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In nature, several ciliated protists possess the remarkable ability to execute ultrafast motions using protein assemblies called myonemes, which contract in response to Ca2+ions. Existing theories, such as actomyosin contractility and macroscopic biomechanical latches, do not adequately describe these systems, necessitating new models to understand their mechanisms. In this study, we image and quantitatively analyze the contractile kinematics observed in two ciliated protists (Vorticella spandSpirostomum sp), and, based on the mechanochemistry of these organisms, we propose a minimal mathematical model that reproduces our observations as well as those published previously. Analyzing the model reveals three distinct dynamic regimes, differentiated by the rate of chemical driving and the importance of inertia. We characterize their unique scaling behaviors and kinematic signatures. Besides providing insights into Ca2+-powered myoneme contraction in protists, our work may also inform the rational design of ultrafast bioengineered systems such as active synthetic cells.

The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeastSchizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak non-ergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, cytoskeletal effects, cell cycle stage and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not “well-mixed” but represents a highly heterogeneous environment in which subcellular components at the 40-nm sizescale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.SignificanceBiophysical properties of the cytoplasm influence many cellular processes, from differentiation to cytoskeletal dynamics, yet little is known about how tightly cells control these properties. We developed a combined experimental and computational approach to analyze cytoplasmic heterogeneity through the lens of diffusion. We find that the apparent cytoplasmic viscosity varies tremendously – over 100-fold within any individual cell, and over 10-fold among individual cells when comparing averages of all particles measured for each cell. The variance was largely independent of temperature, the cytoskeleton, cell cycle stage, and localization, but was magnified under hyperosmotic shock. This suggests that cytoplasmic heterogeneity contributes substantially to biological variability within and between cells, and has significant implications for any cellular process that depends on diffusion.Graphical abstract

International audience; The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functionsin vivoremain poorly understood. Here, we probed the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that both the rates of microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, while a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowdingper se. We reconstituted these effects on microtubulesin vitroby tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of cytoplasm modulates the reactions underlying microtubule dynamic behaviors.

Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-end-binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana. However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution stimulated emission depletion (STED) microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules. This article has an associated First Person interview with the first author of the paper.