Probing of protein localization and shuttling in mitochondrial microcompartments by FLIM with sub-diffraction resolution.
Probing of protein localization and shuttling in mitochondrial microcompartments by FLIM with sub-diffraction resolution.
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DOI：
10.1016/j.bbabio.2016.03.021
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发表时间：
2016-08
期刊：
影响因子：
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通讯作者：
Anna-Carina Söhnel;W. Kohl;I. Gregor;J. Enderlein;Bettina Rieger;K. Busch
中科院分区：
文献类型：
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作者：
Anna-Carina Söhnel;W. Kohl;I. Gregor;J. Enderlein;Bettina Rieger;K. Busch
细胞在代谢上是高度区室化的。特别是，线粒体在其不同的微区室中主持许多重要的反应。然而，由于它们的小尺寸，这些微区室是无法通过常规显微镜。在这里，我们证明了时间相关单光子计数（TCSPC）荧光寿命成像显微镜（FLIM）分类不仅线粒体，但线粒体内不同的微区室。基质中的传感器蛋白具有与膜蛋白处的探针不同的寿命。在线粒体外膜和内膜的定位可以区分寿命的显着差异。该方法足够灵敏，可以监测线粒体微区室内蛋白质位置的变化。蛋白质含量的变化引起的大分子拥挤显着影响的寿命，而氧化条件或生理pH值的变化只有边际效应。我们认为，FLIM是一个通用的和完整的方法来监测时空事件的线粒体。时域中的灵敏度允许获得关于克服衍射限制的亚线粒体定位的大量信息。这篇文章是题为“EBEC 2016：第19届欧洲生物能量学会议，加尔达湖滨，意大利，2016年7月2日至6日”的特刊的一部分，由Paolo Bernardi教授编辑。
The cell is metabolically highly compartmentalized. Especially, mitochondria host many vital reactions in their different microcompartments. However, due to their small size, these microcompartments are not accessible by conventional microscopy. Here, we demonstrate that time-correlated single-photon counting (TCSPC) fluorescence lifetime-imaging microscopy (FLIM) classifies not only mitochondria, but different microcompartments inside mitochondria. Sensor proteins in the matrix had a different lifetime than probes at membrane proteins. Localization in the outer and inner mitochondrial membrane could be distinguished by significant differences in the lifetime. The method was sensitive enough to monitor shifts in protein location within mitochondrial microcompartments. Macromolecular crowding induced by changes in the protein content significantly affected the lifetime, while oxidizing conditions or physiological pH changes had only marginal effects. We suggest that FLIM is a versatile and completive method to monitor spatiotemporal events in mitochondria. The sensitivity in the time domain allows for gaining substantial information about sub-mitochondrial localization overcoming diffraction limitation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.