The organization of the microtubule cytoskeleton and its role on nuclear dynamics in the multinucleate hyphae of "Ashbya gossypii" revealed by live cell imaging and electron microscopy

Nuclear migration is important for normal growth and development of all eukaryotes including filamentous fungi. Among those, Ashbya gossypii, which is evolutionarily related to the budding yeast Saccharomyces cerevisiae, is a particularly attractive organism to study nuclear migration in multi-nucleated, elongated cells (hyphae). Upon hyphal extension, nuclei move toward the growing tip, showing not only long-range migration and asynchronous division, but also extensive nonsynchronized oscillations and occasional bypassing. However, little is known how these processes are coordinated. This thesis is structured in three parts that all share the common purpose to increase our understanding how the particular nuclear dynamics of A. gossypii are controlled with a gene set very similar to the mono-nucleate budding yeast S. cerevisiae. In part I a detailed characterization of the A. gossypii microtubule cytoskeleton is presented. By combining high-resolution electron microscopy analysis of A. gossypii MTOCs and associated microtubules with live cell imaging of GFP-Tub1 labeled microtubules, we found distinct differences to the S. cerevisiae SPBs and a plausible answer to the question how nuclei can behave independently in the continuous cytoplasm of A. gossypii. Part II describes the functional analysis of several A. gossypii MTOC components. Unexpected deletion phenotypes reveal that even though the A. gossypii genome encodes orthologs for all presently known S. cerevisiae SPB genes, their cellular function show distinctive differences to the S. cerevisiae homologs. Our data also suggests that an evolved SPB outer plaque and the cytoplasmic microtubules that it nucleates account for the complexity of nuclear dynamics in A. gossypii. Part III of this thesis deals with the role of the A. gossypii ortholog of Kar9, a protein that is required for the establishment of spindle asymmetry in S. cerevisiae. The presented data indicate that spindle asymmetry and orientation prior to mitosis is not critical in the continuous cytoplasm of A. gossypii.