Descripción del proyecto
Iluminación de moléculas individuales en nuestro mundo celular con escalas temporales cortas y largas
Los esquemas de células de los libros de texto clásicos nos muestran un puñado de orgánulos y prácticamente ninguna molécula en el citoplasma o el nucleoplasma. Sin embargo, las células están repletas de moléculas que interactúan en plazos muy cortos y a distancias cortas o relativamente largas, creando numerosas moléculas a menudo transitorias en cascadas de señalización complejas. El proyecto financiado con fondos europeos HDPROBES está desarrollando dos técnicas extremadamente avanzadas para arrojar luz sobre toda esta actividad en especímenes vivos. Una capturará las moléculas individuales efímeras antes de que desaparezcan y la otra facilitará la obtención de imágenes secuenciales de moléculas individuales para poder realizar un seguimiento de su dinámica sin necesidad de tintes tóxicos. Este dúo dinámico podría revolucionar nuestros conocimientos sobre la señalización celular.
Objetivo
In this proposal, we introduce two new families of probes for live-cell super-resolution microscopy. The first class comprises small-molecule fluorescent sensors for detecting short-lived, small signaling molecules and active enzymes with single-molecule resolution. The spatiotemporal confinement of biological reactive molecules has been hypothesized to regulate various pathological and physiological processes, but the lack of tools to observe directly these microdomains of biochemical activity has precluded the investigation of these mechanisms. The ability to detect small signaling agents and active enzymes with nanometric resolution in intact live specimens will allow us to study the role of compartmentalization in intracellular signaling at an unprecedented resolution. Our studies will focus on detecting elusive reactive oxygen and nitrogen species directly at their sites of endogenous production. We will also investigate the subcellular distribution of protease activity, focusing on its role in non-apoptotic signaling.
The second class of probes encompasses a palette of fluorescent dyes that switch continuously between dark and emissive forms. This dynamic equilibrium will enable the localization of single molecules in a densely labeled field without the need to apply toxic light for photoactivation. Based on a novel switching mechanism, we will prepare dyes of various emission wavelengths that blink in a controlled way. These dyes will allow us to perform, for the first time, super-resolution, multicolor, time-lapse imaging of live specimens over long time. Initial studies will focus on tracking a transcription factor that migrates from the endoplasmic reticulum to the nucleus to initiate a cellular stress response upon protein misfolding. These studies will provide spatiotemporal details of this important translocation, which takes more than one hour to occur and its observation at the single-molecule level is intractable with current super-resolution methods
Ámbito científico
- natural sciencesphysical sciencesopticsmicroscopysuper resolution microscopy
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsprotein folding
- engineering and technologyelectrical engineering, electronic engineering, information engineeringelectronic engineeringsensors
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
Programa(s)
Régimen de financiación
ERC-STG - Starting GrantInstitución de acogida
8006 Zurich
Suiza