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Fluorescent reporters of cardiac electrophysiology provide valuable information on heart cell and tissue function. However, motion artifacts caused by cardiac muscle contraction interfere with accurate measurement of fluorescence signals. Although drugs such as blebbistatin can be applied to stop cardiac tissue from contracting by uncoupling calcium-contraction, their usage prevents the study of excitation-contraction coupling and, as we show, impacts cellular structure. We therefore developed a robust method to remove motion computationally from images of contracting cardiac muscle and to map fluorescent reporters of cardiac electrophysiological activity onto images of undeformed tissue. When validated on cardiomyocytes derived from human induced pluripotent stem cells (iPSCs), in both monolayers and engineered tissues, the method enabled efficient and robust reduction of motion artifact. As with pharmacologic approaches using blebbistatin for motion removal, our algorithm improved the accuracy of optical mapping, as demonstrated by spatial maps of calcium transient decay. However, unlike pharmacologic motion removal, our computational approach allowed direct analysis of calcium-contraction coupling. Results revealed calcium-contraction coupling to be more uniform across cells within engineered tissues than across cells in monolayer culture. The algorithm shows promise as a robust and accurate tool for optical mapping studies of excitation-contraction coupling in heart tissue.
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Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Humanos , Artefactos , Calcio , Programas Informáticos , Calcio de la Dieta , ColorantesRESUMEN
The axoneme is an intricate nanomachine responsible for generating the propulsive oscillations of cilia and flagella in an astonishing variety of organisms. New imaging techniques based on cryoelectron-tomography (cryo-ET) and subtomogram averaging have revealed the detailed structures of the axoneme and its components with sub-nm resolution, but the mechanical function of each component and how the assembly generates oscillations remains stubbornly unclear. Most explanations of oscillatory behavior rely on the dynamic regulation of dynein by some signal, but this may not be necessary if the system of dynein-driven slender filaments is dynamically unstable. Understanding the possibility of instability-driven oscillations requires a multifilament model of the axoneme that accounts for distortions of the axoneme as it bends. Active bending requires forces and bending moments that will tend to change the spacing and alignment of doublets. We hypothesize that components of the axoneme resist and respond to these loads in ways that are critical to beating. Specifically, we propose (i) that radial spokes provide torsional stiffness by resisting misalignment (as well as spacing) between the central pair and outer doublets, and (ii) that the kinematics of dynein arms affect the relationships between active forces and bending moments on deforming doublets. These proposed relationships enhance the ability of theoretical, multifilament models of axonemal beating to generate propulsive oscillatory waveforms via dynamic mechanical instability.
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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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Dineínas , Modelos Biológicos , Axonema/metabolismo , Cilios/metabolismo , Dineínas/química , Flagelos/fisiologíaRESUMEN
Primary cilia are slender cell-surface organelles that project into the intercellular space. In pancreatic beta cells, primary cilia coordinate a variety of cell responses including GPCR signaling, calcium influx, and insulin secretion, along with likely many underappreciated roles in islet development and differentiation. To study cilia function in islet biology, direct visualization of primary cilia by microscopic methods is often a necessary first step. Ciliary abundance, distribution, and morphology are heterogeneous among islet cells and are best visualized by fluorescence microscopy, the tools for which are readily accessible to most researchers. Here we present a collection of fluorescence imaging methods that we have adopted and optimized for the observation of primary cilia in mouse and human islets. These include conventional confocal microscopy using fixed islets and pancreas sections, live-cell imaging with cilia-targeted biosensors and probes, cilia motion recordings, and quantitative analysis of primary cilia waveform in the ex vivo environment. We discuss practical considerations and limitations of our approaches as well as new tools on the horizon to facilitate the observation of primary cilia in pancreatic islets.
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Células Secretoras de Insulina , Islotes Pancreáticos , Animales , Cilios/metabolismo , Humanos , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/diagnóstico por imagen , Ratones , Imagen ÓpticaRESUMEN
Motile cilia beat with an asymmetric waveform consisting of a power stroke that generates a propulsive force and a recovery stroke that returns the cilium back to the start. Cilia are anchored to the cell cortex by basal bodies (BBs) that are directly coupled to the ciliary doublet microtubules (MTs). We find that, consistent with ciliary forces imposing on BBs, bending patterns in BB triplet MTs are responsive to ciliary beating. BB bending varies as environmental conditions change the ciliary waveform. Bending occurs where striated fibers (SFs) attach to BBs and mutants with short SFs that fail to connect to adjacent BBs exhibit abnormal BB bending, supporting a model in which SFs couple ciliary forces between BBs. Finally, loss of the BB stability protein Poc1, which helps interconnect BB triplet MTs, prevents the normal distributed BB and ciliary bending patterns. Collectively, BBs experience ciliary forces and manage mechanical coupling of these forces to their surrounding cellular architecture for normal ciliary beating.
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Cuerpos Basales , Cilios , Cuerpos Basales/metabolismo , Cilios/metabolismo , Microtúbulos/metabolismo , Fenómenos MecánicosRESUMEN
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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Cilióforos , Tetrahymena thermophila , Cuerpos Basales , Cilios , Fenómenos MecánicosRESUMEN
Primary cilia are specialized cell-surface organelles that mediate sensory perception and, in contrast to motile cilia and flagella, are thought to lack motility function. Here, we show that primary cilia in human and mouse pancreatic islets exhibit movement that is required for glucose-dependent insulin secretion. Islet primary cilia contain motor proteins conserved from those found in classic motile cilia, and their three-dimensional motion is dynein-driven and dependent on adenosine 5'-triphosphate and glucose metabolism. Inhibition of cilia motion blocks beta cell calcium influx and insulin secretion. Human beta cells have enriched ciliary gene expression, and motile cilia genes are altered in type 2 diabetes. Our findings redefine primary cilia as dynamic structures having both sensory and motile function and establish that pancreatic islet cilia movement plays a regulatory role in insulin secretion.