Asymmetric cell division (ACD) critically controls the fates of dividing stem cells during normal embryonic development and misregulation of ACD is implicated in tumorigenesis. ?-catenin, a key transcriptional effector of Wnt signaling, is negatively regulated by a destruction complex containing casein kinase I? (CK1?) and two scaffolds, Axin and APC, which trigger ?-catenin degradation. Wnt signaling inhibits this complex through the Frizzled (Fz) receptor and its effector Dishevelled (Dvl), allowing ?-catenin to accumulate. However, the mechanisms that regulate the differential accumulation of ?-catenin after ACD are unclear in any system. Elucidating these mechanisms would provide broadly important insight into cell fate specification. Our objective is to determine the mechanisms of ?-catenin regulation during ACD. C. elegans exhibits Wnt-regulated asymmetric stem cell divisions where only one of the daughter cells activates Wnt target genes. These ACDs thus provide an ideally powerful experimental model for understanding Wnt signaling in an intact organism. We have found that the C. elegans ?-catenin, SYS-1, is regulated by an ortholog of the vertebrate destruction complex component, APR-1/APC, which asymmetrically localizes to one pole of the dividing cell and control asymmetric SYS-1 levels after ACD. We will test the central hypothesis that, because tight control of SYS-1 regulation is required, multiple SYS-1 negative regulatory mechanisms are needed in ACD. Supporting this, we have shown that PRY-1/Axin is required to establish the site of SYS-1 destruction by localizing APR-1/APC. We have also shown that SYS-1 is negatively regulated by localization to mother cell centrosomes during ACD. Our data also implicate an asymmetric nuclear export mechanism in the unsignaled daughter. We propose to:
Aim 1. Determine the mechanism by which asymmetric PRY-1/Axin activity is achieved. To test our hypothesis that Wnt ligands activate a Fz/Dvl-based PRY-1 polarization mechanism, we will: A) determine the location of the functional destruction complex during normal ACD and after optogenetically inducing Wnt expression to alter mother cell polarity, B) examine the ability of Dvl to generate ?signalosomes? and inactivate the destruction complex and C) identify the protein interactions that lead to asymmetric PRY-1/Axin activity.
Aim 2. Determine the mechanism of centrosomal control of SYS-1-dependent cell fate. To test our hypothesis that mother cell SYS-1 localizes to the centrosome and is degraded during ACD, we will A) conduct pulse- chase assays using a photoconvertible SYS-1 to examine centrosomal SYS-1 inheritance, B) genetically place centrosomal regulators in the Wnt pathway, C) examine the whether centrosomal SYS-1 regulation is microtubule-dependent and D) extend our analyses to human ?-catenin.
Aim 3. Determine the role of nuclear export in asymmetric SYS-1 nuclear accumulation. To test the hypothesis that asymmetric SYS-1 nuclear export occurs in the unsignaled daughter we will: A) determine how the nuclear asymmetry mechanism reflects the source of Wnt ligand and B) determine if CKI? generally regulates nuclear export or is specific to SYS-1.
The results of the proposed research are relevant to public health because they are expected provide new insights into the mechanisms by which conserved cell signaling pathways control asymmetric cell division during human development and tumor formation. There is the promise that the results of the proposed studies will contribute to a strong scientific framework for the future development of new drugs targeting the signaling aspects of ?-catenin during defective asymmetric division in human disease. The project is relevant to NIH because we will test specific hypotheses regarding stem cell biology and analyze the results during real time asymmetric divisions, which will elucidate novel mechanisms of Wnt/?-catenin signaling in stem cells that would likely not be adapted without the analysis of C. elegans and mammalian Wnt signaling.
| Lam, Arielle Koonyee; Phillips, Bryan T (2017) Wnt Signaling Polarizes C. elegans Asymmetric Cell Divisions During Development. Results Probl Cell Differ 61:83-114 |
| Baldwin, Austin T; Clemons, Amy M; Phillips, Bryan T (2016) Unique and redundant ?-catenin regulatory roles of two Dishevelled paralogs during C. elegans asymmetric cell division. J Cell Sci 129:983-93 |
