Location: HL 214

Time: 09:00 hrs

BM seminar Christophe Danelon (Delft)
Part 1: Real-time imaging of the effect of Notch activation on cell cycle progression
Part 2: Toward single molecule biophysics of gene expression ballet in liposomes

abstract:
This talk will cover two research projects. I will first present my postdoctoral work performed at the University of Lausanne (Switzerland) on the Notch signalling pathway. Then I will give you an overview of the research activities recently started in my group at the Delft University of Technology, with emphasis to gene expression in liposomes.

Part 1. Notch signalling plays an important role in cell-fate determination and differentiation in mammalian systems. Our goal was to assess how cell cycle is coordinated with Notch activity in keratinocytes. We engineered a squameous carcinoma cell line enabling (i) conditional induction of Notch activation and (ii) in vivo cell cycle imaging. We used the Fucci probes to label individual G1 phase nuclei red and those in S/G2/M phases green, which allowed dual-color time-lapse imaging of cell-cycle progression with or without stimulation of the Notch signalling pathway. Quantitative analysis of single nuclei tracking made it possible to discriminate between early and late stages of each phases and to infer the dynamical nature of the entry into the G1 phase promoted by Notch activation.

Part 2: Cell-free fluorescence imaging of macromolecular complexes or biochemical networks asks for the spatial confinement of the reactants. Lipid vesicle encapsulation offers several experimental advantages and is therefore a simple alternative to surface tethering. We developed a method to produce large vesicles from a variety of phospholipids in physiological buffer. Liposomes were immobilized on glass surfaces and localized with a membrane dye. We succeeded in encapsulating large molecules and showed that liposomes were stable for several days. We employed this method for the functional encapsulation of a minimal gene expression system and imaged in real time the synthesis of autofluorescent proteins in single liposomes.