Coordinated polarity and differentiation of cells in the plane of a tissue layer are essential to the development of multicellular organisms. Arabidopsis thaliana root hairs and trichomes provide model systems to study the pathways that control planar polarity and cell fate specification in plants. A concentration gradient of the plant hormone auxin provides an instructive cue that coordinates polar assembly of signalling complexes at plasma membranes of root epidermal cells; however, knowledge about additional players and cytoskeletal effectors driving cell polarization prior to hair emergence remains limited. On the other hand, epidermal cell fate specification is controlled by a well-characterized gene network of transcription factors that translate positional signals and cell-to-cell communication into tissue-wide patterning. Yet, new components are continuously found to interact with the patterning pathway, shedding light on its connections with diverse developmental processes. This thesis presents the SABRE (SAB) gene as a novel player in planar polarity establishment and root epidermal patterning. SAB is a large protein with sequence similarity to proteins present in all eukaryotes and affects planar polarity as well as orientation of cell divisions and cortical microtubules. Genetic interaction with the microtubule-associated protein gene CLASP further supports involvement of SAB in microtubule arrangement, suggesting a role for this gene in cytoskeletal organisation. Strikingly, SAB also interacts genetically with ACTIN7 (ACT7), and both ACT7 and its modulator ACTIN INTERACTING PROTEIN 1-2 (AIP1-2) contribute to planar polarity of root hair positioning. Cell-file specific expression of AIP1-2 depends on the epidermal-patterning regulator WEREWOLF (WER), revealing a connection between actin organization, planar polarity and cell fate specification. Consistent with this finding, SAB also functions in patterning of the root epidermis by stabilizing cell fate acquisition upstream of the core patterning pathway. These results unveil new roles for SAB in planar polarity and epidermal patterning and suggest that organization of the microtubule and the actin cytoskeleton are important to both planar polarity establishment and cell fate specification. / Samordning av polaritet och differentiering av celler inom ett vävnadslager är avgörande för utvecklingen av multicellulära organismer. Rothår och bladhår hos Arabidopsis thaliana utgör modellsystem för att studera signalvägar som kontrollerar planpolaritet och specifikation av cellers öde hos växter. En koncentrationsgradient av växthormonet auxin ger en instruktiv signal som koordinerar polär hopsättning av signalkomplex vid plasmamembranet i rotepidermisceller; dock är kunskapen om ytterligare aktörer och hur cytoskelettets aktörer påverkar cellpolaritet innan rothår bildas begränsad. Vad gäller differentieringen av epidermala cellers öde kontrolleras dessa genom ett väl karakteriserat nätverk av transkriptionsfaktorer som överför positionssignaler och cell-till-cell kommunikation till vävnadsomfattande mönsterbildning. Fortfarande hittas dock nya komponenter som interagerar med signalvägarna för mönsterbildning, vilket ger nya insikter om dess förbindelser med diverse utvecklingsprocesser. Denna avhandling presenterar genen SABRE (SAB) som en ny aktör i etableringen av planpolaritet och mönsterbildning av rotepidermis. SAB är ett stort protein som har sekvenslikhet med proteiner som finns i alla eukaryoter och det påverkar planpolaritet, orientering av celldelning och kortikala mikrotubler. Genetisk interaktion med genen för det mikrotubuli-associerade proteinet CLASP stärker ytterligare inblandningen av SAB i organiserandet av mikrotubler och antyder att denna gen har en roll i organiserandet av cytoskelettet. Slående är att SAB även interagerar genetiskt med ACTIN7 (ACT7) och att både ACT7 och dess modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2) bidrar till planpolaritet vid positionering av rothår. Cellfils-specifikt uttryck av AIP1-2 beror på den epidermala mönsterbildande genen WEREWOLF (WER), vilket påvisar ett samband mellan organisationen av aktin, planpolaritet och specifikationen av cellers öde. SAB fungerar även i mönsterbildning av rotens epidermis och stabiliserar förvärvet av cellöde uppströms av den centrala signalvägen för mönsterbildning. Dessa resultat visar på nya roller för SAB i planpolaritet och mönsterbildning av epidermis och indikerar att organiseringen av mikrotubler och aktin-cytoskelettet är viktiga både för etablerandet av planpolaritet och för specificeringen av cellers öde.

The actin and microtubule cytoskeleton plays a critical role in the establishment of cell polarity. Cell processes like mitosis and migration rely on the reorganization of the cytoskeleton to properly function. One driver of cell polarity is the formin, Inverted Formin-1 (INF1). INF1 is able to induce F-actin formation, activate the Serum Response Factor (SRF) pathway, stabilize microtubules, associate with microtubules, and disperse the Golgi body. Regulation of INF1 is unique, since it does not possess conserved formin regulatory domains. However, INF1 does possess many potential phosphorylation sites. In this study, we demonstrate that INF1’s ability to induce F-actin stress fibers and activate SRF is inhibited by Microtubule-Affinity Regulating Kinase 2 (MARK2). Inhibition of INF1’s actin polymerization activity by MARK2 likely occurs near INF1’s C-terminus. However, MARK2 was unable to inhibit INF1’s ability to stabilize microtubules, associate with microtubules, and disperse the Golgi. Furthermore, we show that INF1 overexpression is associated with primary cilium absence and in some cases, the presence of long cilia, suggesting that INF1 plays a role in primary cilium formation.

Die selbständige, gerichtete Bewegung von biologischen Zellen ist eine der grundlegendsten und komplexesten Erscheinungen der Natur. In höher entwickelten Lebewesen spielt die Zellbewegung eine wichtige Rolle, z.B. bei der Entwicklung des Organismus, bei der Funktion des Immunsystems aber auch bei der Metastase von Krebszellen. Die physikalischen Prozesse die dieser Fähigkeit zugrunde liegen, sind im Fokus dieser Arbeit. Um besser zu verstehen welche Prozesse im Einzelnen und in welcher Kombination den Zellen erlauben sich gerichtet fortzubewegen, wurde in der vorliegenden Arbeit ein representatives Modellsystem von motilen Zellen untersucht. Fischkeratozyten bewegen sich in vitro regelmäßig und gleichförmig, relativ schnell über die Substratfläche, und stellen aus physikalischer Sicht eine optimierte, sich selbständig bewegende Polymermaschine dar. Um Kräfte in der Bewegungsebene der Zellen zu untersuchen, wurde in der vorliegenden Arbeit eine neuartige, auf dem Rasterkraftmikroskop (RKM) basierende Methode entwickelt. Zusätzlich wurden hochaufgelöste, mit dem Phasenkontrastmikroskop aufgenommene Bilderserien analysiert und die Geschwindigkeitsverteilung in der Zelle durch Korrelationsalgorithmen bestimmt. Die Struktur des Polymernetzwerkes wurde in mit Fluoreszenzfarbstoff markierten Zellen untersucht, und elastische Eigenschaften wurden mit rheologischen RKM-Messungen bestimmt. Traktionskraftmessungen an elastischen Substraten runden das umfassende Bild ab. Durch Veränderung der molekularen Strukturen mit verschiedenen Chemikalien, die unterschiedliche Prozesse im Gesamtsystem stören, konnte nun ein Phasenraum der Kraftgenerierungsprozesse untersucht und unterschiedliche Effekte verschiedenen Prozessen eindeutig zugeordnet werden. Es wurde somit erstmalig experimentell bewiesen, dass die Polymerisation von Aktin die treibende Kraft am vorderen Rand der Zelle ist. Darüber hinaus wurde das Verhalten des Kraftaufbaus mit einem Model beschrieben, das Aufschluss über die Funktionsweise der darunterliegenden Aktinpolymerstrukturens gibt. Desweiteren wurde in der Mitte der Zelle, zwischen vorderem Rand und Zellkörper, erstmalig eine rückwärtsgerichtete Kraft gemessen, die wichtig ist um ein Kräftegleichgewicht zu erstellen. Ein Model das auf entropischen Kräften im Polymersystem basiert, beschreibt diese kontraktilen Kräfte und ordnet sie der Depolymerisation von Aktin zu. Die Bewegung des Zellkörpers wiederum basiert auf dem Zusammenspiel dieser beiden Mechanismen, sowie der Kontraktion von Aktin und Aktinbündeln durch molekulare Motoren. Eine umfassendes Charakterisierung über verschiedene lokale Mechanismen und ihrer Wechselwirkungen konnte somit erstellt werden, und damit das Verständnis der Kraftgenerierung zur Zellbewegung vertieft.

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

[Truncated abstract] Mutations in the skeletal muscle -actin gene (ACTA1) have been shown to be one cause of a broad group of muscle disorders all termed the congenital myopathies. Over 170 different mutations have now been identified across all 6 coding exons of ACTA1 in patients presenting with muscle weakness and any one or more of the following histopathological features: nemaline rods, intranuclear rods, fibre-type disproportion, excess of thin filaments and central cores. While the identification of the causative gene has been of great comfort for affected patients and their families, with pre-natal genetic testing becoming available, the ultimate aim is to develop a therapy for these disorders. Of the therapies currently being explored for the muscular dystrophies, up-regulation of an alternative gene seemed to be one of the most promising avenues for treatment of the ACTA1 diseases. Up-regulation of utrophin, the foetal homologue of dystrophin, has been shown to be a promising therapy for the treatment of Duchenne muscular dystrophy. The main aim of my research was to determine whether up-regulation of cardiac -actin, the predominant -actin expressed in foetal skeletal muscle and in the adult heart, could be used as a therapy for the ACTA1 diseases. A proof-of-concept experiment was performed whereby skeletal muscle -actin knock-out (KO) mice (all of which die by postnatal day 9) were crossed with transgenic mice over-expressing cardiac -actin (known as Coco mice) in postnatal skeletal muscle. ... While patients that are ACTA1 nulls have been identified in a number of mainly consanguineous populations, the majority of ACTA1 mutations result in dominant disease in which the mutant protein interferes with the function of the wild-type skeletal muscle -actin. Research described in this thesis also focuses on characterizing two transgenic mouse models of dominant ACTA1 disease at the ultra-structural, cellular and functional level; this is the first step towards a proof-of-concept experiment to determine whether cardiac -actin up-regulation can dilute out the pathogenesis of dominant ACTA1 disease. It has long been noted that patients with ACTA1 disease do not have ophthalmoplegia, even in the most-severely affected individuals. Protein analysis performed on extraocular muscle (EOM) biopsies obtained from humans, sheep and pigs showed that the EOMs co-express cardiac and skeletal muscle -actin, with cardiac -actin comprising 70 % of the striated -actin pool. Thus we propose that sparing of the EOMs in ACTA1 disease is at least in part due to cardiac -actin diluting out the pathogenesis associated with expression of the mutant skeletal muscle -actin. This finding provides further support for the hypothesis that dilution of mutant skeletal muscle -actin in dominant ACTA1 disease by up-regulation of cardiac -actin may be a viable therapy for this group of devastating muscle diseases. The research contained herein has advanced the understanding of the pathobiology of skeletal muscle -actin diseases and provides strong evidence in support of cardiac -actin up-regulation as a promising therapy for these diseases.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 194-227).

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 79-89). Also available on the World Wide Web.

Thesis (Ph.D.)--University of Western Sydney, 2007. / A thesis presented to the University of Western Sydney, College of Health and Science, School of Natural Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

University, Diss., 2000--München.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 September 20