Nucleotide-free structures of KIF20A illuminate atypical mechanochemistry in this kinesin-6.

KIF20A is a critical kinesin for cell division and a promising anti-cancer drug target. The mechanisms underlying its cellular roles remain elusive. Interestingly, unusual coupling between the nucleotide- and microtubule-binding sites of this kinesin-6 has been reported, but little is known about how its divergent sequence leads to atypical motility properties. We present here the first high-resolution structure of its motor domain that delineates the highly unusual structural features of this motor, including a long L6 insertion that integrates into the core of the motor domain and that drastically affects allostery and ATPase activity. Together with the high-resolution cryo-electron microscopy microtubule-bound KIF20A structure that reveals the microtubule-binding interface, we dissect the peculiarities of the KIF20A sequence that influence its mechanochemistry, leading to low motility compared to other kinesins. Structural and functional insights from the KIF20A pre-power stroke conformation highlight the role of extended insertions in shaping the motor's mechanochemical cycle. Essential for force production and processivity is the length of the neck linker in kinesins. We highlight here the role of the sequence preceding the neck linker in controlling its backward docking and show that a neck linker four times longer than that in kinesin-1 is required for the activity of this motor.

Minimal vertex model explains how the amnioserosa avoids fluidization during Drosophila dorsal closure.

Dorsal closure is a process that occurs during embryogenesis of Drosophila melanogaster. During dorsal closure, the amnioserosa (AS), a one-cell thick epithelial tissue that fills the dorsal opening, shrinks as the lateral epidermis sheets converge and eventually merge. During this process, the aspect ratio of amnioserosa cells increases markedly. The standard 2-dimensional vertex model, which successfully describes tissue sheet mechanics in multiple contexts, would in this case predict that the tissue should fluidize via cell neighbor changes. Surprisingly, however, the amnioserosa remains an elastic solid with no such events. We here present a minimal extension to the vertex model that explains how the amnioserosa can achieve this unexpected behavior. We show that continuous shrink-age of the preferred cell perimeter and cell perimeter polydispersity lead to the retention of the solid state of the amnioserosa. Our model accurately captures measured cell shape and orientation changes and predicts non-monotonic junction tension that we confirm with laser ablation experiments.

Minimal vertex model explains how the amnioserosa avoids fluidization during Drosophila dorsal closure.

Dorsal closure is a process that occurs during embryogenesis of Drosophila melanogaster . During dorsal closure, the amnioserosa (AS), a one-cell thick epithelial tissue that fills the dorsal opening, shrinks as the lateral epidermis sheets converge and eventually merge. During this process, the aspect ratio of amnioserosa cells increases markedly. The standard 2-dimensional vertex model, which successfully describes tissue sheet mechanics in multiple contexts, would in this case predict that the tissue should fluidize via cell neighbor changes. Surprisingly, however, the amnioserosa remains an elastic solid with no such events. We here present a minimal extension to the vertex model that explains how the amnioserosa can achieve this unexpected behavior. We show that continuous shrinkage of the preferred cell perimeter and cell perimeter polydispersity lead to the retention of the solid state of the amnioserosa. Our model accurately captures measured cell shape and orientation changes and predicts non-monotonic junction tension that we confirm with laser ablation experiments. Significance Statement: During embryogenesis, cells in tissues can undergo significant shape changes. Many epithelial tissues fluidize, i.e. cells exchange neighbors, when the average cell aspect ratio increases above a threshold value, consistent with the standard vertex model. During dorsal closure in Drosophila melanogaster , however, the amnioserosa tissue remains solid even as the average cell aspect ratio increases well above threshold. We introduce perimeter polydispersity and allow the preferred cell perimeters, usually held fixed in vertex models, to decrease linearly with time as seen experimentally. With these extensions to the standard vertex model, we capture experimental observations quantitatively. Our results demonstrate that vertex models can describe the behavior of the amnioserosa in dorsal closure by allowing normally fixed parameters to vary with time.

DeepProjection: specific and robust projection of curved 2D tissue sheets from 3D microscopy using deep learning.

The efficient extraction of image data from curved tissue sheets embedded in volumetric imaging data remains a serious and unsolved problem in quantitative studies of embryogenesis. Here, we present DeepProjection (DP), a trainable projection algorithm based on deep learning. This algorithm is trained on user-generated training data to locally classify 3D stack content, and to rapidly and robustly predict binary masks containing the target content, e.g. tissue boundaries, while masking highly fluorescent out-of-plane artifacts. A projection of the masked 3D stack then yields background-free 2D images with undistorted fluorescence intensity values. The binary masks can further be applied to other fluorescent channels or to extract local tissue curvature. DP is designed as a first processing step than can be followed, for example, by segmentation to track cell fate. We apply DP to follow the dynamic movements of 2D-tissue sheets during dorsal closure in Drosophila embryos and of the periderm layer in the elongating Danio embryo. DeepProjection is available as a fully documented Python package.

Multiscale Microrheology Using Fluctuating Filaments as Stealth Probes.

The mechanical properties of soft materials can be probed on small length scales by microrheology. A common approach tracks fluctuations of micrometer-sized beads embedded in the medium to be characterized. This approach yields results that depend on probe size when the medium has structure on comparable length scales. Here, we introduce filament-based microrheology using high-aspect-ratio semiflexible filaments as probes. Such quasi-1D probes are much less invasive than beads due to their small cross sections. Moreover, by imaging transverse bending modes, we simultaneously determine the micromechanical response of the medium on multiple length scales corresponding to the mode wavelengths. We use semiflexible single-walled carbon nanotubes as probes that can be accurately and rapidly imaged based on their stable near-IR fluorescence. We find that the viscoelastic properties of sucrose, polyethylene oxide, and hyaluronic acid solutions measured in this way are in good agreement with those measured by conventional micro- and macrorheology.

Mapping Activity-Dependent Quasi-stationary States of Mitochondrial Membranes with Graphene-Induced Energy Transfer Imaging.

Graphene-induced energy transfer (GIET) was recently introduced for sub-nanometric axial localization of fluorescent molecules. GIET relies on near-field energy transfer from an optically excited fluorophore to a single sheet of graphene. Recently, we demonstrated its potential by determining the distance between two leaflets of supported lipid bilayers. Here, we use GIET imaging for mapping quasi-stationary states of the inner and outer mitochondrial membranes before and during adenosine triphosphate (ATP) synthesis. We trigger the ATP synthesis state in vitro by activating mitochondria with precursor molecules. Our results demonstrate that the inner membrane approaches the outer membrane, while the outer membrane does not show any measurable change in average axial position upon activation. The inter-membrane space is reduced by ∼2 nm. This direct experimental observation of the subtle dynamics of mitochondrial membranes and the change in intermembrane distance upon activation is relevant for our understanding of mitochondrial function.

Length-Selective Dielectrophoretic Manipulation of Single-Walled Carbon Nanotubes.

Single-walled carbon nanotubes (SWNTs) possess unique physical, optical, and electrical properties with great potential for future nanoscale device applications. Common synthesis procedures yield SWNTs with large length polydispersity and varying chirality. Electrical and optical applications of SWNTs often require specific lengths, but the preparation of SWNTs with the desired length is still challenging. Insulator-based dielectrophoresis (iDEP) integrated into a microfluidic device has the potential to separate SWNTs by length. Semiconducting SWNTs of varying length suspended with sodium deoxycholate (NaDOC) show unique dielectrophoretic properties at low frequencies (<1 kHz) that were exploited here using an iDEP-based microfluidic constriction sorter device for length-based sorting. Specific migration directions in the constriction sorter were induced for long SWNTs (≥1000 nm) with negative dielectrophoretic properties compared to short (≤300 nm) SWNTs with positive dielectrophoretic properties. We report continuous fractionation conditions for length-based iDEP migration of SWNTs, and we characterize the dynamics of migration of SWNTs in the microdevice using a finite element model. Based on the length and dielectrophoretic characteristics, sorting efficiencies for long and short SWNTs recovered from separate channels of the constriction sorter amounted to >90% and were in excellent agreement with a numerical model for the sorting process.

Chromophore-Independent Roles of Opsin Apoproteins in Drosophila Mechanoreceptors.

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.

Reply to the 'Comment on "A symmetrical method to obtain shear moduli from microrheology"' by M. Tassieri, Soft Matter, 2018, 14, DOI.

The Comment on our paper introducing "a symmetric method to obtain shear moduli from microrheology" proposes an interpolation method to generate oversampled data from an original time series that are then used to approximate shear moduli at frequencies "beyond the Nyquist frequency." The author states that this can be done without the use of "preconceived fitting functions," implying that the results are unique and reliable. We disagree with these assertions. While it is possible to generate reasonable looking transforms at frequencies above the Nyquist limit by interpolation, any results obtained above the Nyquist limit will be questionable at best. Moreover, while the cubic spline interpolation the author uses may be standard, it constitutes a particular "preconceived" fit and produces oversampled data that are not unique.

Self-organized stress patterns drive state transitions in actin cortices.

Biological functions rely on ordered structures and intricately controlled collective dynamics. This order in living systems is typically established and sustained by continuous dissipation of energy. The emergence of collective patterns of motion is unique to nonequilibrium systems and is a manifestation of dynamic steady states. Mechanical resilience of animal cells is largely controlled by the actomyosin cortex. The cortex provides stability but is, at the same time, highly adaptable due to rapid turnover of its components. Dynamic functions involve regulated transitions between different steady states of the cortex. We find that model actomyosin cortices, constructed to maintain turnover, self-organize into distinct nonequilibrium steady states when we vary cross-link density. The feedback between actin network structure and organization of stress-generating myosin motors defines the symmetries of the dynamic steady states. A marginally cross-linked state displays divergence-free long-range flow patterns. Higher cross-link density causes structural symmetry breaking, resulting in a stationary converging flow pattern. We track the flow patterns in the model actomyosin cortices using fluorescent single-walled carbon nanotubes as novel probes. The self-organization of stress patterns we have observed in a model system can have direct implications for biological functions.

A symmetrical method to obtain shear moduli from microrheology.

Passive microrheology typically deduces shear elastic loss and storage moduli from displacement time series or mean-squared displacements (MSD) of thermally fluctuating probe particles in equilibrium materials. Common data analysis methods use either Kramers-Kronig (KK) transformation or functional fitting to calculate frequency-dependent loss and storage moduli. We propose a new analysis method for passive microrheology that avoids the limitations of both of these approaches. In this method, we determine both real and imaginary components of the complex, frequency-dependent response function χ(ω) = χ'(ω) + iχ''(ω) as direct integral transforms of the MSD of thermal particle motion. This procedure significantly improves the high-frequency fidelity of χ(ω) relative to the use of KK transformation, which has been shown to lead to artifacts in χ'(ω). We test our method on both model and experimental data. Experiments were performed on solutions of worm-like micelles and dilute collagen solutions. While the present method agrees well with established KK-based methods at low frequencies, we demonstrate significant improvement at high frequencies using our symmetric analysis method, up to almost the fundamental Nyquist limit.

Topology determines force distributions in one-dimensional random spring networks.

Networks of elastic fibers are ubiquitous in biological systems and often provide mechanical stability to cells and tissues. Fiber-reinforced materials are also common in technology. An important characteristic of such materials is their resistance to failure under load. Rupture occurs when fibers break under excessive force and when that failure propagates. Therefore, it is crucial to understand force distributions. Force distributions within such networks are typically highly inhomogeneous and are not well understood. Here we construct a simple one-dimensional model system with periodic boundary conditions by randomly placing linear springs on a circle. We consider ensembles of such networks that consist of N nodes and have an average degree of connectivity z but vary in topology. Using a graph-theoretical approach that accounts for the full topology of each network in the ensemble, we show that, surprisingly, the force distributions can be fully characterized in terms of the parameters (N,z). Despite the universal properties of such (N,z) ensembles, our analysis further reveals that a classical mean-field approach fails to capture force distributions correctly. We demonstrate that network topology is a crucial determinant of force distributions in elastic spring networks.

Topology Counts: Force Distributions in Circular Spring Networks.

Filamentous polymer networks govern the mechanical properties of many biological materials. Force distributions within these networks are typically highly inhomogeneous, and, although the importance of force distributions for structural properties is well recognized, they are far from being understood quantitatively. Using a combination of probabilistic and graph-theoretical techniques, we derive force distributions in a model system consisting of ensembles of random linear spring networks on a circle. We show that characteristic quantities, such as the mean and variance of the force supported by individual springs, can be derived explicitly in terms of only two parameters: (i) average connectivity and (ii) number of nodes. Our analysis shows that a classical mean-field approach fails to capture these characteristic quantities correctly. In contrast, we demonstrate that network topology is a crucial determinant of force distributions in an elastic spring network. Our results for 1D linear spring networks readily generalize to arbitrary dimensions.

Mechanical Properties of a Drosophila Larval Chordotonal Organ.

Proprioception is an integral part of the feedback circuit that is essential for locomotion control in all animals. Chordotonal organs perform proprioceptive and other mechanosensory functions in insects and crustaceans. The mechanical properties of these organs are believed to be adapted to the sensory functions, but had not been probed directly. We measured mechanical properties of a particular chordotonal organ-the lateral pentascolopidial (lch5) organ of Drosophila larvae-which plays a key role in proprioceptive locomotion control. We applied tension to the whole organ in situ by transverse deflection. Upon release of force, the organ displayed overdamped relaxation with two widely separated time constants, tens of milliseconds and seconds, respectively. When the muscles covering the lch5 organ were excised, the slow relaxation was absent, and the fast relaxation became faster. Interestingly, most of the strain in the stretched organ is localized in the cap cells, which account for two-thirds of the length of the entire organ, and could be stretched by ∼10% without apparent damage. In laser ablation experiments we found that cap cells retracted by ∼100 µm after being severed from the neurons, indicating considerable steady-state stress and strain in these cells. Given the fact that actin as well as myosin motors are abundant in cap cells, the results point to a mechanical regulatory role of the cap cells in the lch5 organ.

Single-Walled Carbon Nanotubes Probed with Insulator-Based Dielectrophoresis.

Single-walled carbon nanotubes (SWNTs) offer unique electrical and optical properties. Common synthesis processes yield SWNTs with large length polydispersity (several tens of nanometers up to centimeters) and heterogeneous electrical and optical properties. Applications often require suitable selection and purification. Dielectrophoresis is one manipulation method for separating SWNTs based on dielectric properties and geometry. Here, we present a study of surfactant and single-stranded DNA-wrapped SWNTs suspended in aqueous solutions manipulated by insulator-based dielectrophoresis (iDEP). This method allows us to manipulate SWNTs with the help of arrays of insulating posts in a microfluidic device around which electric field gradients are created by the application of an electric potential to the extremities of the device. Semiconducting SWNTs were imaged during dielectrophoretic manipulation with fluorescence microscopy making use of their fluorescence emission in the near IR. We demonstrate SWNT trapping at low-frequency alternating current (AC) electric fields with applied potentials not exceeding 1000 V. Interestingly, suspended SWNTs showed both positive and negative dielectrophoresis, which we attribute to their ζ potential and the suspension properties. Such behavior agrees with common theoretical models for nanoparticle dielectrophoresis. We further show that the measured ζ potentials and suspension properties are in excellent agreement with a numerical model predicting the trapping locations in the iDEP device. This study is fundamental for the future application of low-frequency AC iDEP for technological applications of SWNTs.

Feedback-tracking microrheology in living cells.

Living cells are composed of active materials, in which forces are generated by the energy derived from metabolism. Forces and structures self-organize to shape the cell and drive its dynamic functions. Understanding the out-of-equilibrium mechanics is challenging because constituent materials, the cytoskeleton and the cytosol, are extraordinarily heterogeneous, and their physical properties are strongly affected by the internally generated forces. We have analyzed dynamics inside two types of eukaryotic cells, fibroblasts and epithelial-like HeLa cells, with simultaneous active and passive microrheology using laser interferometry and optical trapping technology. We developed a method to track microscopic probes stably in cells in the presence of vigorous cytoplasmic fluctuations, by using smooth three-dimensional (3D) feedback of a piezo-actuated sample stage. To interpret the data, we present a theory that adapts the fluctuation-dissipation theorem (FDT) to out-of-equilibrium systems that are subjected to positional feedback, which introduces an additional nonequilibrium effect. We discuss the interplay between material properties and nonthermal force fluctuations in the living cells that we quantify through the violations of the FDT. In adherent fibroblasts, we observed a well-known polymer network viscoelastic response where the complex shear modulus scales as G* ∝ (-iω)3/4. In the more 3D confluent epithelial cells, we found glassy mechanics with G* ∝ (-iω)1/2 that we attribute to glassy dynamics in the cytosol. The glassy state in living cells shows characteristics that appear distinct from classical glasses and unique to nonequilibrium materials that are activated by molecular motors.

Sulfo-SMCC Prevents Annealing of Taxol-Stabilized Microtubules In Vitro.

Microtubule structure and functions have been widely studied in vitro and in cells. Research has shown that cysteines on tubulin play a crucial role in the polymerization of microtubules. Here, we show that blocking sulfhydryl groups of cysteines in taxol-stabilized polymerized microtubules with a commonly used chemical crosslinker prevents temporal end-to-end annealing of microtubules in vitro. This can dramatically affect the length distribution of the microtubules. The crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfo-SMCC, consists of a maleimide and an N-hydroxysuccinimide ester group to bind to sulfhydryl groups and primary amines, respectively. Interestingly, addition of a maleimide dye alone does not show the same interference with annealing in stabilized microtubules. This study shows that the sulfhydryl groups of cysteines of tubulin that are vital for the polymerization are also important for the subsequent annealing of microtubules.

Game of Zones: how actin-binding proteins organize muscle contraction.

Locomotion of C. elegans requires coordinated, efficient transmission of forces generated on the molecular scale by myosin and actin filaments in myocytes to dense bodies and the hypodermis and cuticle enveloping body wall muscles. The complex organization of the acto-myosin scaffold with its accessory proteins provides a fine-tuned machinery regulated by effectors that guarantees that sarcomere units undergo controlled, reversible cycles of contraction and relaxation. Actin filaments in sarcomeres dynamically undergo polymerization and depolymerization. In a recent study, the actin-binding protein DBN-1, the C. elegans ortholog of human drebrin and drebrin-like proteins, was discovered to stabilize actin in muscle cells. DBN-1 reversibly changes location between actin filaments and myosin-rich regions during muscle contraction. Mutations in DBN-1 result in mislocalization of other actin-binding proteins. Here we discuss implications of this finding for the regulation of sarcomere actin stability and the organization of other actin-binding proteins.