ABSTRACT
Drosophila border cell clusters model collective cell migration. Airyscan super-resolution microscopy enables fine-scale description of cluster shape and texture. Here we describe how to convert Airyscan images of border cell clusters into 3D models of the surface and detect regions of convex and concave curvature. We use spectral decomposition analysis to compare surface textures across genotypes to determine how genes of interest impact cluster surface geometry. This protocol applies to border cells and could generalize to additional cell types. For complete details on the use and execution of this protocol, please refer to Gabbert et al.1.
ABSTRACT
A common motif in biology is the arrangement of cells into tubes, which further transform into complex shapes. Traditionally, analysis of dynamic tissues has relied on inspecting static snapshots, live imaging of cross-sections or tracking isolated cells in three dimensions. However, capturing the interplay between in-plane and out-of-plane behaviors requires following the full surface as it deforms and integrating cell-scale motions into collective, tissue-scale deformations. Here, we present an analysis framework that builds in toto maps of tissue deformations by following tissue parcels in a static material frame of reference. Our approach then relates in-plane and out-of-plane behaviors and decomposes complex deformation maps into elementary contributions. The tube-like surface Lagrangian analysis resource (TubULAR) provides an open-source implementation accessible either as a standalone toolkit or as an extension of the ImSAnE package used in the developmental biology community. We demonstrate our approach by analyzing shape change in the embryonic Drosophila midgut and beating zebrafish heart. The method naturally generalizes to in vitro and synthetic systems and provides ready access to the mechanical mechanisms relating genetic patterning to organ shape change.
Subject(s)
Drosophila , Zebrafish , AnimalsABSTRACT
Septins self-assemble into polymers that bind and deform membranes in vitro and regulate diverse cell behaviors in vivo. How their in vitro properties relate to their in vivo functions is under active investigation. Here, we uncover requirements for septins in detachment and motility of border cell clusters in the Drosophila ovary. Septins and myosin colocalize dynamically at the cluster periphery and share phenotypes but, surprisingly, do not impact each other. Instead, Rho independently regulates myosin activity and septin localization. Active Rho recruits septins to membranes, whereas inactive Rho sequesters septins in the cytoplasm. Mathematical analyses identify how manipulating septin expression levels alters cluster surface texture and shape. This study shows that the level of septin expression differentially regulates surface properties at different scales. This work suggests that downstream of Rho, septins tune surface deformability while myosin controls contractility, the combination of which governs cluster shape and movement.
Subject(s)
Cell Movement , Drosophila melanogaster , Septins , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Septins/metabolism , Myosins/metabolism , Gene Knockdown Techniques , AnimalsABSTRACT
The actomyosin cytoskeleton is a crucial driver of morphogenesis. Yet how the behavior of large-scale cytoskeletal patterns in deforming tissues emerges from the interplay of geometry, genetics, and mechanics remains incompletely understood. Convergent extension in Drosophila melanogaster embryos provides the opportunity to establish a quantitative understanding of the dynamics of anisotropic non-muscle myosin II. Cell-scale analysis of protein localization in fixed embryos suggests that gene expression patterns govern myosin anisotropy via complex rules. However, technical limitations have impeded quantitative and dynamic studies of this process at the whole embryo level, leaving the role of geometry open. Here, we combine in toto live imaging with quantitative analysis of molecular dynamics to characterize the distribution of myosin anisotropy and the corresponding genetic patterning. We found pair rule gene expression continuously deformed, flowing with the tissue frame. In contrast, myosin anisotropy orientation remained approximately static and was only weakly deflected from the stationary dorsal-ventral axis of the embryo. We propose that myosin is recruited by a geometrically defined static source, potentially related to the embryo-scale epithelial tension, and account for transient deflections by cytoskeletal turnover and junction reorientation by flow. With only one parameter, this model quantitatively accounts for the time course of myosin anisotropy orientation in wild-type, twist, and even-skipped embryos, as well as embryos with perturbed egg geometry. Geometric patterning of the cytoskeleton suggests a simple physical strategy to ensure a robust flow and formation of shape.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Morphogenesis , Myosin Type II/genetics , Myosin Type II/metabolism , Myosins/metabolism , Cytoskeletal Proteins/metabolism , Embryo, Nonmammalian/metabolismABSTRACT
Changes in the geometry and topology of self-assembled membranes underlie diverse processes across cellular biology and engineering. Similar to lipid bilayers, monolayer colloidal membranes have in-plane fluid-like dynamics and out-of-plane bending elasticity. Their open edges and micrometer-length scale provide a tractable system to study the equilibrium energetics and dynamic pathways of membrane assembly and reconfiguration. Here, we find that doping colloidal membranes with short miscible rods transforms disk-shaped membranes into saddle-shaped surfaces with complex edge structures. The saddle-shaped membranes are well approximated by Enneper's minimal surfaces. Theoretical modeling demonstrates that their formation is driven by increasing the positive Gaussian modulus, which in turn, is controlled by the fraction of short rods. Further coalescence of saddle-shaped surfaces leads to diverse topologically distinct structures, including shapes similar to catenoids, trinoids, four-noids, and higher-order structures. At long timescales, we observe the formation of a system-spanning, sponge-like phase. The unique features of colloidal membranes reveal the topological transformations that accompany coalescence pathways in real time. We enhance the functionality of these membranes by making their shape responsive to external stimuli. Our results demonstrate a pathway toward control of thin elastic sheets' shape and topology-a pathway driven by the emergent elasticity induced by compositional heterogeneity.
Subject(s)
Lipid Bilayers , Elasticity , Lipid Bilayers/chemistry , Membranes/metabolism , Normal DistributionABSTRACT
Organ architecture is often composed of multiple laminar tissues arranged in concentric layers. During morphogenesis, the initial geometry of visceral organs undergoes a sequence of folding, adopting a complex shape that is vital for function. Genetic signals are known to impact form, yet the dynamic and mechanical interplay of tissue layers giving rise to organs' complex shapes remains elusive. Here, we trace the dynamics and mechanical interactions of a developing visceral organ across tissue layers, from subcellular to organ scale in vivo. Combining deep tissue light-sheet microscopy for in toto live visualization with a novel computational framework for multilayer analysis of evolving complex shapes, we find a dynamic mechanism for organ folding using the embryonic midgut of Drosophila as a model visceral organ. Hox genes, known regulators of organ shape, control the emergence of high-frequency calcium pulses. Spatiotemporally patterned calcium pulses trigger muscle contractions via myosin light chain kinase. Muscle contractions, in turn, induce cell shape change in the adjacent tissue layer. This cell shape change collectively drives a convergent extension pattern. Through tissue incompressibility and initial organ geometry, this in-plane shape change is linked to out-of-plane organ folding. Our analysis follows tissue dynamics during organ shape change in vivo, tracing organ-scale folding to a high-frequency molecular mechanism. These findings offer a mechanical route for gene expression to induce organ shape change: genetic patterning in one layer triggers a physical process in the adjacent layer - revealing post-translational mechanisms that govern shape change.
Subject(s)
Calcium , Mesoderm , Animals , Calcium/metabolism , Constriction , Drosophila , Mesoderm/metabolism , Morphogenesis/genetics , MusclesABSTRACT
Mixtures of filaments and molecular motors form active materials with diverse dynamical behaviors that vary based on their constituents' molecular properties. To develop a multiscale of these materials, we map the nonequilibrium phase diagram of microtubules and tip-accumulating kinesin-4 molecular motors. We find that kinesin-4 can drive either global contractions or turbulentlike extensile dynamics, depending on the concentrations of both microtubules and a bundling agent. We also observe a range of spatially heterogeneous nonequilibrium phases, including finite-sized radial asters, 1D wormlike chains, extended 2D bilayers, and system-spanning 3D active foams. Finally, we describe intricate kinetic pathways that yield microphase separated structures and arise from the inherent frustration between the orientational order of filamentous microtubules and the positional order of tip-accumulating molecular motors. Our work reveals a range of novel active states. It also shows that the form of active stresses is not solely dictated by the properties of individual motors and filaments, but is also contingent on the constituent concentrations and spatial arrangement of motors on the filaments.
ABSTRACT
Lattices of interacting gyroscopes naturally support band gaps and topologically protected wave transport along material boundaries. Recently the authors and their collaborators found that amorphous arrangements of such coupled gyroscopes also support nontrivial topological phases. In contrast to periodic systems, for which there is a comprehensive understanding and predictive framework for band gaps and band topology, the theory of spectral gaps and topology for amorphous materials remains less developed. Here we use experiments, numerics, and analytic tools to address the relationship between local interactions and nontrivial topology. We begin with a derivation of the equations of motion within the framework of symplectic mechanics. We then present a general method for predicting whether a gap exists and for approximating the Chern number using only local features of a network, bypassing the costly diagonalization of the system's dynamical matrix. Finally we study how strong disorder interacts with band topology in gyroscopic metamaterials and find that amorphous gyroscopic Chern insulators exhibit similar critical behavior to periodic lattices. Our experiments and simulations additionally reveal a topological Anderson insulation transition, wherein disorder drives a trivial phase into a topological one.
ABSTRACT
Nanoparticle monolayer sheets are ultrathin inorganic-organic hybrid materials that combine highly controllable optical and electrical properties with mechanical flexibility and remarkable strength. Like other thin sheets, their low bending rigidity allows them to easily roll into or conform to cylindrical geometries. Nanoparticle monolayers not only can bend, but also cope with strain through local particle rearrangement and plastic deformation. This means that, unlike thin sheets such as paper or graphene, nanoparticle sheets can much more easily conform to surfaces with complex topography characterized by non-zero Gaussian curvature, like spherical caps or saddles. Here, we investigate the limits of nanoparticle monolayers' ability to conform to substrates with Gaussian curvature by stamping nanoparticle sheets onto lattices of larger polystyrene spheres. Tuning the local Gaussian curvature by increasing the size of the substrate spheres, we find that the stamped sheet morphology evolves through three characteristic stages: from full substrate coverage, where the sheet extends over the interstices in the lattice, to coverage in the form of caps that conform tightly to the top portion of each sphere and fracture at larger polar angles, to caps that exhibit radial folds. Through analysis of the nanoparticle positions, obtained from scanning electron micrographs, we extract the local strain tensor and track the onset of strain-induced dislocations in the particle arrangement. By considering the interplay of energies for elastic and plastic deformations and adhesion, we construct arguments that capture the observed changes in sheet morphology as Gaussian curvature is tuned over two orders of magnitude.
ABSTRACT
Conforming materials to rigid substrates with Gaussian curvature-positive for spheres and negative for saddles-has proven a versatile tool to guide the self-assembly of defects such as scars, pleats, folds, blisters, and liquid crystal ripples. Here, we show how curvature can likewise be used to control material failure and guide the paths of cracks. In our experiments, and unlike in previous studies on cracked plates and shells, we constrained flat elastic sheets to adopt fixed curvature profiles. This constraint provides a geometric tool for controlling fracture behaviour: curvature can stimulate or suppress the growth of cracks and steer or arrest their propagation. A simple analytical model captures crack behaviour at the onset of propagation, while a two-dimensional phase-field model with an added curvature term successfully captures the crack's path. Because the curvature-induced stresses are independent of material parameters for isotropic, brittle media, our results apply across scales.