The interior of living cells is a crowded and heterogeneous medium that consists of solid structures and an embedding viscous fluid. In this complex environment, protein transport and interactions are spatially modulated. Further, anomalous protein transport phenomena are observed in living cells that impact on the kinetics of biological reactions and efficiency of target search processes. To understand anomalous protein transport in cells, protein mobility has to be mapped with high resolution on multiple scales. In this thesis, an experimental and theoretical framework for parallelized mobility measurements on multiple length scales by fluorescence correlation spectroscopy with line confocal microscopes is developed. Acquired fluorescence signals are either auto-correlated for protein mobility mapping or cross-correlated to probe the permeability of the intracellular structure. By applying this methodology, the scale-dependent mobility of inert green fluorescent protein (GFP) monomers and multimers is mapped in the cytoplasm and nucleus of living cells. Furthermore, it is retrieved from the time dependence of measured apparent diffusion coefficients that the structure of the intracellular environment appears as that of a porous medium. The methodology developed here yields quantitative mobility and interaction data on multiple scales that can be used in systems biology for understanding the functional organization of living cells.
|Supervisor:||Rippe, PD Dr. Karsten|
|Date of thesis defense:||21 May 2014|
|Date Deposited:||13 Jun 2014 10:29|
|Faculties / Institutes:||Service facilities > German Cancer Research Center (DKFZ)|
570 Life sciences