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.

HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains.

Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.

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.

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.

Quantitative Imaging Analysis of NF-κB for Mathematical Modeling Applications.

Mathematical models can integrate different types of experimental datasets, reconstitute biological systems in silico, and identify previously unknown molecular mechanisms. Over the past decade, mathematical models have been developed based on quantitative observations, such as live-cell imaging and biochemical assays. However, it is difficult to directly integrate next-generation sequencing (NGS) data. Although highly dimensional, NGS data mostly only provides a "snapshot" of cellular states. Nevertheless, the development of various methods for NGS analysis has led to much more accurate predictions of transcription factor activity and has revealed various concepts regarding transcriptional regulation. Therefore, fluorescence live-cell imaging of transcription factors can help alleviate the limitations in NGS data by supplementing temporal information, linking NGS to mathematical modeling. This chapter introduces an analytical method for quantifying dynamics of nuclear factor kappaB (NF-κB) which forms aggregates in the nucleus. The method may also be applicable to other transcription factors regulated in a similar fashion.

Two Closed Conformations of CRAF Require the 14-3-3 Binding Motifs and Cysteine-Rich Domain to be Intact in Live Cells.

The protein rapidly accelerated fibrosarcoma (RAF) is a kinase downstream of the membrane protein RAS in the cellular signal transduction system. In the structure of RAF, the N- and C-terminus domains are connected with a flexible linker. The open/close dynamics and dimerization of RAF are thought to regulate its activity, although the details of these conformations are unknown, especially in live cells. In this work, we used alternating laser excitation to measure cytosolic CRAF in live HeLa cells and obtained single-molecule Förster resonance energy transfer (smFRET) distributions of the structural states. We compared the results for wild-type (WT)-CRAF before and after epidermal growth factor (EGF) stimulation, with mutations of the 14-3-3 binding sites and cysteine-rich domain, and an N-terminus truncation. The smFRET distributions of full-length CRAFs were analyzed by global fitting with three beta distributions. Our results suggested that a 14-3-3 dimer bound to two sites on a single CRAF molecule and induced the formation of the autoinhibitory closed conformation. There were two closed conformations, which the majority of WT-CRAF adopted. These two conformations showed different responsiveness to EGF stimulation.

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.

Insulin signaling shapes fractal scaling of C. elegans behavior.

Fractal scaling in animal behavioral activity, where similar temporal patterns appear repeatedly over a series of magnifications among time scales, governs the complex behavior of various animal species and, in humans, can be altered by neurodegenerative diseases and aging. However, the mechanism underlying fractal scaling remains unknown. Here, we cultured C. elegans in a microfluidic device for 3 days and analyzed temporal patterns of C. elegans activity by fractal analyses. The residence-time distribution of C. elegans behaviors shared a common feature with those of human and mice. Specifically, the residence-time power-law distribution of the active state changed to an exponential-like decline at a longer time scale, whereas the inactive state followed a power-law distribution. An exponential-like decline appeared with nutrient supply in wild-type animals, whereas this decline disappeared in insulin-signaling-defective daf-2 and daf-16 mutants. The absolute value of the power-law exponent of the inactive state distribution increased with nutrient supply in wild-type animals, whereas the value decreased in daf-2 and daf-16 mutants. We conclude that insulin signaling differentially affects mechanisms that determine the residence time in active and inactive states in C. elegans behavior. In humans, diabetes mellitus, which is caused by defects in insulin signaling, is associated with mood disorders that affect daily behavioral activities. We hypothesize that comorbid behavioral defects in patients with diabetes may be attributed to altered fractal scaling of human behavior.

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.

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.

Assessing transfer entropy from biochemical data.

We address the problem of evaluating the transfer entropy (TE) produced by biochemical reactions from experimentally measured data. Although these reactions are generally nonlinear and nonstationary processes making it challenging to achieve accurate modeling, Gaussian approximation can facilitate the TE assessment only by estimating covariance matrices using multiple data obtained from simultaneously measured time series representing the activation levels of biomolecules such as proteins. Nevertheless, the nonstationary nature of biochemical signals makes it difficult to theoretically assess the sampling distributions of TE, which are necessary for evaluating the statistical confidence and significance of the data-driven estimates. We resolve this difficulty by computationally assessing the sampling distributions using techniques from computational statistics. The computational methods are tested by using them in analyzing data generated from a theoretically tractable time-varying signal model, which leads to the development of a method to screen only statistically significant estimates. The usefulness of the developed method is examined by applying it to real biological data experimentally measured from the ERBB-RAS-MAPK system that superintends diverse cell fate decisions. A comparison between cells containing wild-type and mutant proteins exhibits a distinct difference in the time evolution of TE while any apparent difference is hardly found in average profiles of the raw signals. Such a comparison may help in unveiling important pathways of biochemical reactions.

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.

Scanning Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Conformational Dynamics of DNA Holliday Junction from Microsecond to Subsecond.

Single-molecule Förster resonance energy transfer (smFRET) is widely utilized to investigate the structural heterogeneity and dynamics of biomolecules. However, it has been difficult to simultaneously achieve a wide observation time window, a high structure resolution, and a high time resolution with the current smFRET methods. Herein, we introduce a new method utilizing two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) and surface immobilization techniques. This method, scanning 2D FLCS, enables us to examine the structural heterogeneity and dynamics of immobilized biomolecules on a time scale from microsecond to subsecond by slowly scanning the sample stage at the rate of ∼1 µm/s. Application to the DNA Holliday junction (HJ) complex under various [Mg2+] conditions demonstrates that scanning 2D FLCS enables tracking reaction kinetics from 25 µs to 30 ms with a time resolution as high as 1 µs. Furthermore, the high structure resolution of scanning 2D FLCS allows us to unveil the ensemble nature of each isomer state and the heterogeneity of the dynamics of the HJ.

Heterotrimeric Gq proteins act as a switch for GRK5/6 selectivity underlying ß-arrestin transducer bias.

Signaling-biased ligands acting on G-protein-coupled receptors (GPCRs) differentially activate heterotrimeric G proteins and ß-arrestins. Although a wealth of structural knowledge about signaling bias at the GPCR level exists (preferential engagement of a specific transducer), little is known about the bias at the transducer level (different functions mediated by a single transducer), partly due to a poor understanding of GPCR kinase (GRK)-mediated GPCR phosphorylation. Here, we reveal a unique role of the Gq heterotrimer as a determinant for GRK-subtype selectivity that regulates subsequent ß-arrestin conformation and function. Using the angiotensin II (Ang II) type-1 receptor (AT1R), we show that ß-arrestin recruitment depends on both GRK2/3 and GRK5/6 upon binding of Ang II, but solely on GRK5/6 upon binding of the ß-arrestin-biased ligand TRV027. With pharmacological inhibition or genetic loss of Gq, GRK-subtype selectivity and ß-arrestin functionality by Ang II is shifted to those of TRV027. Single-molecule imaging identifies relocation of AT1R and GRK5, but not GRK2, to an immobile phase under the Gq-inactive, AT1R-stimulated conditions. These findings uncover a previously unappreciated Gq-regulated mechanism that encodes GRK-subtype selectivity and imparts distinct phosphorylation-barcodes directing downstream ß-arrestin functions.

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.

PMP2/FABP8 induces PI(4,5)P2-dependent transbilayer reorganization of sphingomyelin in the plasma membrane.

Sphingomyelin (SM) is a mammalian lipid mainly distributed in the outer leaflet of the plasma membrane (PM). We show that peripheral myelin protein 2 (PMP2), a member of the fatty-acid-binding protein (FABP) family, can localize at the PM and controls the transbilayer distribution of SM. Genetic screening with genome-wide small hairpin RNA libraries identifies PMP2 as a protein involved in the transbilayer movement of SM. A biochemical assay demonstrates that PMP2 is a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-binding protein. PMP2 induces the tubulation of model membranes in a PI(4,5)P2-dependent manner, accompanied by the modification of the transbilayer membrane distribution of lipids. In the PM of PMP2-overexpressing cells, inner-leaflet SM is increased whereas outer-leaflet SM is reduced. PMP2 is a causative protein of Charcot-Marie-Tooth disease (CMT). A mutation in PMP2 associated with CMT increases its affinity for PI(4,5)P2, inducing membrane tubulation and the subsequent transbilayer movement of lipids.

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.

p52Shc regulates the sustainability of ERK activation in a RAF-independent manner.

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant but that SHC plays the critical role in the temporal regulation of ERK activation.

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.

Workflows of the Single-Molecule Imaging Analysis in Living Cells: Tutorial Guidance to the Measurement of the Drug Effects on a GPCR.

Single-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein-protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software, named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure ( https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer ).