Quantitative videomicroscopy reveals latent control of cell-pair rotations in vivo.

Collective cell rotations are widely used during animal organogenesis. Theoretical and in vitro studies have conceptualized rotating cells as identical rigid-point objects that stochastically break symmetry to move monotonously and perpetually within an inert environment. However, it is unclear whether this notion can be extrapolated to a natural context, where rotations are ephemeral and heterogeneous cellular cohorts interact with an active epithelium. In zebrafish neuromasts, nascent sibling hair cells invert positions by rotating ≤180° around their geometric center after acquiring different identities via Notch1a-mediated asymmetric repression of Emx2. Here, we show that this multicellular rotation is a three-phasic movement that progresses via coherent homotypic coupling and heterotypic junction remodeling. We found no correlation between rotations and epithelium-wide cellular flow or anisotropic resistive forces. Moreover, the Notch/Emx2 status of the cell dyad does not determine asymmetric interactions with the surrounding epithelium. Aided by computer modeling, we suggest that initial stochastic inhomogeneities generate a metastable state that poises cells to move and spontaneous intercellular coordination of the resulting instabilities enables persistently directional rotations, whereas Notch1a-determined symmetry breaking buffers rotational noise.

Strategies for enhancing automatic fixation detection in head-mounted eye tracking.

Moving through a dynamic world, humans need to intermittently stabilize gaze targets on their retina to process visual information. Overt attention being thus split into discrete intervals, the automatic detection of such fixation events is paramount to downstream analysis in many eye-tracking studies. Standard algorithms tackle this challenge in the limiting case of little to no head motion. In this static scenario, which is approximately realized for most remote eye-tracking systems, it amounts to detecting periods of relative eye stillness. In contrast, head-mounted eye trackers allow for experiments with subjects moving naturally in everyday environments. Detecting fixations in these dynamic scenarios is more challenging, since gaze-stabilizing eye movements need to be reliably distinguished from non-fixational gaze shifts. Here, we propose several strategies for enhancing existing algorithms developed for fixation detection in the static case to allow for robust fixation detection in dynamic real-world scenarios recorded with head-mounted eye trackers. Specifically, we consider (i) an optic-flow-based compensation stage explicitly accounting for stabilizing eye movements during head motion, (ii) an adaptive adjustment of algorithm sensitivity according to head-motion intensity, and (iii) a coherent tuning of all algorithm parameters. Introducing a new hand-labeled dataset, recorded with the Pupil Invisible glasses by Pupil Labs, we investigate their individual contributions. The dataset comprises both static and dynamic scenarios and is made publicly available. We show that a combination of all proposed strategies improves standard thresholding algorithms and outperforms previous approaches to fixation detection in head-mounted eye tracking.

Gaze-angle dependency of pupil-size measurements in head-mounted eye tracking.

Pupillometry - the study of temporal changes in pupil diameter as a function of external light stimuli or cognitive processing - requires the accurate and gaze-angle independent measurement of pupil dilation. Expected response amplitudes often are only a few percent relative to a pre-stimulus baseline, thus demanding for sub-millimeter accuracy. Video-based approaches to pupil-size measurement aim at inferring pupil dilation from eye images alone. Eyeball rotation in relation to the recording camera as well as optical effects due to refraction at corneal interfaces can, however, induce so-called pupil foreshortening errors (PFE), i.e. systematic gaze-angle dependent changes of apparent pupil size that are on a par with typical response amplitudes. While PFE and options for its correction have been discussed for remote eye trackers, for head-mounted eye trackers such an assessment is still lacking. In this work, we therefore gauge the extent of PFE in three measurement techniques, all based on eye images recorded with a single near-eye camera. We present both real world experimental data as well as results obtained on synthetically generated eye images. We discuss PFE effects at three different levels of data aggregation: the sample, subject, and population level. In particular, we show that a recently proposed refraction-aware approach employing a mathematical 3D eye model is successful in providing pupil-size measurements which are gaze-angle independent at the population level.

Spontaneous oscillations of elastic contractile materials with turnover.

Single and collective cellular oscillations driven by the actomyosin cytoskeleton have been observed in numerous biological systems. Here, we propose that these oscillations can be accounted for by a generic oscillator model of a material turning over and contracting against an elastic element. As an example, we show that during dorsal closure of the Drosophila embryo, experimentally observed changes in actomyosin concentration and oscillatory cell shape changes can, indeed, be captured by the dynamic equations studied here. We also investigate the collective dynamics of an ensemble of such contractile elements and show that the relative contribution of viscous and friction losses yields different regimes of collective oscillations. Taking into account the diffusion of force-producing molecules between contractile elements, our theoretical framework predicts the appearance of traveling waves, resembling the propagation of actomyosin waves observed during morphogenesis.

Monitoring actin cortex thickness in live cells.

Animal cell shape is controlled primarily by the actomyosin cortex, a thin cytoskeletal network that lies directly beneath the plasma membrane. The cortex regulates cell morphology by controlling cellular mechanical properties, which are determined by network structure and geometry. In particular, cortex thickness is expected to influence cell mechanics. However, cortex thickness is near the resolution limit of the light microscope, making studies relating cortex thickness and cell shape challenging. To overcome this, we developed an assay to measure cortex thickness in live cells, combining confocal imaging and subresolution image analysis. We labeled the actin cortex and plasma membrane with chromatically different fluorophores and measured the distance between the resulting intensity peaks. Using a theoretical description of cortex geometry and microscopic imaging, we extracted an average cortex thickness of ∼190 nm in mitotic HeLa cells and tested the validity of our assay using cell images generated in silico. We found that thickness increased after experimental treatments preventing F-actin disassembly. Finally, we monitored physiological changes in cortex thickness in real-time during actin cortex regrowth in cellular blebs. Our investigation paves the way to understanding how molecular processes modulate cortex structure, which in turn drives cell morphogenesis.

Coupling a sensory hair-cell bundle to cyber clones enhances nonlinear amplification.

The vertebrate ear benefits from nonlinear mechanical amplification to operate over a vast range of sound intensities. The amplificatory process is thought to emerge from active force production by sensory hair cells. The mechano-sensory hair bundle that protrudes from the apical surface of each hair cell can oscillate spontaneously and function as a frequency-selective, nonlinear amplifier. Intrinsic fluctuations, however, jostle the response of a single hair bundle to weak stimuli and seriously limit amplification. Most hair bundles are mechanically coupled by overlying gelatinous structures. Here, we assayed the effects of mechanical coupling on the hair-bundle amplifier by combining dynamic force clamp of a hair bundle from the bullfrog's saccule with real-time stochastic simulations of hair-bundle mechanics. This setup couples the hair bundle to two virtual hair bundles, called cyber clones, and mimics a situation in which the hair bundle is elastically linked to two neighbors with similar characteristics. We found that coupling increased the coherence of spontaneous hair-bundle oscillations. By effectively reducing noise, the synergic interplay between the hair bundle and its cyber clones also enhanced amplification of sinusoidal stimuli. All observed effects of coupling were in quantitative agreement with simulations. We argue that the auditory amplifier relies on hair-bundle cooperation to overcome intrinsic noise limitations and achieve high sensitivity and sharp frequency selectivity.

Enhancement of sensitivity gain and frequency tuning by coupling of active hair bundles.

The vertebrate inner ear possesses an active process that provides nonlinear amplification of mechanical stimuli. A candidate for this process is active hair bundle mechanics observed, for instance, for hair cells of the bullfrog's sacculus. Hair bundles in various inner ear organs are coupled by overlying membranes. Using a stochastic description of active hair bundle dynamics, we study the consequences of an elastic coupling on the properties of amplification. We report that collective effects in arrays of hair bundles can enhance the amplification gain and the sharpness of frequency tuning as compared with the performance of an isolated hair bundle. We also discuss the transient response elicited by the sudden onset of a periodic stimulus and its relation to temporal integration curves. Simulations of systems with a gradient of intrinsic frequencies show an enhanced amplification gain while preserving a frequency gradient, provided the coupling strength is similar to the hair bundle stiffness. We relate our findings to the situation in the bullfrog's sacculus and the mammalian cochlea.

Local exponents of nonlinear compression in periodically driven noisy oscillators.

Nonlinear compression of periodic signals is a key feature of the active amplifier in inner ear organs of all vertebrates. Different exponents alpha(0) in [-0.88,-0.5] of the sensitivity vs forcing amplitude |chi| approximately f(alpha(0)) have been observed. Here we calculate analytically the local exponent for a generic oscillator, the normal form of a Hopf bifurcation driven by noise and a periodic signal. For weak noise and sufficient distance from the bifurcation on the unstable side, the exponent may be close to -1 for moderate forcing amplitudes beyond linear response. Such strong compression is also found in a model of hair bundle motility.

In Vivo Force Application Reveals a Fast Tissue Softening and External Friction Increase during Early Embryogenesis.

During development, cell-generated forces induce tissue-scale deformations to shape the organism [1,2]. The pattern and extent of these deformations depend not solely on the temporal and spatial profile of the generated force fields but also on the mechanical properties of the tissues that the forces act on. It is thus conceivable that, much like the cell-generated forces, the mechanical properties of tissues are modulated during development in order to drive morphogenesis toward specific developmental endpoints. Although many approaches have recently emerged to assess effective mechanical parameters of tissues [3-8], they could not quantitatively relate spatially localized force induction to tissue-scale deformations in vivo. Here, we present a method that overcomes this limitation. Our approach is based on the application of controlled forces on a single microparticle embedded in an individual cell of an embryo. Combining measurements of bead displacement with the analysis of induced deformation fields in a continuum mechanics framework, we quantify material properties of the tissue and follow their changes over time. In particular, we uncover a rapid change in tissue response occurring during Drosophila cellularization, resulting from a softening of the blastoderm and an increase of external friction. We find that the microtubule cytoskeleton is a major contributor to epithelial mechanics at this stage. We identify developmentally controlled modulations in perivitelline spacing that can account for the changes in friction. Overall, our method allows for the measurement of key mechanical parameters governing tissue-scale deformations and flows occurring during morphogenesis.

Adherens Junction Length during Tissue Contraction Is Controlled by the Mechanosensitive Activity of Actomyosin and Junctional Recycling.

During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. Here, we show that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. We reveal that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis.

Actin cortex architecture regulates cell surface tension.

Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell-cycle progression. We then show that the actin filament length regulators CFL1, CAPZB and DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation.

Patterned Contractile Forces Promote Epidermal Spreading and Regulate Segment Positioning during Drosophila Head Involution.

Epithelial spreading is a fundamental mode of tissue rearrangement occurring during animal development and wound closure. It has been associated either with the collective migration of cells [1, 2] or with actomyosin-generated forces acting at the leading edge (LE) and pulling the epithelial tissue [3, 4]. During the process of Drosophila head involution (HI), the epidermis spreads anteriorly to envelope the head tissues and fully cover the embryo [5]. This results in epidermal segments of equal width that will give rise to the different organs of the fly [6]. Here we perform a quantitative analysis of tissue spreading during HI. Combining high-resolution live microscopy with laser microsurgery and genetic perturbations, we show that epidermal movement is in part, but not solely, driven by a contractile actomyosin cable at the LE. Additional driving forces are generated within each segment by a gradient of actomyosin-based circumferential tension. Interfering with Hedgehog (Hh) signaling can modulate this gradient, thus suggesting the involvement of polarity genes in the regulation of HI. In particular, we show that disruption of these contractile forces alters segment widths and leads to a mispositioning of segments. Within the framework of a physical description, we confirm that given the geometry of the embryo, a patterned profile of active circumferential tensions can indeed generate propelling forces and control final segment position. Our study thus unravels a mechanism by which patterned tensile forces can regulate spreading and positioning of epithelial tissues.

Spontaneous voltage oscillations and response dynamics of a Hodgkin-Huxley type model of sensory hair cells.

We employ a Hodgkin-Huxley type model of basolateral ionic currents in bullfrog saccular hair cells to study the genesis of spontaneous voltage oscillations and their role in shaping the response of the hair cell to external mechanical stimuli. Consistent with recent experimental reports, we find that the spontaneous dynamics of the model can be categorized using conductance parameters of calcium activated potassium, inward rectifier potassium, and mechano-electrical transduction ionic currents. The model is demonstrated to exhibit a broad spectrum of autonomous rhythmic activity, including periodic and quasiperiodic oscillations with two independent frequencies as well as various regular and chaotic bursting patterns. Complex patterns of spontaneous oscillations in the model emerge at small values of the conductance of Ca(2+) activated potassium currents. These patterns are significantly affected by thermal fluctuations of the mechano-electrical transduction current. We show that self-sustained regular voltage oscillations lead to enhanced and sharply tuned sensitivity of the hair cell to weak mechanical periodic stimuli. While regimes of chaotic oscillations are argued to result in poor tuning to sinusoidal driving, chaotically oscillating cells do provide a high sensitivity to low-frequency variations of external stimuli.

WITHDRAWN: Coupled hair bundles could endow the cochlear amplifier with sharp frequency tuning and nonlinear compression.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

WITHDRAWN: Coupled hair bundles could endow the cochlear amplifier with sharp frequency tuning and nonlinear compression.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.