Die vorliegende Arbeit erforscht die Regulation der Expression von Drebrin; DBN (Developmentally Regulated Brain Protein) in Neuronen. DBN ist ein Protein das Actin bindet und Actin-Filamente bündeln kann. Änderungen der Morphologie der Spines verändern die synaptische Aktivität und Plastizität – wichtigen Prozessen bei der Gedächtnisbildung und Alterung des Gehirns, sowie bei geistigen Störungen bzw. Behinderungen. DBN-Expression im Alter und in einigen neurodegenerativen Krankheiten reduziert ist. Eine schwächere Expression von DBN in Spines geht außerdem mit einem Verlust an synaptischen Verbindungen einher, einem gemeinsamen Merkmal von Alterung und neurologischen Störungen wie der Alzheimer Krankheit. Diese Befunde bildeten die Motivation und Grundlage für meine Erforschung der Produktion und Lokalisierung von DBN. In meinem Projekt, habe ich den Effekt der sequenzspezifischen S647-Phosphorylierung von DBN untersucht. Die Arbeit zeigt, dass diese post-translatorische Modifikation die Stabilität von DBN reguliert. Ich habe FUNCAT-PLA und Puro-PLA für die Visualisierung von de novo synthetisierten Proteinen in situ benutzt. Mittels hochauflösender Fluoreszenz-Hybridisierung konnte ich zeigen, dass DBN nicht nur im Zellkörper sondern auch lokal in den Spines translatiert wird. Meine Resultate bieten eine Grundlage für das Verständnis der Regulierung de DBN-Konzentration in Zellen und ermöglichen die weitere Erforschung der Rolle der S647-Phosphorylierung von DBN für die Morphologie von Spines. Die Arbeit bildet außerdem eine experimentelle Plattform für weitere Studien der Rolle von DBN für Spines, sowohl in Bezug auf Stabilität als auch der synaptischen Funktion und Stabilität. / This thesis studies the abundance of the protein Drebrin; DBN (Developmentally Regulated Brain Protein) in neurons, which is an actin-binding protein capable of bundling actin filaments. Synapses in the mammalian brain are formed on tiny protrusions, called dendritic spines. Changes in spine morphology affect synaptic activity and plasticity, which are processes underlying memory formation. DBN abundance plays an important role in regulating dendritic spine morphology. Cognitive decline and neurodegenerative conditions have been shown to be linked with a decrease in DBN levels. A weakening in the expression of this protein in spines is associated with the loss of synaptic connections, a common feature of ageing and neurological disorders such as Alzheimer''s disease. This evidence was the underlying motivation for studying the localization and turnover of DBN. I studied the effect of the site-specific S647 phosphorylation of DBN and found that such post-translational modification regulates protein stability. For the project, I established several novel techniques in our laboratory, including state-of-the-art methods such as FUNCAT-PLA and Puro-PLA for the visualization of de novo synthesized proteins in situ. My results show that DBN translation occurs not only in somata but also locally in the dendrites and spines. The same observation is true for DBN transcripts, which are present both in the soma and dendrites of neurons. These observations suggest that DBN could play an important role during synaptic plasticity. My results allow the future investigation of the potential role of site-specific phosphorylation of DBN in spine morphology. This PhD thesis represents a contribution to better understanding the regulation of DBN abundance. It also provides an experimental platform for additional investigation about the role of DBN in spine morphology, regarding its stability and its correlation with synaptic maintenance and function.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

No description available.

Cell contractility is mainly imagined as a force dipole-like interaction based on actin stress fibers that pull on cellular adhesion sites. Here, we present a different type of contractility based on isotropic contractions within the actomyosin cortex. Measuring mechanosensitive cortical contractility of suspended cells among various cell lines allowed us to exclude effects caused by stress fibers. We found that epithelial cells display a higher cortical tension than mesenchymal cells, directly contrasting to stress fiber-mediated contractility. These two types of contractility can even be used to distinguish epithelial from mesenchymal cells. These findings from a single cell level correlate to the rearrangement effects of actomyosin cortices within cells assembled in multicellular aggregates. Epithelial cells form a collective contractile actin cortex surrounding multicellular aggregates and further generate a high surface tension reminiscent of tissue boundaries. Hence, we suggest this intercellular structure as to be crucial for epithelial tissue integrity. In contrast, mesenchymal cells do not form collective actomyosin cortices reducing multicellular cohesion and enabling cell escape from the aggregates.

Schaut man durch ein Mikroskop auf eine biologische Zelle mit angefärbten Zytoskelett, so erblickt man lange, mehr oder minder gerade Objekte. Mit ziemlicher Sicherheit gehören diese zu einer von drei Arten von Zytoskelettfilamenten -- Aktin- oder Mikrofilamente, Intermediärfilamente und Mikrotubuli. Schon seit mehreren Jahrzehnten versucht man die mechanischen Eigenschaften lebender Zellen nicht nur zu beschreiben, sondern ihr Verhalten von zwei tieferen Ebenen ausgehend zu verstehen: Inwiefern beschreiben die Eigenschaften von Filamentnetzwerken und -gelen die Zellmechanik und, noch tiefgreifender, wie bestimmen eigentlich die einzelnen Filamente die Netzwerkmechanik. Das Verständnis der Mechanik homogener und isotroper, verhedderter als auch quervernetzter Gele ist dabei erstaunlich detailreich, ohne jedoch vollständig dem jüngeren Verständnis von Zellen als glassartige Systeme zu entsprechen. In den letzten Jahren sind daher anisotrope Strukturen mehr und mehr in den Fokus gerückt, die die Bandbreite möglichen mechanischen Verhaltens enorm bereichern. Die vorliegende Arbeit beschäftigt sich mit solch einem hochgradig anisotropen System -- nämlich Aktinbündeln -- unter drei Gesichtspunkten. Mit Hilfe von aktiven Biegedeformationen wird ein funktionales Modul, das eine differentielle Antwort auf verschiedenen Zeitskalen liefert, identifiziert. Es handelt sich um Aktinfilamente, die durch transiente Quervernetzer gebündelt werden. Während sich das System nach kurz anhaltenden Deformation völlig elastisch verhält, sorgt eine Restrukturierung der Quervernetzer während langanhaltender Deformationen für eine plastische Verformung des Bündels. In einem weiteren Aspekt widmet sich die Arbeit der frequenz- und längenabhängigen Biegesteifigkeit. Die Methode des Bündel-Wigglings, das Induzieren von \"Seilwellen\", wird dabei genutzt, um aus der Wellenform die Biegesteifigkeit zu berechnen. Bündel von Aktinbündeln zeigen dabei ein Verhalten, das vom klassischen Worm-like-chain-Modell abweicht und stattdessen durch das Worm-like-bundle-Modell beschrieben werden kann. Der letzte Aspekt dieser Arbeit untersucht den Musterbildungsprozess bei der Entstehung von Aktinbündeln. Gänzlich unerwartet entstehen quasi-isotrope Strukturen mit langreichweitiger Ordnung, wenn der Bündelungsprozess erst nach der Polymerisation von Filamenten frei von zusätzlichen mechanischen Einwirkungen einsetzt. Da dieser Zustand nicht von der klassischen Flüssigkristalltheorie vorhergesagt wird, soll eine Simulation eine Hypothese zum Entstehungsmechanismus testen. Die Annahme einer lateralen Kondensation von Filamenten zu Bündeln reicht demnach aus, um die beobachteten Strukturen zu erzeugen. Diese Arbeit leistet somit einen Beitrag zum Verständnis hochgradig anisotroper Strukturen und deren Überstrukturen, wie sie auch in lebendigen Zellen reichlich vorhanden sind.:1 Actin filament bundles and patterns 1.1 Actin and other cytoskeletal filaments 1.2 Filament and bundle mechanics 1.2.1 Polymer models 1.2.2 Worm-like bundle theory 1.3 Filament bundling 1.4 Active crosslinkers – contraction and pattern formation 2 Materials and Methods, Instruments and Software 2.1 Actin purification and labeling 2.2 Optical tweezers 2.3 Software libraries for instrument integration 3 Bundle mechanics in the time domain 3.1 Bundle formation and sample preparation 3.2 Bundle bending experiment 3.2.1 LabView VI for bundle bending 3.2.2 Bundle bending 3.2.3 Image analysis and data survey 3.3 Bundle workout – Results of the bending experiments 3.3.1 Multiple bends and elastic response 3.3.2 Endurance test – Plastic response after longer holds 3.3.3 Purely elastic depletion-force induced bundles 3.4 Elastic and plastic deformations of F-actin bundles – Discussion 3.4.1 Bundle formation process and bundle thickness 3.4.2 Elastic response 3.4.3 Elastic versus plastic response 3.5 Differential mechanical response – Summary 4 Bundle mechanics in the frequency domain 4.1 Bundle wiggling – the method 4.2 Bundle formation and sample preparation 4.3 Bundle wiggling experiment 4.3.1 LabView VI for bundle wiggling 4.3.2 Wiggling images to wiggle data 4.4 Bundle wiggling – Results 4.4.1 Bead at end 4.4.2 Wiggling bundled bundles 4.4.3 Thick bundles connected to networks 4.5 Frequency-dependent elastic response of bundles – Discussion 5 Actin bundle networks 5.1 Actin condensation in confined environments – The experiment 5.2 Simulating filament condensation 5.2.1 Implementation details 5.3 Simulation of filament condensation to bundle networks 5.4 Condensation drives pattern formation – Discussion 6 Conclusion A Calculations A.1 Subcircular bending arc and radius of curvature A.2 Correction factor for relaxations times of bundles with bead B Protocols B.1 G-actin from rabbit skeletal muscle B.1.1 Acetone powder prep B.1.2 Actin prep B.1.3 Actin gel-filtration B.2 Buffers B.3 NEM-myosin beads B.3.1 NEM-inactivated myosin B.3.2 NEM-myosin coated beads B.4 Bundle preps B.4.1 Depletion force induced and ff-actinin crosslinked bundles B.4.2 Depletion force induced bundles for wiggling experiments Bibliography / Being the most basic unit of living organisms, the cell is a complex entity comprising thousands of different proteins. Yet only very few of which are considered to play a leading part in the cell’s mechanical integrity. The biopolymers actin, intermediate filaments and microtubules constitute the so-called cytoskeleton – a highly dynamic, constantly restructuring scaffold endowing the cell not only with integrity to sustain mechanical perturbations but also with the ability to rapidly reorganize or even drive directed motion. Actin has been regarded to be the protagonist and tremendous efforts have been made to understand passive actin networks using concepts from polymer rheology and statistical mechanics. In bottom-up approaches isotropic, homogeneous actin-gels are well-characterized with rheological methods that measure elastic and viscous properties on different time scales. Cells, however, are not exclusively isotropic networks of any of the mentioned filaments. Rather, actin alone can already be organized into heterogeneous and highly anisotropic structures like bundles. These heterogeneous structures have only come into focus recently with theoretical work addressing bundle networks. and, in the case of the worm-like bundle theory, individual bundles. This work aims at characterizing bundles and bundle-crosslinker systems mechanically in two complementary approaches – in the time as well as in the frequency domain. In addition, it illuminates a bundle formation mechanism that leads to bundle networks displaying higher ordering.:1 Actin filament bundles and patterns 1.1 Actin and other cytoskeletal filaments 1.2 Filament and bundle mechanics 1.2.1 Polymer models 1.2.2 Worm-like bundle theory 1.3 Filament bundling 1.4 Active crosslinkers – contraction and pattern formation 2 Materials and Methods, Instruments and Software 2.1 Actin purification and labeling 2.2 Optical tweezers 2.3 Software libraries for instrument integration 3 Bundle mechanics in the time domain 3.1 Bundle formation and sample preparation 3.2 Bundle bending experiment 3.2.1 LabView VI for bundle bending 3.2.2 Bundle bending 3.2.3 Image analysis and data survey 3.3 Bundle workout – Results of the bending experiments 3.3.1 Multiple bends and elastic response 3.3.2 Endurance test – Plastic response after longer holds 3.3.3 Purely elastic depletion-force induced bundles 3.4 Elastic and plastic deformations of F-actin bundles – Discussion 3.4.1 Bundle formation process and bundle thickness 3.4.2 Elastic response 3.4.3 Elastic versus plastic response 3.5 Differential mechanical response – Summary 4 Bundle mechanics in the frequency domain 4.1 Bundle wiggling – the method 4.2 Bundle formation and sample preparation 4.3 Bundle wiggling experiment 4.3.1 LabView VI for bundle wiggling 4.3.2 Wiggling images to wiggle data 4.4 Bundle wiggling – Results 4.4.1 Bead at end 4.4.2 Wiggling bundled bundles 4.4.3 Thick bundles connected to networks 4.5 Frequency-dependent elastic response of bundles – Discussion 5 Actin bundle networks 5.1 Actin condensation in confined environments – The experiment 5.2 Simulating filament condensation 5.2.1 Implementation details 5.3 Simulation of filament condensation to bundle networks 5.4 Condensation drives pattern formation – Discussion 6 Conclusion A Calculations A.1 Subcircular bending arc and radius of curvature A.2 Correction factor for relaxations times of bundles with bead B Protocols B.1 G-actin from rabbit skeletal muscle B.1.1 Acetone powder prep B.1.2 Actin prep B.1.3 Actin gel-filtration B.2 Buffers B.3 NEM-myosin beads B.3.1 NEM-inactivated myosin B.3.2 NEM-myosin coated beads B.4 Bundle preps B.4.1 Depletion force induced and ff-actinin crosslinked bundles B.4.2 Depletion force induced bundles for wiggling experiments Bibliography

<p>    </p> <p>When mammalian eggs are fertilized, they undergo an egg-to-embryo transition during which different egg activation events take place. Egg activation events include the establishment of blocks to polyspermy, which prevent multiple sperm from fertilizing an egg. One of these blocks to polyspermy occurs at the level of the egg plasma membrane (the membrane block to polyspermy). Previous work in our lab provides evidence that the mammalian membrane block to polyspermy is mediated by sperm-induced calcium signaling and the egg’s actomyosin cytoskeleton (McAvey et al., 2002). This thesis research builds upon this foundation, testing hypotheses about two specific effector molecules, one involved in calcium signaling and one with the actin cytoskeleton, and also developing the use of an actin probe for live-cell imaging, with the goal of imaging actin dynamics in eggs undergoing fertilization. Specifically, we examined the calcium effector molecule Ca2+/Calmodulin-dependent-protein kinase IIg (<strong>CaMKII</strong>g), based on previous studies showing that CaMKII plays a role in the membrane block (Gardner et al., 2007) and that the g isoform of CaMKII is necessary and sufficient for eggs to complete meiosis (Backs et al., 2010). We tested the hypothesis that CaMKIIg would mediate the membrane block to polyspermy but found that egg activation driven by expression of a constitutively active form of CaMKIIg was not sufficient to establish the membrane block. Our studies of the actin cytoskeleton focused on the Arp2/3 complex as a candidate. We tested the hypothesis that Arp2/3, which mediates actin filament branching, was involved in membrane block establishment, building on the finding that disruption of actin with the drug cytochalasin D impairs the membrane block (McAvey et al., 2022). These studies used the Arp2/3 inhibitor CK666, predicting that we would see increased sperm incorporation in CK666-treated eggs. However, an assay of sperm incorporation over time indicated that Arp2/3 may not play a significant role in the membrane block to polyspermy, although follow-up studies will be beneficial. Lastly, the actin probe SiR- Actin was assessed for use on oocytes undergoing live-cell imaging during meiosis I and II. Oocytes were treated with differing concentrations of SiR-Actin and live cell imaged while maturing through meiosis I or completing meiosis II. Higher doses and longer exposure to SiR- Actin caused abnormalities in oocytes during meiosis I but not in eggs completing meiosis II. Together, this work sets the stage of a range of future studies into the mammalian membrane block to polyspermy. </p>

Les bactéries magnétotactiques (MTB) ont la capacité de s'orienter dans un champ magnétique grâce à un organite procaryote constitué d'un nanocristal magnétique biominéralisé et entouré d'une membrane biologique : le magnétosome. La synthèse de cet organite est un processus complexe contrôlé génétiquement par une série de gènes spécifiques aux MTB (les gènes mam) qui sont regroupés sur le chromosome bactérien. Chez la souche modèle Magnetospirillum magneticum AMB-1 cet ensemble de gènes forme un îlot génomique (MAI) auquel s'ajoute un second groupe distinct de 7 gènes homologues aux gènes mam (gènes mam-like) récemment identifié dont le rôle physiologique est très peu caractérisé. Parmi les produits des gènes mam, MamK est impliqué dans l'alignement des magnétosomes. Cette « actin-like » prokaryote qui forme des filaments selon un processus ATP-dépendant a été caractérisée ces dernières années. Dans le MIS de AMB-1, un gène homologue mamK-like a été identifié. Ainsi différentes approches pluridisciplinaires ont été mises en place pour comprendre le rôle de MamK et MamK-like. L'expression des gènes du MIS a été quantifiée. Les souches dépourvues des gènes mamK et mamK-like ainsi que le double mutant ont été obtenues puis phénotypées par différentes techniques d'imagerie. Les interactions entre les deux protéines ont été également testées. Enfin, les deux protéines ont été et leurs propriétés biochimiques caractérisées. L'ensemble de ces données nous permet de proposer un modèle selon lequel MamK et MamK-like participeraient tous deux à l'alignement des magnétosomes bactériens, vraisemblablement par la formation de filaments hybrides. / Magnetotactic bacteria (MTB) have the ability to orient in a magnetic field through a prokaryotic organelle composed of a magnetic nanocrystal surrounded by a biological membrane: the magnetosome. The synthesis of this organelle is a genetically complex process controlled by a series of specific genes (mam genes) grouped together on the bacterial chromosome. In the strain model Magnetospirillum magneticum AMB-1 this set of genes form a genomic island (MAI) and a second distinct group of seven genes homologous to mam genes (mam-like genes) recently identified. The physiological role of this islet magnetosome (MIS) is very little characterized to date.Among the products of mam genes, MamK is involved in the alignment of the magnetosomes. This « actin-like » which forms prokaryote filaments according an ATP - dependent process has been characterized in recent years. In the MIS of AMB-1, a homologous gene mamK-like was identified. And various multidisciplinary approaches have been developed to understand the role of MamK and MamK-like. The MIS gene expression was quantified. The strains lacking genes of mamK, mamK-like and the obtained of double mutant were then phenotyped by different imaging techniques. The interactions between the two proteins were also tested. Finally, the two proteins were overexpressed and their biochemical properties characterized. All of these data allows us to propose a model whereby MamK and MamK-like participate in both the alignment of bacterial magnetosomes, presumably by the formation of hybrid filaments.

<p>The actin cytoskeleton is required for cell expansion and is implicated in cellular responses to the plant growth hormone auxin. However, the molecular and cellular mechanisms that coordinate auxin signaling, cytoskeletal remodeling, and cell expansion are poorly understood. Previous studies have examined actin cytoskeleton responses to long-term auxin treatment, but plants respond to auxin over short timeframes, and growth changes within minutes of exposure to the hormone. To correlate actin arrays with degree of cell expansion, we used quantitative imaging tools to establish a baseline of actin organization, as well as of individual filament behaviors in root epidermal cells under control conditions and after treatment with a known inhibitor of root growth, the auxin indole-3-acetic acid (IAA). We found that cell length was highly predictive of actin array in control roots, and that short-term IAA treatment stimulated denser, more longitudinal, and more parallel arrays by inducing filament unbundling within minutes. By demonstrating that actin filaments were more “organized” after a treatment that stopped elongation, we show there is no direct relationship between actin organization and cell expansion and refute the hypothesis that “more organized” actin universally correlates with more rapidly growing root cells. The plasma membrane-bound auxin transporter AUXIN RESISTANT 1 (AUX1) has previously been shown necessary for archetypal short-term root growth inhibition in the presence of IAA. Although AUX1 was not previously suspected of being upstream of cytoskeletal responses to IAA, we used <i>aux1</i>mutants to demonstrate that AUX1 is necessary for the full complement of actin rearrangements in response to auxin, and that cytoplasmic auxin in the form of the membrane permeable auxin 1‑naphthylacetic acid (NAA) is sufficient to stimulate a partial actin response. Together, these results are the first to quantitate actin cytoskeleton response to short-term auxin treatments and demonstrate that AUX1 is necessary for short-term actin remodeling.</p>

HSP25 ist ein Vertreter der ubiquitär verbreiteten kleinen Hitzeschockproteine, einer Familie innerhalb der großen Klasse der Streßproteine. Es ist an der Vermittlung von zellulärer Streßtoleranz beteiligt, besitzt Chaperoneigenschaften, hemmt die Actinpolymerisation in vitro und ist in der Lage, supramolekulare Komplexe auszubilden. Die hier vorgelegte Arbeit befaßte sich mit der Isolierung, der strukturellen und funktionellen Charakterisierung des Proteins und seinem Vorkommen in verschiedenen Geweben von Ratten mit pathologisch erhöhtem Blutdruck: · Es wurde eine Methode zur schonenden Isolierung von HSP25 aus Ehrlich-Ascites-Tumor (EAT) etabliert. Daraus konnte niedrig- und hochmolekulares Material gewonnen werden. · Unter Anwendung elektronenmikroskopischer und hydrodynamischer Methoden konnte für die hochmolekularen Komplexe des nativen HSP25 ein Strukturmodell abgeleitet wer-den. Es ist durch eine zylinderförmige Struktur aus vier gestapelten Ringen mit je acht HSP25-Monomeren charakterisiert. · Hochmolekulare Komplexe des rekombinanten HSP25 liegen demgegenüber als kom-pakte globuläre Strukturen vor. Elektronenmikroskopische Analysen verschiedener Mutanten und von phosphoryliertem HSP25 zeigen, daß die HSP25-Komplexe mit zu-nehmender Phosphorylierung kleiner werden. Dies belegt einen Zusammenhang zwischen dem Phosphorylierungszustand des Proteins und seiner supramolekularen Organisation. · Mittels Elektronenmikroskopie und Fluoreszenzspektroskopie konnte gezeigt werden, daß nur natives HSP25 aus EAT, aber nicht rekombinantes HSP25 oder HSP25-Mutanten die Actinpolymerisation hemmen. Dies bestätigt den Befund, daß nur unphosphorylierte nati-ve HSP25-Monomere für die Inhibierung der Actinpolymerisation verantwortlich sind. · Es konnten zwei HSP25-Peptide identifiziert werden, die eine Hemmung der Actinpoly-merisation auslösen. Damit konnte erstmals experimentell nachgewiesen werden, daß be-stimmte Sequenzabschnitte von HSP25 eine spezifische Wechselwirkung mit Actin ein-gehen. Mit Hilfe phosphorylierter HSP25-Peptide konnte die Abhängigkeit dieser Reaktion vom Phosphorylierungszustand bestätigt werden. · Vorläufige Ergebnisse mit Zellulose-gebundenen Peptid-Bibliotheken deuten auf eine Wechselwirkung von HSP25 mit einem exponierten Loop in Actin-Domäne IV hin, einem Bereich, der an Actin-Actin-Wechselwirkungen beteiligt ist. · HSP25 hat aufgrund seiner verstärkten Synthese bei vielen pathologischen Prozessen eine medizinische Relevanz. Untersuchungen an verschiedenen Tiermodellen zeigen, daß es bei Hypertonie-belasteten Herzen verstärkt im rechten Ventrikel akkumuliert wird. · Es wird ein Modell vorgestellt, in dem die Struktur-Funktions-Beziehungen für HSP25 zusammengefaßt sind. / HSP25 is a member of the ubiquitous family of small heat shock proteins belonging to the big class of stress proteins. It is related to acquiring of cellular thermotolerance, can act as molecular chaperone, is able to inhibit polymerization of actin in vitro and can form high molecular weight complexes. In this thesis the isolation, structural and functional characteri-zation of this protein as well as its abundance in different tissues of rats suffering on patho-logical forms of hypertension is analyzed: · A method for rapid isolation of HSP25 out of Ehrlich-ascites-tumor (EAT) was estab-lished. From isolated HSP25 low and high molecular weight material could be obtained. · Analysis of high molecular weight complexes by means of electron microscopy and ana-lytical ultracentrifugation results in a structural model characterized by a cylindrical structure composed of four stacked rings each containing eight HSP25 monomers. · High-molecular weight complexes of recombinant HSP25 are organized as compact globular structures. Electron microscopic investigations of different mutants and of in vi-tro phosphorylated HSP25 show a connection between phosphorylational status and su-pramolecular organization of the protein: the higher the degree of phosphorylation the smaller are the HSP35-complexes. · By means of electron microscopy and fluorescence spectroscopy it could be shown that only native HSP25 from EAT but not recombinant HSP25 nor HSP25 mutants inhibit polymerization of actin. This is in agreement with recent results showing that only un-phosphorylated native HSP25 monomers are active inhibitors of actin polymerization. · Two HSP25 derived peptides could be identified as competent inhibitors of actin polym-erization. This is the first experimental evidence for a specific interaction of HSP25 se-quences with actin. This interaction is dependent on the phosphorylational status as con-firmed by phosphorylated HSP25 peptides. · Preliminary results with cellulose bound peptide libraries indicate an interaction of HSP25 with an exposed loop in actin domain IV, an area involved in actin-actin interactions. · HSP25 is of medical relevance because of its increased synthesis in a bright variety of pathological processes. Investigations on different animal models of hypertension show an enhanced accumulation of HSP25 in the right ventricle. · A model is presented summing up the structure-function relationships of HSP25.