Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Graduate Category: Physical and Life Science Degree Level: Graduate, PhD Abstract ID# 1046 A novel myosin light-chain kinase is required for ovulation in the C. elegans spermatheca Charlotte 1Department of Biology, Northeastern University, Boston, MA ZC373.4 is required for proper spatiotemporal calcium signaling B Figure 1: A Cartoon of human (above) and C. elegans (below) myosin light-chain kinase proteins with conserved actin binding, calmodulin binding, and protein kinase domains. B The alignment of the kinase domains (orange in A) for human MYLK and C. elegans ZC373.4 shows a high degree of agreement with darker greys representing greater similarity. C B whole worm A C. elegans reproductive system in Figure 4: A Comparison of calcium D plots normalized to the average intensity detected prior to ovulation (F/FO). The top three plots of ovulations representing worms that have been fed ZC373.4 RNAi. The bottom three plots are WT calcium traces. It is clear that ZC373.4 is required for normal calcium oscillations and oocyte transit. B Knocking down ZC373.4 in worms causes a decrease in average pixel intensity of the 30 frames prior to end ovulation. C Depletion of ZC373.4 also leads to a lower maximum calcium level compared to wild type. D Kymograms of both WT and ZC373.4 RNAi treated worms shows the disruption in normal calcium localization. The distal neck is on the left of each kymogram and the sp-ut valve is on the right. Conclusions and Future Directions ZC373.4 regulates the phosphorylation of MLC A before ovulation B 15 C * 20 Conclusions Based on these data we can conclude that ZC373.4 regulates transit. ZC373.4 is similar to human myosin light-chain kinase and is likely phosphorylating the regulatory MLC in the spermatheca. Additionally, ZC373.4 RNAi results in abnormal calcium signaling during ovulation. . * * * Maximum Intensity (AU) Average Intensity (AU) 15 Ovulation requires a balance of phosphorylation 10 oocyte trapped 5 Future Directions We aim to understand the functions of the other domains of our new myosin light-chain kinase by using CRISPR to delete portions of the C-terminal end which shares little homology to the human MYLK. We also plan to use CRISPR to fluorescently label ZC373.4 with mKate2 and a FLAG tag. We can then perform imaging experiments to determine its localization along with pull downs to determine how it is interacting with other proteins 10 5 Ser19 0 Hypothesis ZC373.4 is the MLCK that phosphorylates the myosin light chain and drives spermatheca cell constriction after ovulation m el -1 1 ZC 37 3. 4 Em pt y el m ZC 37 3. -1 4 1 0 Em pt y Ser19 phosphorylation gives the myosin complex actin binding and ATPase capabilities out start Figure 2: A Time course of a worm treated with ZC373.4 RNAi. The embryo enters and is trapped for nearly an hour while the embryo divides in the spermatheca. B ZC373.4 was depleted using RNAi in worms that suppress the gene ubiquitously (UN1108) and only in the spermatheca (UN1524). Both worm lines express fln-1P::GFP to allow visualization of trapped or empty spermathecae on a plate of freely moving worms. ZC373.4 RNAi caused trapping comparable to plc-1 and fln-1 RNAi, both of which have been shown to be required for successful ovulations1,2. UNC-89 is the only other known MLCK in C. elegans and does not seem to cause trapping. Although LET-502/ROCK can phosphorylate rMLC, it does not cause significant trapping compared to WT. Within the spermatheca Maximum intensity is less in ZC373.4 deficient worms WT Depletion of ZC373.4 causes oocyte trapping in the spermatheca Introduction Coiled-coil tails can form bipolar filaments B A Background calcium is less in ZC373.4 deficient worms ZC373.4 RNAi A spermathecaspecific RNAi Erin J. 1 Cram ZC373.4 shares homology with human myosin lightchain kinase Abstract The C. elegans somatic gonad is essentially a single layer tube of myoepithelial cells that coordinate oocyte maturation and ovulation through the spermatheca, the site of fertilization. Oocyte entry into the spermatheca triggers calcium oscillations that culminate in coordinated actomyosin constriction and expulsion of fertilized embryos. Scaffold proteins, phosphatases, and kinases are required for this coordinated contraction of the actomyosin cassette. To date, there has only been one myosin light-chain kinase (MLCK) identified in the worm and it is not necessary in C. elegans spermathecal actomyosin constriction. In a broad RNAi screen, we have identified a potential MLCK required for successful ovulation, ZC373.4. This MLCK shares key homology to human myosin light-chain kinase (MYLK) domains. Depletion of ZC373.4 via RNAi causes oocyte trapping in the spermatheca. This trapping phenotype is likely due to a lack of coordination between contractions in the distal end and opening of the spermatheca-uterine valve. To further characterize this potential MLCK, ZC373.4 will be fluorescently tagged to identify its localization within the worm. The protein will also be immunoprecipitated to conduct kinase assays to analyze its kinetic activity in vitro. This work will characterize a new MLCK in C. elegans and further elucidate the mechanism by which cells of the somatic gonad control ovulation and spermathecal transit. 1 Kelley , Figure 3: A Preliminary results show phosphorylated MLC (pS20) localizes primarily to the apical surface of the spermatheca. (red = actin; green = phosphorylated MLC) Scale bar = 20μm. B Quantification of empty/WT, ZC373.4, and mel-11 RNAi conditions shows that phosphorylation levels change between the different conditions. C Highest phosphorylation occurs when the myosin phosphatase, MEL-11, is removed. Removal of ZC373.4 results in the least phosphorylation. References 1. Kariya, K. I., Kim Bui, Y., Gao, X., Sternberg, P. W., & Kataoka, T. (2004). Phospholipase C epsilon regulate ovulation in Caenorhabditis elegans. Developmental Biology, 274(1), 201–210. doi:10.1016/j.ydbio. 2004.06.024 2. Kovacevic, I., Orozco, J. M., & Cram, E. J. (2013). Filamin and phospholipase C-ε are required for calcium signaling in the Caenorhabditis elegans spermatheca. PLoS Genetics, 9(5), e1003510. doi:10.1371/ journal.pgen.1003510 NIH R01 GM110268 to E.J.C. funded this work