This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor.

In order for cells to migrate, communicate, and facilitate attachment to the surrounding extraceullar matrix, they must form intricate protein complexes called focal adhesions. The number of identified focal adhesion components continues to grow and the field is an area of active study.Lasp-2 is a member of the nebulin family of actin-binding proteins that has been identified as a member of focal adhesion complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2, the integral focal adhesion proteins, vinculin and paxillin. Interestingly, the interaction of lasp-2 with its binding partners vinculin and paxillin was significantly reduced in presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other, however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1 suggesting the interplay between lasp-2 and lasp-2 could be an adhesion regulatory mechanism. Lasp-2's potential role in metastasis was revealed as overexpression of lasp-2 in SW620 cells, a highly metastatic cancer cell line, increased cell migration, but impeded cell invasion.Lasp-2 transcript and protein is readily detected in neural tissues. Preliminary experiments involving the knockdown of lasp-2- in frog embryos revealed gross morphological abnormalities in the head region as well as the inability to move normally. Neural crest derived melanocytes also failed to migrate normally.Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.

We study a number of non-equilibrium models of interest to both active matter and biological physicists. Using microscopic agent-based simulation as well as numerical integration of stochastic PDEs, we uncover the non-trivial behaviour exhibited when active transport, or an advection field, is added to out of equilibrium systems. When gravity is included in the celebrated Fisher-Kolmogoro Petrovsky Piscouno (F-KPP) equation, to model sedimentation of active bacteria in a container, we observe a discontinuous phase transition between a `sedimentation' and a `growth' phase, which should in principle be observable in real systems. With the addition of multiplicative noise, the resulting model contains, as its limits, both the bacterial sedimentation previously described and the fluctuating hydrodynamic description of Directed Percolation (DP), an important and well-studied non-equilibrium system whose physics incorporate many universal features which are typical of systems with absorbing states. We map out the phase diagram describing all the systems in between these two limiting cases, finding that adding an advection term, however small, immediately lifts the resulting system out of the DP universality class. Furthermore, we find two distinct low-density phases separated by a dynamical phase transition reminiscent of a spinodal transition. Finally, we attempt to improve the current diffusion-limited model for the growth of filopodia, which are intriguing networks of actin fibres used by moving cells to sense their environment. By the addition of directed transport of actin monomers to the fibre tip complex by myosin molecular motors, we show that, under appropriate conditions, the resulting dynamics may be more efficient that transport by diffusion alone, which would result in filopodial lengths better corresponding to experimental observation.

The trans-differentiation of injury-activated fibroblasts to myofibroblasts is a process that provides contractile strength for wound closure. Persistent myofibroblast differentiation, however, is associated with fibrotic pathologies such as organ fibrosis, vascular remodeling, and atherosclerotic plaque formation. Myofibroblasts acquire a contractile phenotype with biochemical properties characteristic of both smooth muscle cells and stromal fibroblasts. The cyto-contractile protein, smooth muscle α-actin (SMαA) is a biomarker of myofibroblast differentiation. Expression of the SMαA gene, ACTA2, is regulated by cis-acting elements and transcription factors that activate or repress the ACTA2 promoter. Purine-rich element binding proteins A (Purα) and B (Purβ) are sequence-specific, single-stranded DNA (ssDNA)/RNA-binding proteins that act as transcriptional repressors of ACTA2 expression. Both Pur proteins interact with the purine-rich strand of a cryptic muscle-CAT (MCAT) enhancer motif in 5'-flanking region of the ACTA2 promoter. Despite significant sequence homology with Purα, Purβ was identified as the dominant repressor of ACTA2 expression in mouse embryonic fibroblasts and vascular smooth muscle cells by virtue of gain-of function and loss-of-function analyses in cultured cells. Biophysical studies indicated that Purβ reversibly self-associates in solution to form a homodimer. Quantitative DNA-binding assays revealed that Purβ interacts with the purine-rich strand of the ACTA2 MCAT motif via a cooperative, multisite binding mechanism to form a high-affinity 2:1 Purβ-ssDNA complex. In this dissertation, a combination of computational, biochemical, and cell-based approaches were employed to elucidate the molecular basis of Purβ repressor interaction with the ACTA2 gene. Limited proteolysis of recombinant mouse Purβ in the presence and absence of the purine-rich strand of the ACTA2 MCAT element led to the identification of a core ssDNA-binding region that retains the ability to dimerize in solution. Knockdown of endogenous Purβ in mouse embryonic fibroblasts via RNA interference induced SMαA expression and conversion to a myofibroblast-like phenotype. To map the specific structural domains in the core region of Purβ that account for its unique ACTA2 repressor and ssDNA-binding functions, computational homology models of the Purβ monomer and dimer were generated based on the x-ray crystal structure of an intramolecular subdomain of Drosophila melanogaster Purα. Empirical biochemical and cell-based analyses of rationally-designed Purβ truncation proteins revealed that the assembled Purβ homodimer is composed of three separate purine-rich ssDNA-binding subdomains. Evaluation of the effects of anionic detergent and high-salt on the binding of Purβ to ssDNA implicated the involvement of hydrophobic and electrostatic interactions in mediating high-affinity nucleoprotein complex formation. This inference was validated by site-directed mutagenesis experiments, which identified several basic amino acid residues required for the ACTA2 repressor activity of Purβ. Collectively, the findings described herein establish the structural and chemical basis for the cooperative interaction of Purβ with the ACTA2 MCAT enhancer and for Purβ-dependent suppression of myofibroblast differentiation.

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...

Neospora caninum é um parasita intracelular obrigatório pertencente ao filo Apicomplexa, conhecido por ser uma das principais causas de aborto parasitário em bovinos e por apresentar transmissão transplacentária. Para locomoverem-se e acessarem o conteúdo intracelular de células hospedeiras, organismos apicomplexas fazem uso de um mecanismo não convencional que se utiliza de uma maquinaria celular cujo papel central é exercido pelo motor actina-miosina, auxiliado por proteínas intermediárias e de acoragem, que realiza a propulsão do parasita na direção do movimento. Para o funcionamento dessa maquinaria, é essencial que actina esteja em sua forma filamentosa (actina-F). Porém, actinas de apicomplexas são conhecidas por serem funcional e estruturalmente não convencionais, formando filamentos pequenos e instáveis in vitro, assim como pelo predomínio de grande maioria de actina monomérica (actin-G) nas células in vivo. Desse modo, para formar e manter actina-F a dinâmica de actina desses organismos requer uma regulação precisa, que, em apicomplexas, é conduzida por um arsenal conhecidamente pequeno de proteínas que se ligam a actina (ABPs). Nosso objetivo neste estudo foi identificar e caracterizar ABPs de N. caninum. Para isso, duas ABPs de N. caninum foram estudadas: fator de despolimerização de actina (NcADF) e proteína associada a ciclase (NcCAP); também, foi gerado e caracterizado soro contra região de actina de N. caninum entre aminoácidos 201 e 310 (anti-NcAct201-310). NcADF (correspondente ao acesso NCLIV_012510 em ToxoDB) foi submetida a caracterização molecular e bioquímica. A sua estrutura terciária foi gerada por modelagem molecular baseada em homologia, apresentando folding conservado, porém com F-loop de menor tamanho, quando comparada a ADF/cofilinas canônicas. A forma recombinante de NcADF foi expressa E. coli BL21 por plasmídeos pET32a(+) e pET28a(+) e solubilizada em tampão desnaturante e nativo, respectivamente. NcADF_pET32 foi purificada e utilizada para geração de soro anti-NcADF, que detectou ambas NcADF recombinantes, assim como proteínas endógenas em western blot 1-D e 2-D com peso molecular e pI próximos aos preditos. O soro anti-NcADF também localizou NcADF difusa no citoplasma, com menos intensidade nos polos de taquizoítas de N. caninum extracelulares. NcADF_pET28 foi purificado na forma nativa e utilizado para caracterização funcional para avaliação de seu papel na dinâmica de actina liofilizada de coelho. Ensaios de cossedimentação, cinética de polimerização e despolimerização, viscosimentria de baixo cisalhamento (queda de bola), estado estacionário e ligação entre actina-G e NcADF, em conjunto, mostraram que NcADF causa despolimerização de actina-F, realiza sequestro de monômeros de actina e quebra de filamentos. NcCAP foi submetida a caracterização molecular e foi identificada como produto de expressão do gene de acesso NCLIV_054140. NcCAP recombinante foi expressa em pET32a(+) e pET28a(+) predominantemente em corpos de inclusão e foi solubilizada em tampão desnaturante. A forma purificada de pET32_NcCAP, identificada por espectrometria de massas, foi utilizada para imunização e o soro resultante detectou NcCAP recombinante e endógena por western blot 1-D e 2-D, apresentando bandas e spots de peso molecular e pI próximos ao esperado. O soro anti-NcCAP também localizou NcCAP em taquizoítas ii extracelulares de N. caninum difusa no citoplasma e/ou com predomínio na região periplasmática da célula. Por fim, o soro anti-NcAct201-310 foi gerado, sendo capaz de detectar proteínas em sua forma nativa e realizar marcação na região periférica e, possivelmente, nuclear de taquizoítas de N. caninum extracelulares. A caracterização de ABPs de N. caninum feita neste trabalho amplia o conhecimento sobre a conservação dessas proteínas ao longo do filo Apicomplexa. Ademais, representa uma contribuição para o entendimento da dinâmica de actina e, por consequência, futuramente, pode colaborar para a elucidação de mecanismos-chave para a sobrevivência e disseminação dos parasitas pelo seu hospedeiro / Neospora caninum is an obligate intracellular parasite that belongs to the phylum Apicomplexa. It is known as one of the main causes of infectious abortion in cows and for its efficient transplacentary transmission. Apicomplexan organisms use a phylum-specific mechanism of invasion and gliding motility, which use an unusual cellular machinery based on an actin myosin motor assisted by intermediary and anchoring proteins that creates the traction force to impulse the parasite forward. Filamentous actin (F-actin) is essential to the appropriate functioning of this machinery, although apicomplexan unconventional actin forms small and unstable filaments in vitro and is found preponderantly as monomer (G-actin) in cells. Thus, the parasites need actin-binding proteins (ABPs) to strictly regulate actin dynamics and to form and maintain F-actin when it is necessary to the cell. Here, we aimed at identifying and characterising ABPs from N. caninum. Two ABPs were characterised: actin-depolymerising factor (NcADF) and cyclase-associated protein (NcCAP) from N. caninum. In addition, a serum against the actin region between amino acids 201 and 310 (anti-NcAct201-310) was raised. NcADF, which corresponds to identification NCLIV_012510 on ToxoDB, was molecular and biochemically characterised. Firstly, the tertiary structure of NcADF was generated by molecular modelling based on homology. Comparing to canonical ADF/cofilins, NcADF presented a conserved folding, albeit its smaller F-loop. The recombinant form of NcADF was expressed in E. coli BL21 using pET32a(+) and pET28a(+) plasmids and solubilized in denaturing and native buffers, respectively. Polyclonal antibodies were raised in mice against purified NcADF_pET32, which was able to detect both forms of recombinant NcADF as well as proteins in 1-D and 2-D western blot with expected molecular weight and isoelectric point (pI). Additionally, NcADF was localised in extracellular N. caninum tachyzoites as a diffuse pattern on cytoplasm with less intensity in both poles. NcADF_pET28 was successfully purified in native form and used for functional characterisation to evaluate the role of recombinant NcADF on lyophilised rabbit actin dynamics. Together, co-sedimentation, polymerisation and depolymerisation kinetic, low shearing viscometry (falling ball), steady state, and G-actin and NcADF binding assays showed that NcADF was able to depolymerise actin-F, sequester actin monomers, and sever filaments. Moreover, NcCAP (identification NCLIV_054140) was also characterised. Recombinant NcCAP was expressed in pET32a(+) and pET28a(+) plasmids predominantly in inclusion bodies and was solubilised in denaturing buffer. NcCAP_pET32 was purified and identified by mass spectrometry. Then, the polyclonal antibodies against this recombinant protein was generated in mice. It was able to detect recombinant and endogenous NcCAP, presenting bands and spots in 1-D and 2-D western blot with molecular weight and pI quite near to the predicted ones. NcCAP was localised as a diffuse pattern on cytoplasm and/or predominantly on periplasmic regions of extracellular taclyzoites of N. caninum. Finally, the serum containing anti-NcAct201-310 polyclonal antibodies was raised in mice. It detected endogenous proteins mainly in native form and localised them on periplasmic and possibly nuclear region in extracellular N. caninum tachyzoites. The characterisation of N. caninum ABPs iv extends our understanding of these proteins conservation and their function throughout the Apicomplexa phylum. Furthrmore, it represents a contribution to the field towards the comprehention of actin dynamics and in the future might provide information for important mechanisms of dissemination and survival of the parasite at its host

Cell death is a key component in development and for the continued renewal of tissues. Phagoptosis is a process in which phagocytes directly lead to the death of other cells. This process of cell death is significantly less characterized when compared to other mechanisms of cell death, such as apoptosis. In the Drosophila ovary, phagoptosis appears to play a key role in the developmental process of oogenesis. Recent studies have shown that genes associated with phagocytosis are required for the programmed death of nurse cells in the Drosophila ovary. Ovaries are made up of 15 nurse cells, a single oocyte, and a layer of follicle cells bordering them. During the process of egg chamber development, all of the nurse cells undergo programmed cell death. During late oogenesis, each nurse cell is surrounded by a group of follicle cells referred to as stretch follicle cells. These stretch follicle cells have recently been implicated as a main promoter of nurse cell phagoptosis. However, an exact mechanism to explain how these stretch follicle cells induce nurse cell death is not fully characterized. To achieve a more detailed understanding of this mechanism, we are examining the function of the cytoskeleton in this process via live imaging. We hypothesize that the follicle cell cytoskeleton plays a significant role in nurse death due to the importance of actin during phagocytosis. Further, we intend to use these live imaging studies to investigate the role of calcium before, during, and after clearance of the nurse cells. Previous studies have shown that calcium bursts within the cell are associated with the initiation of phagocytosis in macrophages, as well as other phagocytic cell types. Studies in this thesis were done by utilizing live imaging and have shown dynamic changes in follicle cell actin before and during the death of nurse cells. These confocal microscopy real time videos have revealed that follicle cell actin polymerizes towards the nurse cell immediately before acidification. Following acidification of the nurse cells, the follicle cell actin changes direction, moving towards the phagocytic follicle cell. Additionally, through live imaging we have observed calcium bursts in the follicle cells immediately before nurse cell death. Overall, this work has provided a more detailed understanding of nurse cell death.

Die Spinale Muskelatrophie (SMA) ist eine häufige autosomal-rezessiv vererbte Erkrankung des motorischen Nervensystems bei Kindern. Ursache der Degeneration von spinalen Motoneuronen ist der homozygote Verlust des SMN- (survival of motoneuron) Gens und ein dadurch bedingter Mangel an SMN-Protein. Untersuchungen an Motoneuronen von Smn-defizienten Mäusen ergaben Störungen des axonalen Längenwachstums aufgrund einer Fehlverteilung des Zytoskelettproteins beta-Aktin und seiner mRNA in den Axonterminalen. Das Axonwachstum wird durch Aktin-Polymerisierung im Wachstumskegel gesteuert. beta-Aktin-mRNA findet sich auch in Axonen, und die lokale Proteinsynthese kann durch neuronale Aktivierung gesteigert werden. Das SMN-Protein ist am axonalen Transport von beta-Aktin beteiligt. In der vorliegenden Arbeit ergaben Western Blot-Analysen in neuralen Stammzellen (NSC) sowie spinalen Motoneuronen in vitro eine Steigerung der SMN-Proteinexpression durch 8-CPT-cAMP. Zur Untersuchung der Auswirkungen der erhöhten SMN-Proteinmenge auf die Pathologie der Motoneurone wurde ein in-vitro-Assay entwickelt, mit dessen Hilfe gezeigt werden konnte, dass eine Behandlung mit 100 µM 8-CPT-cAMP die axonalen Veränderungen isolierter embryonaler Smn-defizienter Motoneurone kompensieren kann. Motoneurone von 14 Tage alten Smn-defizienten und Kontroll-Mausembryonen wurden über sieben Tage hinweg auf einer Matrix aus Poly-Ornithin und Laminin-111 bzw. Laminin-121/221 kultiviert und mit 100µM cAMP und neurotrophen Faktoren behandelt. Nach Fixierung wurden die Zellen mit Antikörpern gegen Islet-1/2, tau und beta-Aktin gefärbt, mit Hilfe eines konfokalen Mikroskops fotografiert und digital vermessen. 8-CPT-cAMP erhöht den beta-Aktin-Gehalt in den axonalen Wachstumskegeln von Smn-defizienten Motoneuronen. Die Größe der Wachstumskegel nimmt durch die Behandlung um das 2-3fache zu und erreicht normale Werte. Auf Laminin-111 bleibt das Längenwachstum der Axone durch 100µM 8-CPT-cAMP unbeeinflusst, auf Laminin-121/221 wird das Längenwachstum normalisiert. Die beta-Aktin-Verteilung innerhalb der Axone und Wachstumskegel von Smn-defizienten Motoneuronen erscheint durch die cAMP-Behandlung nahezu normalisiert. Die Wiederherstellung der beta-Aktin-Verteilung in Wachstumskegeln durch cAMP kann große Auswirkungen auf die Funktionalität der Motoneurone haben. Die Ergebnisse sind möglicherweise ein erster Schritt auf dem Weg zu einer Therapie für die Spinale Muskelatrophie. / Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of alpha-motoneurons in the spinal chord due to low levels of the survival motor neuron (SMN) protein. The genetic cause is the homozygous loss or mutation of the telomeric SMN1 gene and retention of the centromeric SMN2 gene, whose transcripts consist of about 90% truncated and unstable and only 10% functional protein. Motoneurons of Smn-deficient SMN2 transgenic mouse embryos cultured on laminin-1 show abnormalities compared to wildtype controls such as shorter axons, smaller growth cones and a ß-actin protein and mRNA deficit in the distal part of the axon. ß-actin plays a major role in growth cone motility and transmitter release at the presynapse. In addition, SMN works in a complex to transport ß-actin mRNA, which is known to be localized and locally translated in axons and growth cones, along the axon. Local ß-actin protein synthesis can be stimulated by increased neuronal activation. We determined the effects of cAMP on ß-actin localisation in axons as well as on axonal growth parameters in Smn-deficient primary motoneurons. Motoneurons of 14 days old Smn-/-, SMN2 transgenic and wildtype mouse embryos were cultured on laminin for 7 days with 100µM 8-CPT-cAMP and neurotrophic factors BDNF and CNTF. Fluorescence staining and digital measurements revealed a major effect of cAMP treatment on ß-actin distribution and growth cone size, which were restored to normal. Neurite lengths on laminin-111 remained unaffected but were normalized on substrate containing a synapse-specific ß2-laminin isoform. Western blots with neural stem cells (NSC) and heterozygous Smn+/-; SMN2 transgenic motoneurons treated with 100µM cAMP showed a marked upregulation of Smn protein expression. These data point to an important role for cAMP as a possible target of SMA drug therapy.

Die Todesrezeptoren Fas, TRAILR1 und TRAILR2 werden seit einigen Jahren aufgrund ihrer Fähigkeit, Apoptose zu induzieren, als therapeutisch interessantes Ziel bei der Therapie maligner Tumoren angesehen. Gleichzeitig werden immer mehr Entitäten von Tumoren beschrieben, die eine Resistenz gegen die Todesrezeptor-induzierte Apoptose aufweisen. In dieser Konstellation können neben den blockierten proapoptotischen Signalen insbesondere auch Todesrezeptor-assoziierte, protumoral wirksame Signalwege sichtbar werden, die unter anderen Umständen durch die Apoptose maskiert werden. In dieser Arbeit wurde die von FasL- und TRAIL-induzierte Signaltransduktion in einer apoptoseresistenten Variante der kolorektalen Karzinomzelllinie HCT116 untersucht. Eine aktivierende Mutation des PIK3CA-Gens protektiert diese Zellen aufgrund der konstitutiven Aktivierung des onkogenen PI3K/Akt-Signalweges gegenüber Todesrezeptor-vermittelter Apoptose. Durch Vergleich isogener Zelllinien, welche für den PIK3CA-Locus funktionell haploid waren und entweder ein Wildtyp oder ein mutiertes Allel trugen, konnte die Signaltransduktion von Fas und der TRAIL-Todesrezeptoren in apoptoseresistenten Tumorzellen, sowie deren Zusammenspiel mit dem PI3K/Akt-Signalweg im Detail untersucht werden. So wurde in dieser Arbeit gezeigt, dass nach Stimulation der HCT116 PIK3CA-mut protektierten Zellen mit FasL oder TRAIL die initialen Schritte der Apoptoseinduktion durch Todesrezeptoren bis hin zur Bildung des DISC und der Aktivierung von Caspase-8 ungestört vonstatten gehen. Der durch die PIK3CA-Mutation induzierte Schutzmechanismus muss deshalb unterhalb dieser frühen apoptoseinduzierenden Ereignisse wirksam werden. Darüber hinaus zeigte sich, dass Todesliganden in HCT116 PIK3CA-mut Zellen den proinflammatorischen NFκB-Signalweg aktivieren, wohingegen dieser Signalweg in HCT116 PIK3CA-wt Zellen durch die ablaufende Apoptose inhibiert wurde. Während HCT116 PIK3CA-wt Zellen nach Stimulation von Fas oder den TRAIL-Todesrezeptoren morphologisch die klassischen Anzeichen des apoptotischen Zelltods zeigten, veränderten die HCT116 PIK3CA-mut protektierten Zellen ihre Morphologie von einer mesenchymal-länglichen hin zu einer amöboid-abgerundeten Form, die Zellen blieben jedoch vital. Die Änderung der Zellmorphologie konnte mit dem Vorhandensein enzymatisch aktiver Casapse-8 verknüpft werden, generiert durch den Todesrezeptor-assoziierten DISC. Caspase-8 vermittelte die Reorganisation des Aktinzytoskeletts durch Spaltung und der damit einhergehenden Aktivierung von ROCK-1. Blockade der Caspase-8 Aktivierung in HCT116 PIK3CA-mut Zellen durch pharmakologische Inhibitoren oder ektope Überexpression von cFLIPS verhinderte entsprechend den FasL- oder TRAIL-induzierten Übergang zur amöboid-abgerundeten Zellform. Funktionell zeigten die amöboid-abgerundeten HCT116 PIK3CA-mut Zellen im Vergleich zu unstimulierten HCT116 PIK3CA-mut Zellen eine erhöhte Invasivität, was anhand erhöhter Spiegel an Urokinase im Überstand nachgewiesen werden konnte. Diese Arbeit beschreibt mit der Induktion einer amöboid-abgerundeten Zellmorphologie erstmals eine nicht-apoptotische Funktion von Caspase-8 im Kontext der Todesrezeptor-Signaltransduktion, die von der enzymatischen Aktivität abhängig ist. Weiterhin konnte ROCK-1 als Caspase-8 Substrat identifiziert werden. Ob durch die Aktivierung von ROCK-1 und die Reorganisation des Aktinzytoskeletts neben der Ausbildung einer amöboiden Zellmorphologie auch der amöboide Typ der Zellmigration in Gang gesetzt wird, müssen zukünftige Studien zeigen. / During the last years, the death receptors Fas, TRAILR1 and TRAILR2 emerged as promising therapeutic targets in cancer therapy. On the contrary, the number of tumor entities showing resistance against death receptor-induced apoptosis is still rising, thereby limiting the effectiveness of a therapeutic approach. Furthermore, under conditions where death receptor-induced apoptosis is blocked, cell death induction is not the only signal emanating from Fas and TRAIL death receptors. Non-apoptotic and even tumor-promoting signaling pathways may become apparent which are otherwise masked by ongoing apoptosis. This study provides insight in FasL- and TRAIL-induced signaling in an apoptosis resistant variant of HCT116 colorectal cancer cells. An activating mutation in the PIK3CA gene protected these cells against death receptor-induced apoptosis by constitutive activation of the oncogenic PI3K/Akt pathway. Comparing isogenic cell lines either harboring a PIK3CA wild-type allele or an activating mutated allele allowed investigation of signal transduction events associated with Fas and TRAIL death receptors in apoptosis resistant cells as well as their interplay with the PI3K/Akt signaling pathway. Upon stimulation with FasL or TRAIL, PIK3CA-mut protected HCT116 cells were still capable of initiating the first steps of apoptosis induction as was evident from DISC-formation and activating caspase-8. This indicated that the PIK3CA-mut-granted blocking of the apoptotic program must act downstream of these early events. Furthermore, the proinflammatory NFκB pathway was turned on, as demonstrated by the phosphorylation of crucial signaling components and the enhanced expression of NFκB controlled target genes. Activation of the NFκB pathway, however, was masked in HCT116 PIK3CA-wt cells by ongoing apoptosis. After stimulation of Fas or TRAIL death receptors, HCT116 PIK3CA-wt cells exhibited classical morphological apoptotic features. Interestingly, stimulation of HCT116 PIK3CA-mut cells induced transition to an amoeboid-like morphology without affecting the viability. The changes in cellular morphology were crucially dependent on enzymatically active caspase-8 generated at the DISC. Caspase-8 cleaved and thereby activated ROCK-1, a key player in reorganisation of the actin cytoskeleton. Interfering with caspase-8 activation by ectopic expression of cFLIPS or pharmacological inhibitors abrogated the changes in cellular morphology. Additionally, HCT116 PIK3CA-mut cells showed upon FasL- or TRAIL-treatment increased invasiveness, demonstrated by elevated levels of urokinase in the supernatant of the cells. To date, the FasL- or TRAIL-induced transition of HCT116 PIK3CA-mut cells to an amoeboid-like cellular morphology is the first clearly demonstrated non-apoptotic function of caspase-8 in context of death receptor signaling, that is dependent on the enzymatic activity of the molecule. Furthermore, this study identifies ROCK-1 as a novel substrate of caspase-8. Further investigations will have to clarify the role of caspase-8 mediated activation of ROCK-1 and accompanied reorganization of the actin cytoskeleton with respect to the induction of the amoeboid-type of cell migration.