Formation of two microtubule-nucleating sites which perform differently during centrosomal reorganization in a mouse cochlear epithelial cell

This report provides evidence for the formation of a cell surface-associated centrosome with two spatially discrete microtubule-nucleating sites that perform differently; the minus ends of microtubules remain anchored to one site but escape from the other. Centrosomal reorganization in the cells in question, outer pillar cells of the organ of Corti, indicates that its pericentriolar material becomes intimately associated with the plasma membrane at the two nucleating sites. Two large microtubules bundles assemble in each cell. A beam which includes about 1,300 microtubules spans most of the cell apex. It is positioned at right angles to a pillar with about 4,500 microtubules which is oriented parallel to the cell's longitudinal axis. The beam's microtubules elongate from, and remain attached to, a centrosomal region with two centrioles which acts as a microtubule-nucleating site. However, the elongating microtubules do not radiate from the immediate vicinity of the centrioles. During beam assembly, the minus ends of the microtubules are concentrated together close to the plasma membrane (less than 0.2 micron away in many cases) at a site which is located to one side of the cell apex. High concentrations of the pillar's microtubules elongating from one particular site have not been detected. Analyses of pillar assembly indicate that the following sequence of events occurs. Pillar microtubules elongate from an apical cell surface-associated nucleating site, which becomes more distantly separated from the centriolar locality as cell morphogenesis progresses. Microtubules do not accumulate at this apical nucleating site because they escape from it. They migrate down to lower levels in the cell where the mature bundle is finally situated and their plus ends are captured at the cell base.