BBB Seminar - Ilaria Testa
Caught in a moment: investigating the dynamics of proteins at the nanoscale
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SciLife/KTH, Sweden
Formation and dissociation of macromolecular complexes is one of the foundations of all cellular processes. However, state-of-the-art technique does not allow precise, efficient, and specific observation at the same time of arbitrary complex formation, especially in living cells. We developed novel techniques such as STARSS, event triggered STED and MoNaLISA to study the dynamic nature of molecules in living cells beyond the diffraction limit of light. 聽By using fluorescence photo-switching, light patterning and real-time images鈥� computation we provide a new way to monitor elusive cellular process timely and efficiently with time-lapse super resolution imaging.
Molecular assemblies are further investigated by measuring rotational diffusivity. Rotational diffusion provides direct information on the 鈥渕ass spectrum鈥� and local environment of molecular complexes in solution and cells. It is usually measured via fluorescence anisotropy (FA) in steady-state and time-resolved (TR-FA) modes, which probe changes in molecular orientation over a time window defined by the fluorescence lifetime, which is 1-5 ns for commonly used fluorophores. FA is widely used within the life sciences for drug screening applications and binding assays due to its molecular specificity and sensitivity, high throughput, and compatibility with microscopy but only for small molecules (~0.1-30 kDa). Conventional TR-FA cannot reveal binding in most of the human proteome, because the molecular complexes are too large, i.e. they tumble too slowly to be distinguished from stationary within the nanosecond-scale time-window defined by the fluorescence lifetime.
We developed a novel approach named Selective Time-resolved Anisotropy with Reversibly Switchable States (STARSS) to bypass the fundamental limits of fluorescence anisotropy measurements in solution and living cells, extending the observable mass range more than three orders of magnitude and increasing the photo-selection accuracy through fluorescence photo-switching. We used STARSS to investigate viral maturation, chromatin compactization and protein oligomerization in living cells.
颁丑补颈谤辫别谤蝉辞苍:听Clive Bramham, Department of Biomedicine
