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In this work, the role of the TusA protein was investigated for the cell functionality and FtsZ ring assembly in Escherichia coli. TusA is the tRNA-2-thiouridine synthase that acts as a sulfur transferase in tRNA thiolation for the formation of 2-thiouridine at the position 34 (wobble base) of tRNALys, tRNAGlu and tRNAGln. It binds the persulfide form of sulfur and transfers it to further proteins during mnm5s2U tRNA modification at wobble position and for Moco biosynthesis. With this thiomodification of tRNA, the ribosome binding is more efficient and frameshifting is averted during the protein translation. Previous studies have revealed an essential role of TusA in bacterial cell physiology since deletion of the tusA gene resulted in retarded growth and filamentous cells during the exponential growth phase in a rich medium which suddenly disappeared during the stationary phase. This indicates a problem in the cell division process. Therefore the focus of this work was to investigate the role of TusA for cell functionality and FtsZ ring formation and thus the cell separation.
The reason behind the filamentous growth of the tusA mutant strain was investigated by growth and morphological analyses. ΔtusA cells showed a retarded growth during the exponential phase compared to the WT strain. Also, morphological analysis of ΔtusA cells confirmed the filamentous cell shape. The growth and cell division defects in ΔtusA indicated a defect in FtsZ protein as a key player of cell division. The microscopic investigation revealed that filamentous ΔtusA cells possessed multiple DNA parts arranged next to each other. This suggested that although the DNA replication occurred correctly, there was a defect in the step where FtsZ should act; probably FtsZ is unable to assemble to the ring structure or the assembled ring is not able to constrict. All tested mutant strains (ΔtusD, ΔtusE and ΔmnmA) involved in the mnm5s2U34 tRNA modification pathway shared the similar retarded growth and filamentous cell shape like ΔtusA strain. Thus, the cell division defect arises from a defect in mnm5s2U34 tRNA thiolation.
Since the FtsZ ring formation was supposed to be defective in filaments, a possible intracellular interaction of TusA and FtsZ was examined by fluorescent (EGFP and mCherry) fusion proteins expression and FRET. FtsZ expressing tusA mutant (DE3) cells showed a red mCherry signal at the cell poles, indicating that FtsZ is still in the assembling phase. Interestingly, the cellular region of EGFP-TusA fusion protein expressed in ΔtusA (DE3) was conspicuous; the EGFP signal was spread throughout the whole cell and, in addition, a slight accumulation of the EGFP-TusA fluorescence was detectable at the cell poles, the same part of the cell as for mCherry-FtsZ. Thus, this strongly suggested an interaction of TusA and FtsZ.
Furthermore, the cellular FtsZ and Fis concentrations, and their change during different growth phases were determined via immunoblotting. All tested deletion strains of mnm5s2U34 tRNA modification show high cellular FtsZ and Fis levels in the exponential phase, shifting to the later growth phases. This shift reflects the retarded growth, whereby the deletion strains reach later the exponential phase. Conclusively, the growth and cell division defect, and thus the formation of filaments, is most likely caused by changes in the cellular FtsZ and Fis concentrations.
Finally, the translation efficiencies of certain proteins (RpoS, Fur, Fis and mFis) in tusA mutant and in additional gene deletion strains were studied whether they were affected by using unmodified U34 tRNAs of Lys, Glu and Gln. The translation efficiency is decreased in mnm5s2U34 tRNA modification-impaired strains in addition to their existing growth and cell division defect due to the elimination of these three amino acids. Finally, these results confirm and reinforce the importance of Lys, Glu and Gln and the mnm5s2U34 tRNA thiolation for efficient protein translation. Thus, these findings verify that the translation of fur, fis and rpoS is regulated by mnm5s2U34 tRNA modifications, which is growth phase-dependent.
In total, this work showed the importance of the role of TusA for bacterial cell functionality and physiology. The deletion of the tusA gene disrupted a complex regulatory network within the cell, that most influenced by the decreased translation of Fis and RpoS, caused by the absence of mnm5s2U34 tRNA modifications. The disruption of RpoS and Fis cellular network influences in turn the cellular FtsZ level in the early exponential phase. Finally, the reduced FtsZ concentration leads to elongated, filamentous E. coli cells, which are unable to divide.
During an unusually massive filament eruption on 7 June 2011, SDO/AIA imaged for the first time significant EUV emission around a magnetic reconnection region in the solar corona. The reconnection occurred between magnetic fields of the laterally expanding CME and a neighbouring active region. A pre-existing quasi-separatrix layer was activated in the process. This scenario is supported by data-constrained numerical simulations of the eruption. Observations show that dense cool filament plasma was re-directed and heated in situ, producing coronal-temperature emission around the reconnection region. These results provide the first direct observational evidence, supported by MHD simulations and magnetic modelling, that a large-scale re-configuration of the coronal magnetic field takes place during solar eruptions via the process of magnetic reconnection.
We study buckling instabilities of filaments in biological systems. Filaments in a cell are the building blocks of the cytoskeleton. They are responsible for the mechanical stability of cells and play an important role in intracellular transport by molecular motors, which transport cargo such as organelles along cytoskeletal filaments. Filaments of the cytoskeleton are semiflexible polymers, i.e., their bending energy is comparable to the thermal energy such that they can be viewed as elastic rods on the nanometer scale, which exhibit pronounced thermal fluctuations. Like macroscopic elastic rods, filaments can undergo a mechanical buckling instability under a compressive load. In the first part of the thesis, we study how this buckling instability is affected by the pronounced thermal fluctuations of the filaments. In cells, compressive loads on filaments can be generated by molecular motors. This happens, for example, during cell division in the mitotic spindle. In the second part of the thesis, we investigate how the stochastic nature of such motor-generated forces influences the buckling behavior of filaments. In chapter 2 we review briefly the buckling instability problem of rods on the macroscopic scale and introduce an analytical model for buckling of filaments or elastic rods in two spatial dimensions in the presence of thermal fluctuations. We present an analytical treatment of the buckling instability in the presence of thermal fluctuations based on a renormalization-like procedure in terms of the non-linear sigma model where we integrate out short-wavelength fluctuations in order to obtain an effective theory for the mode of the longest wavelength governing the buckling instability. We calculate the resulting shift of the critical force by fluctuation effects and find that, in two spatial dimensions, thermal fluctuations increase this force. Furthermore, in the buckled state, thermal fluctuations lead to an increase in the mean projected length of the filament in the force direction. As a function of the contour length, the mean projected length exhibits a cusp at the buckling instability, which becomes rounded by thermal fluctuations. Our main result is the observation that a buckled filament is stretched by thermal fluctuations, i.e., its mean projected length in the direction of the applied force increases by thermal fluctuations. Our analytical results are confirmed by Monte Carlo simulations for buckling of semiflexible filaments in two spatial dimensions. We also perform Monte Carlo simulations in higher spatial dimensions and show that the increase in projected length by thermal fluctuations is less pronounced than in two dimensions and strongly depends on the choice of the boundary conditions. In the second part of this work, we present a model for buckling of semiflexible filaments under the action of molecular motors. We investigate a system in which a group of motors moves along a clamped filament carrying a second filament as a cargo. The cargo-filament is pushed against the wall and eventually buckles. The force-generating motors can stochastically unbind and rebind to the filament during the buckling process. We formulate a stochastic model of this system and calculate the mean first passage time for the unbinding of all linking motors which corresponds to the transition back to the unbuckled state of the cargo filament in a mean-field model. Our results show that for sufficiently short microtubules the movement of kinesin-I-motors is affected by the load force generated by the cargo filament. Our predictions could be tested in future experiments.
The biological function and the technological applications of semiflexible polymers, such as DNA, actin filaments and carbon nanotubes, strongly depend on their rigidity. Semiflexible polymers are characterized by their persistence length, the definition of which is the subject of the first part of this thesis. Attractive interactions, that arise e.g.~in the adsorption, the condensation and the bundling of filaments, can change the conformation of a semiflexible polymer. The conformation depends on the relative magnitude of the material parameters and can be influenced by them in a systematic manner. In particular, the morphologies of semiflexible polymer rings, such as circular nanotubes or DNA, which are adsorbed onto substrates with three types of structures, are studied: (i) A topographical channel, (ii) a chemically modified stripe and (iii) a periodic pattern of topographical steps. The results are compared with the condensation of rings by attractive interactions. Furthermore, the bundling of two individual actin filaments, whose ends are anchored, is analyzed. This system geometry is shown to provide a systematic and quantitative method to extract the magnitude of the attraction between the filaments from experimentally observable conformations of the filaments.
In the present work, we discuss two subjects related to the nonequilibrium dynamics of polymers or biological filaments adsorbed to two-dimensional substrates. The first part is dedicated to thermally activated dynamics of polymers on structured substrates in the presence or absence of a driving force. The structured substrate is represented by double-well or periodic potentials. We consider both homogeneous and point driving forces. Point-like driving forces can be realized in single molecule manipulation by atomic force microscopy tips. Uniform driving forces can be generated by hydrodynamic flow or by electric fields for charged polymers. In the second part, we consider collective filament motion in motility assays for motor proteins, where filaments glide over a motor-coated substrate. The model for the simulation of the filament dynamics contains interactive deformable filaments that move under the influence of forces from molecular motors and thermal noise. Motor tails are attached to the substrate and modeled as flexible polymers (entropic springs), motor heads perform a directed walk with a given force-velocity relation. We study the collective filament dynamics and pattern formation as a function of the motor and filament density, the force-velocity characteristics, the detachment rate of motor proteins and the filament interaction. In particular, the formation and statistics of filament patterns such as nematic ordering due to motor activity or clusters due to blocking effects are investigated. Our results are experimentally accessible and possible experimental realizations are discussed.