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Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive, despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e., selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically activated processes are localized in space and time yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell-surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single-molecule and single-cell levels, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling. VIDEO ABSTRACT.
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Técnicas Genéticas , Mecanotransdução Celular , Nanopartículas Metálicas , Receptores Notch/metabolismo , Actinas/metabolismo , Caderinas/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Mecanorreceptores/fisiologia , Nanopartículas Metálicas/química , Microesferas , Técnicas de Sonda Molecular , Proteínas Recombinantes de Fusão/metabolismo , Análise Espacial , TempoRESUMO
Understanding the spatial organization of membrane proteins is crucial for unraveling key principles in cell biology. The reaction-diffusion model is commonly used to understand biochemical patterning; however, applying reaction-diffusion models to subcellular phenomena is challenging because of the difficulty in measuring protein diffusivity and interaction kinetics in the living cell. In this work, we investigated the self-organization of the plasmalemma vesicle-associated protein (PLVAP), which creates regular arrangements of fenestrated ultrastructures, using single-molecule tracking. We demonstrated that the spatial organization of the ultrastructures is associated with a decrease in the association rate by actin destabilization. We also constructed a reaction-diffusion model that accurately generates a hexagonal array with the same 130 nm spacing as the actual scale and informs the stoichiometry of the ultrastructure, which can be discerned only through electron microscopy. Through this study, we integrated single-molecule experiments and reaction-diffusion modeling to surpass the limitations of static imaging tools and proposed emergent properties of the PLVAP ultrastructure.
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Proteínas de Transporte , Proteínas de Membrana , Proteínas de Membrana/metabolismo , Difusão , Modelos BiológicosRESUMO
We report a means by which atomic and molecular secondary ions, including cholesterol and fatty acids, can be sputtered through single-layer graphene to enable secondary ion mass spectrometry (SIMS) imaging of untreated wet cell membranes in solution at subcellular spatial resolution. We can observe the intrinsic molecular distribution of lipids, such as cholesterol, phosphoethanolamine and various fatty acids, in untreated wet cell membranes without any labeling. We show that graphene-covered cells prepared on a wet substrate with a cell culture medium reservoir are alive and that their cellular membranes do not disintegrate during SIMS imaging in an ultra-high-vacuum environment. Ab initio molecular dynamics calculations and ion dose-dependence studies suggest that sputtering through single-layer graphene occurs through a transient hole generated in the graphene layer. Cholesterol imaging shows that methyl-ß-cyclodextrin preferentially extracts cholesterol molecules from the cholesterol-enriched regions in cell membranes.
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Membrana Celular/metabolismo , Colesterol/análise , Etanolaminas/análise , Ácidos Graxos/análise , Espectrometria de Massa de Íon Secundário/métodos , Diagnóstico por Imagem , Grafite/química , Simulação de Dinâmica Molecular , Análise de Célula Única/métodos , beta-Ciclodextrinas/químicaRESUMO
In live cells, the plasma membrane is composed of lipid domains separated by hundreds of nanometers in dynamic equilibrium. Lipid phase separation regulates the trafficking and spatiotemporal organization of membrane molecules that promote signal transduction. However, visualizing domains with adequate spatiotemporal accuracy remains challenging because of their subdiffraction limit size and highly dynamic properties. Here, we present a single lipid-molecular motion analysis pipeline (lipid-MAP) for analyzing the phase heterogeneity of lipid membranes by detecting the instantaneous velocity change of a single lipid molecule using the excellent optical properties of nanoparticles, high spatial localization accuracy of single-molecule localization microscopy, and separation capability of the diffusion state of the hidden Markov model algorithm. Using lipid-MAP, individual lipid molecules were found to be in dynamic equilibrium between two statistically distinguishable phases, leading to the formation of small (â¼170 nm), viscous (2.5× more viscous than surrounding areas), and transient domains in live cells. Moreover, our findings provide an understanding of how membrane compositional changes, i.e., cholesterol and phospholipids, affect domain formation. This imaging method can contribute to an improved understanding of spatiotemporal-controlled membrane dynamics at the molecular level.
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Fosfolipídeos , Transdução de Sinais , Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Membranas , Difusão , Bicamadas Lipídicas/metabolismoRESUMO
Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research.
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MicroscopiaRESUMO
Solar-driven reactive oxygen species (ROS) generation is an attractive disinfection technique for cell death and water purification. However, most photocatalysts require high stability in the water environment and the production of ROS with a sufficient amount and diffusion length to damage pathogens. Here, a ROS generation system was developed consisting of tapered crystalline silicon microwires coated with anatase titanium dioxide for a conformal junction. The system effectively absorbed >95% of sunlight over 300-1100 nm, resulting in effective ROS generation. The system was designed to produce various ROS species, but a logistic regression analysis with cellular survival data revealed that the diffusion length of the ROS is â¼9 µm, implying that the most dominant species causing cell damage is H2O2. Surprisingly, a quantitative analysis showed that only 15 min of light irradiation on the system would catalyze a local bactericidal effect comparable to the conventional germicidal level of H2O2 (â¼3 mM).
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Peróxido de Hidrogênio , Luz Solar , Morte Celular , Espécies Reativas de Oxigênio , TitânioRESUMO
Cells use gaseous molecules such as nitric oxide (NO) to transmit both intracellular and intercellular signals. In principle, the endogenous small molecules regulate physiological changes, but it is unclear how randomly diffusive molecules trigger and discriminate signaling programs. Herein, it is shown that gasotransmitters use time-dependent dynamics to discriminate the endogenous and exogenous inputs. For a real-time stimulation of cell signaling, we synthesized a photo-cleavable metal-nitrosyl complex, [CoIII (MDAP)(NO)(CH3 CN)]2+ (MDAP=N,N'-dimethyl-2,11-diaza[3,3](2,6)pyridinophane), which can stably deliver and selectively release NO with fine temporal resolution in the cytosol, and used this to study the extracellular signal-regulated kinases (ERKs), revealing how cells use both exogenous and endogenous NO to disentangle cellular responses. This technique can be to understand how diverse cellular signaling networks are dynamically interconnected and also to control drug delivery systems.
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Cobalto/química , Fotólise , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Sistemas de Liberação de Medicamentos , Modelos MolecularesRESUMO
Monitoring the dynamics of proteins in live cells on appropriate spatiotemporal scales may provide key information regarding long-standing questions in molecular and cellular regulatory mechanisms. However, tools capable of imaging the conformational changes over time have been elusive. Here, we present a single-molecule stroboscopic imaging probes by developing gyroscopic plasmonic nanoparticles, allowing for replication of protein-protein interactions and the conformational dynamics based on rotational and lateral velocities. This study fundamentally monitors the rotational motion of a membrane protein, epidermal growth factor receptor (EGFR), to decipher undiscovered structural dynamics in live cells without any molecular perturbations. This method offers a strategy to visualize assemblies and conformational changes, and provides unique insights into the mechanism underlying the molecular dynamics for receptors.
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Simulação de Dinâmica Molecular , Linhagem Celular , Receptores ErbB/química , Humanos , Ligação Proteica , Conformação ProteicaRESUMO
Precise control over interfacial chemistry between nanoparticles and other materials remains a major challenge that limits broad application of nanotechnology in biology. To address this challenge, we used 'steric exclusion' to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping each QD with a functionalized oligonucleotide. We demonstrated the utility of these QDs as modular and nonperturbing imaging probes by tracking individual Notch receptors on live cells.
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Microscopia de Fluorescência/métodos , Nanopartículas/química , Nanotecnologia/métodos , Pontos Quânticos , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Luz , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/instrumentação , Oligonucleotídeos/química , Oligonucleotídeos Fosforotioatos/química , Distribuição de Poisson , Espalhamento de Radiação , Compostos de Sulfidrila/químicaRESUMO
The material transport system, facilitated by motor proteins, plays a vital role in maintaining a non-equilibrium cellular state. However, understanding the temporal coordination of motor protein activity requires an advanced imaging technique capable of measuring 3D angular displacement in real-time. In this study, a Fourier transform-based plasmonic dark-field microscope has been developed using anisotropic nanoparticles, enabling the prolonged and simultaneous observation of endosomal lateral and rotational motion. A sequence of discontinuous 3D angular displacements has been observed during the pause and run phases of transport. Notably, a serially correlated temporal pattern in the intermittent rotational events has been demonstrated during the tug-of-war mechanism, indicating Markovian switching between the exploitational and explorational modes of motor protein exchange prior to resuming movement. Alterations in transition frequency and the exploitation-to-exploration ratio upon dynein inhibitor treatment highlight the relationship between disrupted motor coordination and reduced endosomal transport efficiency. Collectively, these results suggest the importance of orchestrated temporal motor protein patterns for efficient cellular transport.
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Endossomos , Endossomos/metabolismo , Humanos , Microscopia/métodos , Dineínas/metabolismo , Transporte Biológico/fisiologia , Proteínas Motores Moleculares/metabolismoRESUMO
We have rationally designed a one-dimensional coordination polymer (1D CP), termed 1D-DGIST-18, that exhibits intrinsic structural flexibility. This 1D CP enables its expansion into a three-dimensional network through supramolecular interactions involving coordinated solvents and/or ligands. The strategic selection of solvents for solvent exchange, prior to drying, significantly influences the structures of 1D-DGIST-18 by removing certain coordinating solvents and modulating π-π stacking. Consequently, a hierarchical porosity emerges, ranging from micro- to meso- to macroporous structures, which is attributed to its inherent structural dynamics. Additionally, the formation of excimers endows 1D-DGIST-18, when immersed in acetone, with 'turn-on' fluorescence, as evidenced by fluorescence decay profiles. These structural transitions within 1D-DGIST-18 are further elucidated using single-crystal X-ray diffractometry. The insights from this study provide a foundation for the design of materials with structural dynamics and tunable properties.
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Gold nanoparticles with suitable surface functionalities have been widely used as a versatile nanobioplatform. However, functionalized gold nanoparticles using thiol-terminated ligands have a tendency to aggregate, particularly in many enzymatic reaction buffers containing biological thiols, because of ligand exchange reactions. In the present study, we developed a one-step synthesis of poly(ethylene glycol) (PEG)ylated gold nanoparticles using poly(dimethylaminoethyl methacrylate) (PDMAEMA) in PEG as a polyol solvent. Because of the chelate effect of polymeric functionalities on the gold surface, the resulting PEGylated gold nanoparticles (Au@P-PEG) are very stable under the extreme conditions at which the thiol-monolayer-protected gold nanoparticles are easily coagulated. Using the solvent mixture of PEG and ethylene glycol (EG) and subsequent hydrolysis, gold nanoparticles bearing mixed functionalities of PEG and carboxylate are generated. The resulting particles exhibit selective adsorption of positively charged chymotrypsin (ChT) without nonselective adsorption of bovine serum albumin (BSA). The present nanoparticle system has many advantages, including high stability, simple one-step synthesis, biocompatibility, and excellent binding specificity; thus, this system can be used as a versatile platform for potential bio-related applications, such as separation, sensing, imaging, and assays.
Assuntos
Ácidos Carboxílicos/química , Quimotripsina/análise , Ouro/química , Nanopartículas Metálicas/química , Metacrilatos/química , Nylons/química , Polietilenoglicóis/química , Soroalbumina Bovina/análise , Animais , Bovinos , Técnicas de Química Sintética , Quimotripsina/química , Soroalbumina Bovina/química , Propriedades de SuperfícieRESUMO
We investigated the localized surface plasmon resonances of individual AgAuAg nanorods (NRs) using the dark-field spectro-microscopy technique. We find that the scattering spectra of such hetero-NRs show longitudinal resonance wavelengths that are nearly insensitive to the relative composition of Ag and Au. Instead, the resonance is mostly governed by the overall length of the nanorod. This shows that the plasmons oscillate along the entire length of the NR without the significant perturbation at the Ag-Au interfaces. The results demonstrate that the overall geometry as well as the composition determine the tunability of the hetero-metallic nanostructures, and provide an important design rule for the composition-tunable bimetallic plasmon structures.
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Transporting substances such as gases, nutrients, waste, and cells is the primary function of blood vessels. Vascular cells use membrane proteins to perform crucial endothelial functions, including molecular transport, immune cell infiltration, and angiogenesis. A thorough understanding of these membrane receptors from a clinical perspective is warranted to gain insights into the pathogenesis of vascular diseases and to develop effective methods for drug delivery through the vascular endothelium. This review summarizes state-of-the-art single-molecule imaging techniques, such as super-resolution microscopy, single-molecule tracking, and protein-protein interaction analysis, for observing and studying membrane proteins. Furthermore, recent single-molecule studies of membrane proteins such as cadherins, integrins, caveolins, transferrin receptors, vesicle-associated protein-1, and vascular endothelial growth factor receptor are discussed.
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The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified a subset of fibroblasts and smooth muscle cells which are the major constituents of organ stroma neither proliferative nor senescent in tissues of the elderly, which we termed "mid-old status" cells. Upregulation of pro-inflammatory genes (IL1B and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Remarkably, the microenvironmental change and the functional decline of mid-old cells could be reversed by a young cell-originated protein, SLIT2. Our data identify functional reversion of mid-old cells as a potential method to prevent or ameliorate aspects of aging-related tissue dysfunction.
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Envelhecimento , Senescência Celular , Humanos , Idoso , Senescência Celular/genética , Envelhecimento/genética , Células Epiteliais/fisiologia , Fibroblastos , Miócitos de Músculo LisoRESUMO
Plasmonic nanostructures such as gold nanoparticles are very useful for monitoring chemical reactions because their optical properties are highly dependent upon the environment surrounding the particle surface. Here, we designed the catalytic structure composed of platinized cadmium sulfide with gold domains as a sensitive probe, and we monitored the photocatalytic decomposition of lactic acid to generate hydrogen gas in situ by single-particle dark-field spectroscopy. The plasmon band shift of the gold probe throughout the reaction exhibits significant particle-to-particle variation, and by simulating the reaction kinetics, the rate constant and structural information (including the diffusion coefficient through the shell and the relative arrangement of the active sites) can be estimated for individual catalyst particles. This approach is versatile for the monitoring of various heterogeneous reactions with distinct components at a single-particle level.
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In the present study, we demonstrate the precise tuning of surface plasmon resonance over the full visible range by compositional variation of the nanoparticles. The addition of sulfide ions into the Au@Ag core-shell nanocubes generates stable Au@Ag/Ag(2)S core-shell nanoparticles at room temperature, and the plasmon extinction maximum shifts to the longer wavelength covering the entire visible range of 500-750 nm. Based on the optical property, the Au@Ag core-shell nanocubes are employed as a colorimetric sensing framework for sulfide detection in water. The detection limit is measured to be 10 ppb by UV-vis spectroscopy and 200 ppb by naked eyes. Such nanoparticles would be useful for decoration and sensing purposes, due to their precise color tunability and high stability.
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Ouro/química , Nanopartículas Metálicas/química , Prata/química , Sulfetos/química , Ressonância de Plasmônio de SuperfícieRESUMO
Live video recording of intracellular material transport is a promising means of deciphering the fascinating underlying mechanisms driving life at the molecular level. Such technology holds the key to realizing real-time observation at appropriate resolutions in three-dimensional (3D) space within living cells. Here, we report an optical microscopic method for probing endosomal dynamics with proper spatiotemporal resolution within 3D space in live cells: plasmonic dark-field STORM (pdf-STORM). We first confirmed that pdf-STORM has a spatial resolution comparable to that of scanning electron microscopy. Additionally, by observing two optical probes within a single organelle, we were able to track rotational movements and demonstrate the feasibility of using pdf-STORM to observe the angular displacements of an endosome during a "tug-of-war" over an extended period. Finally, we show various biophysical parameters of the hitherto unelucidated dynamics of endosomes-angular displacement is discontinuous and y-axis movement predominates and follows a long-tail distribution.
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Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.