Basic HTML Version
Poster Full Abstracts - Cell Cycle and Checkpoints
Poster board number is above title. The first author is the presenter
208
284B
The Role of Cyclin B3 in Drosophila Female Meiosis.
Mohammed R Bourouh, Rajdeep Dhaliwal, Andrew Swan. Biological Sciences, University of
Windsor, Windsor, Ontario, Canada.
The meiotic cell cycle is a highly regulated cell division yielding four genetically different gametes. As in mitosis, the meiotic cell cycle is regulated by
Cyclin Dependent Kinases (CDKs). CDKs are activated when bound to their cyclin partners. The type of cyclin bound confers the substrate specificity of the
CDK, but some redundancies exist within cyclin families. In Drosophila, there are three major mitotic cyclins, Cyclin A, B, and B3, whose role in female
meiosis has yet to be characterized. Our research focuses on the characterization of Cyclin B3 in female meiosis. By studying the loss of function phenotype
of Cyclin B3, we found that mutants arrest in anaphase of meiosis I or II. Cyclin B3 null mutants also arrest with elevated levels of Cyclin A and B.
Interestingly, expressing a stable form of Cyclin B3 causes reduced levels of the mitotic cyclins. These results suggest that cyclin B3 could play a role in
APC activation. Furthermore, expressing the stable form of Cyclin B3 causes dramatic microtubule polymerization near the meiotic spindle, as well as near
the cortex of the oocyte, though interestingly not near the male pronucleus. In wild type Drosophila, the mature oocyte arrests in metaphase of meiosis I,
with microtubules forming a network along the cortex of the oocyte. Following egg activation through ovulation, the APC is activated allowing progression
of meiosis, and the cortical microtubule network is broken down. Given our results, we hypothesize that Cyclin B3 functions after egg activation as both an
APC activator, and a regulator of microtubule dynamics.
285C
Checkpoint defects reveal specific requirements for T14 and Y15-mediated Cdk1 inhibitory phosphorylation during Drosophila development.
Joseph O Ayeni
1
, Oindrila Mukherjee
1
, Ramya Varadarajan
1
, David T Stuart
2
, Frank Sprenger
3
, Shelagh Campbell
1
. 1) Department of Biological Sciences,
University of Alberta, Edmonton, Alberta, Canada; 2) Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada; 3) Institute for
Biochemistry, Genetic and Microbiology, NF III, University of Regensburg, 93049 Regensburg, Germany.
During animal development, cell division is dynamically regulated by mechanisms that spatially and temporally control the mitotic regulatory kinase
Cdk1. The activity of Cdk1 is controlled by inhibitory kinases (Wee1 and Myt1) and Cdc25 phosphatases that regulate the phosphorylation state of two
Cdk1 residues: Y15 and T14. Both Wee1 and Myt1 target Y15 whereas only Myt1 can phosphorylate both residues, producing three distinct inhibitory
isoforms. The relevance of different phospho-isoforms catalyzed by the two Cdk1 inhibitory kinases remains unclear, however. To address this question we
examined the developmental significance of Y15 and/or T14 phosphorylation of Cdk1 by expressing Cdk1-VFP phospho-inhibition mutants in wing
imaginal discs and neuroblasts. Expression of completely non-inhibitable Cdk1 caused chromosomal aberrations and cellular defects, resulting in severe
developmental defects. Despite T14 and Y15 phosphorylation having similar inhibitory effects on Cdk1 catalytic activity, mutants affecting these residues
produced distinct cellular effects when expressed. Notably, we found that Y15 phosphorylation of Cdk1 is necessary and sufficient for checkpoint-mediated
G2 phase arrest. Although Myt1-mediated T14 phosphorylation of Cdk1 was neither necessary nor sufficient for checkpoint arrest, this modification could
prevent genome instability associated with completely non-inhibitable Cdk1. This role may underlie some of the unique developmental functions ascribed to
Myt1 kinases in Drosophila, C. elegans, X. laevis and M. muluscus. Our study has therefore provided new insights into conserved Cdk1 regulatory
mechanisms that coordinate cell cycle progression with development processes.
286A
Examination of Arf1 GTPase activity on mitotic events in early
Drosophila
embryos.
Rabab Khodary, Blake Riggs. San Francisco State University
,1600 Holloway Avenue, San Francisco , CA, 94132.
An important aspect of cellular life is the process of cell division and the equal segregation of the genetic material. Crucial to this process, is the assembly
of a microtubule based structure known as the mitotic spindle, which attaches to and segregates the chromosomes to the newly formed daughter cells. There
has been evidence suggesting that Golgi-associated factors including the small GTPase Arf1 , may play a role in mitotic spindle function. However, the
molecular mechanism involved in Golgi membrane factors and mitotic spindle function is poorly understood. We propose that Arf1 GTPase activity plays a
role in mitotic spindle function. To examine Arf1 activity, we injected the small molecule inhibitor of Arf1 activity, Brefeldin A (BFA) into the early
syncytial blastoderm of
Drosophila melanogaster
embryos. Using live fluorescent analysis, BFA was injected into embryos expressing GFP-tubulin / RFP-
Histone prior to entry into mitosis and examined for effects on mitotic spindle assembly and chromosome segregation. We observed defects in spindle sizing
and positioning and a delay in mitotic progression. In addition, injections of BFA into embryos containing a fluorescent marker for the spindle assembly
checkpoint (SAC) showed that this delay is not due to activation of the SAC. Taken together, this suggests that Arf1 activity plays a specific role in proper
spindle positioning. In addition, we plan to examine Arf1 activity by double stranded RNA inhibition of Arf1 in the early embryo, followed by injection of
recombinant Arf1 mutants. These experiments will allow for a detailed examination of changes in Arf1 activity on the effects of mitotic spindle function.
287B
Role of SCF
Skp2
in Maintaining Genome Stability.
Biju Vasavan, Nilanjana Das, Andrew Swan. Biological Sciences, University of Windsor, Windsor,
Ontario.
Polyploidy and genetic instability is characteristic of cancer. Overexpression of Skp2, an F box protein for the SCF-type E3 ligase has been observed in
many types of cancer. Skp2 negatively regulates the cell cycle by ubiquitilating key cell cycle regulators and recognizes its substrates with the help of a Cdk
(cyclin dependent kinase) interacting protein Cks. We have generated null alleles of Skp2 and Cks1 (Cks85A) in Drosophila and have noticed that loss of
either gene results in polyploidy and genetic instability. This indicates that Skp2 has a potential role as a tumour suppressor. Additionally, we see abnormal
accumulation of cells in prometaphase and metapahase, which could be a result of mitotic arrest or delay due to activation of the spindle assembly
checkpoint. We are trying to determine if this is the cause or consequence of polyploidy. Previous studies in human cell lines have shown that Skp2 protects
Cyclin A/Cdk from possible inhibition by p27, an inhibitor of Cdk. We are currently exploring the genetic and physical interaction between Skp2 and Cyclin
A in preventing polyploidy in Drosophila. Deciphering the mechanisms by which SCF
Skp2
prevents genetic instability and tumoriogenesis will be helpful in
future therapeutic research targeting cancer.
288C