Next generation of ultrastable fluorescent proteins for quantitative super-resolution microscopy – STABLE-FP

Super-resolution fluorescence microscopy (“nanoscopy”) has undergone extraordinary development in recent years, illuminating structural cell biology at the nanoscale. Notably, single-molecule localization microscopy (SMLM), the most widely used nanoscopy technique, has expanded its range of applications to address the architecture and dynamics of biomolecules in a quantitative manner, largely based on quantitative Photoactivation Localization Microscopy (qPALM) and single particle tracking PALM (sptPALM). However, these techniques have not yet reached maturity, essentially due to the non-ideal photophysical performance of the used “smart” fluorescent labels, in particular green-to-red photoconvertible fluorescent proteins (PCFPs). Because fluorescent proteins offer genetically encoded labeling, PCFPs still remain the markers of choice for many qPALM and sptPALM applications, but clearly lag behind organic dyes in terms of photostability. Thus, this proposal aims at enhancing the photostability of PCFPs while preserving, or even improving, their monomeric character, folding and maturation.
To achieve this goal we will introduce a paradigm shift in the field of fluorescent protein design by combining investigations of the structural dynamics of PCFPs in realistic environmental conditions with large-scale engineering and screening at the single-molecule (SM) level. The realization of the project will rely on the complementary expertise of our Consortium in structural photophysics, single-molecule localization microscopy and protein engineering. Partner 1 will perform mechanistic investigations of PCFPs that focus on dynamical aspects, notably by engaging NMR studies in addition to kinetic crystallography and optical spectroscopy. This new perspective on FP dynamics will suggest relevant mutation sites to engineer enhanced PCFPs. To investigate these sites, Partner 3 will perform directed-evolution-based engineering while Partner 2 will develop a SM-based high-content screening (HCS-SMLM) platform. This will provide a novel framework, operating in a feedback loop manner, to semi-rationally design ultrastable PCFPs. In addition, our toolbox will enable the investigation of a new concept, that of “PALM acquisition engineering”, which will consist in optimizing illumination conditions and, whenever possible, physicochemical composition of the samples to achieve optimal PCFPs photostability.
Throughout the project, improved variants and imaging protocols will be validated in the frame of two biological applications which will serve as test platforms. The first one is a neurobiology application that concerns the quantification of synaptic proteins. The second one is a microbiology application that addresses the dynamics of nucleoid associated proteins in the radiation resistant bacterium D. radiodurans.

We expect STABLE-FP to address a key expectation of the community, providing photostable PCFPs and new data acquisition protocols suitable for the investigation by qPALM and sptPALM of hitherto poorly accessible biological questions in both eukaryotic and prokaryotic systems.
The project will provide key mechanistic insights into the “dark side” of PCFPs, which has remained largely overlooked thus far due to its intricate nature. We will generate a vast library of variants critical for the advancement of FP research, the characteristics of which will be made available through the design of a comprehensive database.
The development of an innovative HCS-SMLM platform will produce a fully automated environment to screen FP’s photophysical properties at the SM level, applicable in the future to other fluorophores or biological questions.
In conclusion, the project will push the knowledge of FPs beyond the state-of-the-art, making ultrastable PCFPs and novel illumination schemes accessible to the community and eligible for intellectual property.