Bioluminescence

Résumé

vignette|Rendu artistique d'un krill bioluminescent (aquarelle d'Uwe Kils). vignette|Bioluminescence du champignon Panellus stipticus. vignette|Vue rapprochée d'un coléoptère bioluminescent Elateroidea. vignette|Panellus stipticus. La bioluminescence est la production et l'émission de lumière par un organisme vivant via une réaction chimique au cours de laquelle l'énergie chimique est convertie en énergie lumineuse. Le mot a pour origine le terme grec bios signifiant vie et le terme latin lumen, lumière. La bioluminescence est une forme de luminescence, produisant une lumière dite froide car moins de 20 % de la lumière génère de la chaleur. Elle ne doit pas être confondue avec la fluorescence, la phosphorescence de la lumière émise. La bioluminescence peut être générée par des organismes symbiotiques hébergés au sein d'un organisme plus grand. Le composé chimique à l'origine de la luminescence est la luciférine. Celle-ci émet de la lumière en s'oxydant grâce à l'intervention de la

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https://fr.wikipedia.org/wiki/Bioluminescence

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Design and validation of bioorthogonal probes for cellular disease models

Well-validated chemical probes enable testing of biological hypotheses, investigation of target tractability and translatability to the clinical phase. Consequently, these important tool compounds play a key role in the drug discovery process. Moreover, their use for target validation may ultimately help to decrease the attrition rate encountered by new molecular entities in clinical trials. Despite the number of publications describing well-validated chemical probes, for several reasons, it remains a difficult challenge to identify and select the appropriate high quality molecule enabling the desired biological and pharmacological studies in the relevant disease model. Various reports describe sets of principles to be fulfilled by high quality chemical probes, which mainly rely on verifying that chemical probes are potent, engage their intended targets in the relevant cellular model, have sufficient exposure at the desired site of action and express functional pharmacological activities by a selective modulation of their targets. Classical chemical probes include LMW ligands, which inform on the functional consequences of interacting with a particular bio-logical target in a model system. To get information on other parameters (such as sub-cellular distribution of target or compound) different tools may need to be used, often requiring specific chemical functionalization in order to observe the molecule using the currently available technologies. Compared to classical chemical probes, bioorthogonal probes can also be small molecules that elicit a functional response but with the additional advantage of being able to undergo reaction with a variety of chemical reporters (e.g. fluorophores) in situ, in bio-logical model systems such as cells and animals. Therefore, this increases their versatility; for example their visualization using imaging technologies (bioluminescence and fluorescence imaging) can provide important insights on compound permeability, in-tracellular distribution, and potentially co-localization with its protein target in relevant cellular disease models. Hence, the use of a bespoke bioorthogonal probe may answer key biological questions, which are usually only addressed by using multiple classical probes. Herein, we describe the design and the synthesis of bioorthogonal probes to study the inhibition of the p53-Mdm2 protein-protein interaction. Furthermore, we report the development of a set of assays based on fluorescence and bioluminescence techniques enabling the validation of these molecules in a cellular osteosarcoma model. Our approach for chemical probe design and validation in cellular disease models may be applied in principle to a wide range of novel drug discovery projects to provide early mechanistic understanding.

EPFL2019

Chargement

A Biocompatible in Vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice

Ke Cheng, Aurélien Godinat, Douglas Hanahan, Gennady Nikitin

The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin, substrate for firefly luciferase. The success of this "split luciferin" ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a "caged" luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH2-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.

Amer Chemical Soc2013

Chargement

Quantitative studies of gene expression in have shown widespread variability in transcript and protein product abundance at the single-cell level. Such fluctuations in gene expression have been shown to lead to heterogeneous cellular responses and phenotypes, acting in a range of systems, from immunity, cancer to development. To better understand heterogeneity in cellular populations, we developed a novel method for the precise quantification of gene expression in single living cells. Furthermore, we focused on whether and to which extent transcriptional expression profiles are transmitted during cell division. In the first section, we described a new approach for quantitative measurements of gene expression, based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse imaging. Using the currently brightest luminescent reporter, fluctuations of protein expression levels could be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. Moreover, our system allowed measurement of single-cell protein degradation rates in the absence of protein synthesis inhibitors, as well as calculation of absolute synthesis rates over the cell cycle. Finally, the internal tag could be excised by inducible expression of Cre recombinase, which enabled estimation of endogenous mRNA half-lives by monitoring the decay of luminescent signal. Second, to monitor transcriptional activity and quantify memory across multiple endogenous genes in mouse embryonic stem cells, we generated cell lines in which a short-lived luciferase reporter allowed sensitive monitoring of transcriptional kinetics by luminescence imaging at high time resolution over long periods of time. By coupling live single-cell monitoring of transcriptional activity of 9 different promoters with mathematical modelling, we found that different levels of expression fluctuations shape transcriptional memory. Long fluctuations (1-10 generations) and short fluctuations (1-3 h), as well as their amplitudes, vary widely between across different genes and together shape transcriptional memory. Surprisingly, we have shown that there was a large level of gene-specific cell-to-cell synchronicity in transcriptional profiles. Overall, we uncovered that noise and timescales of slow and fast fluctuations, as well as their synchronisation over the cell cycle, defined how transcriptional dynamics were correlated within a lineage of related cells. In conclusion, this thesis described an innovative method that allows measurement of absolute protein expression levels, protein turnover, as well as mRNA half-lives for endogenously expressed genes. Our approach opens new avenues in quantitative studies of gene expression in single living cells. Furthermore, we revealed different levels of transcriptional fluctuations that shape gene specific transcriptional memory and its timescales in mouse embryonic stem cells.

EPFL2018

Chargement

Design and validation of bioorthogonal probes for cellular disease models

EPFL2019

EPFL2018