Our long-term goal is to understand how networks of biochemical reactions cooperate to the maintenance of cellular functional states and cellular homeostasis. More specifically, we are studying how biochemical networks encode for cellular decisions underlying cell and tissue homeostasis and how oncogenes contributed to early tumour initiation and promotion by reprogramming these processes.
We focus our studies on the characterization of checkpoint signalling and the DNA damage response (DDR) with particular interest on their heterogeneous response among clonal population of cells and how oncogenes alter their functioning. We have raised biochemical tools to monitor or alter K-RAS (and its mutants), p53, Caspases and DDR-related kinases.
Single Cell Biochemistry
However, we recognise that state of the art technologies do not permit to characterise a number of biochemical reactions in the living cells thereby limiting our capabilities to establish causal dependencies between the spatiotemporal dynamics of biochemical networks and cellular decisions. Therefore, we devoted significant efforts to develop novel fluorescence-based assays with the aim to enable complex biochemical measurements in single living cells.
Thanks to its low invasiveness, fluorescence microscopy enables the characterization of molecular interactions and other biochemical events (e.g., post-translational modifications) with high spatiotemporal resolution in the living cell. For instance, Foester Resonance Energy Transfer (FRET) can unveil protein-protein interactions or conformational changes in fluorescently tagged proteins by encoding in the properties of fluorescence (lifetime, polarization, colour) biochemical signatures. Thus, we have developed a novel family of FRET pairs that can be used to monitor at least three biochemical reactions simultaneously.
Furthermore, we developed optically responsive protein domains that alter their conformation or elicit interactions upon absorption of light. These novel Optogenetics tools permit us to activate and de-activate with high spatiotemporal resolution the biochemical activity of a specific enzyme or oncogene.
Soon, we’ll disclose more information on this platform.
This is my earlier work on a pH, FRET-based, biosensor done in the laboratory of Prof. Fred Wouters, back then at the European Neuroscience Institute. A good example, in my opinion, on how to rationally engineer the spectral overlap of two fluorophores, in this case fluorescent proteins, to create a biosensor.
“pHlameleons: A Family of FRET-Based Protein Sensors for Quantitative pH Imaging“
Abstract | Intracellular pH is an important indicator for cellular metabolism and pathogenesis. pH sensing in living cells has been achieved using a number of synthetic organic dyes and genetically expressible sensor proteins, even allowing the specific targeting of intracellular organelles. Ideally, a class of genetically encodeable sensors need to cover relevant cellular pH ranges. We present a FRET-based pH sensor platform, based on the pH modulation of YFP acceptor fluorophores in a fusion construct with ECFP. The concurrent loss of the overlap integral upon acidification results in a proportionally reduced FRET coupling. The readout of FRET over the sensitized YFP fluorescence lifetime yields a highly sensitive and robust pH measurement that is self-calibrated. The principle is demonstrated in the existing high-efficiency FRET fusion Cy11.5, and tunability of the platform design is demonstrated by genetic alteration of the pH sensitivity of the acceptor moiety.
This is an earlier collaboration with Dr. Carlos Bertoncini, done in the laboratory of Prof. Clemens Kaminski. A good example of how to modify proteins for sensing biochemical events, in this case oligomerization and aggregation by homoFRET. I looked after the development of the anisotropy detection and homoFRET measurements.
“Towards multiparametric fluorescent imaging of amyloid formation: studies of a YFP model of alpha-synuclein aggregation”
Abstract | Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases. In Parkinson’s disease and several closely related syndromes, the protein α-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.