Final Report Summary - CLATHPOL (Dynamic study of shape and force generation by clathrin polymerization onto lipid membranes in vitro)
The 'CLATHPOL' project aims to investigate the complex interactions driving clathrin coat assembly by developing a bio-mimetic minimal model system. In particular, the project focuses on the physical and biochemical parameters that regulate early phase of clathrin mediated membrane deformation.
The main objectives were assigned to the first phase of the project:
Reconstitution of the minimal model system of clathrin polymerization and deformation using giant unilamellar vesicles.
Investigating the affect of physical parameters such as membrane tension, bending rigidity and urvature on clathrin polymerization.
The first step towards reaching the goals of the project was to reconstitute a working minimal model system so as to visualize clathrin coat polymerization. For this, we first investigated the degree of membrane deformation caused by various adaptors and clathrin mix, in different stoichiometric ratios, on membrane sheets using DIC microscopy. This gave us an estimate of effective protein concentrations that cause optically visible membrane deformation. We then moved to the giant unilamellar vesicle (GUV) model system to be able to visualize directly the binding and polymerization of Clathrin coat by fluorescence imaging. It was found that the minimal lipid requirement for binding of adaptor protein AP180 and Clathrin to membrane was the presence of net negative charge (DOPS, PIP2) on the membrane. Clathrin polymerization was confirmed by fluorescence recovery after photobleaching (FRAP). After reconstituting the AP180 facilitated clathrin polymerization onto GUVs, we confirmed the consequent membrane deformation caused by means of Atomic force microscopy on membrane sheets incubated with the protein mix. Numerous budded structures spanning about 80 nm in diameter and 20 nm in height were observed on the membrane sheet, showing a partially resolved clathrin lattice, thereby confirming the presence of polymerized clathrin coat. Similar observations were made when lipid monolayers were incubated with adaptor/clathrin and imaged by electron microscopy.
The next objective was to look into the role of physical aspects such as membrane tension on the clathrin binding and polymerization. We observe that membrane tension does seem to play a critical role in the binding and polymerization of clathrin. We quantified the polymerization of clathrin under hypo-, iso- and hyper-osmotic conditions and found that clathrin polymerizes most effectively to membranes under low tension causing maximum deformations while it binds significantly strongly to membranes under intermediate tension without causing much deformation. Interestingly, the clathrin binding to membranes under high tension was extremely weak and caused no optically visible deformations.
Further, electron microscopy on ultra thin sections revealed similar observations, thus, by characterizing the degree of deformation at high resolution. We also checked clathrin polymerization and ability to deform membrane is affected by high bending rigidity membrane and interestingly, clathrin failed to polymerize and deform the membrane with high bending rigidity. Finally, micropipette aspiration of the GUVs allowed us to measure precisely the membrane tension regime at which a polymerized clathrin coat seems to rupture. This allows us to show that increased membrane tension fights against polymerization forces of Clathrin, forcing it to polymerize in a flat lattice. It is consistent with recent in vivo data, where clathrin mediated membrane deformation was proposed to be significantly impaired under high membrane tension. Together, We have been able to reconstitute, a minimal model system, based on giant liposomes to study clathrin polymerization and the physical parameters affecting it. To our knowledge, we have for the first time quantitatively measured the tension regimes that allow clathrin polymerization and the force needed to rupture the clathrin coat.
In terms of training, the researcher has trained in bacterial expression, purification and fluorescent labelling of protein of interest and carries out experiments routinely based on model membrane systems such as bilayers, monolayers and giant unilamellar vesicle using fluorescence microscopy and standard image analysis. Researcher also learnt negative staining, ultra thin sectioning and operating transmission electron microscopy. In addition, he also got trained in the micromanipulation of giant liposomes and pulling tubes using optical tweezers.
The main objectives were assigned to the first phase of the project:
Reconstitution of the minimal model system of clathrin polymerization and deformation using giant unilamellar vesicles.
Investigating the affect of physical parameters such as membrane tension, bending rigidity and urvature on clathrin polymerization.
The first step towards reaching the goals of the project was to reconstitute a working minimal model system so as to visualize clathrin coat polymerization. For this, we first investigated the degree of membrane deformation caused by various adaptors and clathrin mix, in different stoichiometric ratios, on membrane sheets using DIC microscopy. This gave us an estimate of effective protein concentrations that cause optically visible membrane deformation. We then moved to the giant unilamellar vesicle (GUV) model system to be able to visualize directly the binding and polymerization of Clathrin coat by fluorescence imaging. It was found that the minimal lipid requirement for binding of adaptor protein AP180 and Clathrin to membrane was the presence of net negative charge (DOPS, PIP2) on the membrane. Clathrin polymerization was confirmed by fluorescence recovery after photobleaching (FRAP). After reconstituting the AP180 facilitated clathrin polymerization onto GUVs, we confirmed the consequent membrane deformation caused by means of Atomic force microscopy on membrane sheets incubated with the protein mix. Numerous budded structures spanning about 80 nm in diameter and 20 nm in height were observed on the membrane sheet, showing a partially resolved clathrin lattice, thereby confirming the presence of polymerized clathrin coat. Similar observations were made when lipid monolayers were incubated with adaptor/clathrin and imaged by electron microscopy.
The next objective was to look into the role of physical aspects such as membrane tension on the clathrin binding and polymerization. We observe that membrane tension does seem to play a critical role in the binding and polymerization of clathrin. We quantified the polymerization of clathrin under hypo-, iso- and hyper-osmotic conditions and found that clathrin polymerizes most effectively to membranes under low tension causing maximum deformations while it binds significantly strongly to membranes under intermediate tension without causing much deformation. Interestingly, the clathrin binding to membranes under high tension was extremely weak and caused no optically visible deformations.
Further, electron microscopy on ultra thin sections revealed similar observations, thus, by characterizing the degree of deformation at high resolution. We also checked clathrin polymerization and ability to deform membrane is affected by high bending rigidity membrane and interestingly, clathrin failed to polymerize and deform the membrane with high bending rigidity. Finally, micropipette aspiration of the GUVs allowed us to measure precisely the membrane tension regime at which a polymerized clathrin coat seems to rupture. This allows us to show that increased membrane tension fights against polymerization forces of Clathrin, forcing it to polymerize in a flat lattice. It is consistent with recent in vivo data, where clathrin mediated membrane deformation was proposed to be significantly impaired under high membrane tension. Together, We have been able to reconstitute, a minimal model system, based on giant liposomes to study clathrin polymerization and the physical parameters affecting it. To our knowledge, we have for the first time quantitatively measured the tension regimes that allow clathrin polymerization and the force needed to rupture the clathrin coat.
In terms of training, the researcher has trained in bacterial expression, purification and fluorescent labelling of protein of interest and carries out experiments routinely based on model membrane systems such as bilayers, monolayers and giant unilamellar vesicle using fluorescence microscopy and standard image analysis. Researcher also learnt negative staining, ultra thin sectioning and operating transmission electron microscopy. In addition, he also got trained in the micromanipulation of giant liposomes and pulling tubes using optical tweezers.