Fluorescence correlation spectroscopy for studying intermediate filament assembly

Abstract

Intermediate filaments play a central role in the cytoskeleton of eukaryotic cells. Together with microtubules and actin filaments they determine the mechanical properties of cells. Microtubules are also the guiding tracks for molecular transport in cells while actin filaments play an essential role in cell motility. The diameter of intermediate filaments lies between the diameter of actin filaments and microtubules which led to their name. There is an enormous genetic variety of intermediate filaments. In humans there are over 70 different genes for intermediate filament proteins. The different types of intermediate filaments are celltype-specific. For example, the intermediate filament protein vimentin is found in cells of mesenchymal origin and keratins occur in epithelial cells. Mutations in genes coding for intermediate filaments are known to cause more than 80 diseases, among them Alexander disease and amyotrophic lateral sclerosis (ALS). Intermediate filaments share a common hierarchical assembly scheme. Several tetramers form unit length filaments (ULFs). The ULFs then anneal longitudinally to form elongated filaments.Studies of the assembly are necessary to understand the structural properties of cells. In this thesis we aim to further understand the assembly of intermediate filaments. We use fluorescence correlation spectroscopy (FCS) which is a versatile technique to study the motility of molecules in solution. During the thesis project, we built a setup suitable for FCS. It allows for detecting the diffusive properties of the different states during the assembly. With the setup we study both the early and the long time scales of the assembly of vimentin filaments. For the long times we employ bulk measurements. To access the early time scales of the assembly, we employ microfluidic techniques. With the microfluidic mixing device we map the temporal axis to a spatial axis. This gives us the possibility to control the interaction of the molecules in a defined manner. We aim to observe the assembly reaction of vimentin intermediate filaments upon the addition of monovalent salt ions. v vi

PEG-mCherry interactions beyond classical macromolecular crowding

“…The mean hydrodynamic radii were calculated by using the Stokes-Einstein equation, assuming spherical particles (see Equation 5), and by using the dimensions of the effective excitation volume, which were estimated from best-fit results to the fluorescence autocorrelation function of a small chromophore with a diffusion coefficient of ∼320 µm 2 /s , ATTO 532. 84 where k B is the Boltzmann constant, T is the absolute temperature, η (% FV O, T ) is the bulk viscosity of the solution of buffer at a given crowder concentration (i.e., % FVO) at a given temperature and R H is the hydrodynamic radius.…”