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1. Intracellular connections between basal bodies promote the coordinated behavior of motile cilia

   https://doi.org/10.1091/mbc.E22-05-0150  

   Soh, Adam W. ; Woodhams, Louis G. ; Junker, Anthony D. ; Enloe, Cassidy M. ; Noren, Benjamin E. ; Harned, Adam ; Westlake, Christopher J. ; Narayan, Kedar ; Oakey, John S. ; Bayly, Philip V. ; et al ( September 2022 , Molecular Biology of the Cell)

   Discher, Dennis (Ed.)

   Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell’s cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces. 

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2. Generation of ciliary beating by steady dynein activity: the effects of inter-filament coupling in multi-filament models

   https://doi.org/10.1098/rsif.2022.0264  

   Woodhams, Louis G. ; Shen, Yenan ; Bayly, Philip V. ( July 2022 , Journal of The Royal Society Interface)

   The structure of the axoneme in motile cilia and flagella is emerging with increasing detail from high-resolution imaging, but the mechanism by which the axoneme creates oscillatory, propulsive motion remains mysterious. It has recently been proposed that this motion may be caused by a dynamic ‘flutter’ instability that can occur under steady dynein loading, and not by switching or modulation of dynein motor activity (as commonly assumed). In the current work, we have built an improved multi-filament mathematical model of the axoneme and implemented it as a system of discrete equations using the finite-element method. The eigenvalues and eigenvectors of this model predict the emergence of oscillatory, wave-like solutions in the absence of dynein regulation and specify the associated frequencies and waveforms of beating. Time-domain simulations with this model illustrate the behaviour predicted by the system's eigenvalues. This model and analysis allow us to efficiently explore the potential effects of difficult to measure biophysical parameters, such as elasticity of radial spokes and inter-doublet links, on the ciliary waveform. These results support the idea that dynamic instability without dynamic dynein regulation is a plausible and robust mechanism for generating ciliary beating. 

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3. Robust acoustic trapping and perturbation of single-cell microswimmers illuminate three-dimensional swimming and ciliary coordination

   https://doi.org/10.1073/pnas.2218951120  

   Cui, Mingyang ; Dutcher, Susan K. ; Bayly, Philip V. ; Meacham, J. Mark ( June 2023 , Proceedings of the National Academy of Sciences)

   We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular algaChlamydomonas reinhardtiiis widely used to investigate the mechanisms underlying ciliary beating and coordination. However, freely swimming cells are difficult to image with sufficient resolution to capture cilia motion, necessitating that the cell body be held during experiments. Acoustic confinement is a compelling alternative to use of a micropipette, or to magnetic, electrical, and optical trapping that may modify the cells and affect their behavior. Beyond establishing our approach to studying microswimmers, we demonstrate a unique ability to mechanically perturb cells via rapid acoustic positioning. 

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4. Self-sustained three-dimensional beating of a model eukaryotic flagellum

   https://doi.org/10.1039/D2SM00514J  

   Rallabandi, Bhargav ; Wang, Qixuan ; Potomkin, Mykhailo ( July 2022 , Soft Matter)

   Flagella and cilia are common features of a wide variety of biological cells and play important roles in locomotion and feeding at the microscale. The beating of flagella is controlled by molecular motors that exert forces along the length of the flagellum and are regulated by a feedback mechanism coupled to the flagella deformation. We develop a three-dimensional (3D) flagellum beating model based on sliding-controlled motor feedback, accounting for both bending and twist, as well as differential bending resistances along and orthogonal to the major bending plane of the flagellum. We show that beating is generated and sustained spontaneously for a sufficiently high motor activity through an instability mechanism. Isotropic bending rigidities in the flagellum lead to 3D helical beating patterns. By contrast, anisotropic flagella present a rich variety of wave-like beating dynamics, including both 3D beating patterns as well as planar beating patterns. We show that the ability to generate nearly planar beating despite the 3D beating machinery requires only a modest degree of bending anisotropy, and is a feature observed in many eukaryotic flagella such as mammalian spermatozoa. 

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5. Evidence for a self‐organized compliant mechanism for the spontaneous steady beating of cilia

   https://doi.org/10.1002/cm.21372  

   Foster, Kenneth W. ; Vidyadharan, Jyothish ; Sangani, Ashok S. ( June 2017 , Cytoskeleton)

   Cilia or eukaryotic flagella are slender 200‐nm‐diameter organelles that move the immersing fluid relative to a cell and sense the environment. Their core structure is nine doublet microtubules (DMTs) arranged around a central‐pair. When motile, thousands of tiny motors slide the DMTs relative to each other to facilitate traveling waves of bending along the cilium's length. These motors provide the energy to change the shape of the cilium and overcome the viscous forces of moving in the surrounding fluid. In planar beating, motors walk toward where the cilium is attached to the cell body. Traveling waves are initiated by motors bending the elastic cilium back and forth, a self‐organized mechanical oscillator. We found remarkably that the energy in a wave is nearly constant over a wide range of (ATP) and medium viscosities and inter‐doublet springs operate only in the central and not in the basal region. Since the energy in a wave does not depend on its rate of formation, the control mechanism is likely purely mechanical. Further the torque per length generated by the motors acting on the doublets is proportional to and nearly in phase with the microtubule sliding velocity with magnitude dependent on the medium. We determined the frequency‐dependent elastic moduli and strain energies of beating cilia. Incorporation of these in an energy‐based model explains the beating frequency, wavelength, limiting of the wave amplitude and the overall energy of the traveling wave. Our model describes the intricacies of the basal‐wave initiation as well as the traveling wave.

    

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