Mitochondria form a network in the cell that rapidly changes through fission, fusion, and motility. Dysregulation of this four-dimensional (4D: x,y,z,time) network is implicated in numerous diseases ranging from cancer to neurodegeneration. While lattice light-sheet microscopy has recently made it possible to image mitochondria in 4D, quantitative analysis methods for the resulting datasets have been lacking. Here we present MitoTNT, the first-in-class software for Mitochondrial Temporal Network Tracking in 4D live-cell fluorescence microscopy data. MitoTNT uses spatial proximity and network topology to compute an optimal tracking assignment. To validate the accuracy of tracking, we created a reaction-diffusion simulation to model mitochondrial network motion and remodeling events. We found that our tracking is >90% accurate for ground-truth simulations and agrees well with published motility results for experimental data. We used MitoTNT to quantify 4D mitochondrial networks from human induced pluripotent stem cells. First, we characterized sub-fragment motility and analyzed network branch motion patterns. We revealed that the skeleton node motion is correlated along branch nodes and is uncorrelated in time. Second, we identified fission and fusion events with high spatiotemporal resolution. We found that mitochondrial skeleton nodes near the fission/fusion sites move nearly twice as fast as random skeleton nodes and that microtubules play a role in mediating selective fission/fusion. Finally, we developed graph-based transport simulations that model how material would distribute on experimentally measured mitochondrial temporal networks. We showed that pharmacological perturbations increase network reachability but decrease network resilience through a combination of altered mitochondrial fission/fusion dynamics and motility. MitoTNT's easy-to-use tracking module, interactive 4D visualization capability, and powerful post-tracking analyses aim at making temporal network tracking accessible to the wider mitochondria research community.

Summary

To expedite the review of semi-automated probability maps of organelles and other features from 3D electron microscopy data we have developed Probability Map Viewer, a Java-based web application that enables the computation and visualization of probability map generation results in near real-time as the data are being collected from the microscope. Probability Map Viewer allows the user to select one or more voxel classifiers, apply them on a sub-region of an active collection, and visualize the results as overlays on the raw data via any web browser using a personal computer or mobile device. Thus, Probability Map Viewer accelerates and informs the image analysis workflow by providing a tool for experimenting with and optimizing dataset-specific segmentation strategies during imaging.

Availability and implementation

https://github.com/crbs/probabilitymapviewer.

Contact

mellisman@ucsd.edu.

Supplementary information

Supplementary data are available at Bioinformatics online.

During migration, eukaryotic cells can continuously change their three-dimensional morphology, resulting in a highly dynamic and complex process. Further complicating this process is the observation that the same cell type can rapidly switch between different modes of migration. Modelling this complexity necessitates models that are able to track deforming membranes and that can capture the intracellular dynamics responsible for changes in migration modes. Here we develop an efficient three-dimensional computational model for cell migration, which couples cell mechanics to a simple intracellular activator-inhibitor signalling system. We compare the computational results to quantitative experiments using the social amoeba Dictyostelium discoideum. The model can reproduce the observed migration modes generated by varying either mechanical or biochemical model parameters and suggests a coupling between the substrate and the biomechanics of the cell.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated MELT motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

Mitochondrial dysfunction is a characteristic trait of human and rodent obesity, insulin resistance and fatty liver disease. Here we show that high-fat diet (HFD) feeding causes mitochondrial fragmentation in inguinal white adipocytes from male mice, leading to reduced oxidative capacity by a process dependent on the small GTPase RalA. RalA expression and activity are increased in white adipocytes after HFD. Targeted deletion of RalA in white adipocytes prevents fragmentation of mitochondria and diminishes HFD-induced weight gain by increasing fatty acid oxidation. Mechanistically, RalA increases fission in adipocytes by reversing the inhibitory Ser637 phosphorylation of the fission protein Drp1, leading to more mitochondrial fragmentation. Adipose tissue expression of the human homolog of Drp1, DNM1L, is positively correlated with obesity and insulin resistance. Thus, chronic activation of RalA plays a key role in repressing energy expenditure in obese adipose tissue by shifting the balance of mitochondrial dynamics toward excessive fission, contributing to weight gain and metabolic dysfunction.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ_377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.