Nucleic Acid-Rich Stress Granules Are Not Merely Crowded Condensates: A Quantitative Raman Imaging Study.

Liquid droplets, formed by intracellular liquid-liquid phase separation (LLPS), are called membraneless organelles. They provide transient enzymatic reaction fields for maintaining cellular homeostasis, although they might transform into aggregates, leading to neurodegenerative diseases. To understand the nature of intracellular droplets, it is crucial to quantify the liquid droplets inside a living cell as well as to elucidate the underlying biological mechanism. In this study, we performed near-infrared fluorescence and Raman imaging to quantify chemical components inside stress granules (SGs) formed by LLPS in living cells. The Raman images reveal that the nucleic acid concentration inside the SGs was more than 20% higher than the surrounding cytoplasm, whereas the lipid concentration was lower. Quantitative Raman intensity analysis using a water Raman band as an internal standard enables in situ concentration determination of nucleic acids in the SGs and other organelles. The intensity of the biomolecular C-H bands relative to the water band indicates that the crowding environment inside the SGs depends on the stress type; under oxidative stress, the inside of the SGs was nearly identical to the outside, whereas it was sparser in hyperosmotic stressed cells, suggesting that the high concentrations of nucleic acids play a pivotal role in maintaining the environments inside the SGs. These results demonstrate that intracellular droplets are not always highly condensed.

A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae.

Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.

A microreactor sealing method using adhesive tape for digital bioassays.

Digital assays using microreactors fabricated on solid substrates are useful for carrying out sensitive assays of infectious diseases and other biological tests. However, sealing of the microchambers using fluid oil is difficult for non-experts, and thus hinders the widespread use of digital microreactor assays. Here, we propose the physical isolation of tiny reactors with adhesive tape (PITAT) using simple, commercially available pressure-sensitive adhesive (PSA) tape as a separator of the microreactors. We confirmed that PSA tape can effectively seal the microreactors and prevent molecules from diffusing out. By testing several types of adhesive tape, we found that rubber-based adhesives are the most suitable for this purpose. In addition, we demonstrated that single-molecule enzyme assays can be successfully performed inside microreactors sealed with PSA tape. The results obtained using PITAT are quantitatively comparable to conventional oil sealing, although it is quick and cost-effective. Finally, we demonstrated that single-particle virus counting of the influenza virus can be achieved using PITAT. Collectively, our results suggest that PITAT may be suitable for use in the design of sensitive tests for infectious diseases at the point of care, where no sophisticated equipment or machines are available.

A novel function of TDIF-related peptides: promotion of axillary bud formation.

Small peptides derived from the CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) gene family play a key role in various cell-cell communications in land plants. Among them, tracheary element differentiation inhibition factor (TDIF; CLE41/CLE44 peptide) and CLE42 peptide of Arabidopsis have almost identical amino acid sequences and act as inhibitors of tracheary element differentiation. In this study, we report a novel function of TDIF and CLE42. We found by the GUS (ß-glucuronidase) reporter gene assay that while CLE41 and CLE44 are expressed preferentially in vascular bundles, CLE42 is expressed strongly in the shoot apical meristem (SAM) and axillary meristems. Overexpression of CLE42 and CLE41 enhanced axillary bud formation in the leaf and cotyledon axils. Before floral transition, the emergence of axillary buds in these plants occurred in an acropetal order. Exogenous supply of either TDIF or CLE42 peptide to the wild type induced similar excess bud emergence. In vascular bundles, the TDIF RECEPTOR (TDR) acts as the main receptor for TDIF. The axillary bud emergence of tdr mutants was little affected by either of the peptides. It was confirmed by scanning electron microscopy that peptide-treated wild-type plants form an axillary meristem-like structure earlier than non-treated plants. SHOOT MERISTEMLESS (STM), a marker gene for meristems, was up-regulated in peptide-treated plants before the axillary meristem becomes morphologically distinguishable. These results indicate that CLE42 peptide and TDIF have an activity to enhance axillary bud formation via the TDR. Judging from its expression pattern, CLE42 may play an important role in the regulation of secondary shoot development.

Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging.

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator "QUEEN", which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells.