Hydrodynamic flow for deterministic sorting of cell-membrane components
The biological membrane, an amphiphilic structure that is the barrier between the cell interior and exterior, is one of the vital building blocks of all cells. Not only does it define the outer boundaries of the cell, it also carries important biological function by virtue of the proteins and other molecules that constitute the membrane, the function of which is in turn intimately coupled to their association with the lipid-based membrane.
That the function of the protein is linked to its amphiphilicity necessitates preservation of the amphiphilic environment when probing the function of membrane associated proteins. The low expression levels of, even overexpressed, membrane proteins in a cell membrane crowded with many different types of proteins presents a barrier for direct studies of this class of proteins in their native membrane environment.
In the work leading up to this thesis, the aim has been to overcome some of these hurdles and enable membrane protein accumulation and purification in a near native cell membrane. In the articles appended to the thesis, steps have been taken towards being able to move, concentrate, purify and in the end visualize single membrane proteins, using a combination of surface-sensitive imaging, microfluidics and hydrodynamic flow. The last development led to the insight that using this approach we were able to determine the exact size of nanometer-sized objects bound to the two-dimensional interface that is the supported membrane by measuring both the nanoparticles' deterministic and stochastic movement.
Looking forward, this thesis work has provided a solid foundation for deterministic sorting of membrane proteins, without the need for detergent solubilization. While this in itself is rather enticing, the possibility to simultaneously determine both the size and the biomolecular content of biological nanoparticles, as demonstrated in the final paper, might help telling whether it is the size, the amount of a specific molecule, or a precise combination of the two, that is decisive for their biological function, such as; infectivity, gene transfer or drug delivery in the context of virions, exosomes and nanoscale drug carriers, respectively.
Native membrane vesicles
Single particle tracking
Supported lipid bilayer