Smart modulation of fluorescent proteins for biosensing applications

The complex processes that take place within the biological cell often involve interactions between multiple molecules, and occur within a specific spatio-temporal context. Fluorescence imaging has become a solid standard for visualizing such processes within the native environment. By using fluorescent labeling strategies, it couples the cellular distribution and activity of biomolecules to a fluorescence-based signal and microscopic readout. The preferred labels for many cell biologists are fluorescent proteins (FPs), being genetically encoded and therefore minimally invasive to the cell. Some of these FPs may be modulated dynamically, on account of their interactions with light, their binding to molecules, or changes in the biochemical environment. This has opened up exciting opportunities for biosensing applications, where these 'smart' dynamic features are converted into a read-out for cellular activity. Furthermore, these smart properties enable optical methods that help to enhance spatial resolution, contrast, or sensitivity. Further progress in cell biology will require new fluorescent protein technologies and bio-imaging approaches to untangle the complex web of intracellular communication networks, its spatio-temporal organization, and advance further downstream translational research. To face this challenge, the aims of this thesis are, first of all, to acquire a deeper understanding of fluorescent protein behavior, which is essential to establish novel approaches for FP modulation; secondly, to use these approaches as an underlying basis for developing new imaging methodologies; and finally, their implementation for novel biosensing applications. To address these aims, three distinct mechanisms to modulate the signals of fluorescent proteins were used throughout this dissertation: light-induced photoswitching in reversibly switchable fluorescent proteins (RSFPs), modulation of the fluorescence through binding-induced effects, and Förster resonance energy transfer. After an introduction to these principles and their application for fluorescent proteins (Chapter 1), the following experimental chapters each implement one or combine multiple of these three strategies to advance the state-of-the-art in fluorescent protein technology and biosensing applications. First, in Chapter 2, I describe the binding of a camelid-derived nanobody to modulate the reversible photoswitching properties of the rsGreen series of RSFPs. By performing an in vitro and in situ characterization, I demonstrate that the rsGreens exist as two inter-converting species with different photoswitching kinetics, and that binding of the nanobody modifies both the spectroscopic and photochromic properties of these RSFPs. Next, in Chapter 3, I expand on the implementation of photoswitching in RSFPs for multiplexed FRET-based biosensing. I demonstrate how the photochromism of a donor fluorophore can be leveraged to quantify the responses of two spectrally overlapping FRET pairs. Using a model for the photoswitching, we validate our method by performing numerical simulations, and confirm its application for live-cell biosensing using a combination of new FRET-based biosensors. Finally, Chapter 4, focuses on binding-induced modulation of a reversible reporter system that is based on an extrinsic fluorescent protein scaffold. Unlike conventional FPs, this class of proteins relies on its fluorescence through the (reversible) binding of exogenously provided chromophores. We assess the application of a split fluorescent reporter, splitFAST, and characterize its performance for the visualization of cell receptor activation and downstream signaling responses. Together, this dissertation demonstrates the versatility of fluorescent proteins for live-cell biosensing applications. By applying a synergistic approach to FP modulation, our results add to the existing body of fluorescent protein knowledge, with particular insights into the photochromism of RSFPs. Our grasp on the properties of FPs also translates to new methodologies, as we show using our unmixing approach, and paves the way for new classes of biosensors that may help to elucidate the complexity of living systems with fluorescence imaging.

Publication year:2021

Accessibility:Open