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Cells are active systems with molecular force generation that drives complex dynamics at the supramolecular scale. We present a quantitative study of molecular motions in cells over times from milliseconds to hours. Noninvasive tracking was accomplished by imaging highly stable near-infrared luminescence of single-walled carbon nanotubes targeted to kinesin-1 motor proteins in COS-7 cells. We observed a regime of active random \"stirring\" that constitutes an intermediate mode of transport, different from both thermal diffusion and directed motor activity. High-frequency motion was found to be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics dominated. In addition to directed transport along microtubules, we observed strong random dynamics driven by myosins that result in enhanced nonspecific transport. We present a quantitative model connecting molecular mechanisms to mesoscopic fluctuations.

Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin–Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions. Significance A single primary cilium extends from the surface of many mammalian cells—often into an aqueous lumen, such as a kidney duct. In kidney epithelial cells, primary cilia are believed to sense fluid flow. This mechanosensory function is critical for proper organ function. Fluid flow is assumed to deflect cilia, leading to activation of transmembrane ion channels. This study defines the mechanical contributions of both bending and pivoting at the base to ciliary deflection. In addition, we report that active intracellular forces drive ciliary pivoting. This cell-directed cilia movement may be important for tuning ciliary mechanosensitivity.

Cells are sensitive to mechanical cues from their environment and at the same time generate and transmit forces to their surroundings. To test quantitatively forces generated by cells not attached to a substrate, we used a dual optical trap to suspend 3T3 fibroblasts between two fibronectin-coated beads. In this simple geometry, we measured both the cells' elastic properties and the force fluctuations they generate with high bandwidth. Cell stiffness decreased substantially with both myosin inhibition by blebbistatin and serum-starvation, but not with microtubule depolymerization by nocodazole. We show that cortical forces generated by non-muscle myosin II deform the cell from its rounded shape in the frequency regime from 0.1 to 10 Hz. The amplitudes of these forces were strongly reduced by blebbistatin and serum starvation, but were unaffected by depolymerization of microtubules. Force fluctuations show a spectrum that is characteristic for an elastic network activated by random sustained stresses with abrupt transitions.

Kinesin‐5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus‐end directed motors. The Saccharomyces cerevisiae kinesin‐5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single‐molecule fluorescence motility assays and live‐cell microscopy. On spindles, Cin8 motors mostly moved slowly (∼25 nm/s) towards the midzone, but occasionally also faster (∼55 nm/s) towards the spindle poles. In vitro , individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus‐end to slow plus‐end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus‐end relative sliding. Between parallel MTs, plus‐end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus‐end motility and affected the ionic strength‐dependent directional switching of Cin8 in vitro . The deletion mutant cells exhibited reduced midzone‐directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8. The yeast kinesin‐5 Cin8 can move bi‐directionally on mitotic spindles and in‐vitro assays provide insights into the molecular basis underlying this directional switching.

Systems in thermodynamic equilibrium are not only characterized by time-independent macroscopic properties, but also satisfy the principle of detailed balance in the transitions between microscopic configurations. Living systems function out of equilibrium and are characterized by directed fluxes through chemical states, which violate detailed balance at the molecular scale. Here we introduce a method to probe for broken detailed balance and demonstrate how such nonequilibrium dynamics are manifest at the mesosopic scale. The periodic beating of an isolated flagellum from Chlamydomonas reinhardtii exhibits probability flux in the phase space of shapes. With a model, we show how the breaking of detailed balance can also be quantified in stationary, nonequilibrium stochastic systems in the absence of periodic motion. We further demonstrate such broken detailed balance in the nonperiodic fluctuations of primary cilia of epithelial cells. Our analysis provides a general tool to identify nonequilibrium dynamics in cells and tissues.

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer’s disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration.

Key Points Single-molecule approaches for studying the dynamic properties of motor proteins have come of age. Recent technical developments allow us to see more details of molecular motions and of the forces that molecules generate. Atomic force microscopy provides the highest available resolution, of about one nanometre, for imaging soft and dynamic motor proteins in action. Recently, significant progress has been made in imaging fragile samples and in high-speed imaging, with rates of up to 25 frames per second being achieved; for example, it is possible to image kinesin motors on top of microtubules with low forces while still being able to resolve single domains of the motor proteins. Fluorescence microscopy is unbeaten in achieving molecular specificity of imaging. Progress has been rapid, especially in single-molecule fluorescence methods. Detectors, which are mostly charged coupled device cameras, are constantly getting more efficient and less noisy. Methods for restricting the sample volume to suppress background noise are becoming increasingly sophisticated. Last, but not least, chemical fluorophores, genetically encoded fluorescent proteins and fluorescent nanoparticles are becoming more versatile, bright and stable. Single-molecule fluorescence experiments in cells remain challenging. Crowding and background fluorescence are difficult to avoid. High hopes are resting on newly developed bright and stable dyes, as well as on fluorescent nanoparticles, especially in the near-infrared spectral range, where cellular background fluorescence is minimal. Optical trapping has been firmly established as a tool of choice when measuring steps or power strokes of motor proteins as well as forces generated by single motors. Optical trapping is being implemented in increasingly sophisticated and powerful ways. The resolution of sub-nanometre steps is possible, time resolution can be as good as microseconds, and controlled forces of piconewtons can be exerted on single molecules in well-controlled geometries. It remains a challenge to apply optical tweezers in cells. Specificity of trapping, as opposed to indiscriminate trapping of various intracellular objects, is hard to achieve, and it is difficult to calibrate force and displacement measurements in cells. Promising developments include the trapping of distinct high-index cellular components, such as lipid droplets, and the use of externally introduced high-index particles, such as gold nanobeads, as well as the exploration of resonantly enhanced trapping. Single-molecule techniques, such as atomic force microscopy, single-molecule fluorescence microscopy and optical tweezers, have helped resolve the mechanisms behind the power strokes, processive steps and forces of cytoskeletal motors. Such techniques might also reveal how motors are integrated into composite mechanical machines to generate complex functions in cells. Much has been learned in the past decades about molecular force generation. Single-molecule techniques, such as atomic force microscopy, single-molecule fluorescence microscopy and optical tweezers, have been key in resolving the mechanisms behind the power strokes, 'processive' steps and forces of cytoskeletal motors. However, it remains unclear how single force generators are integrated into composite mechanical machines in cells to generate complex functions such as mitosis, locomotion, intracellular transport or mechanical sensory transduction. Using dynamic single-molecule techniques to track, manipulate and probe cytoskeletal motor proteins will be crucial in providing new insights.

We tested the mechanical properties of single microtubules by lateral indentation with the tip of an atomic force microscope. Indentations up to ∼3.6 nm, i.e., 15% of the microtubule diameter, resulted in an approximately linear elastic response, and indentations were reversible without hysteresis. At an indentation force of around 0.3 nN we observed an instability corresponding to an ∼1-nm indentation step in the taxol-stabilized microtubules, which could be due to partial or complete rupture of a relatively small number of lateral or axial tubulin-tubulin bonds. These indentations were reversible with hysteresis when the tip was retracted and no trace of damage was observed in subsequent high-resolution images. Higher forces caused substantial damage to the microtubules, which either led to depolymerization or, occasionally, to slowly reannealing holes in the microtubule wall. We modeled the experimental results using finite-element methods and find that the simple assumption of a homogeneous isotropic material, albeit structured with the characteristic protofilament corrugations, is sufficient to explain the linear elastic response of microtubules.

Viral shells are self-assembled protein nanocontainers with remarkable material properties. They combine simplicity of construction with toughness and complex functionality. These properties make them interesting for bionanotechnology. To date we know little about how virus structure determines assembly pathways and shell mechanics. We have here used atomic force microscopy to study structural failure of the shells of the bacteriophage Φ29. We observed rigidity patterns following the symmetry of the capsid proteins. Under prolonged force exertion, we observed fracture along well-defined lines of the 2D crystal lattice. The mechanically most stable building block of the shells was a trimer. Our approach of \"reverse engineering\" the virus shells thus made it possible to identify stable structural intermediates. Such stable intermediates point to a hierarchy of interactions among equal building blocks correlated with distinct next-neighbor interactions. The results also demonstrate that concepts from macroscopic materials science, such as fracture, can be usefully employed in molecular engineering.

X-ray crystallography has revealed an unusual structural element in kinesin-5 motor proteins.