Hajnal A (2002) Mol Cell "Fine-tuning the RAS signaling pathway: Zn(2+) makes the difference."

Hajnal A

[Mol Cell, 2002]

In the May, 2002 issue of Developmental Cell, Bruinsma et al. report that the CDF-1 cation diffusion facilitator protein is required for efficient Ras-mediated signaling in C. elegans. CDF-1 reduces intracellular Zn(2+) levels, indicating an inhibitory effect of Zn(2+) on the Ras pathway.

Marjolein Wildwater et al. (2008) Development & Evolution Meeting "Regulation Of Asymmetric Cell Division During Development Of The Post-Embryonic Seam Cell Lineages"

Wytse Bruinsma, Matilde Galli, Sander Van Den Heuvel, Marjolein Wildwater

[Development & Evolution Meeting, 2008]

During growth and development an animal has to generate a proper number of cells with the right cell fates. One way to simultaneously generate new and different cells is through asymmetric cell division, in which a cell generates two daughters with different fates. Tight regulation of asymmetric cell division frequency is critical for normal development. Defects in this process have major consequences in tissue regeneration and repair and likely contribute to tumour formation. C. elegans hypodermal seam cells are an excellent model system to study the regulatory mechanisms of asymmetric cell division since most seam cells divide in an asymmetric manner once during each larval stage. Interestingly, the majority of seam cells also include a single round of symmetrical division during the second larval stage. To study seam cell divisions in detail we first aimed to set up a method for in vivo time-lapse recording. Although in vivo time-lapse recordings have been extensively used in studies of C. elegans embryonic divisions, a proper method to record larval divisions was never reported. To facilitate filming we generated several strains expressing GFP::H2B (to mark DNA), GFP::PH (to mark membranes), GFP::a-tubulin and GFP::g-tubulin (to mark the spindle) specifically in the seam lineages. We use these tools to study spindle positioning during seam cell division. We could show that spindle poles migrate in a flexible but limited number of directions in both asymmetric and symmetrically dividing seam cells before they end up at the anterior and posterior side of the cell. In the L2 stage, only asymmetric cell division results in two differently sized daughter cells, indicating active spindle positioning. In order to study how division frequency and polarity is controlled in seam cells we have initiated two already successful approaches, a candidate gene approach and a mutagenesis screen. Mutants derived from this latter screen can be subdivided in three different classes: mutants of the first class are affected in division frequency, mutants of the second class are affected in division polarity and mutants of the third class are affected in both. To understand how our candidate genes of interest and our novel mutants control asymmetric seam cell division we are currently introducing reporter constructs into the mutant background to be able to perform in vivo time-lapse studies.

Cirillo, L. et al. (2015) International Worm Meeting "Coordinating Cdk1 and Plk1 activation for mitotic entry in early C. elegans embryos and human cells."

Pindard, L., Thomas, Y., Martino, L., Santamaria, A., Gotta, M., Schwager, F., Van Hove, L., Panbianco, C., Cirillo, L.

[International Worm Meeting, 2015]

Entry into mitosis is regulated by several kinases including Cdk1, Aurora A and Plk1. However, how the activity of these kinases is coordinated in space and time is not fully understood. Our laboratories have recently shown that SPAT-1, an activator of Plk1, is phosphorylated by Cdk-1 at several sites in C. elegans. Our in vivo and in vitro data revealed that phosphorylation of the N-terminal sites is crucial to promote the interaction with PLK-1 and its activation. Together, our results suggest the existence of a positive feedback loop that connects CDK-1 and PLK-1 via SPAT-1 and ensures a robust control of cell division timing during embryonic development.The human ortholog of SPAT-1, Bora, regulates several aspects of mitosis (Bruinsma et al. 2014, Chan et al 2008, Macurek et al. 2008, Seki et al. 2008). Although phosphorylation of Bora is not strictly required to activate Plk1 (Seki et al. 2008, our unpublished data) we find that Cdk1 phosphorylation of Bora greatly increases Plk1 activation in vitro. We have mapped the Cdk1 phosphorylation sites of Bora. Based on the results obtained in C. elegans, we have mutated a number of N-terminal sites and found that Bora ability to activate Plk1 is greatly reduced in vitro. We are currently testing the function of these sites in human cells by monitoring these mutants in vivo. Our data will help to shed light on the activation mechanisms of Plk1 by Bora. Since Plk1 is overexpressed in many cancers, understanding the precise mechanisms of activation of this kinase will help the design of anti-cancer drugs.References:Bruinsma W. et al. J Cell Sci. 2014; Chan EH. et al. Chromosoma. 2008; Macurek L. et al. Nature. 2008; Seki A. et al. Science. 2008.

Dennis Eastburn et al. (2003) International Worm Meeting "Further lessons in the regulation of cell signaling through analyses of suppressors of activated ras."

John Yoder, Mingxue Cui, Min Han, Fang Wang, Dennis Eastburn

[International Worm Meeting, 2003]

Suppression of the Multivulva phenotype of activated ras mutations has been an important assay used in a number of laboratories in identifying and examining factors involved in Ras-mediated cell signaling. We have previously described the studies on a number of genes defined by suppressors of activated ras mutations (sur genes). We will report our recent studies on additional sur genes. The sur-9 gene is defined by a semi-dominant allele, ku258, isolated in a modified screen for suppressors of let-60(n1046). ku258 can also partially suppress a hypomorphic mutation in lin-1. Our results indicate that ku258 is a gf allele resulting from two point mutations in the C. elegans homolog of the transcriptional co-activator p300/CBP, cbp-1. We have shown that these two point mutants cause a near seven-fold increase in the histone acetyltransferase (HAT) activity of recombinant CBP-1 in an in vitro histone acetylation assay. This appears to be the only such HAT activity allele isolated in a CBP/p300 homolog and our results define a negative role for CBP-1 in a Ras signaling event, perhaps by collaborating with LIN-1. The sur-7 gene is defined by a silent mutation with genetic features similar to sur-6 and ksr-1. Our genetic data indicate that these three genes likely act downstream of or in parallel to lin-45 raf. In addition, both sur-6 and sur-7 may function by regulating the activity of ksr-1. Molecular analysis of sur-7 indicates that it likely encodes a protein involved in heavy metal transport with a similar role to that of cdf-1, another gene characterized as a ras suppressor by the Kornfeld laboratory (Bruinsma et al. 2000). However, the structure and expression pattern of SUR-7 indicate that it has a distinct function in regulating cellular heavy metal concentration. Its potential roles on ras signaling are being further investigated. We have made progress in cloning the sur-4 gene which is defined by a semi-dominant suppressor mutation. The gf nature, implied by dosage analysis, suggests that sur-4 acts negatively on ras signaling. Genetic mapping has narrowed the gene to a small chromosomal region containing about 10 transcripts. We have also performed an RNAi feeding screen for suppressors of activated Ras using the Ahringer lab library. A number of genes were found to cause a significant reduction of the Muv phenotype. Further investigation is needed to understand the mechanisms of the suppression phenotypes.