Microtubule-Based Motors in Chromosome Segregation

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9507008 Saunders Recent results have indicated that spindle function in various eukaryotes is controlled by a relative balance between inwardly and outwardly-directed spindle motors. The outwardly-directed motors have recently been identified. The experiments outlined in this proposal are designed to identify the rest of the "inwardly-directed " force generating proteins and to examine the function of these motors in chromosome segregation. Library induced suppression of the kar 3 ts phenotype and the identification of mutants whose spindles do not collapse are used to identify novel members of this group. Mutational analysis and genetic combinations are used to determine if these newly identified motors act antagonistically to the known outwardly-directed motors. New mutants of inwardly-directed motors will be combined with a kar3 disruption allele to determine the "synthetic" phenotype of loss-of-function of all members of this group of mitotic motors. The goal of these mutational analysis experiments will be to determine the functional role of these inwardly-directed motors. The suggested role of these proteins as kinetochore motors an/or inhibitors of spindle elongation is investigated by examination of the effect of loss-of-function on chromosome segregation and the timing and rate of spindle elongation. For this analysis both traditional fixed cell immunocytochemistry and newly-developing real-time analysis of live cells with chromatin and green fluorescent protein- labeled tubulin staining is used. %%% All organisms are composed of microscopic components called cells, analogous to the bricks in a brick building. When the organism grows, or following injury, the mass of organism in most cases is increased not by expansion of the size of existing cells, but by creation of new cells. New cells are created by division of existing cells. The newly dividing cells can grow in size until a second round of division can occur creating four cells where one existed originally. In many unicellular organisms this process will continue indefinitely until limited by availability of nutrients and space. In larger multicellular organisms this process is carefully controlled to prevent the excess proliferation associated with cancer and other growth abnormalities. When cells divide, it is necessary that each daughter cell contain all the components required for survival. Many of the cell components are apparently randomly partitioned, but some of the components are unique and both of the daughter cells requires a certain number of each type. The most well-known example of these unique components are the chromosomes. Each chromosome contains the information to produce proteins, the building blocks of the cell. The amount of each protein made is generally proportional to the number of chromosomes present. Therefore it is critical to that organism that the cells get the right number of each type of chromosome. To achieve this, eukaryotic (non-bacterial) cells utilize a complex and transitory structure called the spindle. The spindle is made of fibers that assemble during cell division. The chromosomes attach to the spindle fibers and are moved to the daughter cells at division in an exquisitely timed and orchestrated process. Recently, this laboratory and others have identified small molecular motors that act within the cell to separate the chromosomes to the daughter cells. The motors are related to other motors discovered previously to move other cellular components. Three different types of motors were identified that can separate the chromosomes, and each can perform the task fairly well by itself. Surprisingly, a second type of motor was identified that acts in an opposite manner to pull chromosomes together. The role of this motor is unknown but it may act to delay the activity of the chromosome separating motors until the appropriate time for cell division. This project identifies and examines thes e motors which pull the chromosomes together. When all of the motors are identified, then their precise role in cell division is determined. This is done by inactivating the motors and asking what the cell is unable to do. Specifically, spindle assembly and stability and chromosome separation are carefully scrutinized. If the cell is unable to perform these function or performs in an abnormal manner, it will suggest when and where the motors work. Understanding the timing and activity of these motors allows a basic understanding of how the cell divides. ***