Neue Werkzeuge für Hydra zur Untersuchung von Rho- und PtdInsP- vermittelten Signalwegen

Table of Contents: Preliminary efforts of the working group showed that the inhibition of different components of the FGFR-Rho-ROCK-Myosin II signaling pathway results in the same phenotype and prevents bud detachment in Hydra. It was demonstrated that budding is a complex, precisely timed process that ultimately results in the accumulation of actin at the bud base, which correlates with myosin II phosphorylation (Holz et al., 2017; Holz et al., 2020). Small Rho- GTPases act as main regulators of actin dynamics and actomyosin contraction (Fagotto, 2014; Fagotto et al., 2013; Menke and Giehl, 2012). A database search of the human RhoA- GTPase using BLAST surprisingly identified four homologous Rho- GTPases in Hydra. Phylogenetic studies showed that Hv_Rho1-3 clearly group within the RhoABC subfamily, while Hv_Rho4 is basal to the subfamily. In-situ-hybridization revealed that the four Rho- GTPases are expressed in morphologically active tissue regions, whereby Hv_Rho1 and Hv_Rho2 in particular are probably involved in cellular processes at the bud base. In addition to immunohistochemical antibody staining, the fusion protein RBD-GFP is effective to detect active RhoA. RBD-GFP specifically binds to active RhoA through the Rho-binding-domain (RBD) (Berger et al., 2009). RBD-GFP was expressed, purified and established as a new method to identify active RhoA- homologous Rho- GTPases in fixed Hydra tissues, such as evaginating bud or tentacle tissue or contracting tissue at the bud base or during regeneration. The in-silico analysis of the Rho- GEFs Kalirin and Trio illustrated a complex and evolutionarily interesting development of the Rho- GEFs in terms of dynamic losses and gains of specific protein domains. Interestingly only one Kalirin- homologous sequence was detectable in Hydra, which possibly mediates the connection between membrane-bound lipids and the Rho- GTPases. In addition to the Rho-ROCK-Myosin II signaling pathway, the involvement of phosphatidylinositol phosphates (PtdInsP) in morphological processes in Hydra was investigated. Generated transgenic Hydra- lines serve as a valuable tool to observe the activity of Phopholipase C (PLC) and PI3- kinase live and in vivo. The use of such transgenic lines demonstrated the activity of PLC at the bud base during budding, as well as a dynamic activity of the PI3- kinase at the interface during regeneration. Furthermore, the PtdInsP- sensors verified the apical localization of PtdIns(4,5)P2 as well as the basal localization of PtdIns(3,4,5)P3. To permit detailed in vivo observation and especially the in vivo microscopy of PtdInsP-GFP- sensors and their cellular localization, different relaxants were tested. Only linalool and benzocaine achieved an effective immobilization for Hydra. Both relaxants showed no significant influence on the process of budding or wound closure after incision. However, analysis of the actin fibers showed that linalool, in contrast to benzocaine, disturbed the polyp's actin cytoskeleton, leading to incorrect fiber orientation and significant reduction of fiber length. Based on existing tools for the analysis of morphogenesis in vertebrates and flies, the fusion protein RBD-GFP and the PtdInsP-GFP- sensors, were established as two interesting methodological tools for Hydra. For the first time, the activity of the Rho- GTPases at the bud base, as well as the cellular localization of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in ectodermal epithelial cells, could be demonstrated. The established methods are useful for detailed investigation of the Rho- GTPases, as well as the PLC and PI3- kinase signaling pathways and their functions during morphogenetic processes in Hydra. Furthermore, an effective immobilization and relaxation protocol that is gentle on actin fibers was developed for Hydra using Benzocaine. In the future, this could be convenient for in vivo microscopy and mechanical manipulation, such as incision of the polyp for regeneration.