RESUMEN
We introduce Cryogenic Optical Localization in 3D (COLD), a method to localize multiple fluorescent sites within a single small protein with Angstrom resolution. We demonstrate COLD by determining the conformational state of the cytosolic Per-ARNT-Sim domain from the histidine kinase CitA of Geobacillus thermodenitrificans and resolving the four biotin sites of streptavidin. COLD provides quantitative 3D information about small- to medium-sized biomolecules on the Angstrom scale and complements other techniques in structural biology.
Asunto(s)
Colorantes Fluorescentes/análisis , Histidina Quinasa/química , Microscopía Fluorescente/métodos , Óptica y Fotónica/métodos , Imagen Individual de Molécula/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biotina/química , Biotina/metabolismo , Cristalografía por Rayos X , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Congelación , Geobacillus/química , Histidina Quinasa/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Espectroscopía de Resonancia Magnética , Óptica y Fotónica/instrumentación , Conformación Proteica , Dominios Proteicos , Procesos Estocásticos , Estreptavidina/metabolismoRESUMEN
Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1-sigma1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1-sigma1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three sigma1 subunit isoforms: A, B and C. The expressions of sigma1A and sigma1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from sigma1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The sigma1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Endosomas/metabolismo , Aprendizaje , Memoria , Vesículas Sinápticas/metabolismo , Complejo 1 de Proteína Adaptadora/análisis , Complejo 1 de Proteína Adaptadora/genética , Animales , Conducta Animal , Células Cultivadas , Clatrina/metabolismo , Femenino , Expresión Génica , Hipocampo/citología , Humanos , Ratones , Ratones Noqueados , Actividad Motora , Neuronas/metabolismo , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
In this work we systematically explored performance of an EM-CCD as a detector for spatially resolved total internal reflection image correlation spectroscopy (TIR-ICS) with respect to adjustable parameters. We show that variations in the observation volume (pixel binning) can be well described by a simple structural term omega. To test the sensitivity of camera-based TIR-ICS we measured diffusion coefficients and particle numbers (PN) of fluorescent probes of different sizes (Fluorospheres, GFP and labeled antibodies) at varying viscosities, concentrations, and sampling rates. TIR-ICS allowed distinguishing between different probe concentrations with differences in PN of 5% and differences of 6% in D by acquiring only 15 independent measurement runs.
Asunto(s)
Inmunoglobulina G/química , Microscopía Fluorescente/instrumentación , Modelos Teóricos , Análisis Espectral/instrumentación , Animales , Difusión , Proteínas Fluorescentes Verdes/química , Técnicas de Dilución del Indicador , Ratones , Microesferas , Tamaño de la Partícula , Conejos , Especificidad de la EspecieRESUMEN
On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR.
Asunto(s)
Axones/metabolismo , Quimografía/métodos , Animales , Cinética , Transporte de Proteínas , Ratas , Reproducibilidad de los Resultados , Sinaptofisina/metabolismoRESUMEN
The advent of super-resolution microscopy opens up the opportunity to study biological structures in unprecedented detail. However, revealing quantitative information about the spatial organization of a set of labeled proteins requires sophisticated analysis. This study introduces a novel robust cluster recognition algorithm based on Delaunay triangulation (CRADT), which can handle complex datasets generated by 3D super-resolution microscopy. This algorithm allows determining volume and shape of protein clusters in 3D. The study demonstrates its performance by applying this algorithm on dual-color 3D super-resolved measurements of mouse hippocampal synapses, stained against the presynaptic active zone marker protein Bassoon and the opposing postsynaptic density protein Homer as well as the exo- and endocytosis machinery proteins Synaptobrevin and Clathrin.