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.

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.

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.

Simultaneous measurement of mass and rotation of trapped absorbing particles in air.

We trap absorbing micro-particles in air by photophoretic forces generated using a single loosely focused Gaussian trapping beam. We measure a component of the radial Brownian motion of a trapped particle cluster and determine the power spectral density, mean squared displacement, and normalized position and velocity autocorrelation functions to characterize the photophoretic body force in a quantitative fashion for the first time. The trapped particles also undergo spontaneous rotation due to the action of this force. This is evident from the spectral density that displays clear peaks at the rotation and the particles' inertial resonance frequencies. We fit the spectral density to the well-known analytical function derived from the Langevin equation, measure the resonance and rotation frequencies, and determine the values for particle mass that we verify at different trapping laser powers with reasonable accuracy.

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'.

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.

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.

Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking.

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.