Description du projet
Éclairer les molécules individuelles de notre monde cellulaire sur des périodes courtes et longues
Les schémas classiques des cellules nous montrent une poignée d’organites et pratiquement aucune molécule dans le cytoplasme ou le nucléoplasme. Cependant, les cellules regorgent de molécules qui interagissent sur des périodes très brèves et sur des distances courtes à relativement longues, créant ainsi de nombreuses molécules, souvent transitoires, dans des cascades de signalisation complexes. Le projet HDPROBES, financé par l’UE, développe deux techniques très avancées destinées à faire la lumière sur cette activité dans des spécimens vivants. L’une permettra de capturer des molécules uniques éphémères avant qu’elles ne disparaissent et l’autre permettra l’imagerie en temps réel de molécules uniques pour en suivre la dynamique sans avoir recours à des colorants toxiques. Ce duo dynamique pourrait révolutionner notre compréhension de la signalisation cellulaire.
Objectif
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
Champ scientifique
- natural sciencesphysical sciencesopticsmicroscopysuper resolution microscopy
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsprotein folding
- engineering and technologyelectrical engineering, electronic engineering, information engineeringelectronic engineeringsensors
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
Programme(s)
Thème(s)
Régime de financement
ERC-STG - Starting GrantInstitution d’accueil
8006 Zurich
Suisse