Younes Farhangi Barooji
A thesis submitted March, 2015 for the degree of Doctor of Philosophy and defended June 22, 2015.
The PhD School of Science
Faculty of Science
Niels Bohr Institute, University of Copenhagen
Seyyed Nader Seyyed Reihani
Poul Martin Bendix
Physical characterization of phospholipid nanotubes and the effect of BAR domain proteins on their mechanical stability
Phospholipid nanotubes are unique assemblies of phospholipid molecules which can be reconstituted in-vitro . They are excellent models for studying the biophysics of nanotubes in living cells. With a radial size of ca. 10 nm, they are well suited for studying size effects and the in uence of curvature on protein binding. In this project, we used various assays to produce nanotubes, which were subsequently size calibrated by using a new calibration scheme based on uorescent intensity from uorophores incorporated in the tubular membrane. The results were used to identify uni- and multilamellar tubes.
The shape dynamics of unilamellar tubes was further quantied by tracing the contour of the nanotubes and calculating the correlation between tangent vectors along the contour. This correlation provides a measure for the tube stiffness, which is called the persistence length. More importantly, it was revealed for the rst time that persistence length scaled linearly with tube radius. The persistence length analysis of lipid tubes can be useful to understand the mechanical properties of tubes in cells.
The assay for quantifying size and persistence length of freely suspended nanotubes was used to measure the effect of laser excitation of uorescent tubes with different radial size. During extended illumination, the stiffness was shown to decrease for membrane tubes in a size specic manner. Identifying the lipid nanotube radius (typically between 10 and 100 nm) by its persistence length and intensity is highly useful for understanding the role of membrane associated proteins in membrane remodeling.
BAR (BinAmphiphysinRvs) domain proteins as a membrane associated proteins play an important role in membrane remodeling, e.g. during the endocytosis process and lopodium formation. Here, the effect of three membranes of the BAR domain family was considered: F-BAR, Arfaptin (classical BAR) and I-BAR. The effect of these proteins on the membrane can be different due to their structure and curvature. We investigated the effect of the F-BAR (Syndapin 1) domain protein on the persistence length of the tubular membranes having different radii. This was achieved by calculating the persistence length of F-BAR coated nanotubes and it was revealed that the stiffness of the F-BAR coated tubular membranes is 5 times higher than protein free tubular membranes. Phospholipid nanotubes (tethers) were also pulled from the vesicle membranes and the curvature sensitivity of the proteins on them was tested against a spectrum of curvatures. We observed that Arfaptin proteins bind very well to the lipid membrane in a low concentration of salt and lead to the tubulation of the membrane.
Finally, The tubulation of membranes by I-BAR proteins was quantied. The persistence length of I-BAR coated phospholipid nanotubes was found to increase by factor 2 in comparison to the protein free tubes having the same size.