Mechanics of Intermediate Filaments

The eukariotic cytoskeleton consists of three major filament types: actin filaments, microtubules and intermediate filaments. While the first two filament types are highly conserved, intermediate filaments vary between cell types. Here, we focus on the intermediate filament vimentin, which occurs mesenchymal cells. A defining feature of intermediate filaments is their assembly process. The filaments assemble in a hierarchical manner from tetrameric complexes of half-staggered antiparalell coiled-coils. These tetrameric structures then align laterally to form unit-length filaments which in turn form filaments by annealing longitudinally. Using fluorescence microscopy techniques we perform experiments on vimentin filaments in vitro: Sub-unit exchange between two differently labeled filament populations is observed  to depend on the filaments cross-section polymorphism. We find that increasing polymorphism leads to a faster sub-unit exchange process. Thus, vimentin intermediate filaments are found to be dynamic structures. Notably these observations are made in the absence of cellular regulatory mechanisms. Aside from the changes in composition of vimentin filaments we observe the mechanical properties of vimentin filaments and make observations on the polymer physical aspects of the filaments. To this end the fluorescently labeled filaments are confined in microchannels with widths in the range of micrometers. We find that vimentin filaments can accurately be described by the worm-like chain model; fluctuations in perpendicular planes decouple. In addition a scaling law describing filaments confined in the Odijk regime is confirmed for polymers with different persistence lengths by the combination of data from vimentin and actin filaments

Vimentin networks at tunable ion-concentration in microfluidic drops

The structure and function of biological systems, for example, cells and proteins, depend strongly on their chemical environment. To investigate such dependence, we design a polydimethylsiloxane-based microfluidic device to encapsulate biological systems in picoliter-sized drops. The content of each individual drop is tuned in a defined manner. As a key feature of our method, the individual chemical composition is determined and related to the drop content. In our case, the drop content is imaged using microscopy methods, while the drops are immobilized to allow for long-time studies. As an application of our device, we study the influence of divalent ions on vimentin intermediate filament networks in a quantitative way by tuning the magnesium concentration from drop to drop. This way we are able to directly image the effect of magnesium on the fluorescently tagged protein in a few hundreds of drops. Our study shows that with increasing magnesium concentration in the drops, the compaction of the networks becomes more pronounced. The degree of compaction is characterized by different morphologies; freely fluctuating networks are observed at comparatively low magnesium concentrations of 5–10 mM, while with increasing magnesium concentration reaching 16 mM they develop into fully aggregated networks. Our approach demonstrates how a systematic study of interactions in biological systems can benefit from the exceptional controllability of microfluidic methods

Helical Superstructure of Intermediate Filaments

International audienc

Sociální a ekonomická analýza rozvoje města Špindlerova Mlýna

The socioeconomic analysis is dedicated to present period. Main point of development is tourist trade, which is supported by cross-border project plans. I evaluate plans, which are dedicated to improve infrastructure. I am concern with the question of financing and the European funds utilization. My other part of the thesis is concentre to cooperation between five Czech municipalities and five Polish municipalities. Their aim is to create "Krkonoše tourist corridor". I also process the information from Questionnaire of inhabitant's satisfaction. My thesis evaluates investment from 2008 and strategic development plan 2007-2010. This strategic plan is dedicated mainly to improve civic amenities, infrastructure development and the new ground plan elaboration. The last part of my work mentions the problems of new highland resort build-up. This plan exceeds the section line of protected territory and it also interferes National park

Detection of contactin-2 in cerebrospinal fluid (CSF) of patients with Alzheimer's disease using Fluorescence Correlation Spectroscopy (FCS)

Objectives Alzheimer's disease (AD) is the most common cause of dementia in the world. As many AD biomarkers occur at rather low abundances in CSF or blood, techniques of very high sensitivity and accuracy are important as diagnostic tools in the clinic. Here, we aimed to provide proof of concept of the use of a single molecule detection technique, Fluorescence Correlation Spectroscopy (FCS) for detection of novel candidate biomarkers for AD. Design and methods FCS detects the diffusion times of the antigen-antibody complexes in highly diluted sample solutions, thus eliminating the need of large sample volumes and allows estimating the concentration of the target antigen. We developed a FCS set-up for contactin-2, a neuronal cell adhesion molecule and a ligand of beta-secretase 1 (BACE1) and amyloid precursor protein (APP), the latter proteins being important players in AD. With this method, we investigated whether contactin-2 concentrations are changed after delayed storage and in patients with Alzheimer's disease. Results The FCS set-up for measuring contactin-2 in CSF had a lower limit of quantification (LLOQ) of 0.2 ng/ml and intra- and inter-assay coefficients of variation (CVs) of 12.2% and 14.6% respectively. Contactin-2 levels were stable up to one week storage of CSF (n = 3) at RT and 4 °C. Further, contactin-2 levels were similar in probable AD patients (n = 34, p = 0.27) compared to patients with subjective cognitive decline (SCD) (n = 11). Conclusions FCS is a sensitive tool, which can be used for detecting biomarkers in the clinical setting using very low sample volumes (10 μl) and can measure proteins in their native conformations in the body fluid