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Membraneless compartments can form in cells through liquidliquid phase separation (see the Perspective by Polymenidou). But what prevents these cellular condensates from randomly fusing together? Using the RNA-binding protein (RBP) Whi3, Langdon et al. demonstrated that the secondary structure of different RNA components determines the distinct biophysical and biological properties of the two types of condensates that Whi3 forms. Several RBPs, such as FUS and TDP43, contain prion-like domains and are linked to neurodegenerative diseases. These RBPs are usually soluble in the nucleus but can form pathological aggregates in the cytoplasm. Maharana et al. showed that local RNA concentrations determine distinct phase separation behaviors in different subcellular locations. The higher RNA concentrations in the nucleus act as a buffer to prevent phase separation of RBPs; when mislocalized to the cytoplasm, lower RNA concentrations trigger aggregation. Science , this issue p. 922 , p. 918 ; see also p. 859 High concentrations of RNA prevent pathological aggregation of RNA binding proteins in the nucleus. Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNA critically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, but a substantial proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 °C, mitochondria were more than 10 °C warmer when the respiratory chain (RC) was fully functional, both in human embryonic kidney (HEK) 293 cells and primary skin fibroblasts. This differential was abolished in cells depleted of mitochondrial DNA or treated with respiratory inhibitors but preserved or enhanced by expressing thermogenic enzymes, such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at or slightly above 50 °C. In view of their potential consequences, these observations need to be further validated and explored by independent methods. Our study prompts a critical re-examination of the literature on mitochondria.

► Assays to quantify cancer biomarkers with high sensitivity and low sample volumes. ► Imaging of cancer cells, tissues, and small animals with multiplexed capabilities. ► Multimodal SERS probes and their potential for clinical translation. Technologies that use surface-enhanced Raman scattering (SERS) have experienced significant growth in biomedical research during the past 4 years. In this review we summarize the progress in SERS for cancer diagnostics, including multiplexed detection and identification of new biomarkers, single-nucleotide polymorphisms, and circulating tumor cells. SERS is also used as a non-invasive tool for cancer imaging with immunoSERS microscopy, histological analysis of biopsies, and in vivo detection of tumors. We discuss the future of SERS probes compatible with multiple imaging modalities and their potential for clinical translation (e.g., endoscope-based and intraoperative imaging as tools for surgical guidance). Moreover, we highlight the potential of SERS agents for targeted drug delivery and photothermal therapy.

Macrophages play crucial roles in protecting our bodies from infection and cancers. As macrophages are multi-functional immune cells, they have diverse plastic subsets, such as M1 and M2, derived from naïve M0 cells. Subset-specific macrophage probes are essential for deciphering and monitoring the various activation of macrophages, but developing such probes has been challenging. Here we report a fluorescent probe, CDr17, which is selective for M1 macrophages over M2 or M0. The selective staining mechanism of CDr17 is explicated as Gating-Oriented Live-cell Distinction (GOLD) through overexpressed GLUT1 in M1 macrophages. Finally, we demonstrate the suitability of CDr17 to track M1 macrophages in vivo in a rheumatoid arthritis animal model. Studying the specific roles of macrophage subsets has been hampered by a lack of subset-specific probes. Here the authors report an M1 selective fluorescent probe named CDr17, and demonstrate the suitability of this probe for tracking M1 macrophages in vivo.

Intracellular viscosity is a fundamental physical parameter that indicates the functioning of cells. In this work, we developed a simple boron-dipyrromethene (BODIPY)-based probe, BTV, for cellular mitochondria viscosity imaging by coupling a simple BODIPY rotor with a mitochondria-targeting unit. The BTV exhibited a significant fluorescence intensity enhancement of more than 100-fold as the solvent viscosity increased. Also, the probe showed a direct linear relationship between the fluorescence lifetime and the media viscosity, which makes it possible to trace the change of the medium viscosity. Furthermore, it was demonstrated that BTV could achieve practical applicability in the monitoring of mitochondrial viscosity changes in live cells through fluorescence lifetime imaging microscopy (FLIM).

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16. Attempts to image activated macrophages in vivo have been hampered by selectivity and delivery problems. Here the authors develop a small molecule fluorescent probe specific to activated M1 and M2 macrophages, identify the orphan receptor Slc18b1/SLC18B1 as the mechanism of uptake, and use it to image atherosclerosis in mice.

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Cucurbitacin B (CuB) isolated from Cucumis melo by using a PC12 cell bioassay system exhibited significant nerve growth factor (NGF)‐mimic or NGF‐enhancing activity in PC12 and primary neuron cells. It was also demonstrated pro‐neurogenesis effects in ICR and APP/PS1 mice and improved memory deficit of APP/PS1 mice. Its possible mechanism includes significant induction of the phosphorylation of glucocorticoid receptor (GR), protein kinase C (PKC), phospholipase C (PLC) and inhibition of cofilin. ChemProteoBase profiling, binding assay and cellular thermal shift assay (CETSA) were used to determine the target protein. Results revealed that CuB could affect actin dynamics as an actin inhibitor but did not bind with GR. The protein level of cofilin in PC12 cells after treating 0.3 μM and different temperatures was significantly higher than that of control group. Other neurotrophic signalling pathways, such as TrkA/TrkB, were analysed with specific inhibitors and Western blot. The inhibitors of TrkA, PLC, PKC, Ras, Raf and ERK1/2 significantly decreased the percentage of PC12 cells with neurite outgrowth and shortened the length of neurite outgrowth induced by CuB. CuB significantly induced the phosphorylation of TrkA, ERK and CREB. The phosphorylation of these proteins was obviously decreased by their specific inhibitors. These results suggest that cofilin is a candidate target protein of CuB in PC12 cells and that the GR/PLC/PKC and TrkA/Ras/Raf/ERK signalling pathways play important roles in the neuroprotective effect of CuB.

Fluorescent bioprobes are invaluable tools for visualizing live cells and deciphering complex biological processes by targeting intracellular biomarkers without disrupting cellular functions. In addition to protein-binding concepts, fluorescent probes utilize various mechanisms, including membrane, metabolism, and gating-oriented strategies. This study introduces a novel fluorescent mechanism distinct from existing ways. Here, we developed a B cell selective probe, CDrB, with unique transport mechanisms. Through SLC-CRISPRa screening, we identified two transporters, SLCO1B3 and SLC25A41, by sorting out populations exhibiting higher and lower fluorescence intensities, respectively, demonstrating contrasting activities. We confirmed that SLCO1B3, with comparable expression levels in T and B cells, facilitates the transport of CDrB into cells, while SLC25A41, overexpressed in T lymphocytes, actively exports CDrB. This observation suggests that SLC25A41 plays a crucial role in discriminating between T and B lymphocytes. Furthermore, it reveals the potential for the reversible localization of SLC25A41 to demonstrate its distinct activity. This study is the first report to unveil a novel strategy of SLC by exporting the probe. We anticipate that this research will open up new avenues for developing fluorescent probes.

This article examines such port competitiveness from the perspective of port efficiency and supply chains. Specifically, studies are examined that investigate port competitiveness from the traditional perspective and from the chain perspective. In doing so, methodological problems of the literature in investigating port competitiveness are deduced.