Spir proteins are the founding members of the novel class of WH2-actin nucleators. A C-terminal modified FYVE zinc finger motif is necessary to target Spir proteins towards intracellular membranes. The function and regulation of the Spir actin organizers at vesicular membranes is almost unknown. Live cell imaging analyses performed in this study show that Spir-2 is localized at tubular vesicles. Cytoplasmic Spir-2-associated vesicles branch and form protrusions, which can make contacts to the microtubule network, where the Spir-2 vesicles stretch and slide along the microtubule filaments. The analysis of living HeLa cells expressing eGFP-tagged Spir-2, Spir-2-ΔKIND and Spir-2-ΔKW (lacking the 4 WH2 domains and the KIND domain) showed Spir-2-associated tubular structures which differ in their length and motility. Throughout the course of that study it could be shown that the tail domain of the actin motor protein myosin Vb, as a force-generating molecule, is colocalizing and co-immunoprecipitating with Spir-2-ΔKW. By using the tail domain of myosin Vb as a dominant negative mutant for myosin Vb-dependent vesicle transport processes it could be shown that Spir-2-ΔKW/MyoVb-cc-tail- associated vesicles exhibit an increased elongation. Moreover, using the microtubule depolymerizing drug nocodazole it could be shown that the elongation and the motility of Spir-2-ΔKW-associated vesicles depends on an intact microtubule cytoskeleton. Motility and morphological dynamics of Spir-2-associated vesicles is therefore dependent on actin, actin motorproteins and microtubule filaments. These results propose a model in which myosin/F-actin forces mediate vesicle branching, allowing the vesicles to move to and in between the microtubule filaments and thereby providing a new degree of freedom in vesicular motility. To determine the exact subcellular localization of Spir-2, colocalization studies were performed. It could be shown that Spir-2 shows a partial colocalization to Rab11a-positive compartments. Furthermore, Spir-2 exhibits an almost identical localization to Arf1 and the Arf1 small G protein but not Rab11a could be immunoprecipitated with Spir-2-ΔKW. This suggests, that Arf1 recruits Spir-2 to Arf1/Rab11a-positive membranes. Another important function of the Spir-2 C-terminus is the membrane targeting by the FYVE domain. By performing a protein-lipid overlay assay, it has been shown that purified GST- and 6xHis-tagged Spir-2-ΔKW bind phosphatidic acid suggesting a mechanism in which Spir-2 is recruited to phosphatidic acid-enriched membranes. To further elucidate the mechanism in which Spir-2 membrane-targeting could be regulated, interaction studies of C-terminal parts of Spir-2 revealed that the Spir-2 proteins interact directly. / Spir Proteine sind die ersten beschriebenen Mitglieder der neuen Klasse der WH2-Aktin Nukleatoren. Ein C-terminaler modifizierter FYVE Zinkfinger ist notwendig um Spir Proteine an intrazelluläre Membranen zu bringen. Die Funktion und die Regulation dieser Aktin Nukleatoren an vesikulären Membranen ist bis jetzt noch nahezu unbekannt. In dieser Studie durchgeführte “Live-cell-Imaging” Experimente zeigten, dass Spir-2 an tubulären Vesikeln lokalisiert ist. Zytoplasmatische Spir-2-assoziierte Vesikel formen Ausläufer, die Kontakte zum Mikrotubuli Netzwerk bilden. Spir-2 Vesikel haben die Fähigkeit sich entlang des Mikrotubuli Zytoskeletts auszudehnen und daran entlang zu gleiten. Die Analyse von lebenden HeLa Zellen, welche eGFP-Spir-2, eGFP-Spir-2-ΔKIND und eGFP-Spir-2-ΔKW (Deletion der 4 WH2 Domänen sowie der KIND Domäne) Fusionsproteine exprimieren, zeigen Spir-2-assoziierte tubuläre Vesikel, die sich in Länge und Beweglichkeit unterscheiden. Während dieser Studie konnte außerdem gezeigt werden, dass die “tail” Domäne des Aktinmotors myosin Vb mit Spir-2-ΔKW kolokalisiert und koimmunopräzipitiert. Die Verwendung der “tail” Domäne als dominant negative Mutante für myosin Vb-abhängigen Vesikeltransport zeigte, dass Spir-2-ΔKW/MyoVb-cc-tail-assoziierte Vesikel eine stark erhöhte Elongation aufweisen. Desweiteren konnte duch die Verwendung von Nocodazol, welches spezifisch Mikrotubulifilamente depolymerisiert, gezeigt werden, dass die Elongation und die Motilität der Spir-2-ΔKW-assoziierten Vesikel von einem intakten Mikrotubuli Zytoskelett abhängig ist. Motilität und morphologische Dynamik der Spir-2-ΔKW-assoziierten Vesikel ist daher abhängig von Aktinfilamenten, Aktin Motorproteinen und Mikrotubulifilamenten. Anhand dieser Ergebnisse lässt sich ein Modell erstellen, in welchem eine Myosin/F-actin induzierte Bewegung eine Verzweigung der Vesikel bewirkt. Dadurch ist eine Bewegung der Vesikel zu Mikrotubulifilamenten aber auch zwischen verschiedenen Mikrotubulifilamenten möglich, welches einen ganz neuen Freiheitsgrad in der vesikulären Bewegung eröffnet. Um die genaue zelluläre Lokalisation von Spir-2 zu analysieren wurden Kolokalisationsstudien durchgeführt. Hierbei konnte gezeigt werden, dass Spir-2 eine partielle Kolokalisation mit Rab11a-positiven Kompartimenten zeigt. Außerdem weist Spir-2 eine nahezu identische Lokalisation zu Arf1 auf. Arf1, aber nicht Rab11a, konnte mit Spir-2-ΔKW koimmunpräzipitiert werden. Arf1 könnte daher für die Rekrutierung von Spir-2 an Arf1/Rab11a-positive Membranen ausschlaggebend sein. Eine weitere wichtige Funktion des Spir-2 C-Terminus ist die Membranlokalisation, welche durch die FYVE Domäne vermittelt wird. Mittels Protein-Lipid Bindungsstudien konnte gezeigt werden, dass aufgereinigte GST- bzw. 6xHis-Spir-2-ΔKW-Fusionsproteine an Phosphatidylsäure binden. Dies deutet darauf hin, dass Spir-2 spezifisch zu Phosphatidylsäure-positiven Membranen rekrutiert wird. Um die weitere Regulation der Spir-2 Membranlokalisation aufzuklären, wurden Protein-Protein-Interaktionsstudien durchgeführt, welche eine direkte Interaktion von Spir-2 Proteinen anhand ihrer C-Termini ergaben.

WASH complex regulates actin dynamics on endosomes by activating the Arp2/3 complex, which subsequently induces generation of branched actin patches. WASH complex is required for proper recycling of many important transmembrane proteins. Although the general physiological function of WASH complex is known, the role of its single subunits have not yet been adequately specified. This work focuses on one of these subunits - protein SWIP. This protein maintains vesicular localization of some WASH complex subunits in the slime mold Dictyostelium discoideum and a point mutation in its sequence causes a severe neurodegenerative disease - autosomal recessive intellectual disorder (ARID). Our results show that SWIP truncation results in its inability to incorporate into WASH complex. However, the C-terminal part of SWIP is able to localize onto intracellular vesicles, which are not WASH complex positive. We have also studied the impact of ARID-causing SWIP mutation, and we show, that it does neither change the protein's ability to bind the complex nor the overall localization of WASH complex.

Microtubular cytoskeleton is involved in many processes in plant cells, including cell division, growth and development. Other proteins enable its functions by modulation of its dynamics and organization and by mediation of functional and structural interaction with other cell structures. Identification of the mediating proteins and the functions of these interactions under specific conditions were the main aims of the thesis. Membrane proteins interacting with microtubules were identified using biochemical methods. Surprisingly, the identified proteins co-sedimenting with microtubules were not members of the "classical" microtubule associated proteins (MAPs). There were enzymes, chaperones and plant specific proteins among them. For further studies, the identified microtubule-associated heat-shock protein 90 (Hsp90_MT) was chosen. Recombinant Hsp90_MT binds directly to microtubules and tubulin dimers in vitro. The ATP-binding pocket is not responsible for this association. In BY-2, Hsp90_MT co-localizes with phragmoplast and cortical microtubules and is involved in microtubule recovery after their depolymerization during cold treatment. In plants, Hsp90 is involved in cell cycle progression, its inhibition causes cell-cycle arrest in G1 phase. Based on literature search for animal proteins...

Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...

2 Abstract ARP2/3 protein complex is formed from seven proteins (ARP2, ARP3 and ARC1- ARPC5) with a relatively conserved structure. ARP2/3 complex branches and nucleates new actin filaments. This thesis focuses on the study of the role and importance of the individual subunits of the complex ARP2/3 in plants. One of the principal aims of this work is to determine whether complex ARP2/3 may at least partially maintain its role when one or more of the subunits are not available. Furthermore if the individual subunits play another, plant-specific role and if the subunits are functionally equivalent in the complex. The main way how to achieve this objective is the analysis of multiple mutants of Arabidopsis thaliana in subunits of ARP2/3 complex. After comparing several phenotypes of mutant lines it is obvious that all the subunits are functionally equivalent. A loss of ARPC5 subunit usually manifests the strongest phenotypic expression. On the contrary, loss ARPC3 and ARPC2b subunits have weak phenotypic manifestations. Because some phenotypes, such as phenotype distorted trichomes was detected only in some mutant lines, whereas the phenotype of faster roots gravitropic response or vacuolar system fragmentation that was detected in all analyzed mutants suggests, that different subunits play varying roles...

In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...

(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...

eIF5A seems to be involved in both, translation initiation and elongation. It was also reported to affect assembly of P-bodies. Given similarities of P-bodies with stress granules (SGs) we decided to test the role of eIF5A in dynamics of heat-induced SGs and its implications for the cell recovery. For the evaluation of eIF5A function in SGs formation was used the temperature- sensitive (ts) mutant eIF5A-3 (C39Y/G118D) cultivated under permissive temperature 25řC and Rpg1-GFP fusion protein as a marker of SGs. The cells were exposed to robust heat shock at 46řC for 10 minutes. The ability of the mutant cells to recover was tested by propidium iodine staining and colony forming units plating. We found that the eIF5A-3 mutant forms heat-induced SGs more loosely aggregated, indicating that the fully functional eIF5A is necessary for SGs assembly. However, it does not seem to affect the rate of SGs dissolution. Survival tests indicate that eIF5A-3 mutant cells are susceptible to dying in a similar way as WT cells; nevertheless, their ability to resume proliferation is significantly better. We also observed a loss of the ts phenotype of the eIF5A-3 mutant. This loss cannot be explained by reversion of mutated eIF5A sequence into normal. Probable cause lies in the adaptive evolution. Our results indicate role of...

Sensory-motile cells fulfill various biological functions ranging from immune activity or wound healing to the formation of the highly complex nervous systems of vertebrates. In the case of neurons, a dynamic structure at the tip of outgrowing processes navigates towards target cells or areas during the generation of neural networks. These fan shaped growth cones are equipped with a highly complex molecular machinery able to detect various external stimuli and to translate them into directed motion. Receptor and adhesion molecules trigger signaling cascades that regulate the dynamics of an internal polymeric scaffold, the cytoskeleton. It plays a crucial role in morphology maintenance as well as in the generation and distribution of growth cone forces. The two major components, actin and microtubules (MTs) connect on multiple levels through interwoven biochemical and mechanical interactions. Actin monomers assemble into semiflexible filaments (F-actin) which in turn are either arranged in entangled networks in the flat outer region of the growth cone (lamellipodium) or in radial bundles termed filopodia. The dynamic network of actin filaments extends through polymerization at the front edge of the lamellipodium and is simultaneously moving towards the center (C-domain) of the growth cone. This retrograde flow (RF) of the actin network is driven by the polymerizing filaments themselves pushing against the cell membrane and the contractile activity of motor proteins (myosins), mainly in the more central transition zone (T-zone). Through transmembrane adhesion molecules, a fraction of the retrograde flow forces is mechanically transmitted to the cellular substrate in a clutch-like mechanism generating traction and moving the GC forward. MTs are tubular polymeric structures assembled from two types of tubulin protein subunits. They are densely bundled in the neurite and at the growth cone “neck” (where the neurite opens out into the growth cone) they splay apart entering the C-domain and more peripheral regions (P-domain). Their advancement is driven by polymerization and dynein motor protein activity. The two subsystems, an extending array of MTs and the centripetal moving actin network are antagonistic players regulating GC morphology and motility. Numerous experimental findings suggest that MTs pushing from the rear interact with actin structures and contribute to GC advancement. Nevertheless, the amount of force generated or transmitted through these rigid structures has not been investigated yet. In the present dissertation, the deformation of MTs under the influence of intracellular load is analyzed with fluorescence microscopy techniques to estimate these forces. RF mechanically couples to MTs in the GC periphery through friction and molecular cross-linkers. This leads to MT buckling which in turn allows the calculation of the underlying force. It turns out that forces of at least act on individual MT filaments in the GC periphery. Compared to the relatively low overall protrusion force of neuronal GCs, this is a substantial contribution. Interestingly, two populations of MTs buckle under different loads suggesting different buckling conditions. These could be ascribed to either the length-dependent flexural rigidity of MTs or local variations in the mechanical properties of the lamellipodial actin network. Furthermore, the relation between MT deformation levels and GC morphology and advancement was investigated. A clear trend evolves that links higher MT deformation in certain areas to their advancement. Interactions between RF and MTs also influence flow velocity and MT deformation. It is shown that transient RF bursts are related to higher MT deformation in the same region. An internal molecular clutch mechanism is proposed that links MT deformation to GC advancement. When focusing on GC dynamics it is often neglected that the retraction of neurites and the controlled collapse of GCs are as important for proper neural network formation as oriented outgrowth. Since erroneous connections can cause equally severe malfunctions as missing ones, the pruning of aberrant processes or the transient stalling of outgrowth at pivotal locations are common events in neuronal growth. To date, mainly short term pausing with minor cytoskeletal rearrangements or the full detachment and retraction of neurite segments were described. It is likely that these two variants do not cover the full range of possible events during neuronal pathfinding and that pausing on intermediate time scales is an appropriate means to avoid the misdetection of faint or ambiguous external signals. In the NG108-15 neuroblastoma cells investigated here, a novel type of collapse was observed. It is characterized by the degradation of actin network structures in the periphery while radial filopodia and the C-domain persist. Actin bundles in filopodia are segmented at one or multiple breaking points and subsequently fold onto the edge of the C-domain where they form an actin-rich barrier blocking MT extension. Due to this characteristic, this type of collapse was termed fold collapse. Possible molecular players responsible for this remarkable process are discussed. Throughout fold collapse, GC C-domain area and position remain stable and only the turnover of peripheral actin structures is abolished. At the same time, MT driven neurite elongation is hindered, causing the GC to stall on a time scale of several to tens of minutes. In many cases, new lamellipodial structures emerge after some time, indicating the transient nature of this collapse variant. From the detailed description of the cytoskeletal dynamics during collapse a working model including substrate contacts and contractile actin-myosin activity is derived. Within this model, the known and newly found types of GC collapse and retraction can be reduced to variations in local adhesion and motor protein activity. Altogether the results of this work indicate a more prominent role of forward directed MT-based forces in neuronal growth than previously assumed. Their regulation and distribution during outgrowth has significant impact on neurite orientation and advancement. The deformation of MT filaments is closely related to retrograde actin flow which in turn is a regulator of edge protrusion. For the stalling of GCs it is not only required that actin dynamics are decoupled from the environment but also that MT pushing is suppressed. In the case of fold collapse, this is achieved through a robust barrier assembled from filopodial actin bundles.

We study the influence of finite shear deformations on the microstructure and rheology of solutions of entangled semiflexible polymers theoretically and by numerical simulations and experiments with filamentous actin. Based on the tube model of semiflexible polymers, we predict that large finite shear deformations strongly affect the average tube width and curvature, thereby exciting considerable restoring stresses. In contrast, the associated shear alignment is moderate, with little impact on the average tube parameters, and thus expected to be long-lived and detectable after cessation of shear. Similarly, topologically preserved hairpin configurations are predicted to leave a long-lived fingerprint in the shape of the distributions of tube widths and curvatures. Our numerical and experimental data support the theory.