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
OBJECTIVE
Why Arabidopsis thaliana?
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.
INTRODUCTION
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.
985.xls
Gene sequence with T-DNA insert
Using multiple sequence alignment, we can track down
the exact ortholog of PICH in A.thaliana.
Multiple Sequence Alignment
Phylogenetic tree of SNF2 helicase proteins
Low effects of aneuploidy in plants
FUTURE DIRECTIONS
METHODS
Comparing karyotypes of normal
human cell with typical cancer cell.
Spindle Assembly Checkpoint (SAC)
Metaphase
Availability of a complete gene knockout through Transfer-DNA
3) Culture pich mutants in medium with microtubule
depolymerizing drugs to verify PICH’s involvement in
the spindle checkpoint of Arabidopsis
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.
PICH: The Tension Sensor of the SAC?
Anaphase
PICH’s characteristic that led to its proposed role in the SAC:
1) PICH accumulates at the centromere during prometaphase and
lasts until anaphase.
2) PICH-positive threads connect sister kinetochores and are
dependant on tension.
3) PICH knockdown with siRNA showed massive missegregation
of chromosomes in mammalian cells which indicates PICH as an
essential protein for the SAC.
1) Observe and compare development and growth of
homozygous pich mutants with wild-type
PICTURES of ho vs. wt
Combine PICH with other checkpoint mutants to
determine a pathway for checkpoint signaling.
If pich + MAD2 = phenotype X:
•pich + mad2 = phenotype X then same pathway
•pich + mad2 = phenotype Y then different pathways
Localize PICH in plant cells
CONCLUSION
RESULTS: Homozygous PICH knockout mutants are not only
viable, but also exhibit no clear phenotype versus wild-type.
2) Examine fertility differences of homozygous pich
mutants by a seed counting assay.
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.
ACKNOWLEDGEMENT
1) Image courtesy of Rhong Li
2) © 1995-2008 by Michael W. Davidson and The Florida State University
3)