Modeling and correction of image drift in dynamic shadowgraphy experiments.

The study of phoretic transport phenomena under non-stationary conditions presents several challenges, mostly related to the stability of the experimental apparatus. This is particularly true when investigating with optical means the subtle temperature and concentration fluctuations that arise during diffusion processes, superimposed to the macroscopic state of the system. Under these conditions, the tenuous signal from fluctuations is easily altered by the presence of artifacts. Here, we address an experimental issue frequently reported in the investigation by means of dynamic shadowgraphy of the non-equilibrium fluctuations arising in liquid mixtures under non-stationary conditions, such as those arising after the imposition or removal of a thermal stress, where experiments show systematically the presence of a spurious contribution in the reconstructed structure function of the fluctuations, which depends quadratically from the time delay. We clarify the mechanisms responsible for this artifact, showing that it is caused by the imperfect alignment of the sample cell with respect to gravity, which couples the temporal evolution of the concentration profile within the sample with the optical signal collected by the shadowgraph diagnostics. We propose a data analysis protocol that enables disentangling the spurious contributions and the genuine dynamics of the fluctuations, which can be thus reliably reconstructed.

Dynamics of anisotropic colloidal systems: What to choose, DLS, DDM or XPCS?

Investigation of the dynamics of colloids in bulk can be hindered by issues such as multiple scattering and sample opacity. These challenges are exacerbated when dealing with inorganic materials. In this study, we employed a model system of Akaganeite colloidal rods to assess three leading dynamics measurement techniques: 3D-(depolarized) dynamic light scattering (3D-(D)DLS), polarized-differential dynamic microscopy (P-DDM), and x-ray photon correlation spectroscopy (XPCS). Our analysis revealed that the translational and rotational diffusion coefficients captured by these methods show a remarkable alignment. Additionally, by examining the q-ranges and maximum volume fractions for each approach, we offer insights into the best technique for investigating the dynamics of anisotropic systems at the colloidal scale.

Pronking and bounding allow a fast escape across a grassland populated by scattered obstacles.

Some quadrupeds have evolved the ability of pronking, which consists in leaping by extending the four limbs simultaneously. Pronking is typically observed in some ungulate species inhabiting grassland populated by obstacles such as shrubs, rocks and fallen branches scattered across the environment. Several possible explanations have been proposed for this peculiar behaviour, including the honest signalling of the fitness of the individual to predators or the transmission of a warning alert to conspecifics, but so far none of them has been advocated as conclusive. In this work, we investigate the kinematics of pronking on a two-dimensional landscape populated by randomly scattered obstacles. We show that when the density of obstacles is larger than a critical threshold, pronking becomes the gait that maximizes the probability of trespassing in the shortest possible time all the obstacles distributed across the distance fled, and thus represents an effective escape strategy based on a simple open-loop control. The transition between pronking and more conventional gaits such as trotting and galloping occurs at a threshold obstacle density and is continuous for a non-increasing monotone distribution of the height of obstacles, and discrete when the distribution is peaked at a non-zero height. We discuss the implications of our results for the autonomous robotic exploration on unstructured terrain.

A planar polarized MYO6-DOCK7-RAC1 axis promotes tissue fluidification in mammary epithelia.

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.

Triap1 upregulation promotes escape from mitotic-slippage-induced G1 arrest.

Drugs targeting microtubules rely on the mitotic checkpoint to arrest cell proliferation. The prolonged mitotic arrest induced by such drugs is followed by a G1 arrest. Here, we follow for several weeks the fate of G1-arrested human cells after treatment with nocodazole. We find that a small fraction of cells escapes from the arrest and resumes proliferation. These escaping cells experience reduced DNA damage and p21 activation. Cells surviving treatment are enriched for anti-apoptotic proteins, including Triap1. Increasing Triap1 levels allows cells to survive the first treatment with reduced DNA damage and lower levels of p21; accordingly, decreasing Triap1 re-sensitizes cells to nocodazole. We show that Triap1 upregulation leads to the retention of cytochrome c in the mitochondria, opposing the partial activation of caspases caused by nocodazole. In summary, our results point to a potential role of Triap1 upregulation in the emergence of resistance to drugs that induce prolonged mitotic arrest.

A planar-polarized MYO6-DOCK7-RAC1 axis promotes tissue fluidification in mammary epithelia.

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodia cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a novel MYO6-DOCK7 axis that is critical for spatially restriction of RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative mode motion in otherwise solid and static carcinoma cell collectives. Highlights: Collective motion of jammed epithelia requires myosin VI activityThe MYO6-DOCK7 axis is critical to restrict the activity of RAC1 in a planar polarized fashionMYO6-DOCK7-RAC1 activation ensures long-range coordination of movements by promoting orientation and persistence of cryptic lamellipodiaMyosin VI overexpression is exploited by infiltrating breast cancer cells.

High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis.

Patients with colorectal liver metastasis (CLM) present with heterogenous clinical outcomes and improved classification is needed to ameliorate the therapeutic output. Macrophages (Mϕ) hold promise as prognostic classifiers and therapeutic targets. Here, stemming from a single-cell analysis of mononuclear phagocytes infiltrating human CLM, we identified two Mϕ markers associated with distinct populations with opposite clinical relevance. The invasive margin of CLM was enriched in pro-inflammatory monocyte-derived Mϕ (MoMϕ) expressing the monocytic marker SERPINB2, and a more differentiated population, tumor-associated Mϕ (TAM), expressing glycoprotein nonmetastatic melanoma protein B (GPNMB). SERPINB2+ MoMϕ had an early inflammatory profile, whereas GPNMB+ TAMs were enriched in pathways of matrix degradation, angiogenesis, and lipid metabolism and were found closer to the tumor margin, as confirmed by spatial transcriptomics on CLM specimens. In a cohort of patients, a high infiltration of SERPINB2+ cells independently associated with longer disease-free survival (DFS; P = 0.033), whereas a high density of GPNMB+ cells correlated with shorter DFS (P = 0.012) and overall survival (P = 0.002). Cell-cell interaction analysis defined opposing roles for MoMϕ and TAMs, suggesting that SERPINB2+ and GPNMB+ cells are discrete populations of Mϕ and may be exploited for further translation to an immune-based stratification tool. This study provides evidence of how multi-omics approaches can identify nonredundant, clinically relevant markers for further translation to immune-based patient stratification tools and therapeutic targets. GPNMB has been shown to set Mϕ in an immunosuppressive mode. Our high dimensional analyses provide further evidence that GPNMB is a negative prognostic indicator and a potential player in the protumor function of Mϕ populations.

Tissue fluidification promotes a cGAS-STING cytosolic DNA response in invasive breast cancer.

The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.

Non-invasive measurement of nuclear relative stiffness from quantitative analysis of microscopy data.

The connection between the properties of a cell tissue and those of the single constituent cells remains to be elucidated. At the purely mechanical level, the degree of rigidity of different cellular components, such as the nucleus and the cytoplasm, modulates the interplay between the cell inner processes and the external environment, while simultaneously mediating the mechanical interactions between neighboring cells. Being able to quantify the correlation between single-cell and tissue properties would improve our mechanobiological understanding of cell tissues. Here we develop a methodology to quantitatively extract a set of structural and motility parameters from the analysis of time-lapse movies of nuclei belonging to jammed and flocking cell monolayers. We then study in detail the correlation between the dynamical state of the tissue and the deformation of the nuclei. We observe that the nuclear deformation rate linearly correlates with the local divergence of the velocity field, which leads to a non-invasive estimate of the elastic modulus of the nucleus relative to the one of the cytoplasm. We also find that nuclei belonging to flocking monolayers, subjected to larger mechanical perturbations, are about two time stiffer than nuclei belonging to dynamically arrested monolayers, in agreement with atomic force microscopy results. Our results demonstrate a non-invasive route to the determination of nuclear relative stiffness for cells in a monolayer.

Multiscale heterogeneous dynamics in two-dimensional glassy colloids.

On approaching the glass transition, a dense colloid exhibits a dramatic slowdown with minute structural changes. Most microscopy experiments directly follow the motion of individual particles in real space, whereas scattering experiments typically probe the collective dynamics in reciprocal space at variable wavevector q. Multiscale studies of glassy dynamics are experimentally demanding and, thus, seldom performed. By using two-dimensional hard-sphere colloids at various area fractions ϕ, we show here that Differential Dynamic Microscopy (DDM) can be effectively used to measure the collective dynamics of a glassy colloid in a range of q within a single experiment. As ϕ is increased, the single decay of the intermediate scattering functions is progressively replaced by a more complex relaxation that we fit to a sum of two stretched-exponential decays. The slowest process, corresponding to the long-time particle escapes from caging, has a characteristic time τs = 1/(DLq2) with diffusion coefficient DL∼(ϕc-ϕ)2.8, and ϕc ≃ 0.81. The fast process exhibits, instead, a non-Brownian scaling of the characteristic time τf(q) and a relative amplitude a(q) that monotonically increases with q. Despite the non-Brownian nature of τf(q), we succeed in estimating the short-time diffusion coefficient Dcage, whose ϕ-dependence is practically negligible compared to the one of DL. Finally, we extend DDM to measure the q-dependent dynamical susceptibility χ4(q, t), a powerful yet hard-to-access multiscale indicator of dynamical heterogeneities. Our results show that DDM is a convenient tool to study the dynamics of colloidal glasses over a broad range of time and length scales.

Bistability of Dielectrically Anisotropic Nematic Crystals and the Adaptation of Endothelial Collectives to Stress Fields.

Endothelial monolayers physiologically adapt to flow and flow-induced wall shear stress, attaining ordered configurations in which elongation, orientation, and polarization are coherently organized over many cells. Here, with the flow direction unchanged, a peculiar bi-stable (along the flow direction or perpendicular to it) cell alignment is observed, emerging as a function of the flow intensity alone, while cell polarization is purely instructed by flow directionality. Driven by the experimental findings, the parallelism between endothelia is delineated under a flow field and the transition of dual-frequency nematic liquid crystals under an external oscillatory electric field. The resulting physical model reproduces the two stable configurations and the energy landscape of the corresponding system transitions. In addition, it reveals the existence of a disordered, metastable state emerging upon system perturbation. This intermediate state, experimentally demonstrated in endothelial monolayers, is shown to expose the cellular system to a weakening of cell-to-cell junctions to the detriment of the monolayer integrity. The flow-adaptation of monolayers composed of healthy and senescent endothelia is successfully predicted by the model with adjustable nematic parameters. These results may help to understand the maladaptive response of in vivo endothelial tissues to disturbed hemodynamics and the progressive functional decay of senescent endothelia.

Deformation profiles and microscopic dynamics of complex fluids during oscillatory shear experiments.

Oscillatory shear tests are widely used in rheology to characterize the linear and non-linear mechanical response of complex fluids, including the yielding transition. There is an increasing urge to acquire detailed knowledge of the deformation field that is effectively present across the sample during these tests; at the same time, there is mounting evidence that the macroscopic rheological response depends on the elusive microscopic behavior of the material constituents. Here we employ a strain-controlled shear-cell with transparent walls to visualize and quantify the dynamics of tracers embedded in various cyclically sheared complex fluids, ranging from almost-ideal elastic to yield stress fluids. For each sample, we use image correlation processing to measure the macroscopic deformation field, and echo-differential dynamic microscopy to probe the microscopic irreversible sample dynamics in reciprocal space; finally, we devise a simple scheme to spatially map the rearrangements in the sheared sample, once again without tracking the tracers. For the yield stress sample, we obtain a wave-vector dependent characterization of shear-induced diffusion across the yielding transition, which is accompanied by a three-order-of-magnitude speed-up of the dynamics and by a transition from localized, intermittent rearrangements to a more spatially homogeneous and temporally uniform activity. Our tracking free approach is intrinsically multi-scale, can successfully discriminate between different types of dynamics, and can be automated to minimize user intervention. Applications are many, as it can be translated to other imaging modes, including fluorescence, and can be used with sub-resolution tracers and even without tracers, for samples that provide intrinsic optical contrast.

Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles.

Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons.

Optimal leap angle of legged and legless insects in a landscape of uniformly distributed random obstacles.

We investigate theoretically the ballistic motion of small legged insects and legless larvae after a jump. Notwithstanding their completely different morphologies and jumping strategies, some legged and legless animals have convergently evolved to jump with a take-off angle of 60°, which differs significantly from the leap angle of 45° that allows reaching maximum range. We show that in the presence of uniformly distributed random obstacles the probability of a successful jump is directly proportional to the area under the trajectory. In the presence of negligible air drag, the probability is maximized by a take-off angle of 60°. The numerical calculation of the trajectories shows that they are significantly affected by air drag, but the maximum probability of a successful jump still occurs for a take-off angle of 59-60° in a wide range of the dimensionless Reynolds and Froude numbers that control the process. We discuss the implications of our results for the exploration of unknown environments such as planets and disaster scenarios by using jumping robots.

Probing roto-translational diffusion of small anisotropic colloidal particles with a bright-field microscope.

Soft and biological materials are often composed of elementary constituents exhibiting an incessant roto-translational motion at the microscopic scale. Tracking this motion with a bright-field microscope becomes increasingly challenging when the particle size becomes smaller than the microscope resolution, a case which is frequently encountered. Here we demonstrate squared-gradient differential dynamic microscopy (SG-DDM) as a tool to successfully use bright-field microscopy to extract the roto-translational dynamics of small anisotropic colloidal particles, whose rotational motion cannot be tracked accurately in direct space. We provide analytical justification and experimental demonstration of the method by successful application to an aqueous suspension of peanut-shaped particles.

Disentangling collective motion and local rearrangements in 2D and 3D cell assemblies.

The accurate quantification of cellular motility and of the structural changes occurring in multicellular aggregates is critical in describing and understanding key biological processes, such as wound repair, embryogenesis and cancer invasion. Current methods based on cell tracking or velocimetry either suffer from limited spatial resolution or are challenging and time-consuming, especially for three-dimensional (3D) cell assemblies. Here we propose a conceptually simple, robust and tracking-free approach for the quantification of the dynamical activity of cells via a two-step procedure. We first characterise the global features of the collective cell migration by registering the temporal stack of the acquired images. As a second step, a map of the local cell motility is obtained by performing a mean squared amplitude analysis of the intensity fluctuations occurring when two registered image frames acquired at different times are subtracted. We successfully apply our approach to cell monolayers undergoing a jamming transition, as well as to monolayers and 3D aggregates that exhibit a cooperative unjamming-via-flocking transition. Our approach is capable of disentangling very efficiently and of assessing accurately the global and local contributions to cell motility.

Multiple dynamic regimes in a coarsening foam.

Intermittent dynamics driven by internal stress imbalances in disordered systems is a fascinating yet poorly understood phenomenon. Here, we study it for a coarsening foam. By exploiting differential dynamic microscopy and particle tracking we determine the dynamical characteristics of the foam at different ages in reciprocal and direct space, respectively. At all wavevectors q investigated, the intermediate scattering function exhibits a compressed exponential decay. However, the access to unprecedentedly small values of q highlights the existence of two distinct regimes for the q-dependence of the foam relaxation rate Γ(q). At high q, Γ(q) ∼ q consistent with directionally-persistent and intermittent bubble displacements. At low q, we find the surprising scaling Γ(q) ∼ q δ , with δ = 1.6 ± 0.2. The analysis of the bubble displacement distribution in real space reveals the existence of a displacement cut-off of the order of the bubble diameter. Introducing such cut-off length in an existing model, describing stress-driven dynamics in disordered systems, fully accounts for the observed behavior in direct and reciprocal space.

Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.