Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor – referred to as pH-Lemon – optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.

Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K⁺, alkaline earth metals including Mg²⁺ and Ca²⁺, and transition metals including Cu⁺/Cu²⁺ and Zn²⁺. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe²⁺/Fe³⁺, Mn²⁺ and Na⁺.

The interplay of metabolic and signaling processes is prerequisite for the functionality of cells. Any disturbances may have severe consequences, resulting in the development of diseases. However, the complex coordination of metabolism and signaling events makes it difficult to decipher the link between molecular irregularities and pathogenesis. An excellent way to provide more clarity is to see into the living cell and watch cellular processes in real-time, with the add-on of being able to manipulate certain processes. Live cell imaging enables us to do exactly that, with steadily improving spatial and temporal resolution. Modern genetically encoded fluorescent probes in combination with state-of-the-art high-resolution imaging devices have proven themselves as a valuable approach for monitoring, manipulating and ultimately understanding the interaction of cell metabolism and signaling. These probes also represent powerful tools for detecting biomarkers of disease, identifying new drug targets and elucidating drug actions at the cellular to the molecular level.

Over the last decades a broad collection of sophisticated fluorescent protein-based probes was engineered with the aim to specifically monitor nitric oxide (NO), one of the most important signaling molecules in biology. Here we report and discuss the characteristics and fields of applications of currently available genetically encoded fluorescent sensors for the detection of NO and its metabolites in different cell types. Because of its radical nature and short half-life, real-time imaging of NO on the level of single cells is challenging. Herein we review state-of-the-art genetically encoded fluorescent sensors for NO and its byproducts such as peroxynitrite, nitrite and nitrate. Such probes enable the real-time visualization of NO signals directly or indirectly on the level of single cells and cellular organelles and, hence, extend our understanding of the spatiotemporal dynamics of NO formation, diffusion and degradation. Here, we discuss the significance of NO detection in individual cells and on subcellular level with genetic biosensors. Currently available genetically encoded fluorescent probes for NO and nitrogen species are critically discussed in order to provide insights in the functionality and applicability of these promising tools. As an outlook we provide ideas for novel approaches for the design and application of improved NO probes and fluorescence imaging protocols.

Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca²⁺ and fundamentally contributing to various signaling pathways. Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine‐tuned by intra‐ and interorganelle H⁺, K⁺, Na⁺, and Ca²⁺ signaling. In this review, we provide an up‐to‐date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting‐edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.

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.