Role of PICH (Plk-1 Interacting Checkpoint “Helicase”) in the Spindle Checkpoints of Plants Ron M. G. Menorca • Joe Ramahi • Pak Kwong • Ravi Maruthachalam • Simon Chan Department of Biological Sciences: Section of Plant Biology University of California, Davis, 95616 985.xls OBJECTIVE Why Arabidopsis thaliana? 3) Culture pich mutants in medium with microtubule depolymerizing drugs to verify PICH’s involvement in the spindle checkpoint of Arabidopsis To study PICH, a recently discovered protein proposed as the tension sensor of the spindle assembly checkpoint, using total gene knockout methods available in the plant model Arabidopsis thaliana. Availability of a complete gene knockout through Transfer-DNA Gene sequence with T-DNA insert INTRODUCTION Using multiple sequence alignment, we can track down the exact ortholog of PICH in A.thaliana. Importance of Correct Chromosome Segregation There are 46 chromosomes in a normal human cell. In cases when a there are more or less than 46 chromosomes (e.g. most cancer cells and down-syndrome) severe, if not lethal, consequences follow. The molecular basis for aneuploidy is still not fully understood, but one common theory is that a defect in one of the mitotic checkpoints of the cell during cell division causes misegregation of chromosomes. Multiple Sequence Alignment Phylogenetic tree of SNF2 helicase proteins Low effects of aneuploidy in plants FUTURE DIRECTIONS Combine PICH with other checkpoint mutants to determine a pathway for checkpoint signaling. METHODS 1) Observe and compare development and growth of homozygous pich mutants with wild-type Comparing karyotypes of normal human cell with typical cancer cell. If pich + MAD2 = phenotype X: pich + mad2 = phenotype X then same pathway pich + mad2 = phenotype Y then different pathways Spindle Assembly Checkpoint (SAC) During metaphase, chromosomes align at the equator and await segragation. The spindle assembly checkpoint, with the use of various proteins, regulates this distribution by sensing the tension exerted by the microtubules and halting anaphase until all chromosomes are correctly attached to opposite poles of the cell. The mechanism for sensing tension for the SAC is still unknown, but it is clear that this plays a major role in preventing aneuploidy. Metaphase PICTURES of ho vs. wt Localize PICH in plant cells CONCLUSION We have found that PICH is not essential for plants to live. Mutants of PICH are healthy and indistinguishable from wild-type. This presents us with two scenarios, either PICH serves a different role in the SAC of plants, or more interestingly, plants have an alternate pathway that can compensate for the loss of this particular protein. Whichever the outcome, learning more about the spindle assembly checkpoint will take us one step closer in solving the mysteries of many devastating deceases. RESULTS: Homozygous PICH knockout mutants are not only viable, but also exhibit no clear phenotype versus wild-type. PICH: The Tension Sensor of the SAC? Anaphase 2) Examine fertility differences of homozygous pich mutants by a seed counting assay. ACKNOWLEDGEMENT PICH’s characteristic that led to its proposed role in the SAC: 1) Image courtesy of Rhong Li 2) © 1995-2008 by Michael W. Davidson and The Florida State University 3) PICH accumulates at the centromere during prometaphase and lasts until anaphase. PICH-positive threads connect sister kinetochores and are dependant on tension. PICH knockdown with siRNA showed massive missegregation of chromosomes in mammalian cells which indicates PICH as an essential protein for the SAC.