The C. elegans Wnt/β-catenin Asymmetry (WβA) pathway utilizes asymmetric regulation of SYS-1/β-catenin and POP-1/TCF coactivators. WβA differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered ‘centrosomal’.
Asymmetric cell division (ACD) allows daughter cells of a polarized mother to acquire different developmental fates. In C. elegans, the Wnt/β-catenin Asymmetry (WβA) pathway oversees many embryonic and larval ACDs; here, a Wnt gradient induces an asymmetric distribution of Wnt signaling components within the dividing mother cell. One terminal nuclear effector of the WβA pathway is the transcriptional activator SYS-1/β-catenin. SYS-1 is sequentially negatively regulated during ACD; first by centrosomal regulation and subsequent proteasomal degradation and second by asymmetric activity of the β-catenin "destruction complex" in one of the two daughter cells, which decreases SYS-1 levels in the absence of WβA signaling. However, the extent to which mother cell SYS-1 influences cell fate decisions of the daughters is unknown. Here, we quantify inherited SYS-1 in the differentiating daughter cells and the role of SYS-1 inheritance in Wnt-directed ACD. Photobleaching experiments demonstrate the GFP::SYS-1 present in daughter cell nuclei is comprised of inherited and de novo translated SYS-1 pools. We used a photoconvertible DENDRA2::SYS-1, to directly observe the dynamics of inherited SYS-1. Photoconversion during mitosis reveals that SYS-1 clearance at the centrosome preferentially degrades older SYS-1, and this accumulation is regulated via dynein trafficking. Photoconversion of the EMS cell during Wnt-driven ACD shows daughter cell inheritance of mother cell SYS-1. Additionally, loss of centrosomal SYS-1 increased inherited SYS-1 and, surprisingly, loss of centrosomal SYS-1 also resulted in increased levels of de novo SYS-1 in both EMS daughter cells. Lastly, we show that daughter cell negative regulation of SYS-1 via the destruction complex member APR-1/APC is key to limit both the de novo and the inherited SYS-1 pools in both the E and the MS cells. We conclude that regulation of both inherited and newly translated SYS-1/β-catenin via centrosomal processing in the mother cell and daughter cell regulation via Wnt signaling are critical to maintain sister SYS-1 asymmetry during ACD.
The C. elegans Wnt/β-catenin Asymmetry (WβA) pathway utilizes asymmetric regulation of SYS-1/β-catenin and POP-1/TCF coactivators. This differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate at birth. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific mobility of centrosomal SYS-1 and ‘traffic jam’ like enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe that depletion of microtubules by Nocodazole treatment or RNAi of putative dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose retrograde microtubule-mediated trafficking enables SYS-1 and negative regulators to enrich at centrosomes, enhancing their interaction and perhaps implicating the centrosome as a mitotic sink for proteins targeted for degradation.
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