Dynamic interplay of microtubule and actomyosin forces drive tissue extension.

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.

Negatively curved cellular membranes promote BAIAP2 signaling hub assembly.

Plasma membrane deformations are associated with curvature-dependent protein enrichment that contributes to a wide array of cellular functions. While the spatio-temporal protein dynamics at membrane indentations is well characterized, relatively little is known about protein kinetics at outwardly deforming membrane sites. This is in part due to the lack of high throughput approaches to systematically probe the curvature-dependence of protein-membrane interactions. Here, we developed a nanopatterned array for multiplexed analysis of protein dynamics at negatively curved cellular membranes. Taking advantage of this robust and versatile platform, we explored how membrane shape influences the prototypic negative curvature sensing protein BAIAP2 and its effector proteins. We find assembly of multi-protein signaling hubs and increased actin polymerization at outwardly deformed membrane sections, indicative of curvature-dependent BAIAP2 activation. Collectively, this study presents technical and conceptual advancements towards a quantitative understanding of spatio-temporal protein dynamics at negatively curved membranes.

Modulation of self-organizing circuits at deforming membranes by intracellular and extracellular factors.

Mechanical forces exerted to the plasma membrane induce cell shape changes. These transient shape changes trigger, among others, enrichment of curvature-sensitive molecules at deforming membrane sites. Strikingly, some curvature-sensing molecules not only detect membrane deformation but can also alter the amplitude of forces that caused to shape changes in the first place. This dual ability of sensing and inducing membrane deformation leads to the formation of curvature-dependent self-organizing signaling circuits. How these cell-autonomous circuits are affected by auxiliary parameters from inside and outside of the cell has remained largely elusive. Here, we explore how such factors modulate self-organization at the micro-scale and its emerging properties at the macroscale.

Systematic analysis of curvature-dependent lipid dynamics in a stochastic 3D membrane model.

To minimize the free energy of the system, lipid membranes display curvature-dependent rearrangements at the local and global scale. The optimal membrane shape is generally approximated by averaging the curvature preference of individual lipids across the whole surface. Potential stress due to imperfections in lipid packing caused by local lipid inhomogeneities, however, is frequently neglected. Here, we developed a stochastic 3D membrane model to investigate the relevance of this parameter for shape-dependent lipid and membrane dynamics. A systematic analysis of the discretized Helfrich type Hamiltonian indicates that stress-energy arising from imperfections in packing is analogous to van der Waals interactions, jointly determining membrane shape and localization of curvature-sensitive lipids based on their relative strengths. Insights from this work can be used to characterize natural and design synthetic agents for membrane-shape changes.

Developmental pruning of sensory neurites by mechanical tearing in Drosophila.

Mechanical forces actively shape cells during development, but little is known about their roles during neuronal morphogenesis. Developmental neurite pruning, a critical circuit specification mechanism, often involves neurite abscission at predetermined sites by unknown mechanisms. Pruning of Drosophila sensory neuron dendrites during metamorphosis is triggered by the hormone ecdysone, which induces local disassembly of the dendritic cytoskeleton. Subsequently, dendrites are severed at positions close to the soma by an unknown mechanism. We found that ecdysone signaling causes the dendrites to become mechanically fragile. Severing occurs during periods of increased pupal morphogenetic tissue movements, which exert mechanical forces on the destabilized dendrites. Tissue movements and dendrite severing peak during pupal ecdysis, a period of strong abdominal contractions, and abolishing ecdysis causes non-cell autonomous dendrite pruning defects. Thus, our data establish mechanical tearing as a novel mechanism during neurite pruning.

Moving through a changing world: Single cell migration in 2D vs. 3D.

Migration of single adherent cells is frequently observed in the developing and adult organism and has been the subject of many studies. Yet, while elegant work has elucidated molecular and mechanical cues affecting motion dynamics on a flat surface, it remains less clear how cells migrate in a 3D setting. In this review, we explore the changing parameters encountered by cells navigating through a 3D microenvironment compared to cells crawling on top of a 2D surface, and how these differences alter subcellular structures required for propulsion. We further discuss how such changes at the micro-scale impact motion pattern at the macro-scale.

Dendrite tapering actuates a self-organizing signaling circuit for stochastic filopodia initiation in neurons.

How signaling units spontaneously arise from a noisy cellular background is not well understood. Here, we show that stochastic membrane deformations can nucleate exploratory dendritic filopodia, dynamic actin-rich structures used by neurons to sample its surroundings for compatible transcellular contacts. A theoretical analysis demonstrates that corecruitment of positive and negative curvature-sensitive proteins to deformed membranes minimizes the free energy of the system, allowing the formation of long-lived curved membrane sections from stochastic membrane fluctuations. Quantitative experiments show that once recruited, curvature-sensitive proteins form a signaling circuit composed of interlinked positive and negative actin-regulatory feedback loops. As the positive but not the negative feedback loop can sense the dendrite diameter, this self-organizing circuit determines filopodia initiation frequency along tapering dendrites. Together, our findings identify a receptor-independent signaling circuit that employs random membrane deformations to simultaneously elicit and limit formation of exploratory filopodia to distal dendritic sites of developing neurons.

A multiplexed phospholipid membrane platform for curvature sensitive protein screening.

The curvature of lipid membranes plays a key role in many relevant biological processes such as membrane trafficking, vesicular budding and host-virus interactions. In vitro studies on the membrane curvature of simplified biomimetic models in the nanometer range are challenging, due to their complicated nanofabrication processes. In this work, we propose a simple and low-cost platform for curvature sensitive protein screening, prepared through scanning probe lithography (SPL) methods, where lipid bilayer patches of different compositions can be multiplexed onto substrate areas with tailored local curvature. The curvature is imposed by anchoring nanoparticles of the desired size to the substrate prior to lithography. As a proof of principle, we demonstrate that a positive curvature membrane sensitive protein derived from the BAR domain of Nadrin2 binds selectively to lipid patches patterned on substrate areas coated with 100 nm nanoparticles. The platform opens up a path for screening curvature-dependent protein-membrane interaction studies by providing a flexible and easy to prepare substrate with control over lipid composition and membrane curvature.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Cellular Membranes, a Versatile Adaptive Composite Material.

Cellular membranes belong to the most vital yet least understood biomaterials of live matter. For instance, its biomechanical requirements substantially vary across species and subcellular sites, raising the question how membranes manage to adjust to such dramatic changes. Central to its adaptability at the cell surface is the interplay between the plasma membrane and the adjacent cell cortex, forming an adaptive composite material that dynamically adjusts its mechanical properties. Using a hypothetical composite material, we identify core challenges, and discuss how cellular membranes solved these tasks. We further muse how pathological changes in material properties affect membrane mechanics and cell function, before closing with open questions and future challenges arising when studying cellular membranes.

Exploring the interdependence between self-organization and functional morphology in cellular systems.

All living matter is subject to continuous adaptation and functional optimization via natural selection. Consequentially, structures with close morphological resemblance repeatedly appear across the phylogenetic tree. How these designs emerge at the cellular level is not fully understood. Here, we explore core concepts of functional morphology and discuss its cause and consequences, with a specific focus on emerging properties of self-organizing systems as the potential driving force. We conclude with open questions and limitations that are present when studying shape-function interdependence in single cells and cellular ensembles.

Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM.

Scanning electron microscopy (SEM) takes advantage of distinct detectors to visualise secondary and back-scattering electrons. Here, we report an integrated approach that relies on these two detection methods to simultaneously acquire correlated information on plasma membrane topography and curvature-sensitive cytosolic protein localization in intact cell samples. We further provide detailed preparation and staining protocols, as well as a thorough example-based discussion for imaging optimisation. Collectively, the presented method enables rapid and precise analysis of cytosolic proteins adjacent to cellular membranes with a resolution of ~100 nm, without time-consuming preparations or errors induced by sequential visualisation present in fluorescence-based correlative approaches.

Receptor-Free Signaling at Curved Cellular Membranes.

Plasma membranes are subject to continuous deformations. Strikingly, some of these transient membrane undulations yield membrane-associated signaling hubs that differ in composition and function, depending on membrane geometry and the availability of co-factors. Here, recent advancements on this ubiquitous type of receptor-independent signaling are reviewed, with a special focus on emerging concepts and technical challenges associated with studying these elusive signaling sites.

ArhGEF37 assists dynamin 2 during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) engages over 30 proteins to secure efficient cargo and membrane uptake. While the function of most core CME components is well established, auxiliary mechanisms crucial for fine-tuning and adaptation remain largely elusive. In this study, we identify ArhGEF37, a currently uncharacterized protein, as a constituent of CME. Structure prediction together with quantitative cellular and biochemical studies present a unique BAR domain and PI(4,5)P2-dependent protein-membrane interactions. Functional characterization yields accumulation of ArhGEF37 at dynamin 2-rich late endocytic sites and increased endocytosis rates in the presence of ArhGEF37. Together, these results introduce ArhGEF37 as a regulatory protein involved in endocytosis.

Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis.

Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis1,2. 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 pathway3,4 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.

Self-organization across scales: from molecules to organisms.

Creating ordered structures from chaotic environments is at the core of biological processes at the subcellular, cellular and organismic level. In this perspective, we explore the physical as well as biological features of two prominent concepts driving self-organization, namely phase transition and reaction-diffusion, before closing with a discussion on open questions and future challenges associated with studying self-organizing systems.This article is part of the theme issue 'Self-organization in cell biology'.

Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing.

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions.

Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations.

Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target.

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.