Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo
Abstract
We present the development and in vivo application of a feedback-based tracking microscope to follow individual mitochondria in sensory neurons of zebrafish larvae with nanometer precision and millisecond temporal resolution. By combining various technical improvements, we tracked individual mitochondria with unprecedented spatiotemporal resolution over distances of >100µm. Using these nanoscopic trajectory data, we discriminated five motional states: a fast and a slow directional motion state in both the anterograde and retrograde directions and a stationary state. The transition pattern revealed that mitochondria predominantly persist in the original direction of travel after a short pause, while transient changes of direction often exhibited longer pauses. Moreover, mitochondria in the vicinity of a second, stationary mitochondria displayed an increased probability to pause. The capability of following and optically manipulating a single organelle with high spatiotemporal resolution in a living organism offers a new approach to elucidating their function in its complete physiological context.
Data availability
The analysis software program is available on Gitlab and the wide-field images and trajectories are available on Zenodo. Source data files have been provided for all the figures.
-
Wide-field Images and TrajectoriesZenodo, 10.5281/zenodo.2813946.
-
Wide-field Images and TrajectoriesZenodo, 10.5281/zenodo.2815430.
-
Wide-field Images and TrajectoriesZenodo, 10.5281/zenodo.281550.
-
Wide-field Images and TrajectoriesZenodo, 10.5281/zenodo.2815703.
-
Wide-field Images and TrajectoriesZenodo, 10.5281/zenodo.2815801.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (SFB1032 (Project B3))
- Thomas Misgeld
- Don C Lamb
Fakultät für Chemie und Pharmazie, Ludwig-Maximilians-Universität München (Center for NanoScience (CeNS) and the BioImaging Network (BIN))
- Don C Lamb
H2020 European Research Council (ERC Grant Agreement n. 616791)
- Thomas Misgeld
German Center for Neurodegenerative Diseases
- Thomas Misgeld
Deutsche Forschungsgemeinschaft (research grants Mi 694/7)
- Thomas Misgeld
- Don C Lamb
Deutsche Forschungsgemeinschaft (Priority Program SPP1710)
- Thomas Misgeld
- Don C Lamb
Deutsche Forschungsgemeinschaft (SFB870 15 (Project A11))
- Thomas Misgeld
- Don C Lamb
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Wehnekamp et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,422
- views
-
- 347
- downloads
-
- 35
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Physics of Living Systems
Bacterial biofilms are communities of bacteria usually attached to solid strata and often differentiated into complex structures. Communication across biofilms has been shown to involve chemical signaling and, more recently, electrical signaling in Gram-positive biofilms. We report for the first time, community-level synchronized membrane potential dynamics in three-dimensional Escherichia coli biofilms. Two hyperpolarization events are observed in response to light stress. The first requires mechanically sensitive ion channels (MscK, MscL, and MscS) and the second needs the Kch-potassium channel. The channels mediated both local spiking of single E. coli biofilms and long-range coordinated electrical signaling in E. coli biofilms. The electrical phenomena are explained using Hodgkin-Huxley and 3D fire-diffuse-fire agent-based models. These data demonstrate that electrical wavefronts based on potassium ions are a mechanism by which signaling occurs in Gram-negative biofilms and as such may represent a conserved mechanism for communication across biofilms.
-
- Cell Biology
- Physics of Living Systems
The Gillespie algorithm is commonly used to simulate and analyze complex chemical reaction networks. Here, we leverage recent breakthroughs in deep learning to develop a fully differentiable variant of the Gillespie algorithm. The differentiable Gillespie algorithm (DGA) approximates discontinuous operations in the exact Gillespie algorithm using smooth functions, allowing for the calculation of gradients using backpropagation. The DGA can be used to quickly and accurately learn kinetic parameters using gradient descent and design biochemical networks with desired properties. As an illustration, we apply the DGA to study stochastic models of gene promoters. We show that the DGA can be used to: (1) successfully learn kinetic parameters from experimental measurements of mRNA expression levels from two distinct Escherichia coli promoters and (2) design nonequilibrium promoter architectures with desired input–output relationships. These examples illustrate the utility of the DGA for analyzing stochastic chemical kinetics, including a wide variety of problems of interest to synthetic and systems biology.