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Mifobio 2023 - Giulia BERTOLIN - Illuminating mitochondrial functions during the cell cycle using
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Giulia BERTOLIN - Illuminating mitochondrial functions during the cell cycle using FRET - based biosensors : Mitochondria are signaling hubs which are key for cell physiology.
At the molecular level, mitochondria are perfectly integrated with other subcellular compartments and machineries. For instance, functions such as mitochondrial morphology and ATP production are known to change according to the cell cycle phase of the cell. However, our view on how the cell cycle machinery can directly shape mitochondrial activities is still fragmentary.
Although biochemical approaches are largely used to investigate mitochondrial functions, genetically-encoded Förster’s Resonance Energy Transfer (FRET) sensors are becoming convenient tools to uncover how mitochondrial activities evolve in space and time. During my talk, I will cover how we discovered the activation of a custom-made FRET biosensor of the cell cycle protein Aurora kinase A/AURKA at mitochondria1–3. I will also describe the experimental and analytical strategies we implemented to increase the dynamic range of the biosensor, and to make it compatible with multiplex FRET strategies relying on Fluorescence Lifetime Imaging Microscopy (FLIM). These strategies include alternative donor/acceptor FRET pairs, and multiple ways to calculate FRET efficiency.
Thanks to the activation of the biosensor at mitochondria, we identified novel roles of AURKA in regulating the turnover of defective organelles and ATP production2,4. Given our interest in the turnover of intracellular material –including mitochondria–we engineered the LC3B biosensor, a genetically-encoded FRET probe to follow this pathway. I will describe notonly the design of the probe, but also a broad-to-sensitive image analysis pipeline to follow clearance by autophagy with an unprecedented spatiotemporal precision. With this tool, we discovered the impact of the cell cycle in regulating turnover by autophagy5. Together, I will highlight how the use of genetically-encoded FRET biosensors pave the way for a better understanding of mitochondrial functions, and show how the cell cycle is a key element regulating the multiple activities of this organelle.
Giulia Bertolin,CNRS, Univ Rennes, IGDR(Institute of Genetics and Development of Rennes),
UMR 6290, F-35000 Rennes, France
At the molecular level, mitochondria are perfectly integrated with other subcellular compartments and machineries. For instance, functions such as mitochondrial morphology and ATP production are known to change according to the cell cycle phase of the cell. However, our view on how the cell cycle machinery can directly shape mitochondrial activities is still fragmentary.
Although biochemical approaches are largely used to investigate mitochondrial functions, genetically-encoded Förster’s Resonance Energy Transfer (FRET) sensors are becoming convenient tools to uncover how mitochondrial activities evolve in space and time. During my talk, I will cover how we discovered the activation of a custom-made FRET biosensor of the cell cycle protein Aurora kinase A/AURKA at mitochondria1–3. I will also describe the experimental and analytical strategies we implemented to increase the dynamic range of the biosensor, and to make it compatible with multiplex FRET strategies relying on Fluorescence Lifetime Imaging Microscopy (FLIM). These strategies include alternative donor/acceptor FRET pairs, and multiple ways to calculate FRET efficiency.
Thanks to the activation of the biosensor at mitochondria, we identified novel roles of AURKA in regulating the turnover of defective organelles and ATP production2,4. Given our interest in the turnover of intracellular material –including mitochondria–we engineered the LC3B biosensor, a genetically-encoded FRET probe to follow this pathway. I will describe notonly the design of the probe, but also a broad-to-sensitive image analysis pipeline to follow clearance by autophagy with an unprecedented spatiotemporal precision. With this tool, we discovered the impact of the cell cycle in regulating turnover by autophagy5. Together, I will highlight how the use of genetically-encoded FRET biosensors pave the way for a better understanding of mitochondrial functions, and show how the cell cycle is a key element regulating the multiple activities of this organelle.
Giulia Bertolin,CNRS, Univ Rennes, IGDR(Institute of Genetics and Development of Rennes),
UMR 6290, F-35000 Rennes, France