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- Bauer, Brigitte, 1978
(author)
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Controlling Chemistry and Membrane Proteome Composition in Nanotube-Vesicle Networks
- 2008
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Doctoral thesis (other academic/artistic)abstract
- 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.
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- Lizana, Ludvig, 1977, et al.
(author)
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Controlling the rates of biochemical reactions and signaling networks by shape and volume changes
- 2008
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:11, s. 4099-4104
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Journal article (peer-reviewed)abstract
- In biological systems, chemical activity takes place in micrometerand nanometer-sized compartments that constantly change in shape and volume. These ever-changing cellular compartments embed chemical reactions, and we demonstrate that the rates of such incorporated reactions are directly affected by the ongoing shape reconfigurations. First, we show that the rate of product formation in an enzymatic reaction can be regulated by simple volume contraction-dilation transitions. The results suggest that mitochondria may regulate the dynamics of interior reaction pathways (e.g., the Krebs cycle) by volume changes. We then show the effect of shape changes on reactions occurring in more complex and structured systems by using biomimetic networks composed of micrometer-sized compartments joined together by nanotubes. Chemical activity was measured by implementing an enzymatic reaction-diffusion system. During ongoing reactions, the network connectivity is changed suddenly (similar to the dynamic tube formations found inside Golgi stacks, for example), and the effect on the reaction is registered. We show that spatiotemporal properties of the reaction-diffusion system are extremely sensitive to sudden changes in network topology and that chemical reactions can be initiated, or boosted, in certain nodes as a function of connectivity. © 2008 by The National Academy of Sciences of the USA.
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