Funded projects

In vitro reconstitution of Myosin IIA-driven membrane fission

2012 - P. Bassereau (UMR 168), B. Goud (UMR144)

Development ofan in vitro system to mimic the MyoIIA–driven fission process. Membrane nanotubes will be pulled from giant unilamellar vesicles (GUVs) using optical tweezers and we will monitor tube stability in the presence of Rab6, MyoIIA, actin and ATP.

1. Isolation of MyoIIA and its binding to membranes using Rab6

2. Formation of GUVs with bound MyoIIA and analysis of fission

3. Phase separation investigation using lipid GUVs to mimick golgi membrane

4. Contractile acto/MyoIIA ring analysis using single-lipid GUVs

Methods/Technologies:

  • Protein purification
  • in vitro protein prenylation
  • GUVs
  • Optical tweezers and force measurement
  • Confocal microscopy to visualize fission
  • Sensitive fluorescent lipids to study phase separation

Keywords: biomimetics, membrane dynamics, fission

figGoudBasserea

A: Schematic representation of MyoIIA recruitment to Giant Unilamellar Vesicles (GUVs)To investigate the role of MyoIIA in membrane fission we develop a minimal in vitro system consisting of giant liposomes (GUVs) coated with MyoIIA. Purified MyoIIA will be recruited to the membrane through its interaction with prenylated Rab6 on the GUVs. Membrane nanotubes will be pulled with optical tweezers in the presence of actin and ATP and tube stability will be monitored to detect fission. B/C: Preliminary results: recruitment of Rab6A to GUVsRab6A was modified with two geranylgeranyl moities and fluorescently labelled with Alexa488. As expected, GDP bound Rab6A shows no binding to membranes (B) whereas the GTP-bound form (C) binds to GUV membranes.

Intracellular membrane compartments communicate through multiple transport pathways that permit constant exchange of macromolecules and lipids. Transport is mediated by vesicles or tubules that bud and fission from one compartment (donor membrane), move within the cytoplasm along cytoskeletal tracks and fuse with the next compartment (acceptor membrane) along a given transport pathway. Each step of intracellular transport is tightly regulated and this regulation is critical for many cellular processes like growth, hormone release, endocytosis and neurotransmission. Perturbations in intracellular trafficking have been implicated in severe pathologies such as Parkinson’s disease and Huntington and intracellular trafficking also plays a significant role in cancer biology.

The aim of this project is to increase our understanding of the regulation of fission in vesicular transport, a step that remains poorly understood. A number of proteins have been identified that seem to be required for membrane fission in vivo, but it is unclear whether they directly participate in fission or if they only have a regulatory role in these processes. Recent results showed an unexpected function of the motor protein Myosin II A (MyoIIA) associated to actin in fission of Rab6 positive vesicles or tubules from the trans-Golgi network. The objective of this project is to reconstitute this fission event with purified components (MyoIIA, Rab6 and actin) using Giant Unilamellar Vesicles (GUVs) and membrane nanotubes that will be pulled from them. This will enable us to elucidate the role of MyoIIA and eventually other proteins in fission and we will be able to investigate the influence of membrane composition on these processes.