoeba.