| Summary: | Microtubules are non-covalent polymers that form an essential part of the cytoskeleton in eukaryotic cells. Alternating phases of growth and shortening are essential for space exploration, force generation and facilitate rearrangements of the microtubule cytoskeleton in response to various stimuli. Microtubule associated proteins regulate filament dynamics and can transport cargoes. The mechanism of how microtubules grow, what triggers the transition from a growing to a shrinking microtubule, and the interplay between the various microtubule-associated proteins is only poorly understood. In vitro reconstitution approaches from purified components in combination with microfluidics techniques and simultaneous multi-colour total internal reflection florescence microscopy were employed to shed new light on the mechanism of microtubule dynamics and the interplay of proteins that bind specifically to growing microtubule ends. Tubulin undergoes conformational changes during incorporation into the polymer. Using a conformation-sensitive designed ankyrin repeat protein probe, it has been found here that these conformational changes occur at much later steps during incorporation into the polymer than previously appreciated. Growing microtubules switch to a rapid shortening phase unless their ends contain a stabilizing structure whose nature is not fully understood. The decay of this stabilizing structure was directly measured by rapid tubulin dilutions and predictions from several theoretical models have been tested. The density of a particular tubulin conformation recognized by microtubule End Binding proteins (EB1/Mal3) could be linked to filament stability. Microtubule end tracking proteins form a dynamic protein interaction network. Here, the molecular mechanism of several main players of these proteins that lead to growing microtubule end accumulation of the motor protein dynein has been elucidated by in vitro reconstitutions. The bottom up approach applied in this thesis yielded new information about fundamental properties of microtubule dynamics and gained new insight into the interplay of an important class of microtubule associated proteins.
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