Abstract
Mitochondria are multifunctional organelles that are crucial to cell homeostasis. They constitute the major site of energy production for the cell, they are key players in signalling pathways using secondary messengers such as calcium, and they are involved in cell death and redox balance paradigms. Mitochondria quickly adapt their dynamics and biogenesis rates to meet the varying energy demands of the cells, both in normal and in pathological conditions. Therefore, understanding simultaneous changes in mitochondrial functions is crucial in developing mitochondria-based therapy options for complex pathological conditions such as cancer, neurological disorders, and metabolic syndromes. To this end, fluorescence microscopy coupled to live imaging represents a promising strategy to track these changes in real time. In this review, we will first describe the commonly available tools to follow three key mitochondrial functions using fluorescence microscopy: Calcium signalling, mitochondrial dynamics, and mitophagy. Then, we will focus on how the development of genetically-encoded fluorescent sensors became a milestone for the understanding of these mitochondrial functions. In particular, we will show how these tools allowed researchers to address several biochemical activities in living cells, and with high spatiotemporal resolution. With the ultimate goal of tracking multiple mitochondrial functions simultaneously, we will conclude by presenting future perspectives for the development of novel genetically-encoded fluorescent biosensors.
Highlights
Mitochondria are essentially known for their role in adenosine triphosphate (ATP) synthesis and contribution to cellular metabolism, yet these organelles regulate a multitude of cellular functions.These functions include Ca2+ buffering, cellular signalling, reactive oxygen species (ROS) production, and apoptotic cell death [1,2,3,4]
Mitochondrial dynamics refers to the balance between mitochondrial fusion and fission events to regulate the size, shape, number, and distribution of mitochondria in cells, and this balance is constantly adjusted in response to physiological cues (Figure 2) [107]
The majority of studies have benefited from imaging probes targeted at mitochondria
Summary
Mitochondria are essentially known for their role in adenosine triphosphate (ATP) synthesis and contribution to cellular metabolism, yet these organelles regulate a multitude of cellular functions. The very first examples of the FRET-based GECIs are cameleons, consisting of blue and green fluorescent proteins, acting as donor and acceptor FRET pairs These fluorophores are connected with a calmodulin domain fused to a myosin light chain kinase M13 [90]. In the case of FRET-based GECIs, Ca2+ binding to the calmodulin domain changes the conformation of the probe, bringing the donor and acceptor pair into close proximity. Compared to FRET-based GECIs, GECO indicators have better signal-to-noise ratios, and they allow for multicolour imaging of Ca2+ ions in different organelles, or in different compartments of an organelle [96] They are currently available with red, blue, and green intensiometric emissions [95]. Ca2+ signalling will deepen our fundamental knowledge about the physiology of the cell, but it will encourage the development of more sophisticated microscopy-based tools
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