My research focuses on the characterization of Mad1, a conserved SAC component, and its genetic or physical interactors. Because I am particularly interested in the role of the SAC pathway in multicellular organisms during development, my laboratory is using C. elegans as a model organism in which to analyze the physiological function of the SAC components at well-defined developmental stages. Although MAD1 in budding yeast is dispensable under normal conditions and is required only for the response to defects in microtubule or kinetochore functions, CeMAD1, the C. elegans homolog of MAD1, has essential roles in maintaining the animal's viability and fertility. This finding indicates that the correct timing of SAC-regulated mitosis is a crucial aspect of C. elegans development. Investigation of the role of MAD1 in a simple multicellular organism in combination with a biochemical approach to identify the physical interactors will provide us with new insight into the physiological function of the SAC pathway that cannot be obtained from studies with monocellular systems.

 

My research addresses the central hypothesis that CeMAD1 is involved in the initial step of the SAC-signaling pathway and that CeMAD1 activity is regulated by multiple pathways that mediate various developmental or environmental cues. My goals are to identify and characterize proteins that regulate SAC activity in multicellular organisms and to elucidate the molecular mechanism by which SAC activity is temporally and spatially regulated during development. To this end, I have been conducting research projects as described below. I am also investigating a molecular link between DNA damage checkpoint and spindle assembly checkpoint.
 

 

Figure 1: A schematic model of the SAC pathway. At the metaphase-anaphase transition, active APC/CCdc20 targets the anaphase inhibitor, securin, for ubiquitin-mediated proteolysis. Separase, activated by the degradation of securin, cleaves a cohesion subunit leading the dissociation of the cohesion complex from chromosomes, thus allowing the sister chromatid separation. MAD1-MAD2 complex specifically localizes to the unattached kinetochore to facilitate MAD2 binding to the APC/C activator, CDC20. MAD2-CDC20 complex constitutes the MCC complex along with BUBR1/MAD3 and BUB3 to inhibit the activity of APC/CCdc20, thus arresting the cell cycle at metaphase. MAD1 recruitment depends on NDC80, the RZZ complex and BUB1. NDC80 functions as part of the microtubule attachment interface.

Analyzing the molecular mechanism by which CeMAD1 causes nutritional signal–induced cell cycle arrest of germ cells will help elucidate the molecular link between the Pten and SAC pathways. The Pten pathway may activate the SAC in response to nutrition deprivation via a mechanism that is distinct from that which mediates spindle damage. This investigation of the role of Pten in the SAC pathway will help increase our understanding of Pten function in maintaining genome stability. Elucidation of the molecular mechanism by which SAC regulates cell cycle progression during embryonic or postembryonic development will also shed light on the essential function of SAC components in multicellular organisms.

 

 

Figure 2:  Molecular link between the Pten-Akt pathway and the SAC pathway in Nutrient signal–dependent cell cycle regulation. When newly hatched C. elegans larvae  (L1 stage larvae) are exposed to the starved condition, the two germ line precursor cells are arrested in prophase. MAD1 mutant worms are defective in this starvation induced prophase arrest of germ cell precursors, resulting in inappropriate germ cell proliferation. Our data suggests that MAD1 is inactive when phosphorylated by Akt kinase, and activated upon dephosphorylation by Pten phosphatase in response to the nutrient deprivation. Left scheme: The proposed model of the link between Pten-Akt [athway and the SAC pathway. Right panels: Immunoflurescence microscope images of wild type and MAD1 mutant worms. Somatic cell nuclei are visualized by staining with DAPI (blue), and germ cell precursors are marked with anti-PGL-1 antibody (red). The starved wild type L1 larva has only two germ cells, but starved MAD1 mutant larva has more than two germ cells due to inappropriate germ cell proliferation.

Our CeMAD3 synthetic lethal screen also identified a Cathepsin L cysteine protease family protein, CPL-1, and a RACK1 (receptor of activated protein kinase C1) family protein, RACK-1, and our functional analysis of these proteins by RNA interference-mediated gene knock down revealed that both proteins are required for the SAC activation in embryonic cells. We will elucidate the molecular mechanisms of the SAC activation by these proteins.

 

 

Figure 3:  C. elegans Spindly protein SPDL-1 localizes to kinetochores during prometaphase and metaphase. Immunofluorescence microscope images of one cell stage embryo in prometaphase (left) and metaphase (right). SPDL-1 (red) and tubulin (green) are immunostained by specific antibodies, and chromosomes (light blue) are stained with DAPI. Note that C. elegans is a holocentric organism, and kinetochore is assembled along the entire length of each mitotic chromosome.

The timing of the sister chromatid separation is regulated by the anaphase-promoting complex (APC) associated with Cdc20. The APC targets the mitotic regulator Securin, and degradation of Securin triggers the onset of anaphase. The SAC inhibits the APC activity via binding to Cdc20. This inhibition stabilizes Securin and delays the onset of anaphase. I identified a novel C. elegans protein IFY-1, which interacts with C. elegans Cdc20 (FZY-1) and separase (SEP-1). IFY-1 is accumulated in one-cell–arrested embryos of an APC-deficient strain. RNA interference (RNAi)–induced reduction of IFY-1 expression causes an embryonic-lethal phenotype. These findings suggest that IFY-1 has a Securin function in the mitotic cell cycle. We generated a transgenic worm strain expressing a functional IFY-1-GFP, and analyzed the developmental stage dependent alteration of IFY-1 localization in germ cells. Our data strongly suggests that IFY-1 is a good indicator of the APC activity and the SAC activity in germ cells undergoing meiosis. Using this IFY-1-GFP expressing worm strain, we will evaluate the requirement of each SAC component for the meiotic progression. We have also identified that a ubiquitin ligase E3 with a HECT domain physically associates with IFY-1, and RNAi mediated knock down of this E3 ligase resulted in an increased IFY-1 level during meiosis II, causing the delay in meiosis II. We are currently investigating how this E3 ligase is integrated into the regulatory mechanism of Securin function. These studies will provide molecular insight into the maintenance of genome integrity during reproduction.

 

 

Figure 4:  Meiotic cell cycle dependent alteration of subcellular localization of IFY-1. Time lapse images of a proximal region of a gonad arm of an adult hermaphrodite expressing IFY-1-GFP (green) and RFP-histone (red). The image sequences are aligned from the top to the bottom. Note that time interval between each sequence are not equal. IFY-1-GFP colocalizes with meiotic chromosomes during prometaphase I and metaphase I, and disappears upon the onset of anaphase I. The intensity of cytoplasmic IFY-1-GFP also drastically diminished immediately after anaphase I.

Experimental data have suggested that the frequency of cells surviving drug treatment increases when the SAC is compromised. However, the relationship between the duration of mitotic delay and cell survival is unknown. It is therefore important to establish the molecular relationship between the SAC pathway and cell death or survival pathways to understand the molecular basis of drug sensitivity of cancer cells. We will study the role of the SAC pathway in activating the cell death pathway in response to chemical or mutational disruption of microtubules, using C. elegans strains in which nonessential SAC genes have been deleted. C. elegans is a useful model because some conserved SAC components are dispensable, and we can analyze the cellular response to antimitotic agents in the absence of functional SAC. Also, the cell death pathway of C. elegans has molecular components similar to those of the apoptotic pathway in mammals.

In budding yeast, the APC targets a yeast Securin Pds1 to trigger the sister chromatid separation. In the presence of DNA damage, the checkpoint kinases Mec1 and Rad53 mediate Pds1 phosphorylation, a finding that suggests that Pds1 is involved not only in the SAC pathway, but also in the DNA damage checkpoint pathway (Cohen-Fix and Koshland, Proc Natl. Acad. Sci. U S A. 1997). Because IFY-1 and Pds1 share a common feature in the SAC pathway, It would be interesting to test whether IFY-1 is a target in the DNA damage checkpoint pathway. This research focus will be initiated by investigating the stability of IFY-1 in DNA damage checkpoint–deficient strains.

 

Although the worm model is one of the most powerful tools for identifying components involved in signaling pathways, it might have some limitations in regard to determining their functional significance in mammals because of the difference in the complexity of the two organisms. Therefore, it would be appropriate to analyze the function of mouse homologs in mouse models. I will generate a knock-out or knock-in mouse, which will be chosen from mutants identified by the genetic screens mentioned above.