Abstract
Mitochondria are cellular organelles with multifaceted tasks and thus composed of different sub-compartments. The inner mitochondrial membrane especially has a complex nano-architecture with cristae protruding into the matrix. Related to their function, the localization of mitochondrial membrane proteins is more or less restricted to specific sub-compartments. In contrast, it can be assumed that membrane proteins per se diffuse unimpeded through continuous membranes. Fluorescence recovery after photobleaching is a versatile technology used in mobility analyses to determine the mobile fraction of proteins, but it cannot provide data on subpopulations or on confined diffusion behavior. Fluorescence correlation spectroscopy is used to analyze single molecule diffusion, but no trajectory maps are obtained. Single particle tracking (SPT) technologies in live cells, such as tracking and localization microscopy (TALM), do provide nanotopic localization and mobility maps of mitochondrial proteins in situ. Molecules can be localized with a precision of between 10 and 20 nm, and single trajectories can be recorded and analyzed; this is sufficient to reveal significant differences in the spatio-temporal behavior of diverse mitochondrial proteins. Here, we compare diffusion coefficients obtained by these different technologies and discuss trajectory maps of diverse mitochondrial membrane proteins obtained by SPT/TALM. We show that membrane proteins in the outer membrane generally display unhindered diffusion, while the mobility of inner membrane proteins is restricted by the inner membrane architecture, resulting in significantly lower diffusion coefficients. Moreover, tracking analysis could discern proteins in the inner boundary membrane from proteins preferentially diffusing in cristae membranes, two sub-compartments of the inner mitochondrial membrane. Thus, by evaluating trajectory maps it is possible to assign proteins to different sub-compartments of the same membrane.
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Acknowledgments
The authors would like to thank Wladislaw Kohl for technical advice and support. We also wish to express our gratitude to Christian Richter for evaluation software. The study was supported by the CRC SFB944 and CiM/Münster.
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Conception and design: K.B., T.A. Acquisition of data: T.A.; K.B. Analysis and interpretation of data: T.A., K.B. Drafting or revising the article: K.B.
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Timo Appelhans declares that he has no conflicts of interest. Karin B. Busch declares that she has no conflicts of interest.
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This article is part of a Special Issue on ‘IUPAB Edinburgh Congress’ edited by Damien Hall
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Appelhans, T., Busch, K.B. Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations. Biophys Rev 9, 345–352 (2017). https://doi.org/10.1007/s12551-017-0287-1
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DOI: https://doi.org/10.1007/s12551-017-0287-1