Microscopie Fonctionnelle du Vivant

Cell death contributes to the maintenance of homeostasis, but mounting evidence has confirmed the involvement of programmed cell death in some diseases. The concept of programmed cell death, which was coined several decades ago to refer to apoptosis, now also encompasses necroptosis, a newly characterized cell death program. Research on programmed cell death has become essential for the development of some new therapies. To study cell death signaling and its molecular mechanisms, new biochemical and fluorogenic approaches have been devised. Here, we first provide an overview of programmed cell death modes and the importance of dynamic cell death studies. Next, we focus on both apoptotic and necroptotic signaling and their mechanisms by providing a systematic review of all the methods and approaches that have been used. We emphasize the contribution of advanced approaches based on fluorescent probes, reporters, and Förster resonance energy transfer (FRET)-based biosensors for studying programmed cell death. Because apoptosis and necroptosis signaling pathways share some effectors molecules, we discuss how these new tools could be used to discriminate between apoptosis and necroptosis. We also describe how we developed specific FRET-based biosensors for detecting necroptosis. Finally, we touch on how dynamic measurement of biomolecules in living models will play a role in personalized prognosis and therapy.

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.