1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells.

Liquid droplets formed inside the cell by liquid-liquid phase separation maintain membrane-less condensates/bodies (or compartments). These droplets are important for concentrating certain molecules and facilitating spatiotemporal regulation of cellular functions. 1,6-hexanediol (1,6-HD), an aliphatic alcohol, inhibits weak hydrophobic protein-protein/protein-RNA interactions required for the droplet formation (droplet melting activity) and is used here to elucidate the formation process of cytoplasmic/nuclear condensates/bodies.

In-cell single-molecule FRET measurements reveal three conformational state changes in RAF protein.

Background: The structures of proteins are intimately related to their functions. Significant efforts have been dedicated to the structural investigation of proteins, mainly those of purified proteins in in vitro environments. Proteins function in living cells and thus protein structures must be regulated by interactions with various molecules, some of which participate in reaction networks, depending on the states, conditions, or actions of the cell.

Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.

Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements.

Condensins under the microscope.

Condensins are key players in mitotic chromosome condensation. Using an elegant combination of state-of-the-art imaging techniques, Walther et al. (2018.

Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy.

In eukaryotic cells, highly condensed inactive/silenced chromatin has long been called "heterochromatin." However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells.

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains.

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells.

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer.

Analyses of mice lacking glycosyltransferase have suggested that their pathological phenotypes are not attributable to the overall change of the sugar modification, but instead the result of changes of the glycan structures on a specific 'target' glycoprotein. Therefore, detecting or monitoring the glycosylation status of a specific protein in living cells is important, but no such methods are currently available. Here we demonstrate the detection of glycoforms of a specific glycoprotein using the fluorescence resonance energy transfer technique.

Live cell single-molecule detection in systems biology.

In this review, we describe technology and use of single-fluorophore imaging and detection in living cells with regard to application in systems biology and medicine. Because all biological reactions occur under aqueous conditions, the realization of single-fluorophore imaging using an optical microscope has led to the direct observation of biological molecules at work. Today, we can observe single molecules in individual living cells and even higher multicellular organisms.

Activation kinetics of RAF protein in the ternary complex of RAF, RAS-GTP, and kinase on the plasma membrane of living cells: single-molecule imaging analysis.

RAS is an important cell signaling molecule, regulating the activities of various effector proteins, including the kinase c-RAF (RAF). Despite the critical function of RAS signaling, the activation kinetics have not been analyzed experimentally in living cells for any of the RAS effectors. Here, we analyzed the kinetics of RAF activation on the plasma membrane in living HeLa cells after stimulation with EGF to activate RAS.

A RasGTP-induced conformational change in C-RAF is essential for accurate molecular recognition.

The dysregulation of Ras-RAF signaling is associated with many types of human cancer. However, the kinetic and dynamic features of the mutual molecular recognition of Ras and RAF remain unknown. Here, we developed a technique for imaging single-pair fluorescence resonance energy transfer in living cells, and coupled this technique to single-molecule kinetic analysis to investigate how C-RAF (a subtype of RAF) molecules distinguish the active form of Ras (RasGTP) from the inactive form (RasGDP).

Single-molecule imaging of fluorescent proteins expressed in living cells.

This chapter focuses on single-molecule imaging (SMI) in living cells using green fluorescent protein (GFP) or its related fluorescent protein tags (GFPs). Use of GFPs is a convenient technique to achieve molecular imaging of most proteins in living cells. However, because of difficulties in preparing samples suitable for SMI and the instability of fluorescence signals, special care is required for SMI using GFPs in living cells.

Reduction-triggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells.

Oligonucleotide-templated reactions are attracting attention as a method for RNA detection in living cells. Previously, a reduction-triggered fluorescence probe has been reported that is based on azide reduction to switch fluorescence on. In this article, we report a more advanced probe, a reduction-triggered fluorescent amplification probe that is capable of amplifying a target signal.

Formation of signal transduction complexes during immobile phase of NGFR movements.

Nerve growth factor (NGF) is a secreted neurotrophin involved in the differentiation, growth, and maintenance of neurons. Here, we have used single-molecule imaging to characterize the behavior of Cy3-tagged NGF after binding to receptor complexes on the surfaces of PC12 cells. We show that NGF-receptor complexes have two distinct diffusive states, characterized as mobile and immobile phase.