Tissue clearing method in visualization of cancer progression and metastasis.

Since various imaging modalities have been developed, cancer metastasis can be detected from an early stage. However, limitations still exist, especially in terms of spatial resolution. Tissue-clearing technology has emerged as a new imaging modality in cancer research, which has been developed and utilized for a long time mainly in neuroscience field. This method enables us to detect cancer metastatic foci with single-cell resolution at whole mouse body/organ level. On top of that, 3D images of cancer metastasis of whole mouse organs make it easy to understand their characteristics. Recently, further applications of tissue clearing methods were reported in combination with reporter systems, labeling, and machine learning. In this review, we would like to provide an overview of this technique and current applications in cancer research and discuss their potentials and limitations.

High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (ΔF/F = 15 to 30 in vitro), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities. We evaluated these biosensors in cultured cells and demonstrated their application in an ex vivo preparation of Drosophila brain tissue. Using these biosensors, we were able to detect glycolytic oscillations, which we analyzed and mathematically modeled.

Biosensor Optimization Using a Förster Resonance Energy Transfer Pair Based on mScarlet Red Fluorescent Protein and an mScarlet-Derived Green Fluorescent Protein.

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca2+, and K+. Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.

The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria.

Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⍺ covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⍺ interaction requires the C-terminal active site of ERO1⍺ and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⍺ complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca2+ flux to maintain bioenergetics in both organelles, while limiting oxidative stress. Therefore, the PERK-ERO1⍺ complex is a key molecular machinery that allows quick metabolic adaptation to ER stress.

Quantification of Intracellular Citrate Concentrations with Genetically Encoded Biosensors.

Citrate is a central intracellular metabolite with roles in a variety of normal and aberrant biological processes. The methods for quantifying citrate concentration in cells can enable the study of the molecular mechanisms of citrate-related biological processes and diseases. Compared to existing analytical methods such as enzymatic assays and mass spectrometry, genetically encoded biosensors based on fluorescent proteins (FPs) offer the advantage of noninvasively tracking intracellular ion and small molecule dynamics with high spatial-temporal resolution. These biosensors are less toxic than chemosensors and can be targeted to specific organelles for subcellular imaging. Here we present a protocol for quantification of cytosolic and mitochondrial citrate in mammalian cells with recently reported genetically encoded biosensors for citrate.