ABSTRACT
The fertilising sperm triggers a transient Ca(2+) increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C(cdc20) inhibitor, links release from metaphase II arrest with the Ca(2+) transient and its degradation is triggered by the Ca(2+)-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca(2+) transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca(2+)-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca(2+) increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Cycle Checkpoints , Meiosis , Ovum/cytology , Phosphoprotein Phosphatases/metabolism , Urochordata/cytology , Urochordata/enzymology , Anaphase-Promoting Complex-Cyclosome/antagonists & inhibitors , Anaphase-Promoting Complex-Cyclosome/metabolism , Animals , Antigens, Polyomavirus Transforming/metabolism , Calcineurin/metabolism , Calcineurin Inhibitors , Calcium/pharmacology , Calcium Signaling/drug effects , Cell Cycle Checkpoints/drug effects , Cyclin B/metabolism , Enzyme Activation/drug effects , Fertilization/drug effects , Mammals/metabolism , Meiosis/drug effects , Metaphase/drug effects , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Ovum/enzymology , Protein Phosphatase 2/metabolism , Rats , Substrate Specificity/drug effects , Urochordata/drug effectsABSTRACT
The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators and is also a kinetochore dynamic protein. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive. Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 °C) and gradually organizes the nuclei in a single monolayer just underneath the cortex. Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.
Subject(s)
Drosophila Proteins/physiology , Drosophila/physiology , Kinetochores/physiology , M Phase Cell Cycle Checkpoints/physiology , Animals , Animals, Genetically Modified , Cdc20 Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Drosophila/cytology , Drosophila/embryology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Spindle Apparatus/genetics , Spindle Apparatus/metabolismABSTRACT
BACKGROUND: Health departments use reactor grids (sex, age, and serologic test for syphilis [STS] titer criteria) to determine which persons to evaluate for untreated syphilis. GOAL: The goal of the study was to assess reactor grid performance in Chicago and reactor grid use nationally in 1999 to 2000. STUDY DESIGN: We reviewed Chicago health department records to identify characteristics of persons with a reactive STS excluded from evaluation by reactor grid criteria and syphilis cases not meeting evaluation criteria. We surveyed health departments regarding reactor grid use. RESULTS: Of persons with a reactive STS, 46% did not meet criteria for health department evaluation, including 62% of men, 29% of women, and 21% with titers > or =1:8. The reactor grid would have excluded 17% of primary syphilis cases. Overall, 82% of health departments use reactor grids. CONCLUSIONS: Reactor grids are widely used and may exclude persons with infectious syphilis from health department evaluation, especially men. The impact of reactor grid use on syphilis control and surveillance in the United States should be evaluated.