RT Dissertation/Thesis T1 The role of the actin binding protein Calponin2 during embryonic development of Xenopus laevis A1 Mantino,Sabrina Maria WP 2022/01/18 AB Despite the abundant variability among adult vertebrate body plans, the developmental steps transforming the single zygote into a multicellular organism of remarkable complexity, are evolutionary highly conserved. Morphogenetic processes such as gastrulation, neural tube closure, body axis extension, neural crest cell migration and organogenesis are thereby at the heart of embryogenesis. Especially the formation of a closed neural tube, which gives rise to the central nervous system, constitutes a fundamental event. Neural tube closure is achieved by convergent extension movements and by apical constriction of neuroepithelial cells. Along with proceeding neurulation, cranial neural cells start to delaminate from the neuroepithelial border. In order to initiate directed migration movements, neural crest cells require polarised cell protrusions and mediate mechanical forces. Changes in cell shape and motility underlying neural tube closure and neural crest cell migration are controlled by specific regulation of the actin cytoskeleton. How these actin dynamics and the myosin-mediated contraction of actin networks are precisely coordinated is not fully understood. In this context, actin filament-associated proteins play an important role for the structural organisation of different actin network types. Calponins constitute an evolutionary highly conserved family of F-actin binding proteins, which are able to influence actin-myosin dynamics and to stabilise actin filaments. Previous studies already demonstrated a role of Calponin proteins in smooth muscle contraction, cell motility and phagocytosis. Vertebrates possess three Calponin isoforms, each displaying specific expression patterns and functions. Calponin2 is expressed in a variety of cell types and several studies performed in vitro indicated that Calponin2 is important for mechanical tension mediation during the course of cell migration. In the early embryo of Xenopus laevis, calponin2 is expressed in tissues that undergo extensive morphogenetic movements and cell migration. This implies an elemental role of Calponin2 for respective morphogenetic steps during embryonic development of this well-established model organism. Within the scope of the present work, the specific function of Calponin2 for dynamic regulation of the actin cytoskeleton was analysed more closely. Localisation of the protein, by utilising a tagged construct, was shown in neural plate cells as well as in migrating neural crest cells. In both cell types, regulated protein degradation occurred, which led to specific expression restricted to the apex of constricting neural plate cells or to forming lamellipodia. Thus, tagged Calponin2 localised to regions of the actin cortex. Loss of Calponin2 function led to defects in neural crest cell specification and migration as well as in convergent extension and apical constriction within the neural plate. All induced phenotypes were rescued by additional calponin2 mRNA injection. In summary, these data demonstrated a specific function of Calponin2 for correct formation of the neural crest as well as for neural tube closure. Furthermore, the precise regulation of protein expression levels, which directly correlated with correct Calponin2 function, was dependent on specific domains that potentially mediate actin-binding. Clik1, Clik2 and the C-terminus were identified as a critical unit regulating protein degradation, both in neural crest cells and neural plate cells. Additionally, it was shown that Calponin2 function for neural apical constriction depends on each of these domains as well. Overall, the degradation of Calponin2, regulated via its F-actin binding, implies a filament stabilising function. Thus, a temporospatial coordination of protein degradation would be necessary to enable dynamic changes of the actin cytoskeleton by a regulated release of actin filaments and to allow the association of other structural effectors during morphogenetic processes of early vertebrate development. K1 Embryologie K1 Neuralleistenzellen K1 Neurulation K1 Morphogenese K1 Zytoskelett PP Hohenheim PB Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim UL http://opus.uni-hohenheim.de/volltexte/2022/1982