Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations

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 subcompartments. 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 spatiotemporal 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.

Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

“…For TOMM20, TIMM23 and ATP5I, we observed substantially shorter T 1/2 recovery times, higher diffusion coefficients and higher mobile fractions than for MIC10 and MIC60, demonstrating that CJ proteins are more restricted in movement compared to membrane proteins of other mitochondrial complexes present in various subcompartments ( Fig 3B, D and E, Appendix Fig S1F). In line with this, MIC60 was reported to show restricted diffusion in the IM compared to OM proteins in another study [59]. To know whether the mobility of these proteins depends on the presence of a fully assembled MICOS complex, which is essential for formation of CJs, we generated MIC13 KO HeLa cells that are well suited for microscopy of mitochondria.…”

Cysteine residues in mitochondrial intermembrane space proteins: more than just import

“…This has been observed for protons that differ in their concentrations at different complexes of the respiratory chain (Rieger et al ., ). Likewise, proteins appear to be actively sorted into either the IBM or the CM, as shown for membrane‐embedded subunits of the respiratory chain and the protein import machinery (Vogel et al ., ; Appelhans and Busch, ; Stoldt et al ., ). Such sorting has also been observed for soluble proteins such as cytochrome c (Scorrano et al ., ), and it is likely that this is also found for other proteins.…”

Analysis of Mitochondrial Dimensions and Cristae Structure in Pluripotent Stem Cells Using Transmission Electron Microscopy

“…protocols and important precautions for Zstack and time-lapse imaging of mitochondria are explained elsewhere (Mitra & Lippincott-Schwartz, 2010). Dynamics of the inner mitochondrial membrane could be inferred by fluorescence recovery after photobleaching (FRAP) experiments using labeled proteins responsible for maintenance of cristae nanostructure (Appelhans & Busch, 2017).…”