RESUMEN
We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.
Asunto(s)
Microscopía Fluorescente/métodos , Línea Celular Tumoral , Humanos , Microtúbulos , Osteosarcoma , Proteínas de Unión al GTP rab/fisiologíaRESUMEN
Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Péptidos de Penetración Celular/análisis , Coloración y Etiquetado/métodos , Cloruro de Amonio/farmacología , Biotina/química , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Péptidos de Penetración Celular/metabolismo , Clorpromazina/farmacología , Citocalasina D/farmacología , Endocitosis/efectos de los fármacos , Filipina/farmacología , Citometría de Flujo , Colorantes Fluorescentes/química , Células HeLa , Humanos , Cinética , Transporte de Proteínas/efectos de los fármacos , Espectrometría de Fluorescencia/métodos , Estreptavidina/química , Succinimidas/química , beta-Ciclodextrinas/farmacologíaRESUMEN
During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 generate DNA double strand breaks within a paired complex (PC) containing two complementary recombination signal sequences (RSSs), the 12RSS and 23RSS, which differ in the length of the spacer separating heptamer and nonamer elements. Despite the central role of the PC in V(D)J recombination, little is understood about its structure. Here, we use fluorescence resonance energy transfer to investigate the architecture of the 23RSS in the PC. Energy transfer was detected in 23RSS substrates in which the donor and acceptor fluorophores flanked the entire RSS, and was optimal under conditions that yield a cleavage-competent PC. The data are most easily explained by a dramatic bend in the 23RSS that reduces the distance between these flanking regions from >160 Å in the linear substrate to <80 Å in the PC. Analysis of multiple fluorescent substrates together with molecular dynamics modeling yielded a model in which the 23RSS adopts a U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule.
Asunto(s)
ADN/química , Proteína HMGB1/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación V(D)J , ADN/metabolismo , División del ADN , Transferencia Resonante de Energía de Fluorescencia/métodos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Espectrometría de FluorescenciaRESUMEN
Intrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson's and Alzheimer's diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense.
Asunto(s)
Amiloide/química , Sinucleínas/química , Proteínas tau/química , Secuencia de Aminoácidos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Método de Montecarlo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson's disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes. Membrane binding is mediated by the first â¼95 residues of aS. We find that nitration of the single tyrosine (Y39) in this domain disrupts binding due to electrostatic repulsion. Moreover, we observe that nitration of the three tyrosines (Y125/133/136) in the C-terminal domain is equally effective in perturbing binding, an intriguing result given that the C-terminus is not thought to interact directly with the lipid bilayer. Our investigations show that tyrosine nitration results in a change of the conformational states populated by aS in solution, with the most prominent changes occurring in the C-terminal region. These results lead us to suggest that nitration of Y125/133/136 reduces the membrane-binding affinity of aS through allosteric coupling by altering the ensemble of conformational states and depopulating those capable of membrane binding. While allostery is a well-established concept for structured proteins, it has only recently been discussed in the context of disordered proteins. We propose that allosteric regulation through modification of specific residues in, or ligand binding to, the C-terminus may even be a general mechanism for modulating aS function.
Asunto(s)
Lípidos de la Membrana/química , Estrés Oxidativo , alfa-Sinucleína/química , Regulación Alostérica , Humanos , Hidrodinámica , Membrana Dobles de Lípidos , Lípidos de la Membrana/metabolismo , Mutación , Nitratos/química , Oxidación-Reducción , Enfermedad de Parkinson/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Tirosina/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
α-Synuclein (αS) is an intrinsically disordered protein whose aggregation into ordered, fibrillar structures underlies the pathogenesis of Parkinson's disease. A full understanding of the factors that cause its conversion from soluble protein to insoluble aggregate requires characterization of the conformations of the monomer protein under conditions that favor aggregation. Here we use single molecule Förster resonance energy transfer to probe the structure of several aggregation-prone states of αS. Both low pH and charged molecules have been shown to accelerate the aggregation of αS and induce conformational changes in the protein. We find that at low pH, the C-terminus of αS undergoes substantial collapse, with minimal effect on the N-terminus and central region. The proximity of the N- and C-termini and the global dimensions of the protein are relatively unaffected by the C-terminal collapse. Moreover, although compact at low pH, with restricted chain motion, the structure of the C-terminus appears to be random. Low pH has a dramatically different effect on αS structure than the molecular aggregation inducers spermine and heparin. Binding of these molecules gives rise to only minor conformational changes in αS, suggesting that their mechanism of aggregation enhancement is fundamentally different from that of low pH.
Asunto(s)
alfa-Sinucleína/química , Fenómenos Biofísicos , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Modelos Moleculares , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , alfa-Sinucleína/genéticaRESUMEN
Interactions between the synaptic protein alpha-Synuclein and cellular membranes may be relevant both to its native function as well as its role in Parkinson's disease. We use single molecule Forster resonance energy transfer to probe the structure of alpha-Synuclein bound to detergent micelles and lipid vesicles. We find evidence that it forms a bent-helix when bound to highly curved detergent micelles, whereas it binds more physiological 100 nm diameter lipid vesicles as an elongated helix. Our results highlight the influence of membrane curvature in determining alpha-Synuclein conformation, which may be important for both its normal and disease-associated functions.
Asunto(s)
Liposomas Unilamelares/química , alfa-Sinucleína/química , Humanos , Enfermedad de Parkinson/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Liposomas Unilamelares/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. More recently, these fibrils have been shown to be useful as structural scaffolds in both natural biological systems and nanotechnology applications. The intense interest in amyloid fibrils has led to the investigation of well-characterized proteins, such as hen egg white lysozyme (HEWL), as model systems to examine structural and mechanistic principles that may be generally applicable to all amyloid fibrils. The purpose of this review is to critically examine the fibril-formation literature of proteins in the lysozyme family with respect to the known structure and folding properties of these proteins. The goal is to identify similarities and differences within the family, examine general misfolding / aggregation principles, and identify key areas of importance for future work on the fibril formation of these proteins.
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Amiloide/química , Amiloide/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/genética , Pliegue de Proteína , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
We describe a comprehensive method for imaging and analysis of local protein dynamics at single sites of exocytosis in living cultured endocrine cells. This method is well suited to quantitatively map the complex dynamics of individual molecules at single sites of vesicle fusion in live cells.
Asunto(s)
Células Endocrinas/metabolismo , Exocitosis , Microscopía Fluorescente/métodos , Animales , Transporte Biológico , Línea Celular , Genes Reporteros , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismoRESUMEN
The control of insulin release from pancreatic beta cells helps ensure proper blood glucose level, which is critical for human health. Protein kinase C has been shown to be one key control mechanism for this process. After glucose stimulation, calcium influx into beta cells triggers exocytosis of insulin-containing dense-core granules and activates protein kinase C via calcium-dependent phospholipase C-mediated generation of diacylglycerol. Activated protein kinase C potentiates insulin release by enhancing the calcium sensitivity of exocytosis, likely by affecting two main pathways that could be linked: (1) the reorganization of the cortical actin network, and (2) the direct phosphorylation of critical exocytotic proteins such as munc18, SNAP25, and synaptotagmin. Here, we review what is currently known about the molecular mechanisms of protein kinase C action on each of these pathways and how these effects relate to the control of insulin release by exocytosis. We identify remaining challenges in the field and suggest how these challenges might be addressed to advance our understanding of the regulation of insulin release in health and disease.
Asunto(s)
Glucemia/metabolismo , Calcio/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Quinasas/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Señalización del Calcio , Diglicéridos/metabolismo , Exocitosis/genética , Regulación de la Expresión Génica , Humanos , Células Secretoras de Insulina/citología , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas Quinasas/metabolismo , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells.
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Exocitosis/fisiología , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Dinaminas/metabolismo , Células Endocrinas/metabolismo , Lípidos , Fusión de Membrana/fisiología , Ratas , Vesículas Secretoras/metabolismoRESUMEN
The aggregation and deposition of the neuronal protein α-synuclein in the substantia nigra region of the brain is a key pathological feature of Parkinson's disease. α-Synuclein assembles from a monomeric state in solution, which lacks stable secondary and tertiary contacts, into highly structured fibrillar aggregates through a pathway which involves the population of multiple oligomeric species over a range of time scales. These features make α-synuclein well suited for study with single-molecule techniques, which are particularly useful for characterizing dynamic, heterogeneous samples. Here, we review the current literature featuring single-molecule fluorescence studies of α-synuclein and discuss how these studies have contributed to our understanding of both its function and its role in disease.
Asunto(s)
Pliegue de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/fisiología , Animales , Humanos , Conformación Proteica , Espectrometría de Fluorescencia/métodos , Relación Estructura-ActividadRESUMEN
The aggregation of the protein α-synuclein (AS) is critical to the pathogenesis of Parkinson's disease. Although generally described as an unstructured monomer, recent evidence suggests that the native form of AS may be an α-helical tetramer which resists aggregation. Here, we show that N-terminal acetylation in combination with a mild purification protocol results in an oligomeric form of AS with partial α-helical structure. N-terminal acetylation of AS could have important implications for both the native and pathological structures and functions of AS. Through our demonstration of a recombinant expression system, our results represent an important step toward biochemical and biophysical characterization of this potentially important form of AS.
Asunto(s)
alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Acetilación , Amiloide/química , Amiloide/metabolismo , Cromatografía en Gel , Dicroismo Circular , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Nanodiscs are a new class of model membranes that are being used to solubilize and study a range of integral membrane proteins and membrane-associated proteins. Unlike other model membranes, the Nanodisc bilayer is bounded by a scaffold protein coat that confers enhanced stability and a narrow particle size distribution. The bilayer diameter can be precisely controlled by changing the diameter of the protein coat. All these properties make Nanodiscs excellent model membranes for single-molecule fluorescence applications. In this chapter, we describe our work using Nanodiscs to apply total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS), and Förster resonance energy transfer (FRET) to study the integral membrane protein cytochrome P450 3A4 and the peripheral membrane-binding proteins islet amyloid polypeptide (IAPP) and alpha-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and facilitates accurate solution-based measurements as well. Nanodiscs also comprise an excellent system to stably immobilize integral membrane proteins in a bilayer without covalent modification, enabling a range of surface-based experiments where accurate localization of the protein of interest is required.