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.

Professor Joe Howard, Yale University, USA

Professor Joe Howard, Yale University, USA

Jonathon (Joe) Howard is best known for his research on motor proteins and the cytoskeleton, and the development of techniques for observing and manipulating individual biological molecules. His research interests include the sensation of forces by specialized receptor cells, the segregation of the chromosomes during mitosis, the motility of cilia, and the branching morphogenesis of neurons. Brought up in Australia, where he studied mathematics and neurobiology, he was professor of Physiology at the University of Washington in Seattle and a Director of the Max Planck Institute of Molecular Cell Biology & Genetics in Dresden, Germany before joining the faculty at Yale. 

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.

Dr Luis Alvarez, Caesar, Germany

Dr Luis Alvarez, Caesar, Germany

Luis Alvarez was born in Spain, Madrid, where he began in 1997 his studies in physics (Universidad Autónoma de Madrid). He continued his studies at the Università degli Studi di Firenze (Italy) during an Erasmus, and he did a Master at the Université Louis Pasteur in Strasbourg (France) in physics of soft and condensed matter. After that, Luis did his PhD in biophysics on the topic of viral infection spreading at the Laboratoire de Physique Statistique (École Normale Supérieure, Paris) under the supervision of Dr Didier Chatenay. Following his PhD, he made a Postdoc in the lab of Professor Benjamin Kaupp at the research centre Jülich (Germany) studying sperm chemotaxis. Since 2010, Luis is a group leader for the 'Biophysics of Cell Motility' at Caesar in Bonn, an institute affiliated with the Max Planck Society. His current interests are understanding cellular information-processing and navigation.

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.

Professor U Benjamin Kaupp, Caesar, Bonn, Germany

Professor U Benjamin Kaupp, Caesar, Bonn, Germany

Professor Kaupp is Emeritus Director at the Center of Advanced European Studies and Research (caesar), an institute of the Max Planck Society, in Bonn, Germany and head of the Department of Molecular Sensory Systems. Professor Kaupp studied Chemistry at the Universities of Tübingen and Berlin. He received his PhD at the Max-Volmer-Institute of the Technical University Berlin and did his 'Habilitation' in Biophysics at the University of Osnabrück in 1983. From 1988 to 2007, Kaupp was director at the Institute of Neuroscience and Biophysics (INB) of the Helmholtz Research Centre Jülich, and from January 2008 to September 2015, he was the managing director of caesar. Professor Kaupp serves on several scientific boards. He is a Scientific Member of the Max Planck Society and a member of the German Academy of Science-Leopoldina. Kaupp`s research priorities lie in the fields of Sensory Systems, Molecular Neurobiology, Chemotaxis, Ion Channels, and Membrane Receptors. His fundamental research results have found their way into many textbooks.

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.

Professor Dave Smith, University of Birmingham, UK

Professor Dave Smith, University of Birmingham, UK

Dave is Professor of Applied Mathematics at the University of Birmingham, UK with primary interests in the fluid mechanics of cilia and flagella. Following PhD study with Professors John Blake and Eamonn Gaffney on mathematical modelling of muco-ciliary transport, Dave was funded as a Medical Research Council Fellow with Centre for Human Reproductive Science with Dr Jackson Kirkman-Brown, combining fluid dynamics and high speed imaging of human sperm interacting with physiological rheology fluids. Alongside this research he pioneered 3D time-resolved models of flow in the left-right organising structure of the vertebrate embryo. Following appointment to a lectureship in 2009, he has been funded by EPSRC, BBSRC and MRC to lead projects ranging from cilia and flagellar flow, to cancer diagnosis and therapy, to pathogen detection. He is currently funded by an EPSRC Healthcare Technologies Challenge Award, and collaborates with experimental groups in Birmingham, Manchester, Nottingham, Edinburgh and Lisbon.

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.

Professor Christoph Schmidt, Georg-August-Universität, Germany and Duke University, USA

Professor Christoph Schmidt, Georg-August-Universität, Germany and Duke University, USA

Professor Schmidt received his Diploma in Physics and his doctorate from the Technical University Munich. Following this he held postdoctoral positions at Harvard University and The Rowland Institute for Science, after which he became Associate Professor of Physics at the University of Michigan, Ann Arbor. He was Professor of Physics at the Vrije Universiteit Amsterdam and a Member of Directorate at the Center for Research on Complex Systems. Currently Professor Schmidt is Professor of Physics at the University of Göttingen and Duke University. Professor Schmidt is a Fellow of the American Physical Society, a Member of the Göttingen Academy of Sciences and Humanities, and received an ERC Advanced Grant in 2014.

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.

Professor Eva Kanso, University of Southern California, USA

Professor Eva Kanso, University of Southern California, USA

Eva Kanso is a professor and the Z.H. Kaprielian Fellow in Aerospace and Mechanical Engineering at the University of Southern California. Prior to joining USC, Kanso held a two-year postdoctoral position in Computing and Mathematical Sciences at Caltech from 2003 to 2005. She received her PhD and MS in Mechanical Engineering as well as her MA in Mathematics from UC Berkeley. Kanso is the recipient of an NSF early CAREER development award in 2007, a Junior Distinguished Alumnus award from the American University of Beirut in 2014 and a USC Graduate Student Mentoring Award in 2016.

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.

Professor Thomas Kiørboe, Danish Technical University, Denmark

Professor Thomas Kiørboe, Danish Technical University, Denmark

Thomas Kiørboe is an Ocean Ecologist, graduated from the University of Copenhagen (PhD 1982; Dr. Scient. 1988), and currently a professor of Ocean Ecology at the Technical University of Demark (DTU) and director of the Centre for Ocean Life (Villum Center of Excellence) at DTU. His main interests are the functional ecology of marine plankton organisms and in achieving a mechanistic understanding of their functioning as well as on their functional roles in pelagic ecosystems. His current work focuses on the small-scale physical interactions between microscopic plankton and their fluid environment, on how these interactions governs the fundamental trade-offs between foraging and surviving, and finally in using this information to build trait-based models of pelagic ecosystems.

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.

Dr Daniela Nicastro, UT Southwestern Medical Center, USA

Dr Daniela Nicastro, UT Southwestern Medical Center, USA

Daniela Nicastro received her PhD in Biology from the Ludwig-Maximilians University in Munich, Germany in 2000. Following 3 years in the lab of Professor Baumeister at the Max-Planck Institute for Biochemistry in Munich (1998-2001), she took a postdoctoral fellow position in the National Center for Research Resources for 3D Electron Microscopy of Cells at the University of Colorado in Boulder. From 2006-2015, she was an Assistant and then tenured Associate Professor of Biology and director of the 'Correlative Light and Electron Microcopy' (CLEM) facility at Brandeis University near Boston. Since July 2015, she is an Associate Professor at the University of Texas Southwestern (UTSW) Medical Center in Dallas with appointments in the Departments for Cell Biology and Biophysics. She has 25 years of experience in electron microscopy of cellular structures and is a leading expert in cellular cryo-electron tomography. The research interest of the Nicastro lab is focused on studying the three-dimensional structure and function of cytoskeletal assemblies, molecular motors, organelles and cells using a combination of cutting-edge methods to elucidate the structure-function relationships of macromolecular complexes in situ, ie in their native environment.

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.

Professor Susan K Dutcher, Washington University in St Louis, USA

Professor Susan K Dutcher, Washington University in St Louis, USA

As a graduate student at the University of Washington, Dutcher worked on the role of cell division cycle genes in spindle pole body duplication with Dr Lee Hartwell. As a postdoctoral fellow at Rockefeller University, she switched to cilia and centrioles in the unicellular alga, Chlamydomonas. She investigates the assembly and function of centrioles and cilia using genetics, biochemistry, light and electron microscopy, and computational biology. Stemming from comparative genomics approaches, the discovery of many cilia/centriole based diseases has helped to illustrate the incredible breath of roles that these organelles play in human health from polycystic kidney disease to respiratory infections to microcephaly to obesity. The key questions include how these microtubule-based structures are assembled and how they function to influence cellular biology, development, and human health. Recently, her lab has become interested in how the substructures in a cilium find the 'right address' in the cilia and centrioles.

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.

Dr Mónica Bettencourt-Dias, Instituto Gulbenkian de Ciência, Portugal

Dr Mónica Bettencourt-Dias, Instituto Gulbenkian de Ciência, Portugal

Mónica Bettencourt-Dias is Director of the Instituto Gulbenkian de Ciência since February 2018. She is also group leader at IGC since 2006. With a PhD in Biochemistry and Molecular Biology by the University College London (UK), Mónica also holds a post graduate degree in Science Communication by the Birkbeck College London. She was awarded two grants from the European Research Council in 2010 and 2015 and was also elected member of the European Molecular Biology Organisation (EMBO).

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.

Professor George Witman, University of Massachussetts Medical School, USA

Professor George Witman, University of Massachussetts Medical School, USA

George Witman is the George F. Booth Chair in the Basic Sciences and a professor in the Division of Cell Biology and Imaging, Department of Radiology, at the University of Massachusetts Medical School. He studies cilia and flagella to understand how they are assembled and function and why defects in specific ciliary proteins cause human diseases. Most of his research utilizes Chlamydomonas reinhardtii, which has major advantages for studies of flagella. Dr Witman pioneered the application of biochemical and molecular approaches to the study of Chlamydomonas flagella.  His current research is focused on discovery and characterization of novel axonemal proteins, and the mechanism of intraflagellar transport (IFT). Advanced techniques used in the Witman lab include super-resolution structured-illumination microscopy, total internal reflection fluorescence microscopy, cryo-electron tomography, and mass spectrometry.

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.

Professor Wallace F Marshall, University of California, San Francisco, USA

Professor Wallace F Marshall, University of California, San Francisco, USA

Wallace Marshall studied Electrical Engineering and Biochemistry at SUNY Stony Brook, and then obtained his PhD at UCSF in the lab of John Sedat, where he analyzed interphase chromatin motion. He then moved to Yale for a postdoc with Joel Rosenbaum where he developed his interest in cilia and flagella. Since 2003 he has been a faculty member at USCF, where he now directs the Center for Cellular Construction. His lab uses a combination of imaging, computational modelling, molecular biology, and genetics, to study fundamental questions of cell geometry, in many cases using cilia and flagella as model organelles.