Neben der Aufgabe, die „Hand-Made“ Klonierungstechnik (HMCTM) erfolgreich in unserem Labor einzuarbeiten, wurden Untersuchungen zur Entwicklungskompetenz und Lebensfähigkeit von Embryonen aus HMCTM durchgeführt. Dabei wurden verschiedene somatische Zellen als Kernspender verwendeten und die Verteilungsmuster von Zytoskelettproteinen (a-Tubulin und F-Actin), die Eigenschaften interzellulärer Kontakte sowie das Auftreten des „Gap-Junction-Proteins Connexin 43 zwischen HMCTM-Embryonen als auch mittels IVP und in vivo produzierte Embryonen verglichen. Die durchschnittliche Gesamteffizienz des Verfahrens lag bei 95% rekonstruierter Embryonen mit 65% mittlerer Teilungsrate. Keine signifikanten Unterschiede wurden zwischen den drei Gruppen von somatischen Zellen in bezug auf Embryorekonstruktionseffizienz, Teilungsrate, Anzahl von >8 Zell Embryonen und Blastozystenrate beobachtet. Von den erzeugten Embryonen erreichten nur 2% das Blastozystenstadium. Viele Embryonen verharrten arretiert zwischen dem 8 und 16 Zell Stadium, was dem Zeitpunkt der Umschaltung vom maternalen auf das embryonale Genom entspricht (TELFORD et al. 1990; MEMILI et al. 1998). Wir erzeugten insgesamt 67 übertragbare Blastozysten von denen 36 auf synchrone Rezipienten transferiert wurden. Die erzeugten 6 Trächtigkeiten entsprechen einer Rate von 17%. Funf Trächtigkeiten gingen verloren und eine führte zur Geburt eines Bullenkalbes - des ersten HMCTM Kalbes in Europa. Die Etablierung der HMCTM Technik in unserem Laboratorium kann damit als erfolgreich betrachtet werden. Die höchste Intensität der a-Tubulinfärbung wurde in klonierten und IVP-Embryonen (ohne signifikante Unterschiede) festgestellt. Im Unterschied dazu war sie in in vivo erzeugten Embryonen signifikant geringer, was auf die möglicherweise ungünstigen Einflüsse der In-vitro-Kulturbedingungen hinweist. Für Actin wurde eine höhere Intensität in in vivo- und IVP-Embryonen (ohne signifikante Unterschiede) festgestellt, während sie in klonierten Embryonen signifikant niedriger war. Das weist auf einen negativen Effekt der Zona pellucida freien HMCTM Methode hin, die für den Kerntransfer verwendet wurde. Spekulativ können die Abweichungen, die in der Verteilung der Zytoskelettproteine beobachtet wurden, als ein Anzeichen für den Entwicklungsblock in den betroffenen Rinderembryonen betrachtet werden (MATSUMOTO et al. 2002). Untersuchungen mittels Western-Blot ergaben, dass in IVP-Embryonen vom 4-Zellstadium bis zum Morulastadium sowohl Connexin-43 als auch a-Tubulin ohne quantitative Unterschiede nachweisbar waren. 8-Zellembryonen aus HMCTM, IVP und in vivo Produktion im Vergleich, zeigten ebenfalls keine quantitativen Unterschiede bezüglich Connexin-43 und a-Tubulin. Diese Ergebnisse entsprechen einer früheren Hypothese das Cx43 Transkripte in in vitro produzierten Rinderembryonen maternalen und embryonalen Ursprungs sein können (WRENZYCKI et al. 1996). Die Ultrastrukturanalyse von 8-Zellembryonen zeigte dass die interzellularen Kontakte zwischen den Blastomeren in bezug auf ihre Länge in HMCTM Embryonen die kürzesten Kontaktzonen (CP) und in in vivo produzierten Embryonen die längsten CP aufwiesen. Während der Abwesenheit von Gap-Junctions (GJ) in den früheren Stufen der Embryogenese können diese CPs eine entscheidende Rolle beim Transfer von Metaboliten zwischen den Zellen und bei der interzellulären Kommunikation bis zum späten Morula- oder frühen Blastozystenstadium, wenn die GJ Bildung einsetzt, übernehmen. Die kürzere CP Länge kann deshalb einer der Gründe für die relativ schlechtere Entwicklungsrate der klonierten Embryonen sein. Detailliertere Untersuchungen zur Bedeutung der Länge der CPs' und ihren funktionellen Aspekten werden zukünftig nötig, um eine genauere Interpretation zu ermöglichen. / Apart from the aim of successfully establishing the Handmade cloning (HMCTM) technique in our laboratory, investigations were conducted to study the developmental competence and viability of the HMCTM -derived embryos while using different somatic cells, and to compare the distribution pattern of cytoskeletal proteins (alpha-tubulin and F-actin) and the nature of cellular contacts, as also the presence of Connexin 43 (gap junction protein) between HMCTM cloned, IVP and in vivo produced embryos. We obtained a 95 % overall average efficiency of formation of the reconstructed embryos with a 65 % average cleavage rate. No significant differences were observed between the three groups of somatic cell donors in terms of embryo reconstruction efficiency, cleavage rate, number of > eight cell stage embryos, and the blastocyst rates. Of the fused couplets, only 2 % reached the blastocyst stage, with many of the embryos arresting between the 8- to 16-cell stage, a stage corresponding to the timing of maternal to embryonic genomic take-over of development in bovine (TELFORD et al. 1990; MEMILI et al. 1998). We also recovered a total of 67 transferable blastocysts, out of which 36 blastocysts were transferred to 36 synchronized recipients resulting in six pregnancies, thereby yielding a 17 % pregnancy rate. Five pregnancies were lost from abortions and one resulted in the birth of a bull calf - the first HMCTM calf born in Europe. The establishment of the HMCTM technique in our lab can thus be regarded as successful. The maximum intensity of tubulin was observed in the cloned and the IVP embryos (with no significant differences) whereas it was significantly lower in the in vivo embryos indicating the adverse effects of in vitro culture conditions. In the case of actin, a higher intensity of staining was observed in the case of in vivo and IVP embryos (with no significant differences) whereas the same was significantly lower in the cloned embryos thereby reflecting the adverse effects of the zona-free technique used in the HMCTM method of SCNT. Speculatively, the deviations/variations observed in the distribution of the cytoskeletal proteins may be an indicator of the developmental arrest in the bovine embryos concerned (MATSUMOTO et al. 2002). Western blot analysis revealed that in the IVP embryos, from 4-cell stage up to the morula stage, both connexin-43 and tubulin were present with no quantitative differences. The 8-cell stage embryos collected from HMCTM cloning, IVP and in vivo production also showed presence of connexin-43 and tubulin, yet again, with no quantitative differences. The results could conform to an earlier hypothesis that Cx43 transcripts in bovine embryos produced in vitro might be of maternal and embryonic origin (WRENZYCKI et al. 1996). Ultrastructural analysis of the 8-cell stage embryos showed that the intercellular contacts between the blastomeres were, in terms of length, of the shortest order in case of the HMCTM cloned embryos whereas these Contact Point (CP) lengths were of the greatest order in vivo produced embryos. In the absence of gap junctions (GJ) in the earlier stages of embryogenesis, these CPs may play a crucial role in the exchange of metabolites and cell-to-cell communication till the late morula or early blastocyst stage when the GJ formation occurs. The shorter CP length may, therefore, be one of the reasons for the relative poor development rate of the cloned embryos. Detailed investigations of the significance of the length of the CPs and their functional aspects would be required in future to facilitate meaningful interpretation.

Pemphigus vulgaris (PV) ist eine blasenbildende Autoimmunerkrankung der Haut. Ein wesentliches Charakteristikum der Erkrankung sind Autoantikörper, welche gegen die humanen Zell-Adhäsionsmoleküle Desmoglein (Dsg) 3 und 1 gerichtet sind und zu zunehmender Zell-Dissoziation der Keratinozyten führen (Akantholyse). Neben der Dsg3-Reorganisation sind zytoskelettale Veränderungen in Form einer ZK-Retraktion und einer Reorganisation des Actin-Zytoskeletts als ein wichtiges Merkmal akantholytischer Zellen beschrieben worden. Dennoch ist der zeitliche Verlauf und die funktionelle Relevanz dieser zytoskelettalen Veränderungen im Vergleich zu anderen Prozessen, wie der Dsg3-Reorganisation oder der Zell-Dissoziation, unklar. In dieser Arbeit wurde daher die Rolle der ZK-Filamente und der Actinfilamente für die PV-Pathogenese untersucht. Inkubation von kultivierten Keratinozyten mit PV-IgG resultierte in einer ZK-Retraktion, welche eng mit dem Beginn der Dsg3-Reorganisation und der Zell-Dissoziation korrelierte. Weiterhin fand sich eine Abhängigkeit der PV-IgG-induzierten ZK-Retraktion und der Zell-Dissoziation von der p38MAPK-Signalkaskade, während die Beteiligung der p38MAPK an der Dsg3-Reorganisation von untergeordneter Rolle zu sein scheint. Übereinstimmend dazu führte eine Überexpression von E-Cadherin zu einer Hemmung der p38MAPK-Aktivierung, der ZK-Retraktion und der Zell-Dissoziation, so dass den Cadherinen eine übergeordnete Rolle in der Vermittlung der PV-Pathogenese zuzukommen scheint. Neben einer ZK-Retraktion zeigten die Zellen als Reaktion auf eine Inkubation mit PV-IgG auch wesentliche Reorganisationen der Actinfilamente, welche ebenfalls eng mit der Dsg3-Reorganisation und der Zell-Dissoziation korrelierten. Darüber hinaus interferierte die pharmakologische Modulation des Actin-Zytoskeletts mit den PV-IgG-Effekten. So führte eine Stabilisierung der Actinfilamente zu einer Reduktion sowohl der Dsg3-Reorganisation als auch der Zell-Dissoziation, während eine Zerstörung der Filamente die Effekte verstärkte. Zur Unterstützung dieser Ergebnisse wurde die Rolle des Actins für die durch Rho-GTPasen vermittelte Hemmung von PV-IgG-Effekten untersucht. Eine Aktivierung der Rho-GTPasen führte neben einer Hemmung PV-IgG-vermittelter Effekte auch zu einer Verstärkung des kortikalen Actin-Rings, während eine Hemmung der Actin-Polymerisation die protektiven Effekte der Rho-GTPasen-Aktivierung aufheben konnte. Zusammenfassend lässt sich sagen, dass die Ergebnisse dieser Arbeit eine übergeordnete Rolle sowohl der desmosomalen als auch der klassischen Cadherine für die PV-Pathogenese zeigen. Daneben scheint auch der Actin-Reorganisation eine wesentliche Position zuzukommen. Die ZK-Retraktion hingegen scheint, zumindest im Bezug auf die Dsg3-Reorganisation, sekundär zu sein, trägt aber möglicherweise im Anschluss an eine p38MAPK-Aktivierung wesentlich zum Verlust der Zell-Zell-Adhäsion bei. / In human autoimmune blistering skin disease pemphigus vulgaris (PV) autoantibodies are mainly directed against keratinocyte cell adhesion molecules desmoglein (Dsg) 3 and 1 and cause keratinocyte cell dissociation (acantholysis). Early ultrastructural work revealed cytokeratin (CK) retraction to be a characteristic hallmark of acantholytic keratinocytes and recent studies reported profound alterations of the actin cytoskeleton. Nevertheless, the temporal sequence and relevance of these cytoskeletal phenomena in pemphigus pathogenesis compared to other events such as Dsg3 reorganisation or keratinocyte dissociation are only poorly understood. We examined roles of CK and actin filaments in PV-IgG-mediated keratinocyte dissociation. Incubation of cells with PV-IgG resulted in a CK retraction which closely correlated with the onset of cell dissociation and Dsg3 reorganisation. Both, PV-IgG-induced CK retraction and cell dissociation were found to be p38MAPK-dependent whereas the contribution of p38MAPK activation for Dsg3 reorganisation seemed to be secondary. According to this, overexpression of E-cadherin prevented PV-IgG-induced p38MAPK activation, cell dissociation and CK retraction. Therefore cadherins seem to have a primary role for PV pathogenesis. Parallel to CK retraction, PV-IgG treatment resulted in striking changes in actin cytoskeleton organization which also closely correlated with cell dissociation and Dsg3 reorganisation.Therefore, we investigated whether pharmacologic manipulation of actin polymerization modulates pathogenic effects of PV-IgG. Pharmacological stabilization of actin filaments significantly blocked cell dissociation and Dsg3 fragmentation whereas actin depolymerisation strongly enhanced pathogenic effects of PV-IgG. To substantiate these findings, we studied whether the protective effects of Rho GTPases, which are potent regulators of the actin cytoskeleton and were shown to be involved in pemphigus pathogenesis, were dependent on modulation of actin dynamics. Activation of Rho-GTPases enhanced the cortical junction-associated actin belt and blunted PV-IgG-induced cell dissociation. However, when actin polymerization was blocked under these conditions the protective effects of Rho-GTPase activation were abrogated. Taken together, these experiments indicate a primary role of both desmosomal and classical cadherins for PV pathogenesis. Furthermore actin reorganization seems to be critical for PV-IgG-induced acantholysis. CK retraction may contribute to p38MAPK-dependent keratinocyte dissociation in pemphigus but appears to be secondary, at least to Dsg3 reorganisation.

Cellulose is the most abundant biopolymer on earth and the main load-bearing structure in plant cell walls. Cellulose microfibrils are laid down in a tight parallel array, surrounding plant cells like a corset. Orientation of microfibrils determines the direction of growth by directing turgor pressure to points of expansion (Somerville et al., 2004). Hence, cellulose deficient mutants usually show cell and organ swelling due to disturbed anisotropic cell expansion (reviewed in Endler and Persson, 2011). How do cellulose microfibrils gain their parallel orientation? First experiments in the 1960s suggested, that cortical microtubules aid the cellulose synthases on their way around the cell (Green, 1962; Ledbetter and Porter, 1963). This was proofed in 2006 through life cell imaging (Paredez et al., 2006). However, how this guidance was facilitated, remained unknown. Through a combinatory approach, including forward and reverse genetics together with advanced co-expression analysis, we identified pom2 as a cellulose deficient mutant. Map- based cloning revealed that the gene locus of POM2 corresponded to CELLULOSE SYNTHASE INTERACTING 1 (CSI1). Intriguingly, we previously found the CSI1 protein to interact with the putative cytosolic part of the primary cellulose synthases in a yeast-two-hybrid screen (Gu et al., 2010). Exhaustive cell biological analysis of the POM2/CSI1 protein allowed to determine its cellular function. Using spinning disc confocal microscopy, we could show that in the absence of POM2/CSI1, cellulose synthase complexes lose their microtubule-dependent trajectories in the plasma membrane. The loss of POM2/CSI1, however does not influence microtubule- dependent delivery of cellulose synthases (Bringmann et al., 2012). Consequently, POM2/CSI1 acts as a bridging protein between active cellulose synthases and cortical microtubules. This thesis summarizes three publications of the author, regarding the identification of proteins that connect cellulose synthases to the cytoskeleton. This involves the development of bioinformatics tools allowing candidate gene prediction through co-expression studies (Mutwil et al., 2009), identification of candidate genes through interaction studies (Gu et al., 2010), and determination of the cellular function of the candidate gene (Bringmann et al., 2012). / Zellulose ist das abundanteste Biopolymer der Erde und verleiht pflanzlichen Zellwänden ihre enorme Tragkraft. Mit der Reißfestigkeit von Stahl umwickeln Zellulosefibrillen pflanzliche Zellwände wie ein Korsett. Die Orientierung der Zellulosefibrillen bestimmt zugleich die Wachstumsrichtung, indem sie den Zellinnendruck (Turgor) in die entsprechende Ausdehnungsrichtung dirigiert (Somerville et al.,2004).Folglich zeigen Mutanten mit gestörter Zellulosesynthese oft geschwollene Organe und Zellen, die sich nicht mehr gerichtet ausdehnen können (zusammengefasst von Endler und Persson,2011). Wie aber erhalten die Zellulosefibrillen ihre parallele Orientierung? Erste Experimente aus den1960ern führten zur Vermutung, kortikale Mikrotubuli leiten die Zellulosesynthasen auf ringförmigen Bahnen um die Zellen herum (Green, 1962; Ledbetter and Porter, 1963). Diese Theorie wurde 2006 mit Hilfe moderner mikroskopischer Methoden bestätigt (Paredez et al., 2006). Wie jedoch dieser Leitmechanismus funktioniert, blieb bisher unentdeckt. Durch die Kombination verschiedener genetischer und bioinformatischer Methoden, konnten wir pom2 als Zellulose defiziente Mutante identifizieren. Die Ermittlung des Genlocus durch Map-based cloning zeigte, dass es sich bei POM2 um CELLULOSE SYNTHASE INTERACTING 1 (CSI1) handelt, ein Gen, dessen korrespondierendes Protein, wie vorher von uns gezeigt, mit dem zytosolischen Teil der primären Zellulosesynthasen interagiert (Gu et al., 2010). Durch ausführliche zellbiologische Charakterisierung von POM2/CSI1 konnten wir seine zelluläre Funktion entschlüsseln. Mit Hilfe konfokaler Spinning- Disc-Mikroskopie konnten wir zeigen, dass in Abwesenheit von POM2/CSI1, Zellulosesynthasen von den Mikrotubuli- Bahnen abweichen. Der ebenfalls von den Mikrotubuli abhängige Transport der Zellulosesynthasen zur Zellmembran hingegen, war nicht beeinflusst (Bringmann et al., 2012). Demzufolge ist POM2/CSI1 das gesuchte Bindeglied zwischen aktiven Zellulosesynthasen und Mikrotubuli. In dieser Dissertationsschrift werden drei Publikationen des Autors zusammengefasst, die wa ̈hrend der Arbeit an der Dissertiation entstanden sind. Sie beinhalten die Entwicklung bioinformatischer Methoden zur Ko- Expressionsanalyse, um Kandidatengene zu ermitteln (Mutwil et al., 2009), die Identifikaton des Kandidatengens POM2/CSI1 in einer Interaktionsstudie (Gu et al., 2010), sowie die Bestimmung der zellula ̈ren Funktion des korrespondieren- den Proteins POM2/CSI1 (Bringmann et al., 2012).

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.

Microtubule associated molecular motors are involved in a multitude of fundamental cellular processes such as intracellular transport and spindle positioning. During these movements multiple motor proteins often work together and are, therefore, able to exert high forces. Thus force generation and sensing are common mechanisms for controlling motor driven movement. These mechanisms play a pivotal role when motor proteins antagonize each other, e.g. to facilitate oscillations of the spindle or the nucleus. Single motor proteins have been characterized in depth over the last two decades, our understanding of the collective behavior of molecular motors remains, however, poor. Since motor proteins often cooperate while they walk along microtubules, it is necessary to describe their collective reaction to a load quantitatively in order to understand the mechanism of many motor-driven processes. I studied the antagonistic action of many molecular motors (of one kind) in a gliding geometry. For this purpose I crosslinked two microtubules in an antiparallel fashion, so that they formed \"doublets\". Then I observed the gliding motility of these antiparallel doublets and analyzed the gliding velocity with respect to the relative number of motors pulling or pushing against each other. I observed that the antiparallel doublets gliding on conventional kinesin-1 (from Drosophila melanogaster) as well as cytoplasmic dynein (from Saccharomyces cerevisae) exhibited two distinct modes of movement, slow and fast, which were well separated. Furthermore I found a bistability, meaning, that both kinds of movement, slow and fast, occurred at the same ratio of antagonizing motors. Antiparallel doublets gliding on the non-processive motor protein Ncd (the kinesin-14 from D. melanogaster) showed, however, no bistability. The collective dynamics of all three motor proteins were described with a quantitative theory based on single-motor properties. Furthermore the response of multiple dynein motors towards an external, well-defined load was measured in a gliding geometry by magnetic tweezing. Examples of multi-motor force-velocity relationships are presented and discussed. I established, furthermore, a method for counting single surface immobilized motors to guide the evaluation of the tweezing experiments.

Neben der Aufgabe, die „Hand-Made“ Klonierungstechnik (HMCTM) erfolgreich in unserem Labor einzuarbeiten, wurden Untersuchungen zur Entwicklungskompetenz und Lebensfähigkeit von Embryonen aus HMCTM durchgeführt. Dabei wurden verschiedene somatische Zellen als Kernspender verwendeten und die Verteilungsmuster von Zytoskelettproteinen (a-Tubulin und F-Actin), die Eigenschaften interzellulärer Kontakte sowie das Auftreten des „Gap-Junction-Proteins Connexin 43 zwischen HMCTM-Embryonen als auch mittels IVP und in vivo produzierte Embryonen verglichen. Die durchschnittliche Gesamteffizienz des Verfahrens lag bei 95% rekonstruierter Embryonen mit 65% mittlerer Teilungsrate. Keine signifikanten Unterschiede wurden zwischen den drei Gruppen von somatischen Zellen in bezug auf Embryorekonstruktionseffizienz, Teilungsrate, Anzahl von >8 Zell Embryonen und Blastozystenrate beobachtet. Von den erzeugten Embryonen erreichten nur 2% das Blastozystenstadium. Viele Embryonen verharrten arretiert zwischen dem 8 und 16 Zell Stadium, was dem Zeitpunkt der Umschaltung vom maternalen auf das embryonale Genom entspricht (TELFORD et al. 1990; MEMILI et al. 1998). Wir erzeugten insgesamt 67 übertragbare Blastozysten von denen 36 auf synchrone Rezipienten transferiert wurden. Die erzeugten 6 Trächtigkeiten entsprechen einer Rate von 17%. Funf Trächtigkeiten gingen verloren und eine führte zur Geburt eines Bullenkalbes - des ersten HMCTM Kalbes in Europa. Die Etablierung der HMCTM Technik in unserem Laboratorium kann damit als erfolgreich betrachtet werden. Die höchste Intensität der a-Tubulinfärbung wurde in klonierten und IVP-Embryonen (ohne signifikante Unterschiede) festgestellt. Im Unterschied dazu war sie in in vivo erzeugten Embryonen signifikant geringer, was auf die möglicherweise ungünstigen Einflüsse der In-vitro-Kulturbedingungen hinweist. Für Actin wurde eine höhere Intensität in in vivo- und IVP-Embryonen (ohne signifikante Unterschiede) festgestellt, während sie in klonierten Embryonen signifikant niedriger war. Das weist auf einen negativen Effekt der Zona pellucida freien HMCTM Methode hin, die für den Kerntransfer verwendet wurde. Spekulativ können die Abweichungen, die in der Verteilung der Zytoskelettproteine beobachtet wurden, als ein Anzeichen für den Entwicklungsblock in den betroffenen Rinderembryonen betrachtet werden (MATSUMOTO et al. 2002). Untersuchungen mittels Western-Blot ergaben, dass in IVP-Embryonen vom 4-Zellstadium bis zum Morulastadium sowohl Connexin-43 als auch a-Tubulin ohne quantitative Unterschiede nachweisbar waren. 8-Zellembryonen aus HMCTM, IVP und in vivo Produktion im Vergleich, zeigten ebenfalls keine quantitativen Unterschiede bezüglich Connexin-43 und a-Tubulin. Diese Ergebnisse entsprechen einer früheren Hypothese das Cx43 Transkripte in in vitro produzierten Rinderembryonen maternalen und embryonalen Ursprungs sein können (WRENZYCKI et al. 1996). Die Ultrastrukturanalyse von 8-Zellembryonen zeigte dass die interzellularen Kontakte zwischen den Blastomeren in bezug auf ihre Länge in HMCTM Embryonen die kürzesten Kontaktzonen (CP) und in in vivo produzierten Embryonen die längsten CP aufwiesen. Während der Abwesenheit von Gap-Junctions (GJ) in den früheren Stufen der Embryogenese können diese CPs eine entscheidende Rolle beim Transfer von Metaboliten zwischen den Zellen und bei der interzellulären Kommunikation bis zum späten Morula- oder frühen Blastozystenstadium, wenn die GJ Bildung einsetzt, übernehmen. Die kürzere CP Länge kann deshalb einer der Gründe für die relativ schlechtere Entwicklungsrate der klonierten Embryonen sein. Detailliertere Untersuchungen zur Bedeutung der Länge der CPs'' und ihren funktionellen Aspekten werden zukünftig nötig, um eine genauere Interpretation zu ermöglichen. / Apart from the aim of successfully establishing the Handmade cloning (HMCTM) technique in our laboratory, investigations were conducted to study the developmental competence and viability of the HMCTM -derived embryos while using different somatic cells, and to compare the distribution pattern of cytoskeletal proteins (alpha-tubulin and F-actin) and the nature of cellular contacts, as also the presence of Connexin 43 (gap junction protein) between HMCTM cloned, IVP and in vivo produced embryos. We obtained a 95 % overall average efficiency of formation of the reconstructed embryos with a 65 % average cleavage rate. No significant differences were observed between the three groups of somatic cell donors in terms of embryo reconstruction efficiency, cleavage rate, number of > eight cell stage embryos, and the blastocyst rates. Of the fused couplets, only 2 % reached the blastocyst stage, with many of the embryos arresting between the 8- to 16-cell stage, a stage corresponding to the timing of maternal to embryonic genomic take-over of development in bovine (TELFORD et al. 1990; MEMILI et al. 1998). We also recovered a total of 67 transferable blastocysts, out of which 36 blastocysts were transferred to 36 synchronized recipients resulting in six pregnancies, thereby yielding a 17 % pregnancy rate. Five pregnancies were lost from abortions and one resulted in the birth of a bull calf - the first HMCTM calf born in Europe. The establishment of the HMCTM technique in our lab can thus be regarded as successful. The maximum intensity of tubulin was observed in the cloned and the IVP embryos (with no significant differences) whereas it was significantly lower in the in vivo embryos indicating the adverse effects of in vitro culture conditions. In the case of actin, a higher intensity of staining was observed in the case of in vivo and IVP embryos (with no significant differences) whereas the same was significantly lower in the cloned embryos thereby reflecting the adverse effects of the zona-free technique used in the HMCTM method of SCNT. Speculatively, the deviations/variations observed in the distribution of the cytoskeletal proteins may be an indicator of the developmental arrest in the bovine embryos concerned (MATSUMOTO et al. 2002). Western blot analysis revealed that in the IVP embryos, from 4-cell stage up to the morula stage, both connexin-43 and tubulin were present with no quantitative differences. The 8-cell stage embryos collected from HMCTM cloning, IVP and in vivo production also showed presence of connexin-43 and tubulin, yet again, with no quantitative differences. The results could conform to an earlier hypothesis that Cx43 transcripts in bovine embryos produced in vitro might be of maternal and embryonic origin (WRENZYCKI et al. 1996). Ultrastructural analysis of the 8-cell stage embryos showed that the intercellular contacts between the blastomeres were, in terms of length, of the shortest order in case of the HMCTM cloned embryos whereas these Contact Point (CP) lengths were of the greatest order in vivo produced embryos. In the absence of gap junctions (GJ) in the earlier stages of embryogenesis, these CPs may play a crucial role in the exchange of metabolites and cell-to-cell communication till the late morula or early blastocyst stage when the GJ formation occurs. The shorter CP length may, therefore, be one of the reasons for the relative poor development rate of the cloned embryos. Detailed investigations of the significance of the length of the CPs and their functional aspects would be required in future to facilitate meaningful interpretation.

Microtubule associated molecular motors are involved in a multitude of fundamental cellular processes such as intracellular transport and spindle positioning. During these movements multiple motor proteins often work together and are, therefore, able to exert high forces. Thus force generation and sensing are common mechanisms for controlling motor driven movement. These mechanisms play a pivotal role when motor proteins antagonize each other, e.g. to facilitate oscillations of the spindle or the nucleus. Single motor proteins have been characterized in depth over the last two decades, our understanding of the collective behavior of molecular motors remains, however, poor. Since motor proteins often cooperate while they walk along microtubules, it is necessary to describe their collective reaction to a load quantitatively in order to understand the mechanism of many motor-driven processes. I studied the antagonistic action of many molecular motors (of one kind) in a gliding geometry. For this purpose I crosslinked two microtubules in an antiparallel fashion, so that they formed \"doublets\". Then I observed the gliding motility of these antiparallel doublets and analyzed the gliding velocity with respect to the relative number of motors pulling or pushing against each other. I observed that the antiparallel doublets gliding on conventional kinesin-1 (from Drosophila melanogaster) as well as cytoplasmic dynein (from Saccharomyces cerevisae) exhibited two distinct modes of movement, slow and fast, which were well separated. Furthermore I found a bistability, meaning, that both kinds of movement, slow and fast, occurred at the same ratio of antagonizing motors. Antiparallel doublets gliding on the non-processive motor protein Ncd (the kinesin-14 from D. melanogaster) showed, however, no bistability. The collective dynamics of all three motor proteins were described with a quantitative theory based on single-motor properties. Furthermore the response of multiple dynein motors towards an external, well-defined load was measured in a gliding geometry by magnetic tweezing. Examples of multi-motor force-velocity relationships are presented and discussed. I established, furthermore, a method for counting single surface immobilized motors to guide the evaluation of the tweezing experiments.:1 Introduction to the functions of molecular motors 1 1.1 How molecular motors move 1 1.1.1 Of muscles and molecules 1 1.1.2 Kinesin-1, the working horse of single-molecule research 3 1.1.3 Kinesin-14, an unusual kinesin with a new twist 6 1.1.4 Cytoplasmic dynein, the molecule with many qualities 7 1.2 Structure and function of microtubules 8 1.3 The directionality of molecular motors 9 1.4 Force regulation in cell biology via molecular motors 10 1.4.1 Bidirectional cargo transport 10 1.4.2 Dynein drives intracellular oscillations 13 1.4.3 Control of spindle length 15 2 Introduction to the collective behavior of molecular motors in vitro 19 2.1 Cooperativity of molecular motors 19 2.2 How multiple motors work against a load 21 2.2.1 Theoretical concepts 21 2.2.2 Optical tweezing of multiple motors 22 2.2.3 Alternative experimental approaches 23 2.2.4 Membrane tube dynamics 24 2.3 Antagonizing molecular motors 25 2.3.1 Competition between dissimilar motors 25 2.3.2 Competition between identical motors 26 2.4 Aim of the project 28 3 Characterization of molecular motors 31 3.1 Results: The run length of processive motors 31 3.1.1 Run length of kinesin-1 at different ATP concentrations 31 3.1.2 The run length of cytoplasmic dynein 34 3.2 Results for multi-motor gliding assays 37 3.2.1 The effect of ATP on the gliding motility 37 3.2.2 The effect of temperature on the gliding motility 39 3.2.3 Bead transport does not influence gliding motility 42 3.3 Discussion 43 4 Magnetic tweezing of multiple molecular motors 45 4.1 Concepts of the magnetic tweezing setup 45 4.1.1 Theoretical concepts 45 4.1.2 Implementation 48 4.1.3 Calibration 51 4.2 Results of multi-motor force measurements 53 4.2.1 External force leads to microtubule re-orientation 53 4.2.2 Cytoplasmic dynein is able to withstand high opposing loads 55 4.2.3 Force-velocity curves at very low motor densities 56 4.2.4 Averaging of multi-motor force-velocity relationships 58 4.3 Discussion 60 5 Reconstitution of antagonizing motor activity 63 5.1 The doublet assay 63 5.2 Experimental results of the doublet assay 65 5.2.1 Kinesin-1 driven doublets move in discrete velocity regimes 65 5.2.2 Velocity affects the shape of the bistability curve 68 5.2.3 Dynein\'s processivity allows bistability at low velocity 69 5.2.4 Ncd does not exhibit a bistability curve 70 5.3 Theoretical results of the doublets assay 71 5.3.1 General concepts 71 5.3.2 Theory for processive motors 73 5.3.3 Theory for non-processive motors 75 5.3.4 The emergence of bistability 78 5.3.5 Model for single-motor force-velocity relationships 81 5.4 Comparison between theoretical and experiment results 83 5.5 Discussion 87 6 Materials and Methods 91 6.1 List of chemicals and equipment 91 6.2 Buffer recipes 92 6.3 Protein purification 93 6.4 Preparation of microtubules 95 6.5 Preparation of flow cells 96 6.6 Fluorescence microscopy 98 6.7 Errors computation 100 6.8 Software 100 7 References 103 8 Acknowledgement 113

Anders als menschengemachte Maschinen verfügen Zellen über keinen festgeschriebenen Bauplan und die Positionen einzelner Elemente sind häufig nicht genau festgelegt, da die Moleküle diffusiven Zufallsbewegungen unterworfen sind. Darüber hinaus sind einzelne Bauteile auch nicht auf eine einzelne Funktion festgelegt, sondern können parallel in verschiedene Prozesse einbezogen sein. Basierend auf Selbstorganisation und Selbstassemblierung muß die Organisation von Anordnung und Funktion einer lebenden Zelle also bereits in ihren einzelnen Komponenten inhärent enthalten sein. Die intrazelluläre Organisation wird zum großen Teil durch ein internes Biopolymergerüst reguliert, das Zytoskelett. Biopolymer-Netzwerke und –Fasern durchdringen die gesamte Zelle und sind verantworlich für mechanische Integrität und die funktionale Architektur. Unzählige essentielle biologische Prozesse hängen direkt von einem funktionierenden Zytoskelett ab. Die vorliegende Arbeit zielt auf ein besser Verständnis und den Nachbau zweier verschiedener funktionaler Module lebender Zellen anhand stark reduzierter Modellsysteme. Als zentrales Element wurde Aktin gewählt, da dieses Biopolymer eine herausragende Rolle in nahezu allen eukaryotischen Zellen spielt. Mit dem ersten Modellsystem wird der bewegliche Aktin-Polymerfilm an der Vorderkante migrierender Zellen betrachtet. Die wichtigsten Elemente dieser hochdynamischen Netzwerke sind bereits bekannt und wurden in dieser Arbeit benutzt um ein experimentelles Modellsystem zu etablieren. Vor allem aber lieferten detailierte Computersimulationen und ein mathematisches Modell neue Erkenntnisse über grundlegende Organisationsprinzipien dieser Aktinnetzwerke. Damit war es nicht nur möglich, experimentelle Daten erfolgreich zu reproduzieren, sondern das Entstehen von Substrukturen und deren Charakteristika auf proteinunabhängige, generelle Mechanismen zurückzuführen. Das zweite studierte System betrachtet die Selbstassemblierung von Aktinnetzwerken durch entropische Kräfte. Aktinfilamente aggregieren hierbei durch Kondensation multivalenter Ionen oder durch Volumenausschluss hochkonzentrierter inerter Polymere. Ein neu entwickelter Experimentalaufbau bietet die Möglichkeit in gut definierten zellähnlichen Volumina, Konvektionseinflüsse zu umgehen und Aggregationseffekte gezielt einzuschalten. Hierbei wurden neuartige, regelmäßige Netzwerkstrukturen entdeckt, die bislang nur im Zusammenhang mit molekularen Motoren bekannt waren. Es konnte ferner gezeigt werden, dass die Physik der Flüssigkristalle entscheidend zu weiteren Variationen dieser Netzwerke beiträgt. Dabei wird ersichtlich, dass entstehende Netzwerke in ihrer Architektur direkt die zuvor herrschenden Anisotropien der Filamentlösung widerspiegeln.

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.

Um Beweise für eine mögliche Mikrokompartimentierung der Glykolyse im pflanzlichen System zu erhalten, sollten in der vorliegenden Arbeit Protein-Protein-Interaktionen der cytosolischen Mais-Aldolase mit anderen Proteinen experimentell nachgewiesen werden. Die in Tieren bekannte Interaktion des glykolytischen Enzyms Aldolase mit Aktin, einem Bestandteil des Cytoskeletts, wurde für Pflanzen in vitro durch Copolymerisationsversuche bestätigt. Die Bindung pflanzlicher Aldolase an Aktinfilamente wurde anders als im tierischen System durch das Substrat Fructose-1,6-bisphosphat auch in hohen Konzentrationen (10 mM) nicht vollständig verhindert, sondern führte lediglich zu einer um 50% verringerten Bindung. Eine ebenfalls hemmende Wirkung auf die Bindung der Aldolase an Aktin wiesen Fructose-6-phosphat und Fructose-2,6-bisphosphat in Konzentrationen von 10 mM auf. Ein eindeutiger Einfluss des Redox-Milieus auf die Aldolase-Aktin-Bindung konnte nicht nachgewiesen werden. Mit Hilfe des im Rahmen dieser Arbeit etablierten Hefe-2-Hybrid-Systems wurden weitere Interaktionspartner der Aldolase identifiziert. Insgesamt wurden neun mögliche Protein-Protein-Interaktionen nachgewiesen, bei denen es sich jedoch zum Teil um falsch-positive Interaktionen handeln kann. Neben einigen noch unbekannten Proteinen konnten Interaktionen mit einem Translations-Initiationsfaktor und dem spannungsabhängigen Anionenkanalprotein VDAC nachgewiesen werden. In Bindeversuchen auf Grundlage der Affinitätschromatographie mit den rekombinanten Proteinen VDAC und Aldolase wurde ein weiterer Hinweis auf eine Interaktion zwischen VDAC und Aldolase erhalten. Aufgrund unspezifischer Bindungen der Aldolase an die Affinitätsmatrix konnte mit dieser Methode jedoch keine eindeutige Verifizierung der Interaktion erzielt werden. Eine eindeutige Bestätigung der Interaktionen zwischen Aldolase und Aktin sowie zwischen Aldolase und VDAC erfolgte durch Far-Western-Blots .