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
-
- 2,394
- views
-
- 344
- downloads
-
- 34
- 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
-
- Computational and Systems Biology
- Physics of Living Systems
B-cell repertoires are characterized by a diverse set of receptors of distinct specificities generated through two processes of somatic diversification: V(D)J recombination and somatic hypermutations. B-cell clonal families stem from the same V(D)J recombination event, but differ in their hypermutations. Clonal families identification is key to understanding B-cell repertoire function, evolution, and dynamics. We present HILARy (high-precision inference of lineages in antibody repertoires), an efficient, fast, and precise method to identify clonal families from single- or paired-chain repertoire sequencing datasets. HILARy combines probabilistic models that capture the receptor generation and selection statistics with adapted clustering methods to achieve consistently high inference accuracy. It automatically leverages the phylogenetic signal of shared mutations in difficult repertoire subsets. Exploiting the high sensitivity of the method, we find the statistics of evolutionary properties such as the site frequency spectrum and dN/dS ratio do not depend on the junction length. We also identify a broad range of selection pressures spanning two orders of magnitude.
-
- Physics of Living Systems
We propose the Self Returning Excluded Volume (SR-EV) model for the structure of chromatin based on stochastic rules and physical interactions. The SR-EV rules of return generate conformationally defined domains observed by single-cell imaging techniques. From nucleosome to chromosome scales, the model captures the overall chromatin organization as a corrugated system, with dense and dilute regions alternating in a manner that resembles the mixing of two disordered bi-continuous phases. This particular organizational topology is a consequence of the multiplicity of interactions and processes occurring in the nuclei, and mimicked by the proposed return rules. Single configuration properties and ensemble averages show a robust agreement between theoretical and experimental results including chromatin volume concentration, contact probability, packing domain identification and size characterization, and packing scaling behavior. Model and experimental results suggest that there is an inherent chromatin organization regardless of the cell character and resistant to an external forcing such as RAD21 degradation.