In Vitro and In Vivo macromolecular dynamics : from biofilaments to living cells

Abstract

Studying macromolecules and living cells dynamics in situ significantly contribute to the understanding of various biological processes in living organisms. The biopolymer actin is one of the major building blocks of the cytoskeleton and is further crucial for numerous biological processes. Numerous mechanical responses of the cell including deformation and movement are based on physical properties of cytoskeletal networks, which are influenced by chemical gradients and modulate cytoskeletal functionality. In order to analyze the formation and properties of actin networks in concentration gradients, we developed multi-height microfluidic devices with diffusion-controlled microchambers. This unique approach enables for creating flow-free, steady state concentration gradients of different profiles, such as linear or step-like. Specific features of actin networks emerging in defined gradients are investigated. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity. Furthermore, we study the actin filaments as a model system for semiflexible polymers in microfluidic flow. Filamentous actin facing hydrodynamic forces undergo conformational transitions and analyzing their behavior provides a better understanding of non-Newtonian fluids in microchannels and in living organisms. We introduce a microfluidic device with wide and narrow channel segments, resulting in flow fields of spatially varying flow strength. These structured microchannels with alternating high- and low-velocity segments generate non-equilibrium and non-stationary alternating stretch-coil and coil-stretch transitions of fluorescently labeled actin filaments. We study the conformational transitions of filaments with different contour lengths and at different flow velocities. When the filament enters the wider section of the channel they coil under compression, whereas they are starching with a suppression of thermal fluctuations in the extensional regime during reentering the narrow part of the channel. Actin filaments exposed to hydrodynamic forces in structured microchannels with high- and low-velocity segments were characterized by center of mass velocity changes, the evolution of end-to-end distances and bending energies of the filament passing through the channel. Another biopolymer being essential for all known forms of life is DNA. We study the reversible process of DNA packing and unpacking, which is crucial for cell functioning. In eukaryotic cells, the DNA is wrapped around histone proteins, creating repeatable subunits called nucleosomes, which are then further folded into the chromosomes. For the experiments, histones were replaced by a positively charged, nearly spherical and biocompatible polyamidoamine (PAMAM) dendrimers of generation 6 (G6). In analogy to the histone, PAMAM G6 forms complexes with the DNA through sequence-independent, electrostatic interaction between the negatively charged nucleic acid and the protonated, positively charged dendrimer. We analyze the DNA / PAMAM G6 complex organization at different pH of the solution. Moreover, we study DNA decondensation, which is essential for processes such as transcription, replication and repair. DNA unwrapping was initiated by the DNA / PAMAM G6 complex interaction with heparin, which is highly negatively charged and serves as the competitive agent for DNA. First, DNA compaction and decompaction measurements were performed in glass capillaries using small angle X-ray scattering (SAXS), where we successfully analyzed structural changes of the DNA / PAMAM G6 complexes. Furthermore, specially developed microfluidic devices allow the measurement of the reaction dynamics of these processes. Using X-ray compatible, hydrodynamic focusing microfluidic devices with chevron/herringbone structures, we analyzed the real-time dynamics of DNA release from artificial gene carriers at different heparin concentrations. In this thesis, studies of live cell X-ray imaging are also discussed. Visualization of nanoscale features in living cells is very desirable for investigations of intracellular structures. We use X-ray ptychography to directly explore the dynamics of unstained living fission yeast Schizosaccharomyces pombe cells during meiosis in a natural, aqueous environment. X-ray imaging techniques allow us to investigate soft matter of several micrometers thickness in hydrated states without labeling at nanoscale resolution. We show that it is possible to make a sequence of X-ray images of living cells, which was not feasible so far and additionally, visualize the dynamic changes. Cells were alive even after several ptychographic X-ray scans and we obtained a sequence of X-ray images of individual living fission yeast, which allowed us to visualize and examine the meiotic nuclear oscillations and autophagic cell death subsequently induced by the ionizing radiation. Furthermore, the accumulated radiation after each scan allowed for a precise determination of the critical X-ray doses of autophagic vacuole formation and the lethal dose for fission yeast. This method enables looking at living biological samples and processes in a time-resolved label-free setting.