Research

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We moved to Paris !

 

Please note that our laboratory moved to Paris in 2017. We are now continuing or work at the Institut Pasteur. Our unit Membrane Biochemistry and Transport is embedded in the department of Cell Biology and Infection.

 

You can visit our homepage following this link: 

https://research.pasteur.fr/en/team/membrane-biochemistry-and-transport/



Our mission:

Our group Molecular Membrane and Organelle Biology aims to understand how cellular organelles are being shaped. For many years membranes have been seen as passive boundaries of cells and organelles, which merely provide mechanical support and a two-dimensional interaction surface for proteins. However, it became apparent that the shape of organelles is closely intertwined with organelle-function and cellular homeostasis. We seek to understand the shape-function relationship of organelles at a molecular level.

 

Our focus:

Most cellular organelles are not de novo synthesized, but multiplied by expansion and division. Autophagosomes are the exception of this rule. Autophagy is a highly conserved catabolic pathway which delivers surplus or damaged cytoplasmic material to lysosomes for degradation. During this process, a cup-shaped membrane is nucleated de novo and expands to capture cargo. After sealing, a double-membrane surrounded autophagosome is being formed. This process involves highly coordinated membrane fusion and shaping processes, which are poorly understood at the molecular level. More than 30 proteins are involved in coordinating autophagosome-biogenesis and we aim to decipher their individual contributions to this demanding membrane remodeling pathway.

Biogenesis of an autophagosome: small donor vesicles fuse to give rise to a cup-shaped precursor membrane, termed phagophore. The phagophore expands to capture cytoplasmic cargo. After sealing, the double membrane surrounded autophagosome is formed. Zoom Image
Biogenesis of an autophagosome: small donor vesicles fuse to give rise to a cup-shaped precursor membrane, termed phagophore. The phagophore expands to capture cytoplasmic cargo. After sealing, the double membrane surrounded autophagosome is formed. [less]

 

Our methods:

We are using a synthetic biology approach to recapitulate the formation of autophagosomes in the test tube. Synthetic biology is a rather new field in life sciences, which is based on the idea to identify a minimal set of components that is needed to recapitulate biological processes in vitro using purified components in a well-defined environment. Two major components need to be combined to reproduce autophagosome biogenesis in vitro: purified proteins and model membranes. 

We recombinantly express the proteins of interest in E.coli, S.cerevisiae or insect cells and purify them to homogeneity using chromatography-based techniques. To analyze the function of these proteins with fluorescence-based techniques, we introduce single cysteines at defined positions within the protein and conjugate small fluorophores to them. This has the advantage that perturbations of the protein structure are minimized and the full biological activity of the protein is retained. 

Model membranes come in two major flavors: supported and freestanding membranes. They are produced from synthetic lipids or lipid extracts of cells. We use supported lipid bilayers (SLBs) to study protein dynamics and their spatial organization on membranes. Combining SLBs with TIRF-microscopy allows us to detect proteins at a single-molecule level and to study their dynamic association, dissociation, and lateral movement. Atomic force microscopy is applied to visualize proteins and protein-networks on membranes with high spatial resolution. Vesicles of different sizes (small, large, and giant unilamellar vesicles) are used for biochemical assays and confocal fluorescence microscopy.

These combined approaches allow us to decipher the molecular mechanisms that drive autophagosome biogenesis. Moreover, we use these insights to make predictions that can be tested in vivo to validate our model. Cell biological experiments involve the generation of mutants and their biochemical characterization, as well as live-cell imaging approaches.  

Fluorescent Recovery after Photobleaching (FRAP) experiment of a protein on GUV-membranes.

A fluorescent labeled protein (green) was bound to GUV membranes (red). In the first part of the movie, recovery of membrane-fluorescence (red) is shown. Rapid recovery of the fluorescence after photobleaching indicate that lipids diffuse freely. The second part of the movie shows recovery of protein fluorescence. Virtually no fluorescence recovers, suggesting that the protein is entirly immobile.
 
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