During cell division, chromosome pairs are collected on a spindle shaped microtubule scaffold. The pairs are then separated to the two poles of the spindle. Subsequently the poles move further apart, taking the chromosomes with them, and the cell membrane constricts into the spindle midplane, pinching the cell into two daughters each with an exact copy of the genetic material and half of the cytoplasm of the original. This description is a greatly simplified version of a very complicated and tightly regulated process. The microtubules are the central players; they define the basic spindle architecture, they are the tracks that guide chromosomes to their destinations at the various stages and they are the substrates that molecular motors use to push the spindle poles apart before the cytoplasm is divided. The process is of fundamental importance for both healthy and diseased cells. As cancer cells are dividing uncontrollably, the spindle is a common target for anti-cancer drugs. The proposed work is a series of structure determinations aimed at visualizing how critically important proteins and macromolecular complexes interact at foci of activity on the microtubules. The regions of interest are the plus ends of kinetochore and interpolar microtubules. At the kinetochore, coordinated microtubule assembly and disassembly result in chromosome congregation followed by separation of the chromosome pairs. Of particular interest are protein complexes responsible for chromosome attachment to disassembling microtubule plus ends. At the overlap region of interpolar microtubules, crosslinking proteins and motors determine and regulate spindle length. These crucial protein complexes will be studied. We will continue work on a protein complex that nucleates microtubule branches. Finally, we will extend recently completed work on tubulin ligases - enzymes that make posttranslational modifications to microtubules. The structure determinations will be carried out by cryo-electron microscopy and image analysis. The structural results we obtain will provide the basis for a mechanistic understanding of some of the most important events taking place on spindle microtubules during cell division.