This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The mitotic spindle, a bipolar microtubule array, plays a critical role in chromosome segregation during mitosis. Conventionally, the centrosome is considered to be th emajor microtubule nucleation center in the spindle. However, through live imaging and functional studies, several laboratories including ours have recently proposed a new model in which many spindle microtubules are generated inside the spindle, and therefore, minus-ends of the microtubules are distributed throughout the spindle. Furthermore, we have identified a protein complex 'augmin'that is responsible for the intra-spindle microtubule nucleation, in which new microtubules are possibly nucleated from the existing microtubules (""""""""branched nucleation"""""""") (Goshima et al. 2007, 2008;Uehara et al. 2009, 2010). However, these models have not been proven, because the distribution of minus-ends have not been determined by electron microscopy in human cells. In this study, we would like to test the models through directly visualizing the minus-ends of spindle microtubules using electron tomography. Specifically, we wish to perform 1) Tomographic reconstruction of 3-D data and 3-D modeling for the metaphase spindle of human U2OS cells. 2) the same analysis for the U2OS cells depleted of augmin, which resulted in reduced microtubule numbers in the metaphase spindle, and compare with the control cells.
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