DHA-containing phospholipids control membrane fusion and transcellular tunnel dynamics.

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (DHA-PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses showed that human umbilical vein endothelial cells (HUVECs) subjected to a DHA diet undergo a 6-fold enrichment in DHA-PLs at the plasma membrane (PM) at the expense of monounsaturated oleic acid-containing PLs (OA-PLs). Consequently, DHA-PL enrichment at the PM induces a reduction in cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM lead to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PL levels in membranes affect cell biomechanical properties.

Cell Tracking Profiler - a user-driven analysis framework for evaluating 4D live-cell imaging data.

Accurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function in vivo Here, by using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm, we established a 'ground truth' for muSC behaviour. This revealed that segmentation and tracking algorithms from commonly used programs are error-prone, leading us to develop a fast semi-automated image analysis pipeline that allows user-defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) is a package that runs two existing programs, HK Means and Phagosight within the Icy image analysis suite, to enable user-managed cell tracking from 3D time-lapse datasets to provide measures of cell shape and movement. We demonstrate how CTP can be used to reveal changes to cell behaviour of muSCs in response to manipulation of the cell cytoskeleton by small-molecule inhibitors. CTP and the associated tools we have developed for analysis of outputs thus provide a powerful framework for analysing complex cell behaviour in vivo from 4D datasets that are not amenable to straightforward analysis.

Icy: an open bioimage informatics platform for extended reproducible research.

Current research in biology uses evermore complex computational and imaging tools. Here we describe Icy, a collaborative bioimage informatics platform that combines a community website for contributing and sharing tools and material, and software with a high-end visual programming framework for seamless development of sophisticated imaging workflows. Icy extends the reproducible research principles, by encouraging and facilitating the reusability, modularity, standardization and management of algorithms and protocols. Icy is free, open-source and available at http://icy.bioimageanalysis.org/.

Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH.

Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

Real-time analysis of the behaviour of groups of mice via a depth-sensing camera and machine learning.

Preclinical studies of psychiatric disorders use animal models to investigate the impact of environmental factors or genetic mutations on complex traits such as decision-making and social interactions. Here, we introduce a method for the real-time analysis of the behaviour of mice housed in groups of up to four over several days and in enriched environments. The method combines computer vision through a depth-sensing infrared camera, machine learning for animal and posture identification, and radio-frequency identification to monitor the quality of mouse tracking. It tracks multiple mice accurately, extracts a list of behavioural traits of both individuals and the groups of mice, and provides a phenotypic profile for each animal. We used the method to study the impact of Shank2 and Shank3 gene mutations-mutations that are associated with autism-on mouse behaviour. Characterization and integration of data from the behavioural profiles of Shank2 and Shank3 mutant female mice revealed their distinctive activity levels and involvement in complex social interactions.

Mapping molecular assemblies with fluorescence microscopy and object-based spatial statistics.

Elucidating protein functions and molecular organisation requires to localise precisely single or aggregated molecules and analyse their spatial distributions. We develop a statistical method SODA (Statistical Object Distance Analysis) that uses either micro- or nanoscopy to significantly improve on standard co-localisation techniques. Our method considers cellular geometry and densities of molecules to provide statistical maps of isolated and associated (coupled) molecules. We use SODA with three-colour structured-illumination microscopy (SIM) images of hippocampal neurons, and statistically characterise spatial organisation of thousands of synapses. We show that presynaptic synapsin is arranged in asymmetric triangle with the 2 postsynaptic markers homer and PSD95, indicating a deeper localisation of homer. We then determine stoichiometry and distance between localisations of two synaptic vesicle proteins with 3D-STORM. These findings give insights into the protein organisation at the synapse, and prove the efficiency of SODA to quantitatively assess the geometry of molecular assemblies.