Genetic and Cellular Analysis of Anoxia-Induced Cell Cycle Arrest in Caenorhabditis elegans

The soil-nematode Caenorhabditis elegans survives oxygen deprivation (anoxia < 0.001 kPa of O2, 0% O2) by entering into a state of suspended animation during which cell cycle progression at interphase, prophase and metaphase stage of mitosis is arrested. I conducted cell biological characterization of embryos exposed to various anoxia exposure times, to demonstrate the requirement and functional role of spindle checkpoint gene san-1 during brief anoxia exposure. I conducted a synthetic lethal screen, which has identified genetic interactions between san-1, other spindle checkpoint genes, and the kinetochore gene hcp-1. Furthermore, I investigated the genetic and cellular mechanisms involved in anoxia-induced prophase arrest, a hallmark of which includes chromosomes docked at the nuclear membrane. First, I conducted in vivo analysis of embryos carried inside the uterus of an adult and exposed to anoxic conditions. These studies demonstrated that anoxia exposure prevents nuclear envelope breakdown (NEBD) in prophase blastomeres. Second, I exposed C. elegans embryos to other conditions of mitotic stress such as microtubule depolymerizing agent nocodazole and mitochondrial inhibitor sodium azide. Results demonstrate that NEBD and chromosome docking are independent of microtubule function. Additionally, unlike anoxia, exposure to sodium azide causes chromosome docking in prophase blastomeres but severely affects embryonic viability. Finally, to identify the genetic mechanism(s) of anoxia-induced prophase arrest, I conducted extensive RNA interference (RNAi) screen of a subset of kinetochore and inner nuclear membrane genes. RNAi analysis has identified the novel role of 2 nucleoporins in anoxia-induced prophase arrest

Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest

BACKGROUND: The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O(2)) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber. RESULTS: Embryos exposed to brief periods to anoxia (30 minutes) contain prophase blastomeres with chromosomes in close proximity to the nuclear membrane, condensation of interphase chromatin and metaphase blastomeres with reduced spindle microtubules density. Embryos exposed to longer periods of anoxia (1–3 days) display several characteristics including interphase chromatin that is further condensed and in close proximity to the nuclear membrane, reduction in spindle structure perimeter and reduced localization of SAN-1 at the kinetochore. Additionally, we show that the spindle checkpoint protein SAN-1 is required for brief periods of anoxia-induced cell cycle arrest, thus demonstrating that this gene product is vital for early anoxia responses. In this report we suggest that the events that occur as an immediate response to brief periods of anoxia directs cell cycle arrest. CONCLUSION: From our results we conclude that the sub-nuclear characteristics of embryos exposed to anoxia depends upon exposure time as assayed using brief (30 minutes), intermediate (6 or 12 hours) or long-term (24 or 72 hours) exposures. Analyzing these changes will lead to an understanding of the mechanisms required for initiation and maintenance of cell cycle arrest in respect to anoxia exposure time as well as order the events that occur to bring about anoxia-induced cell cycle arrest

Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

<p>Abstract</p> <p>Background</p> <p>The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role <it>san-1</it>, the MAD3 homologue, has in <it>Caenorhabditis elegans </it>embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable <it>san-1(ok1580) </it>deletion mutant.</p> <p>Results</p> <p>The <it>san-1(ok1580) </it>animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of <it>san-1(ok1580) </it>embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of <it>san-1(ok1580) </it>embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of <it>san-1(ok1580);hcp-1(RNAi) </it>embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the <it>san-1(ok1580);hcp-1(RNAi) </it>animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of <it>mdf-2(RNAi);hcp-1(RNAi) </it>embryos but not <it>bub-3(RNAi);hcp-1(RNAi) </it>embryos was also compromised. Finally, we found that <it>san-1(ok1580) </it>and <it>bub-3(RNAi)</it>, but not <it>hcp-1(RNAi) </it>embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein.</p> <p>Conclusion</p> <p>Together, these data suggest that in the <it>C. elegans </it>embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that <it>san-1(ok1580);hcp-1(RNAi) </it>animals had a severe viability defect whereas in the <it>san-1(ok1580);hcp-2(RNAi) </it>and <it>san-1(ok1580);hcp-2(ok1757) </it>animals the viability defect was not as severe suggesting that <it>hcp-1 </it>and <it>hcp-2 </it>are not completely redundant.</p

Studying synthetic lethal interactions in the zebrafish system: insight into disease genes and mechanisms

The post-genomic era is marked by a pressing need to functionally characterize genes through understanding gene-gene interactions, as well as interactions between biological pathways. Exploiting a phenomenon known as synthetic lethality, in which simultaneous loss of two interacting genes leads to loss of viability, aids in the investigation of these interactions. Although synthetic lethal screening is a powerful technique that has been used with great success in many model organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans, this approach has not yet been applied in the zebrafish, Danio rerio. Recently, the zebrafish has emerged as a valuable system to model many human disease conditions; thus, the ability to conduct synthetic lethal screening using zebrafish should help to uncover many unknown disease-gene interactions. In this article, we discuss the concept of synthetic lethality and provide examples of its use in other model systems. We further discuss experimental approaches by which the concept of synthetic lethality can be applied to the zebrafish to understand the functions of specific genes

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Cellular and genetic analysis supports the notion that NPP-16/NUP50 and CDK-1 function to reversibly arrest prophase blastomeres in Caenorhabditis elegans embryos exposed to anoxia. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo

Genetic analysis of the spindle checkpoint genes , , and the CENP-F homologues and in -2

DIC microscopy. Phenotypes observed include a larvae arrest or slow growth phenotypes (A). For or animals phenotypes included abnormal cell morphology in the mitotic region of the gonad (white arrow head), abnormal cell morphology in the meiotic region of the gonad (white arrow), and abnormal oocyte morphology (B, black arrow head). Animals could produce sperm (B, black arrows), but the spermatheca often appeared abnormal as indicated by sperm localization. The position of the vulva is indicated by a V. Bar = 25 μm.<p><b>Copyright information:</b></p><p>Taken from "Genetic analysis of the spindle checkpoint genes , , and the CENP-F homologues and in "</p><p>http://www.celldiv.com/content/3/1/6</p><p>Cell Division 2008;3():6-6.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2265278.</p><p></p

Genetic analysis of the spindle checkpoint genes , , and the CENP-F homologues and in -5

S assayed. The MPM-2 antibody recognizes mitotic proteins located at the kinetochore and centrioles. Two typical metaphase blastomeres are shown. The metaphase blastomere shown in Q-T is of abnormal structure. Bar = 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Genetic analysis of the spindle checkpoint genes , , and the CENP-F homologues and in "</p><p>http://www.celldiv.com/content/3/1/6</p><p>Cell Division 2008;3():6-6.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2265278.</p><p></p

Genetic analysis of the spindle checkpoint genes , , and the CENP-F homologues and in -1

Ability to hatch (white filled bars) and the ability to develop to adulthood after three days on food (black filled bars). The animals were given four days to develop to adulthood. The data obtained is from four independent experiments with > 200 total animals assayed. Error bar is standard deviation.<p><b>Copyright information:</b></p><p>Taken from "Genetic analysis of the spindle checkpoint genes , , and the CENP-F homologues and in "</p><p>http://www.celldiv.com/content/3/1/6</p><p>Cell Division 2008;3():6-6.</p><p>Published online 4 Feb 2008</p><p>PMCID:PMC2265278.</p><p></p