Project description
Lighting up single molecules in our cellular world over short and long timescales
Classic textbook schematics of cells show us a handful of organelles and virtually no molecules in the cytoplasm or nucleoplasm. However, cells are packed with molecules that interact over very short timescales and over short to relatively long distances, creating numerous, often transient molecules in complex signalling cascades. The EU-funded HDPROBES project is developing two highly advanced techniques to shed light on all this activity in live specimens. One will capture ephemeral single molecules before they disappear and the other will enable time-lapse imaging of single molecules to follow their dynamics without the need for toxic dyes. This dynamic duo could revolutionise our understanding of cellular signalling.
Objective
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
Fields of science
- natural sciencesphysical sciencesopticsmicroscopysuper resolution microscopy
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsprotein folding
- engineering and technologyelectrical engineering, electronic engineering, information engineeringelectronic engineeringsensors
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
Programme(s)
Topic(s)
Funding Scheme
ERC-STG - Starting GrantHost institution
8006 Zurich
Switzerland