Bioelectric control of locomotor gaits in the walking ciliate Euplotes.

Diverse animal species exhibit highly stereotyped behavioral actions and locomotor sequences as they explore their natural environments. In many such cases, the neural basis of behavior is well established, where dedicated neural circuitry contributes to the initiation and regulation of certain response sequences. At the microscopic scale, single-celled eukaryotes (protists) also exhibit remarkably complex behaviors and yet are completely devoid of nervous systems. Here, to address the question of how single cells control behavior, we study locomotor patterning in the exemplary hypotrich ciliate Euplotes, a highly polarized cell, which actuates a large number of leg-like appendages called cirri (each a bundle of ∼25-50 cilia) to swim in fluids or walk on surfaces. As it navigates its surroundings, a walking Euplotes cell is routinely observed to perform side-stepping reactions, one of the most sophisticated maneuvers ever observed in a single-celled organism. These are spontaneous and stereotyped reorientation events involving a transient and fast backward motion followed by a turn. Combining high-speed imaging with simultaneous time-resolved electrophysiological recordings, we show that this complex coordinated motion sequence is tightly regulated by rapid membrane depolarization events, which orchestrate the activity of different cirri on the cell. Using machine learning and computer vision methods, we map detailed measurements of cirri dynamics to the cell's membrane bioelectrical activity, revealing a differential response in the front and back cirri. We integrate these measurements with a minimal model to understand how Euplotes-a unicellular organism-manipulates its membrane potential to achieve real-time control over its motor apparatus.

Mechanisms and functions of multiciliary coordination.

Ciliated organisms are present in virtually every branch of the eukaryotic tree of life. In diverse systems, cilia operate in a coordinated manner to drive fluid flows, or even propel entire organisms. How do groups of motile cilia coordinate their activity within a cell or across a tissue to fulfil essential functions of life? In this review, we highlight the latest developments in our understanding of the mechanisms and functions of multiciliary coordination in diverse systems. We explore new and emerging trends in bioimaging, analytical, and computational methods, which together with their application in new model systems, have conspired to deliver important insights into one of the most fundamental questions in cellular dynamics.

Active oscillations in microscale navigation.

Living organisms routinely navigate their surroundings in search of better conditions, more food, or to avoid predators. Typically, animals do so by integrating sensory cues from the environment with their locomotor apparatuses. For single cells or small organisms that possess motility, fundamental physical constraints imposed by their small size have led to alternative navigation strategies that are specific to the microscopic world. Intriguingly, underlying these myriad exploratory behaviours or sensory functions is the onset of periodic activity at multiple scales, such as the undulations of cilia and flagella, the vibrations of hair cells, or the oscillatory shape modes of migrating neutrophils. Here, I explore oscillatory dynamics in basal microeukaryotes and hypothesize that these active oscillations play a critical role in enhancing the fidelity of adaptive sensorimotor integration.

Methods and Measures for Investigating Microscale Motility.

Motility is an essential factor for an organism's survival and diversification. With the advent of novel single-cell technologies, analytical frameworks, and theoretical methods, we can begin to probe the complex lives of microscopic motile organisms and answer the intertwining biological and physical questions of how these diverse lifeforms navigate their surroundings. Herein, we summarize the main mechanisms of microscale motility and give an overview of different experimental, analytical, and mathematical methods used to study them across different scales encompassing the molecular-, individual-, to population-level. We identify transferable techniques, pressing challenges, and future directions in the field. This review can serve as a starting point for researchers who are interested in exploring and quantifying the movements of organisms in the microscale world.

Phenotyping single-cell motility in microfluidic confinement.

The movement trajectories of organisms serve as dynamic read-outs of their behaviour and physiology. For microorganisms this can be difficult to resolve due to their small size and fast movement. Here, we devise a novel droplet microfluidics assay to encapsulate single micron-sized algae inside closed arenas, enabling ultralong high-speed tracking of the same cell. Comparing two model species - Chlamydomonas reinhardtii (freshwater, 2 cilia), and Pyramimonas octopus (marine, 8 cilia), we detail their highly-stereotyped yet contrasting swimming behaviours and environmental interactions. By measuring the rates and probabilities with which cells transition between a trio of motility states (smooth-forward swimming, quiescence, tumbling or excitable backward swimming), we reconstruct the control network that underlies this gait switching dynamics. A simplified model of cell-roaming in circular confinement reproduces the observed long-term behaviours and spatial fluxes, including novel boundary circulation behaviour. Finally, we establish an assay in which pairs of droplets are fused on demand, one containing a trapped cell with another containing a chemical that perturbs cellular excitability, to reveal how aneural microorganisms adapt their locomotor patterns in real-time.

A minimal robophysical model of quadriflagellate self-propulsion.

Locomotion at the microscale is remarkably sophisticated. Microorganisms have evolved diverse strategies to move within highly viscous environments, using deformable, propulsion-generating appendages such as cilia and flagella to drive helical or undulatory motion. In single-celled algae, these appendages can be arranged in different ways around an approximately 10 µm long cell body, and coordinated in distinct temporal patterns. Inspired by the observation that some quadriflagellates (bearing four flagella) have an outwardly similar morphology and flagellar beat pattern, yet swim at different speeds, this study seeks to determine whether variations in swimming performance could arise solely from differences in swimming gait. Robotics approaches are particularly suited to such investigations, where the phase relationships between appendages can be readily manipulated. Here, we developed autonomous, algae-inspired robophysical models that can self-propel in a viscous fluid. These macroscopic robots (length and width = 8.5 cm, height = 2 cm) have four independently actuated 'flagella' (length = 13 cm) that oscillate under low-Reynolds number conditions (Re∼O(10-1)). We tested the swimming performance of these robot models with appendages arranged two distinct configurations, and coordinated in three distinct gaits. The gaits, namely the pronk, the trot, and the gallop, correspond to gaits adopted by distinct microalgal species. When the appendages are inserted perpendicularly around a central 'body', the robot achieved a net performance of 0.15-0.63 body lengths per cycle, with the trot gait being the fastest. Robotic swimming performance was found to be comparable to that of the algal microswimmers across all gaits. By creating a minimal robot that can successfully reproduce cilia-inspired drag-based swimming, our work paves the way for the design of next-generation devices that have the capacity to autonomously navigate aqueous environments.

A new kind of beat.

New mathematical model reveals how the flagella of some single-celled algae generate a lasso-like beat pattern that propels the cell through water.

Control of Helical Navigation by Three-Dimensional Flagellar Beating.

Helical swimming is a ubiquitous strategy for motile cells to generate self-gradients for environmental sensing. The model biflagellate Chlamydomonas reinhardtii rotates at a constant 1-2 Hz as it swims, but the mechanism is unclear. Here, we show unequivocally that the rolling motion derives from a persistent, nonplanar flagellar beat pattern. This is revealed by high-speed imaging and micromanipulation of live cells. We construct a fully 3D model to relate flagellar beating directly to the free-swimming trajectories. For realistic geometries, the model reproduces both the sense and magnitude of the axial rotation of live cells. We show that helical swimming requires further symmetry breaking between the two flagella. These functional differences underlie all tactic responses, particularly phototaxis. We propose a control strategy by which cells steer toward or away from light by modulating the sign of biflagellar dominance.

Intracellular coupling modulates biflagellar synchrony.

Beating flagella exhibit a variety of synchronization modes. This synchrony has long been attributed to hydrodynamic coupling between the flagella. However, recent work with flagellated algae indicates that a mechanism internal to the cell, through the contractile fibres connecting the flagella basal bodies, must be at play to actively modulate flagellar synchrony. Exactly how basal coupling mediates flagellar coordination remains unclear. Here, we examine the role of basal coupling in the synchronization of the model biflagellate Chlamydomonas reinhardtii using a series of mathematical models of decreasing levels of complexity. We report that basal coupling is sufficient to achieve inphase, antiphase and bistable synchrony, even in the absence of hydrodynamic coupling and flagellar compliance. These modes can be reached by modulating the activity level of the individual flagella or the strength of the basal coupling. We observe a slip mode when allowing for differential flagellar activity, just as in experiments with live cells. We introduce a dimensionless ratio of flagellar activity to basal coupling that is predictive of the mode of synchrony. This ratio allows us to query biological parameters which are not yet directly measurable experimentally. Our work shows a concrete route for cells to actively control the synchronization of their flagella.

Origins of eukaryotic excitability.

All living cells interact dynamically with a constantly changing world. Eukaryotes, in particular, evolved radically new ways to sense and react to their environment. These advances enabled new and more complex forms of cellular behaviour in eukaryotes, including directional movement, active feeding, mating, and responses to predation. But what are the key events and innovations during eukaryogenesis that made all of this possible? Here we describe the ancestral repertoire of eukaryotic excitability and discuss five major cellular innovations that enabled its evolutionary origin. The innovations include a vastly expanded repertoire of ion channels, the emergence of cilia and pseudopodia, endomembranes as intracellular capacitors, a flexible plasma membrane and the relocation of chemiosmotic ATP synthesis to mitochondria, which liberated the plasma membrane for more complex electrical signalling involved in sensing and reacting. We conjecture that together with an increase in cell size, these new forms of excitability greatly amplified the degrees of freedom associated with cellular responses, allowing eukaryotes to vastly outperform prokaryotes in terms of both speed and accuracy. This comprehensive new perspective on the evolution of excitability enriches our view of eukaryogenesis and emphasizes behaviour and sensing as major contributors to the success of eukaryotes. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

Functional exploration of heterotrimeric kinesin-II in IFT and ciliary length control in Chlamydomonas.

Heterodimeric motor organization of kinesin-II is essential for its function in anterograde IFT in ciliogenesis. However, the underlying mechanism is not well understood. In addition, the anterograde IFT velocity varies significantly in different organisms, but how this velocity affects ciliary length is not clear. We show that in Chlamydomonas motors are only stable as heterodimers in vivo, which is likely the key factor for the requirement of a heterodimer for IFT. Second, chimeric CrKinesin-II with human kinesin-II motor domains functioned in vitro and in vivo, leading to a ~ 2.8 fold reduced anterograde IFT velocity and a similar fold reduction in IFT injection rate that supposedly correlates with ciliary assembly activity. However, the ciliary length was only mildly reduced (~15%). Modeling analysis suggests a nonlinear scaling relationship between IFT velocity and ciliary length that can be accounted for by limitation of the motors and/or its ciliary cargoes, e.g. tubulin.

On the unity and diversity of cilia.

Cilia are specialized cellular organelles that are united in structure and implicated in diverse key life processes across eukaryotes. In both unicellular and multicellular organisms, variations on the same ancestral form mediate sensing, locomotion and the production of physiological flows. As we usher in a new, more interdisciplinary era, the way we study cilia is changing. This special theme issue brings together biologists, biophysicists and mathematicians to highlight the remarkable range of systems in which motile cilia fulfil vital functions, and to inspire and define novel strategies for future research. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Reorganization of complex ciliary flows around regenerating Stentor coeruleus.

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such as Chlamydomonas. Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliate Stentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring in Stentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Synchrony and symmetry-breaking in active flagellar coordination.

Living creatures exhibit a remarkable diversity of locomotion mechanisms, evolving structures specialized for interacting with their environment. In the vast majority of cases, locomotor behaviours such as flying, crawling and running are orchestrated by nervous systems. Surprisingly, microorganisms can enact analogous movement gaits for swimming using multiple, fast-moving cellular protrusions called cilia and flagella. Here, I demonstrate intermittency, reversible rhythmogenesis and gait mechanosensitivity in algal flagella, to reveal the active nature of locomotor patterning. In addition to maintaining free-swimming gaits, I show that the algal flagellar apparatus functions as a central pattern generator that encodes the beating of each flagellum in a network in a distinguishable manner. The latter provides a novel symmetry-breaking mechanism for cell reorientation. These findings imply that the capacity to generate and coordinate complex locomotor patterns does not require neural circuitry but rather the minimal ingredients are present in simple unicellular organisms. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Ciliate Biology: The Graceful Hunt of a Shape-Shifting Predator.

Even single-celled eukaryotes are capable of highly complex behaviors. A new study reveals how one unicellular predator actively manipulates and remodels its unique cytoskeletal morphology to achieve rapid shape changes and a remarkable hunting strategy.

Coordination of eukaryotic cilia and flagella.

Propulsion by slender cellular appendages called cilia and flagella is an ancient means of locomotion. Unicellular organisms evolved myriad strategies to propel themselves in fluid environments, often involving significant differences in flagella number, localisation and modes of actuation. Remarkably, these appendages are highly conserved, occurring in many complex organisms such as humans, where they may be found generating physiological flows when attached to surfaces (e.g. airway epithelial cilia), or else conferring motility to male gametes (e.g. undulations of sperm flagella). Where multiple cilia arise, their movements are often observed to be highly coordinated. Here I review the two main mechanisms for motile cilia coordination, namely, intracellular and hydrodynamic, and discuss their relative importance in different ciliary systems.

Time Irreversibility and Criticality in the Motility of a Flagellate Microorganism.

Active living organisms exhibit behavioral variability, partitioning between fast and slow dynamics. Such variability may be key to generating rapid responses in a heterogeneous, unpredictable environment wherein cellular activity effects continual exchanges of energy fluxes. We demonstrate a novel, noninvasive strategy for revealing nonequilibrium control of swimming-specifically, in an octoflagellate microalga. These organisms exhibit surprising features of flagellar excitability and mechanosensitivity, which characterize a novel, time-irreversible "run-stop-shock" motility comprising forward runs, knee-jerk shocks with dramatic beat reversal, and long stops during which cells are quiescent yet continue to exhibit submicron flagellar vibrations. Entropy production, associated with flux cycles arising in a reaction graph representation of the gait-switching dynamics, provides a direct measure of detailed balance violation in this primitive alga.

Coordinated beating of algal flagella is mediated by basal coupling.

Cilia and flagella often exhibit synchronized behavior; this includes phase locking, as seen in Chlamydomonas, and metachronal wave formation in the respiratory cilia of higher organisms. Since the observations by Gray and Rothschild of phase synchrony of nearby swimming spermatozoa, it has been a working hypothesis that synchrony arises from hydrodynamic interactions between beating filaments. Recent work on the dynamics of physically separated pairs of flagella isolated from the multicellular alga Volvox has shown that hydrodynamic coupling alone is sufficient to produce synchrony. However, the situation is more complex in unicellular organisms bearing few flagella. We show that flagella of Chlamydomonas mutants deficient in filamentary connections between basal bodies display markedly different synchronization from the wild type. We perform micromanipulation on configurations of flagella and conclude that a mechanism, internal to the cell, must provide an additional flagellar coupling. In naturally occurring species with 4, 8, or even 16 flagella, we find diverse symmetries of basal body positioning and of the flagellar apparatus that are coincident with specific gaits of flagellar actuation, suggesting that it is a competition between intracellular coupling and hydrodynamic interactions that ultimately determines the precise form of flagellar coordination in unicellular algae.

Rhythmicity, recurrence, and recovery of flagellar beating.

The eukaryotic flagellum beats with apparently unfailing periodicity, yet responds rapidly to stimuli. Like the human heartbeat, flagellar oscillations are now known to be noisy. Using the alga C. reinhardtii, we explore three aspects of nonuniform flagellar beating. We report the existence of rhythmicity, waveform noise peaking at transitions between power and recovery strokes, and fluctuations of interbeat intervals that are correlated and even recurrent, with memory extending to hundreds of beats. These features are altered qualitatively by physiological perturbations. Further, we quantify the recovery of periodic breaststroke beating from transient hydrodynamic forcing. These results will help constrain microscopic theories on the origins and regulation of flagellar beating.

Flagellar synchronization through direct hydrodynamic interactions.

Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties.DOI: http://dx.doi.org/10.7554/eLife.02750.001.