How a cell can produce two daughter cells with different identities is the fundamental question we study using the Drosophila adult sensory organ (SO) lineage as a model system. SO precursors (SOP) are epithelial cells present in a single layer neuroepithelium on the dorsal thorax of Drosophila. SOP undergoes a series of four ACD in which a mother cell gives rise to two daughter cells. At each division, the acquisition of cell identity is controlled by the differential activation of Notch. Notch is activated by Delta (Dl) present on the surface of adjacent cells. Our work contributed to show that sorting of Notch and Dl along the apico-basal axis at cytokinesis is important for proper Notch activation. We also identified novel regulators of Notch signaling including membrane traffic regulators and regulators of epithelial cell cytokinesis. This led us to propose a working hypothesis according which Notch is activated at the level of the novel E-Cad-based adhesive contact that assembles between SOP daughter cells at cytokinesis. We are currently investigating the interplay between the mechanics of epithelial cell cytokinesis and the spatio-temporal control of Notch activation in flies.
1- How is Epithelial Tissue Integrity Preserved Throughout Cytokinesis until Abscission? As epithelial cells divide, they form a novel interface onto which adhesive contacts are form, apical-basal polarity is transmitted from mother to daughter cells, and chemical barrier needs to be preserved. Concomitantly, cell-cell communication takes place on this novel membrane interface. How is this complex series of events coordinated throughout cytokinesis? Our previous work contributed to show that epithelial cells divide with and against their neighbors and form a long adhesive interface. Our current work, based on cutting-edge quantitative live-imaging and electron microscopy (Fig. 3) aims at investigating how septate junctions are remodeled to preserve the paracellular/chemical barrier function of epithelia throughout cytokinesis. We also investigate the role of the ESCRT machinery and of components of septate junction in epithelial cells en route to abscission. This task is a prerequisite for a comparative analysis with the cytokinesis of SOP dividing asymmetrically and exhibiting Notch-dependent binary cell fate decision during cytokinesis.
2- Interplay between SOP Cytokinesis and Activation of the Notch Pathway? To directly address when and where the productive ligand/receptor activation takes place during SOP cytokinesis, our work currently investigate:
2-1 Dynamics of cell polarity markers and Notch components during SOP cytokinesis Live imaging and EM approaches are applied to define epithelial cell polarity transmission, protein composition and apicobasal polarity of the novel SOP daughter cell interface that we hypothesize serves as a launching platform for Notch activation (Fig.4). Genome editing with photoconvertible probes is developed to define the active pool of Notch.
2-2 membrane traffic regulators Time-lapse imaging, genome editing combined with optogenetics are currently employed to study the respective functions of membrane traffic regulator identified in (Le Bras et al, 2012) in Notch signaling.
2-3 AurA, asymmetric cell division and temporal control of Notch activation The mitotic kinase Aurora A (AurA) regulates asymmetric cell division by phosphorylation of the Par Complex. We found that AurA also phosphorylates Numb to regulate the endocytic trafficking of Notch. By adopting a KO/KI approach for AurA and Numb, our work aims to determine the roles of AurA in symmetric versus asymmetric cell division
3- Mechanotransduction or How Tensile Forces Impact on Notch-dependent Fate Acquisition? In 2013, a turn to biophysical approaches was made through collaboration with the team of Dr. Yusuke Toyama, Mechanobiology Institute (Singapore) and the hiring of a biophysicist Mathieu Pinot. Notch signaling and mechanical forces are intrinsically linked: (1) signaling takes place in epithelial cells that are subjected to tensile forces transmitted by the adherens junctions, (2) tensiles forces are applied to novel membrane interface at cytokinesis (our data), (3) forces are associated to membrane deformability during ligand endocytosis, (4) pulling forces are exerted on Notch and induce conformational change needed for receptor activation (Dr. G. Weinmaster’s lab) (5) and acto-myosin cytoskeleton dynamics that are source of tensile forces are different in SOP versus epidermal cells (our data). By multi-disciplinary approaches coupling fly genetics, cell biology and biophysical methodologies consisting of nano-laser ablation techniques (Fig. 5) and micro-rheology using optical tweezers our current work investigate the link between mechanical forces and Notch signaling
We want to decipher the interplay between cell polarity, membrane trafficking, cell cycle and cellular mechanics in Notch-dependent fate acquisition. Based on the importance of Notch signaling in human pathology, including tumorigenesis, on a medium term, we aim to develop a genetically tractable mammalian cell system setup such as stem-cell based organoid to further investigate this interplay in a pathological context.
Our research is currently funded by the CNRS, ANR Programme Blanc ApiNotch 2012-2015, La ligue Nationale contre le Cancer-Equipe Labellisée