Tissue morphogenesis requires tight coordination between biochemical signaling and mechanical forces that sculpt cells and tissues. While actomyosin networks are well-established force generators, microtubule-based mechanics have recently emerged as crucial contributors to tissue remodeling. Yet, how dynamic microtubules, whose plus ends undergo compression-induced catastrophes that limit their load-bearing capacity, generate forces in vivo remains unclear. Here, we identify Orbit, the Drosophila cytoplasmic linker-associated protein (CLASP) homolog, as a key factor that stabilizes non-centrosomal microtubule plus ends in vivo, enabling them to sustain mechanical loads. In the pupal wing epithelium, these Orbit-stabilized, planar-polarized microtubules are consistent with a role in counteracting actomyosin contractility and promoting tissue elongation. Loss of Orbit increases catastrophe frequency and disrupts epithelial elongation, whereas Orbit overexpression enhances microtubule rescues by suppressing catastrophes, thereby promoting cell anisotropy and tissue extension. Moreover, Orbit-mediated stabilization is sufficient to induce microtubule-dependent, filopodia-like protrusions independent of actin. Together, these findings establish CLASP-dependent microtubule stabilization as a key mechanism linking polymerization dynamics to epithelial morphogenesis.

Mechanical stimuli, particularly laminar blood flow, play a crucial role in shaping the vascular system. Changes in the rate of blood flow manifest in altered shear stress, which activates signaling cascades that drive vascular remodeling. Consistently, dysregulation of the endothelial response to fluid shear forces and aberrant flow patterns both lead to pathological conditions, including impaired blood vessel development and atherosclerosis. Despite its importance, the mechanisms driving the coordinated cell behavior underlying vascular remodeling are not fully understood. Combining classical cell biological approaches with advanced image analysis, mathematical modeling, biomimetic strategies, and in vivo studies, we identify the planar cell polarity (PCP) protein Vangl1 as an enforcer of flow-dependent cell dynamics in the vascular system. We demonstrate that shear stress triggers the relocation of Vangl1 from an internal reservoir to the plasma membrane at the initiation of cell remodeling. Membrane enrichment of Vangl1 is mediated by a Coronin1C-dependent shift in the equilibrium between endo- and exocytosis and results in the spatial reorganization of another essential PCP protein, Frizzled6 (Fzd6). The resulting mutual exclusion of the core PCP proteins Fzd6 and Vangl1 augments differential junctional and cytoskeletal dynamics along the flow axis. Loss of Vangl1 limits the ability of endothelial cells to respond to shear forces in a coordinated fashion, resulting in irregular cell alignment along the flow direction and erroneous vessel sprouting. Together, these studies introduce core PCP signaling as a determinant of collective cell dynamics and organization of the vascular system. ### Competing Interest Statement The authors have declared no competing interest. The data that support the findings of this study are available from the corresponding authors upon reasonable request. No restrictions apply.

In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. We show that wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, we confirm that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces. Actomyosin and microtubule-based forces are both important for tissue development, but how these systems interact in space and time remains unclear. Here, the authors study fly wing epithelium growth and determine aspects driving cell shape that are driven by microtubule or actomyosin-generated forces.

In vivo, cells experience complex tissue environments with various chemical and physical features. They sense and respond to tissue morphology and mechanical properties and adjust their behavior and function based on the surrounding. In contrast to the free environment experienced on 2D substrates commonly used in research, the 3D natural environment represents a major physical obstacle for cells. Here, cells are usually confined either by the extracellular matrix (ECM) or neighboring cells. The importance of such confinements has been demon-strated in the past decades by showing its influence on cell decision-making in many vital biological processes such as migration, division and cytoskeletal reorganization. Despite these insights, the sheer level of complexity faced when studying cell biological questions in biomimetic confined situations, led to an indispensable need for a 3D system which can simulate the in vivo confined condition, while being capable of providing microenvironments with different chemical and physical properties for the cells and capturing the mechanical forces and properties of the studied biological sample. Here we introduce a microconfiner that finally provides a new imaging capacity, namely the confine-ment force microscopy (CFM). We are able to adjust the confinement level in real time during microscopy while measuring not only the the cellular traction but also the cellular compression forces. Furthermore, the chemical and physical properties of the microenvironment can be optimized for the respective questions. We demonstrate the power of this confinement system by the mechanical response of cells, migration analysis of immune cells, the timed force generation during durotaxis driven adhesion switching and the viscoelastic properties of cancer tissue.

In this contribution we investigate in mathematical modeling and efficient simulation of biological cells with a particular emphasis on effective modeling of structural properties that originate from active forces generated from polymerization and depolymerization of cytoskeletal components. In detail, we propose a nonlinear continuum approach to model microtubule-based forces which have recently been established as central components of cell mechanics during early fruit fly wing development. The model is discretized in space using the finite-element method. Although the individual equations are decoupled by a semi-implicit time discretization, the discrete model is still computationally demanding. In addition, the parameters needed for the effective model equations are not easily available and have to be estimated or determined by repeatedly solving the model and fitting the results to measurements. This drastically increases the computational cost. Reduced basis methods have been used successfully to speed up such repeated solves, often by several orders of magnitude. However, for the complex nonlinear models regarded here, the application of these model order reduction methods is not always straight-forward and comes with its own set of challenges. In particular, subspace construction using the Proper Orthogonal Decomposition (POD) becomes prohibitively expensive for reasonably fine grids. We thus propose to combine the Hierarchical Approximate POD, which is a general, easy-to-implement approach to compute an approximate POD, with an Empirical Interpolation Method to efficiently generate a fast to evaluate reduced order model. Numerical experiments are given to demonstrate the applicability and efficiency of the proposed modeling and simulation approach.

When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia‐like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca 2+ , likely to enhance the LD–organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress‐induced lipotoxicity.

In the developing Drosophila abdomen, the epithelial tissue displays extensive cytoskeletal remodeling. In stark contrast to the spatio-temporal control of the actin cytoskeleton, the regulation of microtubule architecture during epithelial morphogenesis has remained opaque. In particular, its role in cell motility remains unclear. Here, we show that minus-end binding protein Patronin is required for organizing microtubule arrays in histoblast cells that form the Drosophila abdomen. Loss of Patronin results in a dorsal cleft, indicating the compromised function of histoblasts. We further show that Patronin is polarized in these cells and is required for the formation of highly dynamic non-centrosomal microtubules in the migrating histoblasts. Thus, our study demonstrates that regulation of microtubule cytoskeleton through Patronin mediates epithelium remodeling.

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.© 2020 Kesarwani et al. PMID: 32886100 Funding information This work was supported by: DBT-Wellcome Trust India Alliance, India Grant ID: IA/I/14/2/501533 DBT-Wellcome Trust India Alliance, India Grant ID: IA/E/15/1/502339 Wellcome Trust, United Kingdom

During morphogenesis, tissues undergo extensive remodeling to get their final shape. Such precise sculpting requires the application of forces generated within cells by the cytoskeleton and transmission of these forces through adhesion molecules within and between neighboring cells. Within individual cells, microtubules together with actomyosin filaments and intermediate filaments form the composite cytoskeleton that controls cell mechanics during tissue rearrangements. While studies have established the importance of actin-based mechanical forces that are coupled via intercellular junctions, relatively little is known about the contribution of other cytoskeletal components such as microtubules to cell mechanics during morphogenesis. In this review the focus is on recent findings, highlighting the direct mechanical role of microtubules beyond its well-established role in trafficking and signaling during tissue formation.

The biophysical and biochemical properties of live tissues are important in the context of development and disease. Methods for evaluating these properties typically involve destroying the tissue or require specialized technology and complicated analyses. Here, we present a novel, noninvasive methodology for determining the spatial distribution of tissue features within embryos, making use of nondirectionally migrating cells and software we termed "Landscape," which performs automatized high-throughput three-dimensional image registration. Using the live migrating cells as bioprobes, we identified structures within the zebrafish embryo that affect the distribution of the cells and studied one such structure constituting a physical barrier, which, in turn, influences amoeboid cell polarity. Overall, this work provides a unique approach for detecting tissue properties without interfering with animal's development. In addition, Landscape allows for integrating data from multiple samples, providing detailed and reliable quantitative evaluation of variable biological phenotypes in different organisms.

To reach their destination, migrating cells rely on polarized signal inputs to align the direction of their motion. However, navigational guidance cues are not always present, which establishes the need for exploratory search mechanisms in cells seeking signal inputs. Here, we investigate how non-Brownian search patterns emerge in adherent vertebrate cells. Combining experimental and theoretical analysis, we demonstrate that nanoscale plasma membrane deformations nucleate a mechanochemical feedback loop that mediates longevity of the cell's leading edge, a necessary requirement for directed cell migration. We further observe stochastic transitions between phases of random and persistent cell motion, whereby the mechanochemical circuit augments cell persistence and search area. Collectively, these findings are consistent with a self-organizing system for a superdiffusive pattern of motion that is spontaneously employed by migratory cells in the absence of external signal inputs.

Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis(1,2). However, unlike for actin-based mechanical forces, our knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. Here, we establish microtubules as central components of cell mechanics during tissue morphogenesis. We find that individual cells are mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. We further establish that the Fat planar cell polarity (Ft-PCP) signalling pathway(3,4) couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels.

The ability of cells to polarize is an intrinsic property of almost all cells and is required for the devlopment of most multicellular organisms. To develop cell polarity, cells integrate various signals derived from intrinsic as well as extrinsic sources. In the recent years, cell-cell adhesion receptors have turned out as important regulators of cellular polarization. By interacting with conserved cell polarity proteins, they regulate the recruitment of polarity complexes to specific sites of cell-cell adhesion. By initiating intracellular signaling cascades at those sites, they trigger their specific subcellular activation. Not surprisingly, cell-cell adhesion receptors regulate diverse aspects of cell polarity, including apico-basal polarity in epithelial and endothelial cells, front-to-rear polarity in collectively migrating cells, and planar cell polarity during organ development. Here, we review the recent developments highlighting the central roles of cell-cell adhesion molecules in the development of cell polarity. (C) 2017 Elsevier Ltd. All rights reserved.

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension-retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

Planar cell polarity, the polarization of cells within the plane of the epithelium, orthogonal to the apical‐basal axis, is essential for a growing list of developmental events, and – over the last 15 years – has evolved from a little‐studied curiosity in Drosophila to the subject of a substantial research enterprise. In that time, it has been recognized that two molecular systems are responsible for polarization of most tissues: Both the “core” Frizzled system and the “global” Fat/Dachsous/Four‐jointed system produce molecular asymmetry within cells, and contribute to morphological polarization. In this review, we discuss recent findings on the molecular mechanism that links “global” directional signals with local coordinated polarity.

In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations. DOI: 10.7554/eLife.03116.001

Planar cell polarity (PCP) signaling controls the polarization of cells within the plane of an epithelium. Two molecular modules composed of Fat(Ft)/Dachsous(Ds)/Four-jointed(Fj) and a 'PCP-core' including Frizzled(Fz) and Dishevelled(Dsh) contribute to polarization of individual cells. How polarity is globally coordinated with tissue axes is unresolved. Consistent with previous results, we find that the Ft/Ds/Fj-module has an effect on a MT-cytoskeleton. Here, we provide evidence for the model that the Ft/Ds/Fj-module provides directional information to the core-module through this MT organizing function. We show Ft/Ds/Fj-dependent initial polarization of the apical MT-cytoskeleton prior to global alignment of the core-module, reveal that the anchoring of apical non-centrosomal MTs at apical junctions is polarized, observe that directional trafficking of vesicles containing Dsh depends on Ft, and demonstrate the feasibility of this model by mathematical simulation. Together, these results support the hypothesis that Ft/Ds/Fj provides a signal to orient core PCP function via MT polarization.