Unity and diversity of ciliary systems in locomotion and transport

The regular, snake-like beating of sperm flagella and the breaststroke-like swimming of cilia require spatially and temporally coordinated activity of the axonemal dyneins. It is thought that coordination is mediated by stresses or strains that build up within the moving axoneme, but it is not known which components of stress or strain are involved, nor how they feed back on the dyneins. To answer this question, the group has measured the beating patterns of isolated, reactivate axonemes of the unicellular alga Chlamydomonas reinhardtii. The group compared the measurements in wild-type and mutant cells with models derived from different feedback mechanisms. They found that regulation by changes in axonemal curvature was the only mechanism that accords with the measurements. The group suggests that distortions due to bending of twisted axonemes may provide a mechanism by which the motors sense curvature. To facilitate modelling studies of axonemal beats, the group have published a simplified version of their model.

Biological microswimmers are propelled by the beat of thin filaments protruding from the cell body – called cilia or flagella. One of the best-characterised flagellated microswimmers are sperm from sea urchin. To find the egg, sperm register egg-derived chemicals and translate the sensory flow into steering. Even though sperm navigation often occurs in three dimensions (3D), our knowledge is mostly restricted to the two dimensional (2D) scenario. For a better understanding of sperm navigation in 3D, the group employs caged chemoattractants to establish rapidly well-defined chemical gradients and digital holographic microscopy to resolve the 3D swimming path and the underlying 3D flagellar beat. They also apply these photonic techniques to advance our understanding of the differences among sperm species. Sperm species not only vary at the level of signalling components but also vary in shape and motility behaviours. These differences might be attributed to the different conditions under which fertilisation happens. For example, marine invertebrates, such as echinoderms or tunicates, release their gametes (eggs and sperm) into the ocean (external fertilisers). In contrast, mammalian species release sperm in the reproductive tract of the female, where they interact with the lining of the surrounding tissue (internal fertilisers). The group characterises sperm motility in 3D for internal and external fertilisers to better understand sperm behaviours across different species.

Sperm from marine invertebrates navigate to the egg in a chemoattractant gradient. The sperm flagellum serves as an antenna that registers the chemoattractant, as a motor that propels the cell, and as a rudder that steers sperm in the chemical landscape. Sperm are exquisitely sensitive: they can register the binding of a single chemoattractant molecule and translate binding events into a Ca²⁺ response that controls the flagellar beat and thereby the steering response. Professor Kaupp will discuss the cGMP-signalling pathway that endows sperm with single-molecule sensitivity.

The dynamics of cellular responses, including changes in voltage, pH_i, and Ca²⁺, is optically recorded in motile sperm. The group finds that, during navigation, sperm perform a surprisingly rich variety of computational operations; they can count, differentiate, integrate, and reset the signalling pathway. Furthermore, Professor Kaupp will decipher how such cell algebra is embodied by biochemical and electrical mechanisms.

The organised internal left-right asymmetry of the developing vertebrate embryo is initiated by asymmetric fluid dynamics (‘nodal flow’) in an enclosed transient organising structure – the ventral node in mice, Kupffer’s vesicle of zebrafish, and primary node of humans. Rotating cilia, tilted with respect to already-established axes, cause leftward transport of particles, including for example morphogen-carrying vesicles. Mechanical viscous forces may also play a critical role – either through mechanosensory effects or stress-induced exocytosis. Despite years of research, the precise nature of the conversion of the nodal flow to asymmetric development is unresolved; one key part of the challenge is that due to the microscopic nature of the system it is challenging to measure flow velocities and flow-induced stresses directly. Professor Smith will discuss methods for mathematical modelling of the flow which integrate experimental data on the shape of the organising structure with data on the cilia beat pattern and frequency. These models enable the extraction of mechanical parameters such as the time-varying flow field and the viscous forces, in addition to understanding how the flow affects the release, transport and capture of particles. This talk will focus on two specific issues: (1) cell-level modelling of stress-induced release of vesicular particles, (2) organ-level modelling of how elongation of the structure, and the convexity/concavity of the floor, modify particle paths across species, and possible implications. The open-source simulation framework, based on the NEAREST numerical scheme for the discretization of the regularized stokeslet boundary integral equation, is highly extensible to other ciliated systems.

Primary cilia of kidney epithelial cells are believed to act as mechanosensors for kidney-duct fluid flow. Genetic defects localising to the primary cilia are connected to polycystic kidney disease (PKD), a fatal monogenic disorder. Mechanical parameters such as flexural rigidity must modulate ciliary mechanosensation. It has been shown that there is a basal rotation component to ciliary deflection, which has the potential to affect aspects of mechanosensation. To investigate this aspect of ciliary mechanical response, the group has deflected primary cilia of Madin-Darby canine kidney (MDCK-II) cells with an optical trap and recorded their relaxation dynamics with video microscopy. Professor Schmidt presents a simple mechanical model including basal compliance, basal drag, and flexural rigidity that describes the bi-exponential relaxation behaviour seen in the data. The measured values imply a mixing of relaxation mode time scales, depending on cilia length, and suggest a possible mechanical filtering mechanism in the cilium's response.

Motile cilia are micron-scale hair-like protrusions from epithelial cells that beat collectively to transport fluid. Individual cilia are driven into oscillatory motion by dynein molecular motors acting on an intricate structure of microtubule doublets referred to as the central axoneme. On the tissue level, the coordinated beating of cilia serves diverse biological functions, from mucociliary clearance in the airways to cerebrospinal fluid transport in the brain ventricles. Yet the relationship between cilia structure and organisation and their biological function remains elusive. Here, Professor Kanso will present a series of physics-based models that take into account minimal cilia features in order to examine: (1) the emergence of self-sustained oscillations in individual cilia; (2) the coordinated beating of neighboring cilia; and (3) the role of cilia-driven flows in transport and mixing. Professor Kanso will conclude by commenting on the implications of these models to understanding the biophysical mechanisms underlying the interaction of ciliated tissues with microbial partners.

Many aquatic unicellular protists are equipped with flagella and cilia that facilitate not only motility of the cells but – maybe more importantly – their capture and handling of prey. These flagellates and ciliates typically feed on sub-micron sized bacteria and other small cells, and their fundamental problem in encountering prey is that viscosity impedes predator-prey contact at the small Reynolds numbers at which they operate. The cilia or flagella essentially create a flow of water past the cell that, one way or another, facilitate predator-prey contact, often in ways that are not understood. Feeding currents also disturb the water, thereby attracting flow-sensing predators. Therefore, the flagellation as well as flagella beat patterns and kinematics in free-living protists must have evolved to optimise the fundamental trade-off between resource acquisition (near field flow) and survival (far field flow). Professor Kiørboe will first describe two examples where we now understand the fluid dynamics of prey encounter, namely filter feeding in choanoflagellates and interception feeding in dinoflagellates. He will then show flow fields created by a large range of protists with diverse flagellation and kinematics, and demonstrate the feeding-survival trade-off. Professor Kiørboe will use simple analytical as well as CFD models to provide a mechanistic understanding of the observations. Simple point-force models often provide good descriptions of far-field flow patterns and, hence, predation risk, but the near-cell flow field, essential for prey capture, relies on observations or CFD.

Motile cilia and flagella are highly conserved organelles composed of more than 600 different proteins that are essential for the normal development and health of many eukaryotes including humans. The group integrates cryo-electron tomography with EM-visible labelling, comparative genetics and biochemical methods to reveal the in situ 3D structure of cilia, and the mechanism and regulation of their motility. Intact ciliated cells or isolated axonemes are rapidly frozen to provide outstanding sample preservation, and both high structural and time resolution of dynamic cellular processes. This study of active sea urchin sperm flagella directly visualised the macromolecular complexes and their structural changes during flagellar beating. The group resolved distinct conformations of dynein motors and regulators, and showed that many of them are distributed in bend-direction-dependent fashion in active flagella. These results provide direct evidence for the conformational switching predicted by the 'switch-point-hypothesis'. However, they also reveal a fundamentally different mechanism of generating motility by inhibiting, rather than activating, selected dyneins causing an asymmetric distribution of force and thus bending. This provides a new understanding of the distinct roles played by various dyneins and regulators in ciliary motility and suggest a molecular mechanism for robust beating.

The VFL3 gene in Chlamydomonas is required for the assembly of the centrin-based distal striated fiber (DSF) that tethers the two mature basal bodies. A nonsense mutation in VFL3 leads to the loss of the DSF and to a variable number of basal bodies and cilia. VFL3 has other localisations and functions. The CCDC61 protein was crystalised. The first ~630 amino acids form seven β-sheets that dimerise and require F128 and D129 (FD). The C-terminal domain forms a coiled coil that requires 5 basic residues to dimerise. The tagged wild-type transgene localises at the basal bodies and probasal bodies as four dots. The signal is distal to BLD10/CEP135. The FD mutant transgene shows partial rescue of the mutant phenotypes. The 5E mutant, which changes the 5 basic residues to glutamates, fails to rescue the phenotypes. The basal body localisation in both mutants is diffuse, and four distinct four dots are not observed. The VFL3 protein also localises to the proximal region of the cilia. This ciliary localisation occurs in both wild-type and the FD mutant but not in the 5E mutant. The VFL3 locus encodes at least two mRNA isoforms. The short form makes a 30kD protein, and it fails to rescue the mutant phenotypes. It has an asymmetric distribution between two basal bodies. The group hypothesises that this short version may play a role in licensing basal body duplication and provide another important layer of regulation for basal body duplication/assembly that is similar to the mini CEP135.

The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrioles can also serve as a template for cilia formation, which plays an important role in signalling and motility. Dr Bettencourt-Dias will focus this talk on the regulation of the centriole structure, in particular on variations in the structure that naturally occur to ensure the formation of different cilia types, as well as variations that occur in disease, and are associated with abnormal cell division.

Nearly all motile cilia have a '9+2' axoneme containing a central apparatus (CA), consisting of two central microtubules with projections, that is essential for motility. To date, only 22 proteins are known to be CA components. To identify new candidate CA proteins, the group used quantitative mass spectrometry to compare the proteomes of isolated axonemes of wild-type Chlamydomonas and a CA-less mutant. The group identified 44 novel candidate CA proteins, of which 14 are conserved in humans. To validate the results, five of the latter were studied more closely. They obtained mutants defective for these proteins, and rescued the mutants with genes expressing tagged versions of the proteins. Some of the mutants had distinct and novel phenotypes, demonstrating that the disrupted proteins have important and specific roles in the control of ciliary motility. The group then used structured-illumination microscopy to localise the tagged proteins within the axoneme. The group found that all five are indeed CA components; thus, it is likely that most of the other candidates also are CA proteins. Many of the 44 proteins could be assigned to specific locations within the CA. These results reveal that the CA is far more compositionally complex than previously recognised, and provide a greatly expanded knowledge base for studies to understand the architecture of the CA and how it functions. The discovery of the new conserved CA proteins will facilitate genetic screening to identify patients with a form of primary ciliary dyskinesia that has been difficult to diagnose.

The motile and sensory functions of cilia and flagella require them to be of defined length, but the mechanisms by which such a complex machine is built to a particular length are not obvious. Quantitative imaging has shown that intraflagellar transport is regulated as a function of length, raising the question of how this system senses ciliary length. The group has tested three classes of mechanisms based on time of flight, diffusion, and calcium signalling, using a combination of quantitative live-cell imaging, Chlamydomonas genetics, and computational modelling. Two additional control elements, the length dependent loading of cargo and the transcriptional regulation of ciliary proteins during growth will also be discussed and integrated into a complete length control system.