Kayo Hibino

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. Azidomethyl fluorescein was newly synthesized and introduced into a probe. Azido-masked fluorescein on the probe showed a strong turn-on fluorescence signal upon oligonucleotide-templated Staudinger reduction. The catalytic reaction of the probe offered a turnover number of 50 as fluorescence readout within 4 h. Finally, probes were introduced into human leukemia HL-60 cells by use of streptolysin O pore-forming peptide. We successfully detected and quantitated the 28S rRNA and beta-Actin mRNA signal above the background by flow cytometry. In addition, the same RNA targets were imaged by fluorescence microscopy. The data suggest that a reduction-triggered amplification probe may be a powerful tool in analyzing the localization, transcription, or processing of RNA species in living eukaryotic cells.

Single-Molecule Imaging of Signaling Molecules in Living Cells

Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, we extended this technology to real-time observation of complexes of fluorescent dye-labeled epidermal growth factor (EGF) and its receptor (EGFR). Detection of single molecules was confirmed by single-step photobleaching. Using the single-molecule technique, the process of dimerization, a key step of EGFR signal transduction was observed. In addition to EGFR, single-molecules of fluorescently labeled nerve growth factor, GFP fusion forms of small G proteins and Raf1 kinase, and a fluorescent analogue of cAMP were observed in living cells.

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). Functional fragments of C-RAF containing the Ras-binding domains did not detect the switch in Ras activity in living cells as efficiently as did C-RAF. Single-molecule analysis showed that RasGDP associates with closed-conformation C-RAF, whereas the association of C-RAF with RasGTP immediately triggers the open RAF conformation, which induces an effective interaction between C-RAF and RasGTP. Spontaneous conformational changes from closed C-RAF to the open form rarely occur in quiescent cells. The conformational change in C-RAF is so important to Ras-RAF molecular recognition that C-RAF mutants lacking the conformational change cannot distinguish between RasGDP and RasGTP. The manipulation of the conformation of an effector molecule is a newly identified function of RasGTP.

Conversion of graded phosphorylation into switch-like nuclear translocation via autoregulatory mechanisms in ERK signalling

The phosphorylation cascade in the extracellular signal-regulated kinase (ERK) pathway is a versatile reaction network motif that can potentially act as a switch, oscillator or memory. Nevertheless, there is accumulating evidence that the phosphorylation response is mostly linear to extracellular signals in mammalian cells. Here we find that subsequent nuclear translocation gives rise to a switch-like increase in nuclear ERK concentration in response to signal input. The switch-like response disappears in the presence of ERK inhibitor, suggesting the existence of autoregulatory mechanisms for ERK nuclear translocation involved in conversion from a graded to a switch-like response. In vitro reconstruction of ERK nuclear translocation indicates that ERK-mediated phosphorylation of nucleoporins regulates ERK translocation. A mathematical model and knockdown experiments suggest a contribution of nucleoporins to regulation of the ERK nuclear translocation response. Taken together, this study provides evidence that nuclear translocation with autoregulatory mechanisms acts as a switch in ERK signalling.

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

Background: RAF is activated in the ternary complex with RAS and undetermined kinase. Results: The elementary reaction network and kinetic parameters of molecular interactions and phosphorylation of RAF were determined. Conclusion: Two RAS-binding domains of RAF coordinately work to phosphorylate RAF efficiently. Significance: Activation of RAS effector was first analyzed quantitatively using single-molecule imaging in live cells. 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. RAF is recruited by the active form of RAS (RAS-GTP) from the cytoplasm to the plasma membrane through two RAS-binding sites (the RAS-binding domain and the cysteine-rich domain (CRD)) and is activated by its phosphorylation by still undetermined kinases on the plasma membrane. Using single-molecule imaging, we measured the dissociation time courses of GFP-tagged molecules of wild type RAF and fragments or mutants of RAF containing one or two of the three functional domains (the RAS-binding domain, the CRD, and the catalytic domain) to determine their interaction with membrane components. Each molecule showed a unique dissociation time course, indicating that both its interaction with RAS-GTP and its phosphorylation by the kinases are rate-limiting steps in RAF activation. Based on our experimental results, we propose a kinetic model for the activation of RAF. The model suggests the importance of the interaction between RAS-GTP and CRD for the effective activation of RAF, which is triggered by rapid RAS-GTP-induced conformational changes in RAF and the subsequent presentation of RAF to the kinase. The model also suggests necessary properties of the kinases that activate RAF.

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. Using single‐molecule imaging, we can determine the absolute values of kinetic and dynamic parameters of molecular reactions as a whole and during fluctuations and distribution. In addition, identification of the coordinate of single molecules has enabled super‐localization techniques to virtually improve spatial resolution of optical microscopy. Single‐molecule detection that depends on point detection instead of imaging is also useful in detecting concentrations, diffusive movements, and molecular interactions in living cells, especially in the cytoplasm. The precise and absolute values of positional, kinetic, and dynamic parameters that are determined by single‐molecule imaging and detection in living cells constitute valuable data on unitary biological reactions, because they are obtained without destroying the integrity of complex cellular systems. Moreover, most parameters that are determined by single‐molecule measurements can be substituted directly into equations that describe kinetic and dynamic models in systems biology and medicine. WIREs Syst Biol Med 2012, 4:183–192. doi: 10.1002/wsbm.161

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. Techniques for vector preparation, protein expression, sample preparation, microscopy, and image processing for SMI of GFPs in living cells are discussed in this chapter, along with examples of imaging applications. Double labeling of single molecules and single-pair fluorescent resonance energy transfer (spFRET) are possible in living cells using GFP and YFP as fluorescent tags. The limitations of SMI using GFPs are also discussed.

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 in vitro 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. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

Single-Molecule Imaging Measurements of Protein-Protein Interactions in Living Cells

Even though we have several techniques, represented by the electron microscopy, to obtain images of single molecules, in this chapter, we use ‘single-molecule imaging’ (SMI) for a lim‐ ited means—that is, imaging of fluorescently labeled biological molecules at work for ana‐ lyzing their behaviors. To observe biological molecules at work, imaging in aqueous conditions is essential. Therefore, optical microscopy is the main technology in SMI. Fluores‐ cence labeling is good to use for imaging in optical microscopy, because it allows high con‐ trast and selective imaging of molecules that we are interested in. SMI provides information of dynamics and kinetics of molecular reactions. In 1995, two groups firstly and independ‐ ently realized SMI of biological molecules in aqueous conditions [1,2]. In the early days, SMI was used mainly for the in vitro studies of protein motors [1,2] and metabolic enzymes [3]. Detection of enzymatic reaction (reaction kinetics) [1,3] and detection of protein dynamics (lateral and rotational movements) [2] have been the two main usages of SMI since the first development of this technology. After that, the application of SMI has been extended, and in 2000, SMI became to be used in living cells [4,5].

Single-Molecule Analysis of Molecular Recognition Between Signaling Proteins RAS and RAF

Single-molecule kinetic and dynamic analyses of the biochemical reactions in living cells are one of the most useful methods of investigating the molecular mechanisms of cellular reactions, especially those involved in signal transduction on the plasma membrane. Here, we focus on single-molecule analyses of the intracellular signaling from RAS to RAF, which occurs on the plasma membrane. RAS and RAF are cell signaling proteins that regulate various cellular behaviors, including cell fate determination for proliferation and differentiation. Using single-molecule techniques in living cells, including single-pair Forster resonance energy transfer (FRET) detection and single-molecule tracking, we directly measured the elementary processes in the reactions between RAS and RAF, including the dissociation of RAS and RAF, the conformational changes in RAF, and the diffusion of RAS and RAF along the plasma membrane. Based on the results of these measurements, we discuss how the mutual molecular recognition between RAS and RAF facilitates accurate signal transduction.