Eukaryotic cells are compartmentalized into membrane-bound organelles, ensuring highly specialized and essential functions such as protection of the genome, protein synthesis and packaging, intracellular calcium signalling... Most organelles harbour a complex morphology, which is an important structural feature for the achievement of their assigned function. Moreover, some organelles are constituted of distinct subcompartments exhibiting specific structures associated to specialized functions. Visualizing the fine structure of organelles is thus essential to understand many aspects of their associated functions. Importantly, organelles architecture is highly dynamic and can be subjected to dramatic rearrangements in response to cellular changes like cell division or differentiation. The methods available so far to render organelle morphology require highly specialized techniques and are most often incompatible with high-rate image acquisition. The aim of this proposal is to develop a method to distinguish organelle substructures in live cells using a simple bi-dimensional confocal imaging setup. Fluorescent probes of organelle morphology will be designed based on the striking membrane sensing properties of a family of amphitpathic helices called ALPS motifs. To establish the usefulness of these tools, we propose to study the dynamics of the nuclear envelope (NE) and endoplasmic reticulum (ER) during mitosis.
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