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Stieß, Michael (2010): Studies on microtubule nucleation during axon growth. Dissertation, LMU München: Faculty of Biology
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Abstract

Neurons are the signaling cells of the nervous system. To propagate signals, neurons elongate several neurites, which differentiate into a single axon and several dendrites during development. Among the factors that contribute to this differentiation process, the cytoskeleton and in particular the microtubules play a key role. For instance, the growth of the axon and the dendrites depends on dynamic microtubules and requires the formation of new microtubules. The centrosome is regarded as the primary source of microtubules in axonal and dendritic growth and has been proposed to direct axon formation. However, while microtubule nucleation from centrosomes enables efficient spindle-pole organization and cytokinesis during cell division, it is difficult to reconcile the distinct microtubule array in branching axons, dendrites and spines with such focal microtubule assembly. Thus, the exact role of the centrosome and centrosomal microtubule nucleation in axon growth is still unclear. To address this question, my doctoral research focused on where microtubules are generated in developing neurons and what role centrosomal microtubule nucleation plays in axonal growth. Using rodent hippocampal neurons in culture as a model system, I found that the centrosome loses its function as a microtubule organizing center (MTOC) during neuronal development. The microtubule nucleating factor gamma-tubulin was depleted from the centrosome. Consequently, after depolymerization with nocodazole, microtubules did not regrow at the centrosome at later stages of development. Nevertheless, acentrosomal microtubule nucleation still occurred. Furthermore, axonal growth was unchanged after the centrosome has lost its activity. Moreover, when the axon was lesioned in mature neurons, a new axon grew out in the absence of centrosomal gamma-tubulin. As axons grow in mature neurons without a functional centrosome, I next asked the question of whether axon growth requires centrosomal microtubule nucleation in earlier stages of development, when the centrosome still functions as a MTOC. With the use of a two-photon laser ablation setup, the centrosome was removed in neurons that just started to form an axon. Intriguingly, the neurons retained the ability to grow an axon when the centrosome had been ablated by a laser. Thus, loss of centrosomal microtubule nucleation is not a limiting factor for axon growth and regeneration. My research implies that acentrosomal microtubule assembly is a key feature to establish the sophisticated cytoskeleton of neurons, which is the source for their complex morphology. While the centrosome is necessary for cell cycle progression and neurogenesis, neuronal differentiation requires sophisticated architectural changes that may be incompatible with a large microtubule network emanating from a focal point. Thus, acentrosomal microtubule nucleation may be a key feature during differentiation of neuronal, but also of non-neuronal cells. Dismantling the centrosome and decentralizing microtubule nucleation may be essential to enable axon branching, dendrite formation and spine generation.