Bionic tracking: using eye tracking to track biological cells in virtual reality

We present Bionic Tracking, a novel method for solving biological cell tracking problems with eye tracking in virtual reality using commodity hardware. Using gaze data, and especially smooth pursuit eye movements, we are able to track cells in time series of 3D volumetric datasets. The problem of tracking cells is ubiquitous in developmental biology, where large volumetric microscopy datasets are acquired on a daily basis, often comprising hundreds or thousands of time points that span hours or days. The image data, however, is only a means to an end, and scientists are often interested in the reconstruction of cell trajectories and cell lineage trees. Reliably tracking cells in crowded three-dimensional space over many time points remains an open problem, and many current approaches rely on tedious manual annotation or curation. In the Bionic Tracking approach, we substitute the usual 2D point-and-click interface for annotation or curation with eye tracking in a virtual reality headset, where users follow cells with their eyes in 3D space in order to track them. We detail the interaction design of our approach and explain the graph-based algorithm used to connect different time points, also taking occlusion and user distraction into account. We demonstrate Bionic Tracking using examples from two different biological datasets. Finally, we report on a user study with seven cell tracking experts, highlighting the benefits and limitations of Bionic Tracking compared to point-and-click interfaces

A Protocol to Quantify Cellular Morphodynamics: From Cell Labelling to Automatic Image Analysis

Overview of the First Eukaryome Congress at Insitut Pasteur. Paris, October 16–18, 2019.International audienceCellular morphodynamics can be used as markers for many physiological and pathological processes. This protocol provides a step-by-step guide to identify variations in motility and morphology within (or across) cell populations using non-invasive live imaging and reproducible image analysis techniques such as segmentation and tracking. Detailed instructions cover all the way from cell culturing and labelling to automatic image and statistical analyses, including the definition of multiple descriptors that characterise the shape and movement of cells in a quantitative manner. All methods are available as free open-source software and illustrated by video tutorials

Astroglial ER-mitochondria calcium transfer mediates endocannabinoid-dependent synaptic integration

Intracellular calcium signaling underlies the astroglial control of synaptic transmission and plasticity. Mitochondria-endoplasmic reticulum contacts (MERCs) are key determinants of calcium dynamics, but their functional impact on astroglial regulation of brain information processing is unexplored. We found that the activation of astrocyte mitochondrial-associated type-1 cannabinoid (mtCB1) receptors determines MERC-dependent intracellular calcium signaling and synaptic integration. The stimulation of mtCB1 receptors promotes calcium transfer from the endoplasmic reticulum to mitochondria through a specific molecular cascade, involving the mitochondrial calcium uniporter (MCU). Physiologically, mtCB1-dependent mitochondrial calcium uptake determines the dynamics of cytosolic calcium events in astrocytes upon endocannabinoid mobilization. Accordingly, electrophysiological recordings in hippocampal slices showed that conditional genetic exclusion of mtCB1 receptors or dominant-negative MCU expression in astrocytes blocks lateral synaptic potentiation, through which astrocytes integrate the activity of distant synapses. Altogether, these data reveal an endocannabinoid link between astroglial MERCs and the regulation of brain network functions

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair

International audienceDifferent mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock