Date : 17/06/2011

Internship proposal for : Master 1 or Master 2

Laboratory
Subcellular structure and cellular dynamic
UMR 144 CNRS-Institut Curie
26 rue d'Ulm 75248 Paris cedex 05
Website : http://umr144.curie.fr/en/research-groups/molecular-mechanisms-intracellular-transport-bruno-goud/molecular-mechanisms-intrace
Main discipline : Cell Biology
Lab director : Bruno Goud

Mentor
Jean-Baptiste Manneville
email : This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
phone : 01 56 24 65 64

Subjects
1.: intracellular transport
2.: membrane deformation
3.: diacylglycerol

Tools and methodologies
1.: giant unilamellar vesicles
2.: micropipette
3.: optical tweezers

Summary of lab's interests

Coat proteins play a critical role during the initial stages of intracellular transport. Coats allow the formation of transport intermediates by inducing membrane deformation and participate in molecular sorting. The physical mechanisms of budding of transport intermediates and of molecular sorting are still poorly understood. Most in vitro models developed previously are based on the use of small liposomes (radius of about 100 nm) visualized by electron microscopy, which does not allow a dynamic study of budding and sorting. We have developed, in collaboration with the group of Patricia Bassereau (UMR168, CNRS-Institut Curie), several in vitro model systems to visualize by real time optical microscopy the dynamics of protein coats. These assays are based on giant unilamellar vesicles (GUV, typically 10 µm radius) and lipid nanotubes (radius of about 20 nm) pulled from giant vesicles by kinesin molecular motors (1-3) or by optical tweezers (3,4).

Summary of project

The project will focus on the COPI coat. The COPI coat is involved in retrograde transport from Golgi apparatus to the endoplasmic reticulum. In collaboration with the group of Bruno Antonny (IPMC, Nice Sophia-Antipolis), we have developed fluorescent versions of the different molecular components of the COPI coat (Arf1, coatomer and ArfGAP1 and its ALPS motifs). We have recently reconstituted the binding of the COPI coat to giant vesicles and showed that a low membrane tension facilitates the deformation induced by COPI (5). This minimal system includes the proteins Arf1 and coatomer and, although it is sufficient to induce membrane deformation, it does not allow membrane fission. In a first part of the project, we want to characterize in more details the dynamics of membrane deformation, measure the forces induced by the COPI coat assembly and characterize the threshold tension below which deformation is effective. Measurements will be made using optical tweezers coupled to micropipette aspiration and confocal microscopy as described in (4). In a second part of the project, we will complicate the system to reconstitute membrane fission events. Several candidates will be tested to identify the minimal fission machinery that generates COPI-coated vesicles: actin polymerization, phase separation, or proteins or lipids that have been in volved in fission of COPI vesicles in vivo, such as ArfGAP1, BARS-50 and diacylglycerols. References : (1) Roux et al., Proc Natl Acad Sci U S A, 2002 99 5394-9 (2) Leduc et al., Proc Natl Acad Sci U S A, 2004 101 17096-101 (3) Roux et al., EMBO J, 2005 24 1537-45 (4) Sorre et al., Proc Natl Acad Sci U S A, 2009 106 5622-5626 (5) Manneville et al., Proc Natl Acad Sci U S A, 2008 105 16946-16951