On the unity and diversity of cilia

Abstract

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’.

1. Introduction

Several hundreds of years have passed since Antoni van Leeuwenhoek first observed the enigmatic motion of ciliated animalcules and spermatozoa, and communicated his findings in a series of letters to the Royal Society [1]. Since then, we have come to realize the importance and ubiquity of these tiny hair-like organelles for an ever-increasing number of processes. Motile cilia and eukaryotic flagella enable sperm navigation, locomotion of microswimmers and circulation of respiratory and cerebrospinal fluid. Ciliated structures occur in organisms that span several orders of magnitude in size, complexity and over 1 billion years of evolutionary history. Despite this functional diversity, the structure of the cilium is remarkably conserved [2–4]. Cilia have ancient evolutionary origins in unicellular microorganisms [5,6] that typically actuate and coordinate these whip-like appendages to effect crawling, feeding, swimming and navigation through aqueous habitats. In this regard, cross-species studies that adopt a comparative approach have yielded important insights into ciliary structure and function [7].

Throughout the history of cilium research, there have been multiple cases where findings in one species have proven consequential in a number of distantly related systems spanning the entire eukaryotic diversity. Comparative approaches have not only driven molecular discoveries, but also our understanding of the static and dynamic aspects of cilia. In fact, we owe much of our knowledge of basic ciliary biology and motility to Chlamydomonas reinhardtii, an easy-to-culture, genetically tractable green alga dubbed the ‘green yeast’ [8]. For instance, the phenomenon of intraflagellar transport (IFT)—the bidirectional transport of proteins inside cilia—was discovered in Chlamydomonas [9]. IFT has since been shown to be present in most ciliated organisms [10,11], and is critical to build cilia and to control length, ciliary sensing and many signalling processes [12]. Likewise, key axonemal structures were studied in the first instance in this green alga [13–15]. The cilium has an irrefutable place in human health and disease, with ciliary dysfunctions implicated in diverse pathologies including Kartagener's syndrome, loss of male fertility owing to immotile sperm [16] or polycystic kidney disease [17]. Recent trends towards increasingly quantitative analysis of motile ciliary dynamics have enabled more efficient and improved diagnosis and detection of ciliopathic phenotypes. For example, techniques for particle motion detection in physics have been adapted to detect changes in human bronchial ciliary beating in primary ciliary dyskinesia [18]. Live-cell imaging and hydrodynamic modelling have yielded key insights into the human sperm in fertility research [19].

Cilia are not only important for human health, but are involved in the reproduction process of the vast majority of eukaryotes (e.g. [20]), and ciliary beating and sensing are essential for motility, feeding and predator avoidance behaviours in many aquatic organisms [21–23]. Understanding the unique ecology of small ciliated organisms from the bottom-up is a prerequisite to understanding the structure and function of marine food webs [24].

Yet despite decades of research, we as a community still do not have conclusive answers to fundamental questions such as how a cilium beats or how multiple cilia coordinate their activity. Diverse methodologies have been accumulated, including cryogenic electron microscopy (cryoEM), high-speed imaging technologies, theoretical models and computational tools. These technological innovations have enabled ever-greater temporal resolution of ciliary dynamics, and ever-finer spatial resolution of the ciliary architecture, yet still lacking is the effective integration of the two. The adoption of these state-of-the-art methods for the study of ciliary dynamics by a larger number of laboratories remains an important challenge. What is really needed is a holistic and fundamentally interdisciplinary approach to understanding several aspects of ciliary function and control. In this timely issue and the associated scientific meeting, we brought together researchers that would not typically interact, with the aim of facilitating and catalysing a fruitful exchange of ideas between these disparate communities.

2. Ciliary systems in locomotion and transport

The movement of motile cilia in a fluid generates a flow, which either transports fluid across surfaces or else leads to self-propulsion. Several key papers from recent years have revealed unifying themes in the biophysics of cilia-generated flows.

Processes as diverse as the generation of complex flows in the cerebrospinal fluid of the vertebrate brain [25–28], the exchange of nutrients and dissolved gases in reef corals [29] or the active recruitment of symbiotic Vibrio bacteria in a squid [30] all rely on extended ciliated surfaces displaying complex ciliary dynamics. At the single-cell or single-cilium level, assorted motor proteins and structural components generate and regulate the beat pattern [31]. The resulting beat dynamics are highly sensitive not only to intracellular signalling processes, but also respond actively to extrinsic factors such as mechanical shearing, temperature, light and chemical signals. By incorporating optogenetic methods for delivering targeted perturbations, recent biophysical investigations were able to reconstruct how sperm swim and steer towards gradients of chemoattractant [32]. There is rapid progress in capturing the near-native structure of cilia with cryoEM tomography [33,34]. Early research efforts attempted to reconcile this structural information with observed patterns of ciliary beating, by invoking different physical models of how beating emerges from cooperative dynein activity at the microscale [35,36]. More recently, new theoretical models have emerged, incorporating curvature control, dynein inhibition and instability-driven mechanisms [37–40].

There are also numerous systems in which ciliated structures mediate both locomotion and transport. For example, at the size and complexity scale of marine larvae, this swimming-feeding trade-off becomes particularly significant. New insights into the pathways and control architectures used by marine organisms for regulating the activity of extended ciliated bands and surfaces have been made possible by detailed reconstructions of whole-organism neurocircuitry by serial EM [41]. Delving deeper into the developmental and molecular diversity of cilia and basal bodies may help us understand how different types of cilia came to be specialized for different functions, even within the same organism [42].

3. Purpose of this theme issue

In March 2019, a Theo Murphy International Scientific meeting on ‘Unity and Diversity of Ciliary Systems for Locomotion and Transport’ was held. Participants discussed important advances in the field and exchanged technologies, hypotheses and approaches, and discovered unique synergies between laboratories working with very different systems. The emerging consensus was that a broad, comparative approach was necessary to discover universal principles that underpin the dynamics and regulation of ciliary systems. This special issue highlights the diversity represented by speakers at the meeting, both in terms of their scientific expertise but also in the variety of their study systems which range from the mammalian brain through to zooplankton and to unicellular green algae.

Thus far, the field of cilia research has placed a strong emphasis on the molecular biology of cilia, understandable given their importance in, for example, hedgehog signalling, development and ciliopathies. By contrast, the dynamic aspects of ciliary motility are often under-represented in the programmes of many topical meetings on cilia. Similarly, while several recent thematic issues and books discuss the molecular biology of cilia, less attention has been given to ciliary dynamics and coordination, with no collection of articles devoted to the intersection of these two disparate but intrinsically coupled themes. This thematic issue seeks to fill a gap in the existing literature, collating in one volume a number of case studies and results that will help to improve our understanding of the dichotomy between the biological and physical aspects of ciliary motility.

The objectives of this issue are threefold: (i) to identify and highlight the range and diversity of biological phenomena which rely on cilia, (ii) to explore novel, cross-disciplinary approaches to studying cilia, and (iii) to reveal universal biophysical mechanisms that underlie ciliary form and function. This synergistic interplay is already in evidence in several of the articles in this issue.

4. Structure and overview of contributions

We organize this issue broadly into the following four sections. We begin by considering the structure and assembly of a single cilium or flagellum. The organism of choice in this first group of three papers is the biflagellate green alga Chlamydomonas, further highlighting its importance as a model species. In her contribution [43], Dutcher reviews asymmetries in the Chlamydomonas flagellar axoneme, which are sometimes overlooked. These include proximal/distal differences, radial/doublet specific differences as well as differences between the two flagella—termed cis and trans, which are thought to underly the cell's ability to phototax [44]. Despite these intrinsic differences, the two Chlamydomonas flagella have apparently equal length. Length regulation in Chlamydomonas (and indeed many organisms) is a remarkable feat of natural bioengineering and is a phenomenon that continues to fascinate biologists [45,46] and, more recently, mathematicians [47]. Using Chlamydomonas mutants which assemble abnormally long flagella, Wemmer et al. [48] shed new light on this process by first confirming that the rate at which IFT particles enter the flagellum is length-dependent. However, when compared with a kinetic model, the authors also show that increased IFT injection cannot be the sole mechanism for the increase in flagellum length. Despite the broad conservation of cilium ultrastructure and IFT, axonemal composition and structure show species-specific differences. Zhao et al. [49] used comparative genomics to reveal the unity and diversity of the central apparatus—a major component of ‘9 + 2’ cilia [50]. Across diverse phylogenetic groups, more exotic axonemal permutations exist (e.g. ‘9 + 0’, ‘9 + 1’). The new results identified a number of central apparatus proteins that are conserved throughout eukaryotic evolution. They also identified a separate group of central apparatus proteins that are only present in a group of green algae, the Volvocales. The authors discuss possible functional roles of these elusive proteins, such as to enable central pair rotation, or even phototaxis.

The above studies highlight the structural complexity of the cilium, but physical models are necessary to parse the molecular information, and to interpret the localization and function of each component within the overall ciliary architecture. Our next group of papers focuses on ciliary dynamics. Man et al. [51] incorporate microscale motor activity into a sliding control model and examine regimes in which the model filament exhibits spontaneous oscillations. The model displays high sensitivity to boundary conditions (whether hinged or clamped), showing, in particular, that clamped filaments can propagate bending waves bidirectionally. Theoretical modelling of this kind can help explain not only how ciliary beating is generated but also how cells enact symmetry-breaking at the microscale to control and select for distinct beat patterns, which remains very much an open problem. In pathogenic flagellates including Trypanosoma and Leishmania, proximal/distal asymmetries appear to be critical for the ability to switch between tip-to-base and base-to-tip beating waveforms [52]. Meanwhile, sperm rely on a different kind of symmetry-breaking to change its swimming direction, as discussed in this new review by Gong et al. [53]. In many species, sperm cells are steered effectively towards the egg by activating asymmetric flagellar beating modes which exhibit non-zero average curvatures. Compared to uniflagellates, multiflagellates achieve directional steering and spatio-temporal symmetry-breaking in a radically different way. Wan [54] elaborates on the case of unicellular algae that coordinate multiple basally coupled appendages to pattern self-movement, showing that gait control in these organisms is an active process. Particular attention is paid to gait mechanosensitivity—where the beat pattern changes as a direct result of mechanical perturbations. More generally, a delicate balance exists between extrinsic and intrinsic motility control in different ciliary systems [55]. In a new research contribution, Hamilton et al. [56] use data gathered from different species of organisms (ranging from unicellular and multicellular green algae to humans) and a hydrodynamic rower model to further discriminate between mutual interciliary coupling, versus the susceptibility for cilia to be entrained by external flows. The authors emphasize the need to account for the underlying single-cilium dynamics when evaluating the relative importance of these two effects.

As we move to multiciliated systems containing many thousands of beating cilia, the intricate interplay between ciliary polarity, complex geometries and ciliary flows becomes increasingly apparent. Our third group of papers explores this multiscale problem and highlights how the global positioning of cilia with oriented beating planes shapes complex physiological flows. We begin with ciliary beating in Stentor—a unicellular protist that feeds using an anterior oral apparatus. Wan et al. [57] take advantage of Stentor's unique regenerative capabilities to study the de novo assembly of a new oral apparatus, and the emergence of long-range metachronal coordination of the oral cilia across micrometric distances. In many animals, ciliated surfaces can also become internalized. Nawroth et al. [58] review the biomechanics of mucociliary clearance in the human airways, and discuss how this may be modified in clinical phenotypes. Detailed mathematical modelling is suggested to provide a benchmark for decoupling the hierarchical aspects of these structure–function relationships to target the integration between the molecular machinery, microenvironment and chemical signalling or feedback. The fluid-filled ventricles of the mammalian brain are also abundant in cilia, which must coordinate to propel streams of cerebrospinal fluid along the ventricular walls. Recent experimental and computational innovations have enabled increasingly detailed and quantitative visualization of these flows, which are highly dynamic, even in brain explants. Eichele et al. [59] detail the types, alignment and putative transport functions of cilia existing in the ependyma. Over the course of evolution, nervous systems arose to coordinate ciliary locomotion over even longer distances, adding a new dimension to controlling ciliated structures [41,60]. Marinkovic et al. [61] review the anatomy, ciliomotor circuitry and feeding systems in marine invertebrates, focusing on the neurons that innervate ciliated cells, and the diverse physiological cues that modulate ciliary activity through the release of neurotransmitters and neuropeptides. Complex cilia-generated flows are revealed through these and other recent studies to have key functional roles in transporting and sorting material and nutrients in a variety of contexts. In the embryonic node, motile cilia are necessary for left–right symmetry-breaking, but the mechanism relating biased transport to biased gene expression has remained mysterious. In a new study, Gallagher et al. [62] simulated different flow and particle deposition patterns in an idealized mouse node. In particular, the authors' results favour a uniformly random release model (rather than cilia-localized release), in which even a single beating cilium is sufficient to produce biased transport.

Thus, cilia do not only generate flows, but flows in internal body cavities can also act as patterning and signalling systems during development. In the final set of papers, the dialogue between simulations and experimental observations continues with an opinion piece by Cartwright et al. [63] that surveys general mechanisms of cilia-mediated sensing in left–right organizers. The question of whether nodal cilia are chemosensing or mechanosensing [64] remains open, and indeed, Cartwright et al. suggest that it may be species-dependent. Extending this comparative theme, Ringers et al. [65] review the identity and function of motile cilia in different animal models (zebrafish, frog, mouse, human), and in diverse sensory organs (nose, ear, brain, spinal cord). Given their multifarious roles in development and sensation, disentangling how nervous systems manipulate ciliary beating will be critical to understanding the onset of ciliopathies. The fish otic vesicle represents a particularly elegant ciliated system where an inertial, biomineral mass termed the otolith, mediates mechanosensation in the inner ear. Whitfield [66] unravels the fascinating process by which otolith assembly begins at the tips of immotile kinocilia, but correct otolith placement and subsequent development requires both tether hair cells and motile cilia. Similarities between the canonical vertebrate hair cell and the mechanosensory cells of diverse marine invertebrates suggest a common evolutionary origin. Bezares-Calderón et al. [67] review and pieces together the often fragmentary literature on the astonishing diversity of mechanosensory cilia in aquatic animals. The authors discuss a rich tapestry of behaviours where ciliary motility and sensing are inextricably coupled, such as in the motor cilia used for swimming and feeding, and in mechanosensory organs necessary for detecting fluid pressures and shear flows.

5. Discussion and outlook

Cilia are truly fascinating entities. They are highly active, display nonlinear dynamics, interesting geometries and interact collectively within novel topologies and geometries in novel ways. This is a field that welcomes the involvement of more physicists and theoretical modelling, but fundamentally, future progress and new insights will require an interdisciplinary approach. Recent technological advances in genetics and biological imaging have revolutionized our understanding of the molecular biology of the cilium, and given us unprecedented insight into the pathways underlying ciliary signalling, yet there is comparatively less emphasis on the physical aspects of ciliary motility and behaviour.

This volume and its associated meeting represented an invaluable opportunity to bring together disparate communities of scientists. This included experts on cilia biology, ultrastructure and proteome, and theoreticians specializing in hydrodynamic modelling. Diverse topics discussed highlight for the biologists the importance of adopting physical or quantitative approaches. Meanwhile, physicists should benefit from a greater appreciation of the biological detail that will be necessary to inform the next generation of theoretical models. Crudely speaking, once we have acquired a ‘parts list’, the logical next step forward is to understand how the ‘parts come together’ for the regulation of complex ciliary activity. This will require a truly transdisciplinary effort. The second key theme which we wish to emphasize here is the value of comparative studies. The basic unit that is the motile cilium, with its meshwork of microtubules and motor proteins, has remained largely conserved since the first eukaryotic cells and unites single-celled algae, marine invertebrates or large mammals. Despite the common ultrastructure, different species exhibit significant diversity in the control mechanisms and ciliary proteomes reflecting unique evolutionary histories. In animals, the emergence of neuronal mechanisms dedicated to regulating ciliary activity enabled greater spatio-temporal precision of control.

This special issue of Philosophical Transactions B offers a unique take on this highly active and growing interdisciplinary field. We hope that this collection of papers provides a valuable resource for the community to appreciate the importance and ubiquity of cilia and cilia-mediated processes in living systems. The insights herein provided should help motivate new research themes in understanding the fundamentals of cilia biology. We hope that readers will, as we have, enjoy finding out more about the fascinating unity and diversity of this ancient organelle.

Data accessibility

This article has no additional data.

Competing interests

We declare we have no competing interests.

Funding

This study was financially supported by the Royal Society.