Browsing by Subject "Cytoskeleton"

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Entstehung und Morphogenese des Vorderhirns - Die Rolle des mit Mikrotubuli assoziierten Proteins Hmmr in Xenopus laevis
(2020) Nickel, Angela; Feistel, Kerstin
The anlage of the central nervous system is formed during early embryonic development. The neuroectoderm establishes the neural plate which folds up to form the neural tube, a process that requires extensive cell rearrangements. During further embryogenesis the anterior part of the neural tube develops into the brain while the posterior part forms the spinal cord. Disturbances during neural tube closure (NTC) lead to severe developmental aberrations. Occurrence of specific neural tube defects indicates a distinct regulation of NTC along the anterior-posterior axis. For example, the severe malformation craniorachischisis is characterized by a failure to close the neural tube from hindbrain levels onwards, while the forebrain region develops normally. This distinct regulation presents itself in the wildtype in a delay between cranial and caudal NTC. While the mechanisms leading to posterior NTC are quite well understood, the morphological processes at the future forebrain level are largely unknown. The aim of this dissertation was to identify cell and tissue morphogenetic processes which are required for the formation and development of the anterior neural tube. As the underlying changes in cell shape as well as cell migration depend on the regulation of the cytoskeleton, the role of the microtubule-associated protein Hmmr was analyzed in the model organism Xenopus laevis. HMMR is a breast cancer susceptibility gene with described roles mainly in the tumor context, regulating cell motility and maintenance of mitotic spindle integrity. In Xenopus, gain as well as loss of function of hmmr delayed NTC and led to defects during further forebrain development. Loss of hmmr impaired separation of telencephalic hemispheres, resembling the human malformation “middle interhemispheric variant of holoprosencephaly”. Failure of ventricle separation could be traced back to disturbed roof plate formation. This was due to impaired NTC resulting from a lack of neural cell convergence. Tissue convergence at the forebrain level is mediated by radial intercalation (RI). During the required regulation of cell polarization and elongation via the microtubule cytoskeleton, hmmr cooperated with the core component of the planar cell polarity (PCP) pathway vangl2, which had been solely characterized as a factor for posterior NTC so far. In addition, experiments with hmmr deletion constructs missing functional domains at the amino- and/or carboxyl-terminus, revealed that elongation and intercalation are distinct processes which are regulated differentially via specific domains of Hmmr. RI required direct binding of Hmmr to microtubules, suggesting that Hmmr influences intercalation movements by regulating dynamic instability of microtubules. RI is essential for mesenchymal to epithelial transition (MET), a physiological morpho- genetic process, which is also involved in establishing tumor metastases in a pathological context. MET is regulated by concerted interaction of canonical Wnt / beta-Catenin and non- canonical Wnt / PCP signaling. Further tissue-specific loss of function experiments uncovered a general role for hmmr in Wnt-modulated RI / MET processes during gastrulation as well as during pronephros and tailbud development in Xenopus. The results suggest that Hmmr regulates microtubule dynamics. Since canonical as well as non-canonical Wnt signaling have been associated with microtubules, hmmr could act as a molecular switch to regulate the activity and interplay of two signaling pathways. This thesis thus identified a new physiological role for the microtubule-binding protein Hmmr, which up to now had been mainly studied in the cancer context. It was shown that Hmmr-mediated RI is a major driving force for anterior NTC. In addition, Hmmr was identified as an essential regulator of microtubule-dependent Wnt signaling in MET processes.
The role of the actin binding protein Calponin2 during embryonic development of Xenopus laevis
(2021) Mantino, Sabrina Maria; Feistel, Kerstin
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.