Stochastic force dynamics of the model microswimmer Chlamydomonas reinhardtii: Active forces and energetics.

We study the stochastic force dynamics of a model microswimmer (Chlamydomonas reinhardtii), using a combined experimental, theoretical, and numerical approach. While swimming dynamics have been extensively studied using hydrodynamic approaches, which infer forces from the viscous flow field, we directly measure the stochastic forces generated by the microswimmer using an optical trap via the photon momentum method. We analyze the force dynamics by modeling the microswimmer as a self-propelled particle, à la active matter, and analyze its energetics using methods from stochastic thermodynamics. We find complex oscillatory force dynamics and power dissipation on the order of 10^{6}k_{B}T/s(∼fW).

Quantifying the non-equilibrium activity of an active colloid.

Active matter systems exhibit rich emergent behavior due to constant injection and dissipation of energy at the level of individual agents. Since these systems are far from equilibrium, their dynamics and energetics cannot be understood using the framework of equilibrium statistical mechanics. Recent developments in stochastic thermodynamics extend classical concepts of work, heat, and energy dissipation to fluctuating non-equilibrium systems. We use recent advances in experiment and theory to study the non-thermal dissipation of individual light-activated self-propelled colloidal particles. We focus on characterizing the transition from thermal to non-thermal fluctuations and show that energy dissipation rates on the order of ∼kBT s-1 are measurable from finite time series data.

Active diffusion in oocytes nonspecifically centers large objects during prophase I and meiosis I.

Nucleus centering in mouse oocytes results from a gradient of actin-positive vesicle activity and is essential for developmental success. Here, we analyze 3D model simulations to demonstrate how a gradient in the persistence of actin-positive vesicles can center objects of different sizes. We test model predictions by tracking the transport of exogenous passive tracers. The gradient of activity induces a centering force, akin to an effective pressure gradient, leading to the centering of oil droplets with velocities comparable to nuclear ones. Simulations and experimental measurements show that passive particles subjected to the gradient exhibit biased diffusion toward the center. Strikingly, we observe that the centering mechanism is maintained in meiosis I despite chromosome movement in the opposite direction; thus, it can counteract a process that specifically off-centers the spindle. In conclusion, our findings reconcile how common molecular players can participate in the two opposing functions of chromosome centering versus off-centering.

Small-scale displacement fluctuations of vesicles in fibroblasts.

The intracellular environment is a dynamic space filled with various organelles moving in all directions. Included in this diverse group of organelles are vesicles, which are involved in transport of molecular cargo throughout the cell. Vesicles move in either a directed or non-directed fashion, often depending on interactions with cytoskeletal proteins such as microtubules, actin filaments, and molecular motors. How these proteins affect the local fluctuations of vesicles in the cytoplasm is not clear since they have the potential to both facilitate and impede movement. Here we show that vesicle mobility is significantly affected by myosin-II, even though it is not a cargo transport motor. We find that myosin-II activity increases the effective diffusivity of vesicles and its inhibition facilitates longer states of non-directed motion. Our study suggests that altering myosin-II activity in the cytoplasm of cells can modulate the mobility of vesicles, providing a possible mechanism for cells to dynamically tune the cytoplasmic environment in space and time.

Active Mechanics Reveal Molecular-Scale Force Kinetics in Living Oocytes.

Active diffusion of intracellular components is emerging as an important process in cell biology. This process is mediated by complex assemblies of molecular motors and cytoskeletal filaments that drive force generation in the cytoplasm and facilitate enhanced motion. The kinetics of molecular motors have been precisely characterized in vitro by single molecule approaches, but their in vivo behavior remains elusive. Here, we study the active diffusion of vesicles in mouse oocytes, where this process plays a key role in nuclear positioning during development, and combine an experimental and theoretical framework to extract molecular-scale force kinetics (force, power stroke, and velocity) of the in vivo active process. Assuming a single dominant process, we find that the nonequilibrium activity induces rapid kicks of duration τ ∼ 300 µs resulting in an average force of F ∼ 0.4 pN on vesicles in in vivo oocytes, remarkably similar to the kinetics of in vitro myosin-V. Our results reveal that measuring in vivo active fluctuations allows extraction of the molecular-scale activity in agreement with single-molecule studies and demonstrates a mesoscopic framework to access force kinetics.

Active cell mechanics: Measurement and theory.

Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

Active diffusion positions the nucleus in mouse oocytes.

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

Active transport of vesicles in neurons is modulated by mechanical tension.

Effective intracellular transport of proteins and organelles is critical in cells, and is especially important for ensuring proper neuron functionality. In neurons, most proteins are synthesized in the cell body and must be transported through thin structures over long distances where normal diffusion is insufficient. Neurons transport subcellular cargo along axons and neurites through a stochastic interplay of active and passive transport. Mechanical tension is critical in maintaining proper function in neurons, but its role in transport is not well understood. To this end, we investigate the active and passive transport of vesicles in Aplysia neurons while changing neurite tension via applied strain, and quantify the resulting dynamics. We found that tension in neurons modulates active transport of vesicles by increasing the probability of active motion, effective diffusivity, and induces a retrograde bias. We show that mechanical tension modulates active transport processes in neurons and that external forces can couple to internal (subcellular) forces and change the overall transport dynamics.

Measuring nonequilibrium vesicle dynamics in neurons under tension.

Vesicle transport in neurons is a highly complex nonequilibrium process. Their subcellular environment is undergoing constant fluctuations from thermal energy and molecular motors. Vesicle transport is an interplay between random motion (passive) and directed motion (active) driven by molecular motors along cytoskeletal filaments. It has been shown that growth, guidance, and vesicle dynamics of neurons is affected by mechanical tension. Here we present a method to analyze vesicle transport via a temporal Mean Square Displacement (tMSD) analysis while applying mechanical strain to neurons. The tMSD analysis allows characterization of active and passive vesicle motion as well as many other parameters including: power law scaling, velocity, direction, and flux. Our results suggest: (1) The tMSD analysis is able to capture vesicle motion alternating between passive and active states, and indicates that vesicle motion in Aplysia neurons is primarily passive (exhibiting active motion for ~8% of the time). (2) Under mechanical stretch (increased neurite tension), active transport of vesicles increases to ~13%, while vesicle velocity remains unchanged. (3) Upon unstretching (decreased tension), the level of active transport returns to normal but vesicle velocity decreases. These results suggest that vesicle transport in neurons is highly sensitive to mechanical stimulation. Our method allows precise characterization of vesicle dynamics in response to applied mechanical strain.

Cardiac myocytes' dynamic contractile behavior differs depending on heart segment.

Cardiac myocytes originating from different parts of the heart exhibit varying morphology and ultrastructure. However, the difference in their dynamic behavior is unclear. We examined the contraction of cardiac myocytes originating from the apex, ventricle, and atrium, and found that their dynamic behavior, such as amplitude and frequency of contraction, differs depending on the heart segment of origin. Using video microscopy and high-precision image correlation, we found that: (1) apex myocytes exhibited the highest contraction rate (∼17 beats/min); (2) ventricular myocytes exhibited the highest contraction amplitude (∼5.2 micron); and (3) as myocyte contraction synchronized, their frequency did not change significantly, but the amplitude of contraction increased in apex and ventricular myocytes. In addition, as myocyte cultures mature they formed contractile filaments, further emphasizing the difference in myocyte dynamics is persistent. These results suggest that the dynamic behavior (in addition to static properties) of myocytes is dependent on their segment of origin.

Myoblast morphology and organization on biochemically micro-patterned hydrogel coatings under cyclic mechanical strain.

Mechanical forces and geometric constraints play critical roles in determining cell functionality and tissue development. Novel experimental methods are essential to explore the underlying biological mechanisms of cell response. We present a versatile method to culture cells on adhesive micro-patterned substrates while applying long-term cyclic tensile strain (CTS). A polydimethysiloxane (PDMS) mold is coated with a cell repulsive NCO-sP(EO-stat-PO) hydrogel which in turn is covalently patterned by fibronectin using micro-contact printing. This results in two-dimensional, highly selective cell-adhesive micro-patterns. The substrates allow application of CTS to adherent cells for more than 4 days under cell culture conditions without unspecific adhesion. The applicability of our system is demonstrated by studying the adaptive response of C2C12 skeletal myoblasts seeded on fibronectin lines with different orientations relative to the strain direction. After application of CTS (amplitude of 7%, frequency of 0.5 Hz) we find that actin fiber organization is dominantly controlled by CTS. Nuclei shape is predominantly affected by the constraint of the adhesive lines, resulting in significant elongation. Morphologically, myotube formation was incomplete after 4 days of culture, but actin striations were observed exclusively on the 45 degrees line patterns subjected to CTS, the direction of maximum shear strain.