Timothy P. Stearns
Microtubule Organization in Yeast and Animal cells
All cells are polar, and the key determinant of this polarity is the cytoskeleton. Microtubules are unique among the cytoskeletal filaments in that their structure, number, and orientation are controlled by an organizing center. This microtuble organizing center, or centrosome, nucleates the assembly of microtubules from soluble tubulin subunits and maintains an attachment to one end of the resulting polymer. In addition, the centrosome duplicates once per cell cycle, so that there is one centrosome in an interphase cell and two centrosomes in a mitotic cell, where they form the two poles of the mitotic spindle.
The centrosome is a complex organelle, and to understand how it works it will be necessary to define its components and determine how they work together. One of the key components is gamma-tubulin, a special tubulin that is localized to the centrosome and required for centrosome function. Gamma-tubulin is highly conserved, allowing the use of diverse approaches to determine its function and identify the proteins that it interacts with. First, we are studying the Saccharomyces cerevisiae gamma-tubulin gene, TUB4. Tub4p is localized to the yeast spindle pole body, TUB4 is essential for viability, and tub4 mutations affect the function of the spindle pole body. Specifically, conditional-lethal mutations result in a monopolar mitotic spindle. Electron microscopy reveals that this is due to a defect in spindle pole body duplication; the new spindle pole body of a duplicated pair does not nucleate microtubules and does not separate from the old spindle pole body.
The second approach is biochemical and involves purification both of gamma-tubulin in its native form from the cytoplasm of cells, and gamma-tubulin by itself, using a baculovirus overexpression system. Cytoplasmic gamma-tubulin is in the form of a 25S complex that contains gamma-tubulin and several other proteins. A cell line expressing an epitope-tagged gamma-tubulin has been created, allowing purification of the complex. To identify the activities of gamma-tubulin itself, epitope-tagged gamma-tubulin has been overexpressed in and purified from insect cells; this gamma-tubulin is monomeric. Both the complex and the monomeric form are being examined for activities in in vitro assays for centrosome and microtubule function.