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Cells. 2019 Dec 6;8(12). pii: E1583. doi: 10.3390/cells8121583.

Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation.

Author information

1
Gottfried Schatz Research Center, Chair of Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.
2
Division of Cardiology, Medical University of Graz, 8010 Graz, Austria.
3
BioTechMed Graz, Mozartgasse 12/II, 8010 Graz, Austria.

Abstract

Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic β-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR (mitochondrial sirtuin 4 tripartite abundance reporter), a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles.

KEYWORDS:

array confocal laser scanning microscopy; fluorescence microscopy; fluorescent protein; genetically encoded sensor; mitochondrial protein import; self-complementing split FP technology; sfCherry2; sfGFP; sirtuin 4; structural illumination microscopy

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