Enhanced transcriptional heterogeneity mediated by NF-κB super-enhancers.

The transcription factor NF-κB, which plays an important role in cell fate determination, is involved in the activation of super-enhancers (SEs). However, the biological functions of the NF-κB SEs in gene control are not fully elucidated. We investigated the characteristics of NF-κB-mediated SE activity using fluorescence imaging of RelA, single-cell transcriptome and chromatin accessibility analyses in anti-IgM-stimulated B cells. The formation of cell stimulation-induced nuclear RelA foci was abolished in the presence of hexanediol, suggesting an underlying process of liquid-liquid phase separation. The gained SEs induced a switch-like expression and enhanced cell-to-cell variability in transcriptional response. These properties were correlated with the number of gained cis-regulatory interactions, while switch-like gene induction was associated with the number of NF-κB binding sites in SE. Our study suggests that NF-κB SEs have an important role in the transcriptional regulation of B cells possibly through liquid condensate formation consisting of macromolecular interactions.

TRPV4-dependent Ca2+ influx determines cholesterol dynamics at the plasma membrane.

The activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable nonselective cation channel, are controlled by its surrounding membrane lipids (e.g., cholesterol, phosphoinositides). The transmembrane region of TRPV4 contains a cholesterol recognition amino acid consensus (CRAC) motif and its inverted (CARC) motif located in the plasmalemmal cytosolic leaflet. TRPV4 localizes in caveolae, a bulb-shaped cholesterol-rich domain at the plasma membrane. Here, we visualized the spatiotemporal interactions between TRPV4 and cholesterol at the plasma membrane in living cells by dual-color single-molecule imaging using total internal reflection fluorescence microscopy. To this aim, we labeled cholesterol at the cytosolic leaflets of the plasma membrane using a cholesterol biosensor, D4H. Our single-molecule tracking analysis showed that the TRPV4 molecules colocalize with D4H-accessible cholesterol molecules mainly in the low fluidity membrane domains in which both molecules are highly clustered. Colocalization of TRPV4 and D4H-accessible cholesterol was observed both inside and outside of caveolae. Agonist-evoked TRPV4 activation remarkably decreased colocalization probability and association rate between TRPV4 and D4H-accessible cholesterol molecules. Interestingly, upon TRPV4 activation, the particle density of D4H-accessible cholesterol molecules was decreased and the D4H-accessible cholesterol molecules in the fast-diffusing state were increased at the plasma membrane. The introduction of skeletal dysplasia-associated R616Q mutation into the CRAC/CARC motif of TRPV4, which reduced the interaction with cholesterol clusters, could not alter the D4H-accessible cholesterol dynamics. Mechanistically, TRPV4-mediated Ca2+ influx and the C-terminal calmodulin-binding site of TRPV4 are essential for modulating the plasmalemmal D4H-accessible cholesterol dynamics. We propose that TRPV4 remodels its surrounding plasmalemmal environment by manipulating cholesterol dynamics through Ca2+ influx.

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids.

The cytoplasmic domain of receptor tyrosine kinases (RTKs) plays roles as a kinase and a protein scaffold; however, the allocation of these two functions is not fully understood. Here, we analyzed the assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied members of RTKs, by combining single-pair fluorescence resonance energy transfer (FRET) imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (T654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias toward oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of T654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which T654 phosphorylation causes a switch of the major function of EGFR from kinase-activating dimers to scaffolding oligomers.

Amyloid conformation-dependent disaggregation in a reconstituted yeast prion system.

Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

A SLiM-dependent conformational change in baculovirus IE1 controls its focus formation ability.

The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in hr-dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but hr-dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.

A novel sterol-binding protein reveals heterogeneous cholesterol distribution in neurite outgrowth and in late endosomes/lysosomes.

We identified a mushroom-derived protein, maistero-2 that specifically binds 3-hydroxy sterol including cholesterol (Chol). Maistero-2 bound lipid mixture in Chol-dependent manner with a binding threshold of around 30%. Changing lipid composition did not significantly affect the threshold concentration. EGFP-maistero-2 labeled cell surface and intracellular organelle Chol with higher sensitivity than that of well-established Chol probe, D4 fragment of perfringolysin O. EGFP-maistero-2 revealed increase of cell surface Chol during neurite outgrowth and heterogeneous Chol distribution between CD63-positive and LAMP1-positive late endosomes/lysosomes. The absence of strictly conserved Thr-Leu pair present in Chol-dependent cytolysins suggests a distinct Chol-binding mechanism for maistero-2.

Origin of diverse phosphorylation patterns in the ERBB system.

Intercellular signals induce various cellular responses, including growth, proliferation, and differentiation, via the dynamic processes of signal transduction pathways. For cell fate decisions, ligand-binding induces the phosphorylation of ERBB receptors, which in turn activate downstream molecules. The ERBB family includes four subtypes, which diverged through two gene duplications from a common ancestor. Differences in the expression patterns of the subtypes have been reported between different organs in the human body. However, how these different expression properties influence the diverse phosphorylation levels of ERBB proteins is not well understood. Here we study the origin of the phosphorylation responses by experimental and mathematical analyses. The experimental measurements clarified that the phosphorylation levels heavily depend on the ERBB expression profiles. We developed a mathematical model consisting of the four subtypes as monomers, homodimers, and heterodimers and estimated the rate constants governing the phosphorylation responses from the experimental data. To understand the origin of the diversity, we analyzed the effects of the expression levels and reaction rates of the ERBB subtypes on the diversity. The difference in phosphorylation rates between ERBB subtypes showed a much greater contribution to the diversity than did the dimerization rates. This result implies that divergent evolution in phosphorylation reactions rather than in dimerization reactions after whole genome duplications was essential for increasing the diversity of the phosphorylation responses.

In-Cell Single-Molecule Analysis of Molecular State and Reaction Kinetics Coupling.

Cellular signaling is regulated by the spatiotemporal dynamics and kinetics of molecular behavior. To investigate the mechanisms at the molecular level, fluorescence single-molecule analysis is an effective method owing to the direct observation of individual molecules in situ in cells and the results in quantitative information about the behavior. The integration of machine learning into this analysis modality enables the acquisition of behavioral features at all time points of all molecules. As a case study, we described a hidden Markov model-based approach to infer the molecular states of mobility and clustering for epidermal growth factor receptor (EGFR) along a single-molecule trajectory. We reveal a scheme of the receptor signaling through the dynamic coupling of the mobility and clustering states under the influence of a local membrane structure. As the activation process progressed, EGFR generally converged to an immobile cluster. This state exhibited high affinity with a specific cytoplasmic protein, shown by two-color single-molecule analysis, and could be a platform for downstream signaling. The method was effective for elucidating the biophysical mechanisms of signaling regulation when comprehensive analysis is possible for a huge number and multiple molecular species in the signaling pathway. Thus, a fully automated system for single-molecule analysis, in which indispensable expertise was replicated using artificial intelligence, has been developed to enable in-cell large-scale analysis. This system opens new single-molecule approaches for pharmacological applications as well as the basic sciences.

Comparative Analysis of Single-Molecule Dynamics of TRPV1 and TRPV4 Channels in Living Cells.

TRPV1 and TRPV4, members of the transient receptor potential vanilloid family, are multimodal ion channels activated by various stimuli, including temperature and chemicals. It has been demonstrated that TRPV channels function as tetramers; however, the dynamics of the diffusion, oligomerization, and endocytosis of these channels in living cells are unclear. Here we undertook single-molecule time-lapse imaging of TRPV1 and TRPV4 in HEK 293 cells. Differences were observed between TRPV1 and TRPV4 before and after agonist stimulation. In the resting state, TRPV4 was more likely to form higher-order oligomers within immobile membrane domains than TRPV1. TRPV1 became immobile after capsaicin stimulation, followed by its gradual endocytosis. In contrast, TRPV4 was rapidly internalized upon stimulation with GSK1016790A. The selective loss of immobile higher-order oligomers from the cell surface through endocytosis increased the proportion of the fast-diffusing state for both subtypes. With the increase in the fast state, the association rate constants of TRPV1 and TRPV4 increased, regenerating the higher-order oligomers. Our results provide a possible mechanism for the different rates of endocytosis of TRPV1 and TRPV4 based on the spatial organization of the higher-order structures of the two TRPV channels.

DNA-Based Synthetic Growth Factor Surrogates with Fine-Tuned Agonism*.

Designing synthetic surrogates of functional proteins is an important, albeit challenging, task in the field of chemistry. A strategy toward the design of synthetic agonists for growth factor or cytokine receptors that elicit a desired signal activity has been in high demand, as such ligands hold great promise as safer and more effective therapeutics. In the present study, we used a DNA aptamer as a building block and described the strategy-guided design of a synthetic receptor agonist with fine-tuned agonism. The developed synthetic partial agonist can regulate therapeutically relevant cellular activities by eliciting fine-tuned receptor signaling.

Resolvin E3 attenuates allergic airway inflammation via the interleukin-23-interleukin-17A pathway.

We investigated the effects of resolvin E (RvE) 1, RvE2, and RvE3 on IL-4- and IL-33-stimulated bone marrow-derived dendritic cells (BMDCs) from house dust mite (HDM)-sensitized mice. We also investigated the role of RvE3 in a murine model of HDM-induced airway inflammation. In vitro, BMDCs from HDM-sensitized mice were stimulated with IL-4 and IL-33 and then treated with RvE1, RvE2, RvE3, or vehicle. RvE1, RvE2, and RvE3 suppressed IL-23 release from BMDCs. In vivo, RvE3 administrated to HDM-sensitized and challenged mice in the resolution phase promoted a decline in total numbers of inflammatory cells and eosinophils, reduced levels of IL-23 and IL-17 in lavage fluid, and suppressed IL-23 and IL-17A mRNA expression in lung and peribronchial lymph nodes. RvE3 also reduced resistance in the lungs of HDM-sensitized mice. A NanoBiT ß-arrestin recruitment assay using human embryonic kidney 293 cells revealed that pretreatment with RvE3 suppressed the leukotriene B4 (LTB4)-induced ß-arrestin 2 binding to LTB4 receptor 1 (BLT1R), indicating that RvE3 antagonistically interacts with BLT1R. Collectively, these findings indicate that RvE3 facilitates the resolution of allergic airway inflammation, partly by regulating BLT1R activity and selective cytokine release by dendritic cells. Our results accordingly identify RvE3 as a potential therapeutic target for the management of asthma.-Sato, M., Aoki-Saito, H., Fukuda, H., Ikeda, H., Koga, Y., Yatomi, M., Tsurumaki, H., Maeno, T., Saito, T., Nakakura, T., Mori, T., Yanagawa, M., Abe, M., Sako, Y., Dobashi, K., Ishizuka, T., Yamada, M., Shuto, S., Hisada, T. Resolvin E3 attenuates allergic airway inflammation via the interleukin-23-interleukin-17A pathway.

Transcriptionally inducible Pleckstrin homology-like domain, family A, member 1, attenuates ErbB receptor activity by inhibiting receptor oligomerization.

Feedback control is a key mechanism in signal transduction, intimately involved in regulating the outcome of the cellular response. Here, we report a novel mechanism by which PHLDA1, Pleckstrin homology-like domain, family A, member 1, negatively regulates ErbB receptor signaling by inhibition of receptor oligomerization. We have found that the ErbB3 ligand, heregulin, induces PHILDA1 expression in MCF-7 cells. Transcriptionally-induced PHLDA1 protein directly binds to ErbB3, whereas knockdown of PHLDA1 increases complex formation between ErbB3 and ErbB2. To provide insight into the mechanism for our time-course and single-cell experimental observations, we performed a systematic computational search of network topologies of the mathematical models based on receptor dimer-tetramer formation in the ErbB activation processes. Our results indicate that only a model in which PHLDA1 inhibits formation of both dimers and tetramer can explain the experimental data. Predictions made from this model were further validated by single-molecule imaging experiments. Our studies suggest a unique regulatory feature of PHLDA1 to inhibit the ErbB receptor oligomerization process and thereby control the activity of receptor signaling network.

Lipid-Protein Interplay in Dimerization of Juxtamembrane Domains of Epidermal Growth Factor Receptor.

Transmembrane (TM) helix and juxtamembrane (JM) domains (TM-JM) bridge the extracellular and intracellular domains of single-pass membrane proteins, including epidermal growth factor receptor (EGFR). TM-JM dimerization plays a crucial role in regulation of EGFR kinase activity at the cytoplasmic side. Although the interaction of JM with membrane lipids is thought to be important to turn on EGF signaling, and phosphorylation of Thr654 on JM leads to desensitization, the underlying kinetic mechanisms remain unclear. In particular, how Thr654 phosphorylation regulates EGFR activity is largely unknown. Here, combining single-pair FRET imaging and nanodisc techniques, we showed that phosphatidylinositol 4,5-bis phosphate (PIP2) facilitated JM dimerization effectively. We also found that Thr654 phosphorylation dissociated JM dimers in the membranes containing acidic lipids, suggesting that Thr654 phosphorylation electrostatically prevented the interaction with basic residues in JM and acidic lipids. Based on the single-molecule experiment, we clarified the kinetic pathways of the monomer (inactive state)-to-dimer (active state) transition of JM domains and alteration in the pathways depending on the membrane lipid species and Thr654 phosphorylation.

A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

Single-molecule imaging and manipulation of biomolecular machines and systems.

BACKGROUND: Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. SCOPE OF REVIEW: We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. MAJOR CONCLUSIONS: Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. GENERAL SIGNIFICANCE: Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy.

Organization and functions of the nucleolus is maintained by mobilities and interactions of nucleolar factors. Because the nucleolus is a densely packed structure, molecular crowding effects determined by the molecular concentrations and mobilities in the nucleolus should also be important for regulating nucleolar organization and functions. However, such molecular property of nucleolar organization is not fully understood. To understand the biophysical property of nucleolar organization, the diffusional behaviors of inert green fluorescent protein (GFP) oligomers with or without nuclear localization signals (NLSs) were analyzed under various conditions by fluorescence correlation spectroscopy. Our result demonstrates that the mobility of GFPs inside the nucleolus and the nucleoplasm can be represented by single free diffusion under normal conditions, even though the mobility in the nucleolus is considerably slower than that in the chromatin region. Moreover, the free diffusion of GFPs is found to be significantly size- and NLS-dependent only in the nucleolus. Interestingly, the mobility in the nucleolus is highly sensitive to ATP depletion, as well as actinomycin D (ActD) treatment. In contrast, the ultra-structure of the nucleolus was not significantly changed by ATP depletion but was changed by ActD treatment. These results suggest that the nucleolus behaves similarly to an open aqueous-phase medium with an increased molecular crowding effect that depends on both energy and transcription.

Raman spectral dynamics of single cells in the early stages of growth factor stimulation.

Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used the Raman spectra of single cells to trace their internal dynamics during the early stages of growth factor stimulation. This method allows nondestructive and inclusive time-series analyses of chemical compositions of the same single cells. Applying a Gaussian mixture model to the major principal components of the single-cell Raman spectra, we detected the dynamics of the chemical states in MCF-7 cancer-derived cells in the absence and presence of differentiation and proliferation factors. The dynamics displayed characteristic variations according to the functions of the growth factors. In the differentiation pathway, the chemical composition changed directionally between multiple states, including both reversible and irreversible state transitions. In contrast, in the proliferation pathway, the chemical composition was homogenized into a single state. The differentiation factor also stimulated fluctuations in the chemical composition, whereas the proliferation factor did not.

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells.

Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) EMBO J 25:4215-4222], suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low- and high-affinity sites of HRG association on the cell surface.

Raman and autofluorescence spectrum dynamics along the HRG-induced differentiation pathway of MCF-7 cells.

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.