Controlling Chemistry and Membrane Proteome Composition in Nanotube-Vesicle Networks

Doctoral thesis, 2008

This thesis presents a combination of experimental and theoretical techniques to elucidate the dynamics of chemical reactions in confined biological systems and bridges the gap between biomimetic and biological systems by using cell membranes as simplified biomimetic devices. These are in turn used and analyzed to draw conclusions about the cellular membrane makeup. Cells constantly undergo changes in shape and volume while biochemical reactions occur inside. In these confined spaces, the influence of geometry and structural dynamics on reaction behavior is an important factor which has to be taken into account to get a more complete understanding of dynamic cellular processes. The use of biomimetic nanotube-vesicle networks (NVN) have generated important knowledge about membrane behavior, and reaction and transport phenomena in small-scale systems. In this work, they are a helpful tool to study the reaction-diffusion behavior of enzymatic reactions. Investigations of reactions in different static network geometries imply that the geometry in which a reaction takes place can control the behavior of a catalytic reaction. Also, a high sensitivity of a reaction-diffusion system to changes in network topology is shown, implying that chemical reactions can be readily induced or boosted in certain nodes as a function of connectivity. Such changes in connectivity are related to the dynamic tube formations found inside Golgi stacks, for example. The relationship between enzymatic reaction rate, and volume fluctuations is shown by demonstrating that reactions can be turned on and off just by changing compartment volume. In order to add functionality to NVNs, a method to construct NVNs from the cell plasma membrane (PM) has been employed. The membrane is taken from unilamellar PM protrusions and possesses the native composition of membrane proteins (MP) and lipids from the PM. This enables functional studies of plasma membrane constituents, e.g. transport activity of MPs, but also reaction-diffusion behavior in a cell-like environment. In order to perform such studies, but also in the view of drug discovery, it is crucial to know which MPs reside in the plasma membrane. Cells are able to release the PM in form of micron-sized vesicles for which a purification protocol was developed. The membrane protein content was analyzed by exposing plasma membrane vesicles (PMVs) to proteolytic digestion of the embedded membrane proteins and analysis of peptides by mass spectrometry. More than 90% of the identified proteins are annotated to the PM which presents an unprecedented degree of purity in PM proteome analysis. PMVs originate solely from the PM, providing a platform for proteomic and functional studies, where the possibility to control MP composition via e.g. recombinant or overexpression of proteins is especially exciting. PMVs can be regarded as a versatile simplistic cell model, enabling studies of more complex cellular processes.