Shedding light on mitochondrial outer-membrane permeabilization and membrane potential: State of the art methods and biosensors.

Membrane structural integrity is essential for optimal mitochondrial function. These organelles produce the energy needed for all vital processes, provided their outer and inner membranes are intact. This prevents the release of mitochondrial apoptogenic factors into the cytosol and ensures intact mitochondrial membrane potential (ΔΨm) to sustain ATP production. Cell death by apoptosis is generally triggered by outer mitochondrial membrane permeabilization (MOMP), tightly coupled with loss of ΔΨ m. As these two processes are essential for both mitochondrial function and cell death, researchers have devised various techniques to assess them. Here, we discuss current methods and biosensors available for detecting MOMP and measuring ΔΨ m, focusing on their advantages and limitations and discuss what new imaging tools are needed to improve our knowledge of mitochondrial function.

Cristae shaping and dynamics in mitochondrial function.

Mitochondria are multifunctional organelles of key importance for cell homeostasis. The outer mitochondrial membrane (OMM) envelops the organelle, and the inner mitochondrial membrane (IMM) is folded into invaginations called cristae. As cristae composition and functions depend on the cell type and stress conditions, they recently started to be considered as a dynamic compartment. A number of proteins are known to play a role in cristae architecture, such as OPA1, MIC60, LETM1, the prohibitin (PHB) complex and the F1FO ATP synthase. Furthermore, phospholipids are involved in the maintenance of cristae ultrastructure and dynamics. The use of new technologies, including super-resolution microscopy to visualize cristae dynamics with superior spatiotemporal resolution, as well as high-content techniques and datasets have not only allowed the identification of new cristae proteins but also helped to explore cristae plasticity. However, a number of open questions remain in the field, such as whether cristae-resident proteins are capable of changing localization within mitochondria, or whether mitochondrial proteins can exit mitochondria through export. In this Review, we present the current view on cristae morphology, stability and composition, and address important outstanding issues that might pave the way to future discoveries.

PIM2 kinase has a pivotal role in plasmablast generation and plasma cell survival, opening up novel treatment options in myeloma.

The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.

Chlamydia trachomatis development requires both host glycolysis and oxidative phosphorylation but has only minor effects on these pathways.

The obligate intracellular bacteria Chlamydia trachomatis obtain all nutrients from the cytoplasm of their epithelial host cells and stimulate glucose uptake by these cells. They even hijack host ATP, exerting a strong metabolic pressure on their host at the peak of the proliferative stage of their developmental cycle. However, it is largely unknown whether infection modulates the metabolism of the host cell. Also, the reliance of the bacteria on host metabolism might change during their progression through their biphasic developmental cycle. Herein, using primary epithelial cells and 2 cell lines of nontumoral origin, we showed that between the 2 main ATP-producing pathways of the host, oxidative phosphorylation (OxPhos) remained stable and glycolysis was slightly increased. Inhibition of either pathway strongly reduced bacterial proliferation, implicating that optimal bacterial growth required both pathways to function at full capacity. While we found C. trachomatis displayed some degree of energetic autonomy in the synthesis of proteins expressed at the onset of infection, functional host glycolysis was necessary for the establishment of early inclusions, whereas OxPhos contributed less. These observations correlated with the relative contributions of the pathways in maintaining ATP levels in epithelial cells, with glycolysis contributing the most. Altogether, this work highlights the dependence of C. trachomatis on both host glycolysis and OxPhos for efficient bacterial replication. However, ATP consumption appears at equilibrium with the normal production capacity of the host and the bacteria, so that no major shift between these pathways is required to meet bacterial needs.

Sparse denoising and adaptive estimation enhances the resolution and contrast of fluorescence emission difference microscopy based on an array detector.

An array detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high-resolution image by linearly combining sub-detector images, especially the fluorescence emission difference (FED) technique. To improve the resolution and contrast of FED microscopy based on an array detector, we propose to associate sparse denoising with spatial adaptive estimation. We show on both calibration slides and real data that our approach applied to the full stack of spatially reassigned detector signals, enables us to achieve a higher reconstruction performance in terms of resolution, image contrast, and noise reduction.

Mitochondrial transport, partitioning, and quality control at the heart of cell proliferation and fate acquisition.

Mitochondria are essential to cell homeostasis, and alterations in mitochondrial distribution, segregation, and turnover have been linked to complex pathologies such as neurodegenerative diseases and cancer. Understanding how these functions are coordinated in specific cell types is a major challenge to discover how mitochondria globally shape cell functionality. In this review, we first describe how mitochondrial transport and dynamics are regulated throughout the cell cycle in yeast and in mammals. Second, we explore the functional consequences of mitochondrial transport and partitioning on cell proliferation, fate acquisition, and stemness and on the way cells adapt their metabolism. Finally, we focus on how mitochondrial clearance programs represent a further layer of complexity for cell differentiation or in the maintenance of stemness. Defining how mitochondrial transport, dynamics, and clearance are mutually orchestrated in specific cell types may help our understanding of how cells can transition from a physiological to a pathological state.

Quantitative dSTORM super-resolution microscopy localizes Aurora kinase A/AURKA in the mitochondrial matrix.

BACKGROUND INFORMATION: Mitochondria are dynamic organelles playing essential metabolic and signaling functions in cells. Their ultrastructure has largely been investigated with electron microscopy (EM) techniques. However, quantifying protein-protein proximities using EM is extremely challenging. Super-resolution microscopy techniques as direct stochastic optical reconstruction microscopy (dSTORM) now provide a fluorescent-based, quantitative alternative to EM. Recently, super-resolution microscopy approaches including dSTORM led to valuable advances in our knowledge of mitochondrial ultrastructure, and in linking it with new insights in organelle functions. Nevertheless, dSTORM is mostly used to image integral mitochondrial proteins, and there is little or no information on proteins transiently present at this compartment. The cancer-related Aurora kinase A/AURKA is a protein localized at various subcellular locations, including mitochondria. RESULTS: We first demonstrate that dSTORM coupled to GcoPS can resolve protein proximities within individual submitochondrial compartments. Then, we show that dSTORM provides sufficient spatial resolution to visualize and quantify the most abundant pool of endogenous AURKA in the mitochondrial matrix, as previously shown for overexpressed AURKA. In addition, we uncover a smaller pool of AURKA localized at the OMM, which could have a potential functional readout. We conclude by demonstrating that aldehyde-based fixatives are more specific for the OMM pool of the kinase instead. CONCLUSIONS: Our results indicate that dSTORM coupled to GcoPS colocalization analysis is a suitable approach to explore the compartmentalization of non-integral mitochondrial proteins as AURKA, in a qualitative and quantitative manner. This method also opens up the possibility of analyzing the proximity between AURKA and its multiple mitochondrial partners with exquisite spatial resolution, thereby allowing novel insights into the mitochondrial functions controlled by AURKA. SIGNIFICANCE: Probing and quantifying the presence of endogenous AURKA - a cell cycle-related protein localized at mitochondria - in the different organelle subcompartments, using quantitative dSTORM super-resolution microscopy.

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

Insights into the non-mitotic functions of Aurora kinase A: more than just cell division.

AURKA is a serine/threonine kinase overexpressed in several cancers. Originally identified as a protein with multifaceted roles during mitosis, improvements in quantitative microscopy uncovered several non-mitotic roles as well. In physiological conditions, AURKA regulates cilia disassembly, neurite extension, cell motility, DNA replication and senescence programs. In cancer-like contexts, AURKA actively promotes DNA repair, it acts as a transcription factor, promotes cell migration and invasion, and it localises at mitochondria to regulate mitochondrial dynamics and ATP production. Here we review the non-mitotic roles of AURKA, and its partners outside of cell division. In addition, we give an insight into how structural data and quantitative fluorescence microscopy allowed to understand AURKA activation and its interaction with new substrates, highlighting future developments in fluorescence microscopy needed to better understand AURKA functions in vivo. Last, we will recapitulate the most significant AURKA inhibitors currently in clinical trials, and we will explore how the non-mitotic roles of the kinase may provide new insights to ameliorate current pharmacological strategies against AURKA overexpression.

Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance.

Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified. We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.

Sex-specific loss of mitochondrial membrane integrity and mass in the auditory brainstem of a mouse model of Fragile X syndrome.

Sound sensitivity is one of the most common sensory complaints for people with autism spectrum disorders (ASDs). How and why sounds are perceived as overwhelming by affected people is unknown. To process sound information properly, the brain requires high activity and fast processing, as seen in areas like the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem. Recent work has shown dysfunction in mitochondria, which are the primary source of energy in cells, in a genetic model of ASD, Fragile X syndrome (FXS). Whether mitochondrial functions are also altered in sound-processing neurons, has not been characterized yet. To address this question, we imaged the MNTB in a mouse model of FXS. We stained MNTB brain slices from wild-type and FXS mice with two mitochondrial markers, TOMM20 and PMPCB, located on the Outer Mitochondrial Membrane and in the matrix, respectively. These markers allow exploration of mitochondrial subcompartments. Our integrated imaging pipeline reveals significant sex-specific differences in the degree of mitochondrial length in FXS. Significant differences are also observable in the overall number of mitochondria in male FXS mice, however, colocalization analyses between TOMM20 and PMPCB reveal that the integrity of these compartments is most disrupted in female FXS mice. We highlight a quantitative fluorescence microscopy pipeline to monitor mitochondrial functions in the MNTB from control or FXS mice and provide four complementary readouts. Our approach paves the way to understanding how cellular mechanisms important to sound encoding are altered in ASDs.

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells.

Although several mechanisms of macroautophagy/autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to its activation, the ATG4B protease primes the key autophagy player MAP1LC3B/LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster's resonance energy transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine-tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image makes it possible to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are pools of unprimed LC3B upon ATG4B downregulation, and the priming of the biosensor is abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Moreover, we screened for commercially-available ATG4B inhibitors, and illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Finally, we uncovered the CDK1-dependent regulation of the ATG4B-LC3B axis at mitosis. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.Abbreviations: Aqua: aquamarine; ATG: autophagy related; AURKA: aurora kinase A; BafA1: bafilomycin A1; CDK1: cyclin dependent kinase 1; DKO: double knockout; FLIM: fluorescence lifetime imaging microscopy; FP: fluorescence protein; FRET: Förster's resonance energy transfer; GABARAP: GABA type A receptor-associated protein; HBSS: Hanks' balanced salt solution; KO: knockout; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSC: NSC 185058; PE: phosphatidylethanolamine; SKO: single knockout; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type; ZPCK: Z-L-phe chloromethyl ketone.

Aurora kinase A/AURKA functionally interacts with the mitochondrial ATP synthase to regulate energy metabolism and cell death.

Cancer cells often hijack metabolic pathways to obtain the energy required to sustain their proliferation. Understanding the molecular mechanisms underlying cancer cell metabolism is key to fine-tune the metabolic preference of specific tumors, and potentially offer new therapeutic strategies. Here, we show that the pharmacological inhibition of mitochondrial Complex V delays the cell cycle by arresting breast cancer cell models in the G0/G1 phase. Under these conditions, the abundance of the multifunctional protein Aurora kinase A/AURKA is specifically lowered. We then demonstrate that AURKA functionally interacts with the mitochondrial Complex V core subunits ATP5F1A and ATP5F1B. Altering the AURKA/ATP5F1A/ATP5F1B nexus is sufficient to trigger G0/G1 arrest, and this is accompanied by decreased glycolysis and mitochondrial respiration rates. Last, we discover that the roles of the AURKA/ATP5F1A/ATP5F1B nexus depend on the specific metabolic propensity of triple-negative breast cancer cell lines, where they correlate with cell fate. On one hand, the nexus induces G0/G1 arrest in cells relying on oxidative phosphorylation as the main source of energy. On the other hand, it allows to bypass cell cycle arrest and it triggers cell death in cells with a glycolytic metabolism. Altogether, we provide evidence that AURKA and mitochondrial Complex V subunits cooperate to maintain cell metabolism in breast cancer cells. Our work paves the way to novel anti-cancer therapies targeting the AURKA/ATP5F1A/ATP5F1B nexus to lower cancer cell metabolism and proliferation.

Approaches to monitor ATP levels in living cells: where do we stand?

ATP is the most universal and essential energy molecule in cells. This is due to its ability to store cellular energy in form of high-energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, and signaling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolyzed with a spatiotemporal resolution is necessary to understand its functions both in physiological and in pathological contexts. In this review, first we will describe the organization of the electron transport chain and ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths, and weaknesses. Finally, we will explore the palette of genetically encoded biosensors designed for microscopy-based approaches, and show how their spatiotemporal resolution opened up the possibility to follow ATP levels in living cells.

3D quantification of autophagy activation and autophagosome-to-mitochondria recruitment in a Drosophila model of Parkinson's disease.

Here, we describe a protocol for comprehensive quantification of autophagosome recruitment to mitochondria as an early step in mitophagy. Data collected using this protocol can be useful in the study of neurodegenerative disease, cancer, and metabolism-related disorders using models in which co-expression of mito-GFP and mCherry-Atg8a is feasible. This protocol has the advantage of assessment in an in vivo model organism (Drosophila melanogaster), where tissue-specific mitophagy can be investigated. For complete details on the use and execution of this protocol, please refer to (Cackovic et al., 2018).

tdLanYFP, a Yellow, Bright, Photostable, and pH-Insensitive Fluorescent Protein for Live-Cell Imaging and Förster Resonance Energy Transfer-Based Sensing Strategies.

Yellow fluorescent proteins (YFPs) are widely used as optical reporters in Förster resonance energy transfer (FRET)-based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pH values. Here, we characterize the yellow fluorescent protein tdLanYFP, derived from the tetrameric protein from the cephalochordate Branchiostoma lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133,000 mol-1·L·cm-1, it is, to our knowledge, the brightest dimeric fluorescent protein available. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and in live cells. As a consequence, tdLanYFP allows imaging of cellular structures with subdiffraction resolution using STED nanoscopy and is compatible with the use of spectromicroscopies in single-molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pH values. Finally, we show that tdLanYFP is a valuable FRET partner either as a donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFP a very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging including FRET experiments at acidic pH.

Mitochondrial Aurora kinase A induces mitophagy by interacting with MAP1LC3 and Prohibitin 2.

Epithelial and haematologic tumours often show the overexpression of the serine/threonine kinase AURKA. Recently, AURKA was shown to localise at mitochondria, where it regulates mitochondrial dynamics and ATP production. Here we define the molecular mechanisms of AURKA in regulating mitochondrial turnover by mitophagy. AURKA triggers the degradation of Inner Mitochondrial Membrane/matrix proteins by interacting with core components of the autophagy pathway. On the inner mitochondrial membrane, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2, which triggers mitophagy in a PARK2/Parkin-independent manner. The formation of the tripartite complex is induced by the phosphorylation of PHB2 on Ser39, which is required for MAP1LC3 to interact with PHB2. Last, treatment with the PHB2 ligand xanthohumol blocks AURKA-induced mitophagy by destabilising the tripartite complex and restores normal ATP production levels. Altogether, these data provide evidence for a role of AURKA in promoting mitophagy through the interaction with PHB2 and MAP1LC3. This work paves the way to the use of function-specific pharmacological inhibitors to counteract the effects of the overexpression of AURKA in cancer.

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging.

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells.

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded Förster's Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

Imaging Mitochondrial Functions: from Fluorescent Dyes to Genetically-Encoded Sensors.

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.