@phdthesis{Yildiz2023,
  author    = {Yildiz, Tugba},
  title     = {Dissecting the role of the TusA protein for cell functionality and FtsZ ring assembly in Escherichia coli},
  doi       = {10.25932/publishup-61713},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-617135},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XI, 171},
  year      = {2023},
  abstract  = {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.},
  language  = {en}
}
@phdthesis{Kraikivski2005,
  author    = {Kraikivski, Pavel},
  title     = {Non-equilibrium dynamics of adsorbed polymers and filaments},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5979},
  school      = {Universit{\"a}t Potsdam},
  year      = {2005},
  abstract  = {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.},
  subject      = {Polymere},
  language  = {en}
}
@phdthesis{Gutjahr2007,
  author    = {Gutjahr, Petra},
  title     = {Conformations of semiflexible polymers and filaments},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-15918},
  school      = {Universit{\"a}t Potsdam},
  year      = {2007},
  abstract  = {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.},
  language  = {en}
}
@phdthesis{Baczyński2009,
  author    = {Baczyński, Krzysztof Konrad},
  title     = {Buckling instabilities of semiflexible filaments in biological systems},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-37927},
  school      = {Universit{\"a}t Potsdam},
  year      = {2009},
  abstract  = {In dieser Arbeit werden Knickinstabilit{\"a}ten von Filamenten in biologischen Systemen untersucht. Das Zytoskelett von Zellen ist aus solchen Filamenten aufgebaut. Sie sind f{\"u}r die mechanische Stabilit{\"a}t der Zelle verantwortlich und spielen eine große Rolle bei intrazellul{\"a}ren Transportprozessen durch molekulare Motoren, die verschiedene Lasten wie beispielsweise Organellen entlang der Filamente des Zytoskeletts transportieren. Filamente sind semiflexible Polymere, deren Biegeenergie {\"a}hnlich groß ist wie die thermische Energie, so dass sie auch als elastische Balken auf der Nanoskala gesehen werden k{\"o}nnen, die signifikante thermische Fluktuationen zeigen. Wie ein makroskopischer elastischer Balken k{\"o}nnen auch Filamente eine mechanische Knickinstabilit{\"a}t unter Kompression zeigen. Im ersten Teil dieser Arbeit wird untersucht, wie diese Instabilit{\"a}t durch thermische Fluktuationen der Filamente beeinflusst wird. In Zellen k{\"o}nnen Kompressionskr{\"a}fte durch molekulare Motoren erzeugt werden. Das geschieht zum Beispiel w{\"a}hrend der Zellteilung in der mitotischen Spindel. Im zweiten Teil der Arbeit untersuchen wir, wie die stochastische Natur einer von Motoren generierten Kraft die Knickinstabilit{\"a}t von Filamenten beeinflusst. Zun{\"a}chst stellen wir kurz das Problem von Knickinstabilit{\"a}ten auf der makroskopischen Skala dar und f{\"u}hren ein Modell f{\"u}r das Knicken von Filamenten oder elastischen St{\"a}ben in zwei Raumdimensionen und in Anwesenheit thermischer Fluktuationen ein. Wir pr{\"a}sentieren eine analytische L{\"o}sung f{\"u}r Knickinstabilit{\"a}ten in Anwesenheit thermischer Fluktuationen, die auf einer Renormierungsgruppenrechnung im Rahmen des nichtlinearen Sigma-Models basiert. Wir integrieren die kurzwelligen Fluktuationen aus, um eine effektive Theorie f{\"u}r die langwelligen Moden zu erhalten, die die Knickinstabilit{\"a}t bestimmen. Wir berechnen die {\"A}nderung der kritischen Kraft f{\"u}r die Knickinstabilit{\"a}t und zeigen, dass die thermischen Fluktuationen in zwei Raumdimensionen zu einer Zunahme der kritischen Kraft f{\"u}hren. Außerdem zeigen wir, dass thermische Fluktuationen im geknickten Zustand zu einer Zunahme der mittleren projizierten L{\"a}nge des Filaments in Richtung der wirkenden Kraft f{\"u}hren. Als Funktion der Konturl{\"a}nge des Filaments besitzt die mittlere projizierte L{\"a}nge eine Spitze an der Knickinstabilit{\"a}t, die durch thermische Fluktuationen abgerundet wird. Unser Hauptresultat ist die Beobachtung, dass ein geknicktes Filament unter dem Einfluss thermischer Fluktuationen gestreckt wird, d.h. dass seine mittlere projizierte L{\"a}nge in Richtung der Kompressionskraft auf Grund der thermischen Fluktuationen zunimmt. Unsere analytischen Resultate werden durch Monte-Carlo Simulationen der Knickinstabilit{\"a}t semiflexibler Filamente in zwei Raumdimensionen best{\"a}tigt. Wir f{\"u}hren auch Monte-Carlo Simulationen in h{\"o}heren Raumdimensionen durch und zeigen, dass die Zunahme der projizierten L{\"a}nge unter dem Einfluss thermischer Fluktuationen weniger ausgepr{\"a}gt ist und stark von der Wahl der Randbedingungen abh{\"a}ngt. Im zweiten Teil der Arbeit formulieren wir ein Modell f{\"u}r die Knickinstabilit{\"a}t semiflexibler Filamente unter dem Einfluss molekularer Motoren. Wir untersuchen ein System, in dem sich eine Gruppe von Motoren entlang eines fixierten Filaments bewegt, und dabei ein zweites Filament als Last tr{\"a}gt. Das Last-Filament wird gegen eine Wand gedr{\"u}ckt und knickt. W{\"a}hrend des Knickvorgangs k{\"o}nnen die Motoren, die die Kraft auf das Filament generieren, stochastisch von dem Filament ab- und an das Filament anbinden. Wir formulieren ein stochastisches Model f{\"u}r dieses System und berechnen die "mean first passage time", d.h. die mittlere Zeit f{\"u}r den {\"U}bergang von einem Zustand, in dem alle Motoren gebundenen sind zu einem Zustand, in dem alle Motoren abgebunden sind. Dieser {\"U}bergang entspricht auch einem {\"U}bergang aus dem gebogenen zur{\"u}ck in einen ungebogenen Zustand des Last-Filaments. Unser Resultat zeigt, dass f{\"u}r gen{\"u}gend kurze Mikrotubuli die Bewegung der Motoren von der durch das Last-Filament generierten Kraft beeinflusst wird. Diese Ergebnisse k{\"o}nnen in zuk{\"u}nftigen Experimenten {\"u}berpr{\"u}ft werden.},
  language  = {en}
}