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Cell polarity is required for asymmetric division, a mechanism to generate cell diversity by distributing fate determinants unequally to daughter cells. The establishment of polarity requires the evolutionarily conserved partitioning-defective (PAR) proteins as well as the actin cytoskeleton. In Caenorhabditis elegans one-cell embryos, the PAR proteins are segregated into an anterior (PAR-3, PAR-6) and a posterior (PAR-1, PAR-2) corticaldomain. The formation of PAR polarity correlates with anterior-posterior differences in the contractile activity of the cortex, known as "contractile polarity". It is thought that regulation of contractile polarity controls the establishment of PAR polarity, but detailed evidence to support this idea is lacking. To investigate how modulation of the actomyosin cytoskeleton affects polarity establishment, the acto-myosin cytoskeleton was perturbed by RNA-mediated interference (RNAi) of two Rho GTPases, CDC-42 and RHO-1. To examine how Rho GTPases are implemented in actin remodeling, it is important to analyze how their activity is controlled and how different activities affect polarity formation. The role of two putative Rho GTPase regulators, the Rho GTPase exchange factor (GEF) ECT-2 and the Rho GTPase activating protein (GAP) K09H11.3 were analyzed with respect to polarity formation. The formation of polarity was analyzed by using GFP-labeled proteins, and several different tracking methods were used to investigate the establishment of contractile and PAR polarity in more detail.This study demonstrates that both RHO-1 and CDC-42 are involved in polarity establishment in C. elegans embryos. But importantly, both act by different mechanisms. RHO-1 organizes the acto-myosin cytoskeleton into a contractile network, and therefore is essential for the formation of contractile polarity. The organization of the acto-myosin cytoskeleton is critical to ensure proper PAR protein distribution. Furthermore, a balance of RHO-1 activity by the GEF ECT-2 and the GAP K09H11.3 appears to be important for cortical contractility, for PAR protein domain size and for mutual exclusion of the PAR proteins. Although CDC-42 was shown to be a universal regulator of the actin cytoskeleton, CDC-42 acts downstream of contractile polarity. CDC-42 is required for linking PAR-6 to the cortex. In the absence of RHO-1 and ECT-2, PAR-6 and CDC-42 are not localized to the anterior cortex. This suggests that RHO-1, by organizing the actomyosin cytoskeleton into a contractile network, regulates the segregation of CDC-42 to the anterior cortex, and concomitantly PAR-6 localization. This study shows that the distribution of PAR is related to cortical activity and supports the model that the actin cytoskeleton plays an important role in polarity establishment.

Chlamydien sind obligat intrazelluläre Bakterien, die sich in einem membranumschlossenen Kompartiment namens Inklusion vermehren. Nach Infektion fragmentiert der Golgi-Apparat der Wirtszelle in kleine Membranstapel. Dies verbessert die Aufnahme von Sphingolipiden und ist deshalb für die chlamydiale Vermehrung essentiell. Die infektionsinduzierte Golgi-Fragmentierung geschieht nach Spaltung des Golgi-Matrix-Proteins Golgin-84. In dieser Arbeit konnte, durch den Vergleich mit bekannten Substraten und Inhibitorstudien, die chlamydiale Protease CPAF (Chlamydia protease-like activity factor) als das Enzym identifiziert werden, das diese Spaltung induziert, abhängig von der Anwesenheit zweier Rab-Proteine, Rab6 und Rab11, die den zellulären Vesikeltransport kontrollieren und zur Inklusion rekrutiert werden. Die Fragmentierung des Golgi-Apparates verhinderte dessen Relokalisierung während der Zellpolarisierung nach Einbringen eines migratorischen Stimulus. Sowohl infizierte als auch Golgin-84-depletierte Zellen migrierten langsamer und randomisiert in einem Motilitätsassay. Die Relokalisierung des Golgi-Apparates konnte durch seine Stabilisierung mittels WEHD oder Rab-Depletion wieder gewonnen werden, was die Zellmotilität teilweise wieder herstellte. Darüber hinaus konnte gezeigt werden, dass die Infektion außer der Golgi-Reorientierung die Signaltransduktion durch GTPasen beeinflusst. Die Aktivität von Cdc42 in infizierten Zellen war erhöht und die Interaktionen mit vielen ihrer Effektoren laut quantitativer Massenspektrometrie stark verändert. Die Ergebnisse dieser Arbeit zeigen, dass CPAF die für Chlamydien lebenswichtige Golgin-84 Prozessierung und Fragmentierung des Golgi-Apparates auslöst. Dies verringert die Mobilität der Wirtszelle, vor allem da der Golgi-Apparat während der Polarisierung nicht mehr ausgerichtet werden kann, des Weiteren durch Modulierung der Protein-Protein-Interaktionen von Cdc42. / Chlamydia are obligate intracellular human pathogens that proliferate inside a membrane-bound compartment called the inclusion. In infected cells, the Golgi apparatus is fragmented into small ministacks that are aligned around the inclusion. This facilitates uptake of host cell sphingolipids and is essential for chlamydial development. Infection-induced Golgi fragmentation happens after processing of the Golgi matrix protein golgin-84. This work could, via comparison with well-known substrates and inhibitor studies, identify the chlamydial protease CPAF (Chlamydia protease-like activity factor) as the enzyme accountable for this cleavage. Golgi Fragmentation depended on two Rab proteins, Rab6 and Rab11, which control vesicle transport and are recruited to the Chlamydia inclusion. As a consequence of Golgi fragmentation, cells lost the capacity to reorient the Golgi apparatus during polarization after a migratory stimulus. Both infected and golgin-84 depleted cells with a permanently fragmented Golgi apparatus displayed decelerated and furthermore randomized migration in a motility assay. Relocalization of the Golgi apparatus could be restored via stabilizing WEHD treatment or Rab depletion which partly rescued cell motility. Moreover, it could be shown that migration signaling via small GTPases was influenced by Chlamydia infection. Infected cells exhibited activation of the small polarity GTPase Cdc42. Numerous interactions with downstream effectors were strongly altered in infected cells according to quantitative mass spectrometry. Particularly, the binding of Cdc42 to migration-associated effectors was decreased. The results of this work show that CPAF, by processing of golgin-84, induces Golgi fragmentation which is vitally important for Chlamydia. This disturbs host cell motility because the Golgi apparatus cannot be reoriented during polarization and, additionally, via the modulation of protein-protein-interactions of Cdc42.