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. 2025 Feb 6;36(3):ar25. doi: 10.1091/mbc.E24-10-0441

SYS-1/beta-catenin inheritance and regulation by Wnt signaling during asymmetric cell division

Maria F Valdes Michel a, Bryan T Phillips a,b,*
Editor: Jeff Hardinc
PMCID: PMC11974967  PMID: 39813084

Abstract

Asymmetric cell division (ACD) allows daughter cells of a polarized mother to acquire different developmental fates. In Caenorhabditis elegans, the Wnt/β-catenin Asymmetry (WβA) pathway regulates 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 that newly localized centrosomal SYS-1 depends on dynein trafficking. Photoconversion of DENDRA2::SYS-1 in the EMS cell during Wnt-driven ACD shows daughter cell inheritance of mother cell SYS-1. Additionally, disrupting centrosomal SYS-1 localization in mother cells 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. Last, we show that negative regulation of SYS-1 in daughter cells 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 via centrosomal processing in the mother cell and daughter cell regulation via Wnt signaling are critical to maintain sister SYS-1 asymmetry during ACD.

  • Wnt/beta-catenin signaling controls cell fate decisions in many diverse contexts but mother cell-to-daughter cell inheritance of the pathway's namesake transcription factor, beta-catenin, is unclear.

  • Wnt-regulated C. elegans asymmetric cell divisions are here leveraged to interrogate this problem using a combination of photobleaching and photoconversion studies. We find that mother cell beta-catenin is inherited and visibly accumulates in daughter cells and affects the balance of de novo beta-catenin regulation by Wnt signaling.

  • These studies indicate that proper Wnt/beta-catenin signaling during asymmetric cell division requires tight control by dividing mother cells to prevent inappropriate accumulation in both daughter cells.

INTRODUCTION

Asymmetric cell division (ACD) controls cell fate specification during development by increasing cell diversity in developing tissues, including such diverse examples as Caenorhabditis elegans embryos, Drosophila neuroblasts, and mammalian lung epithelial cells (Bertrand and Hobert, 2009; Neumuller and Knoblich, 2009; Chhabra and Booth, 2021; Kochendoerfer et al., 2021). During adulthood, ACD maintains tissue integrity by promoting stem cell self-renewal while producing cells that will further differentiate to repopulate dying or degraded cells (Yamashita et al., 2010). Given that ACDs of stem cells maintain the ability to replenish damaged or growing tissues even postdevelopment, tight regulation of these stem cell niches is needed to prevent tumorigenesis. Accordingly, pathways that regulate ACD are implicated in the increased risk of cancer (Morrison and Kimble, 2006). The same signaling pathways often regulate of both homeostatic and developmental ACDs, thus elucidating the mechanisms regulating ACD will contribute to the understanding of cell fate specification during development and illuminate the mechanism by which tumors develop (Bajaj et al., 2015).

The Wnt/β-catenin pathway is a well-conserved signaling pathway that regulates stem cell maintenance, cell renewal and differentiation, cell polarity, and ACD (Ring et al., 2014; Chen et al., 2022; Habib and Acebrón, 2022; Hayat et al., 2022). In this pathway, Wnt ligands activate a signal transduction cascade that stabilizes the coactivator β-catenin, which translocates into the nucleus, binds TCF transcription factors and initiates transcription of Wnt target genes. In the absence of Wnt ligand, β-catenin is degraded by a “destruction complex” comprised of casein kinase 1α (CK1α), glycogen synthase kinase 3β (GSK3β), and two scaffolds, Axin and adenomatous polyposis coli (APC). In the absence of β-catenin, TCF acts as a transcriptional repressor of Wnt-target genes (Lin et al., 1998; MacDonald et al., 2009; Murgan et al., 2015). Aberrant regulation of Wnt signaling leads to developmental cell specification defects and is a common theme of cancer biology. Tumor sequencing further reveals frequent inactivating mutations in APC and activating mutations in β-catenin (Giles et al., 2003; Obrador-Hevia et al., 2010; Qu et al., 2016). Constitutive Wnt pathway activity is present in 90% of colorectal cancer cases. This may be impacted by changes in the cell polarity and in ACD, which leads to an increase in the cellular proliferation of intestinal epithelial stem cells (Giles et al., 2003; Quyn et al., 2010). Though regulation of β-catenin is essential for proper Wnt signaling, how the mother cell distributes β-catenin into the daughter cells and how β-catenin is differentially regulated during ACD remains unclear.

The Wnt/β-catenin asymmetry (WβA) pathway in C. elegans controls many embryonic and larval ACDs through regulation of the β-catenin, SYS-1 (Kidd et al., 2005; Huang et al., 2007; Phillips and Kimble, 2009). The WβA pathway is a branched pathway specialized for ACD that uses a β-catenin paralogue, WRM-1 and SYS-1, in each branch (Rocheleau et al., 1999; Lo et al., 2004; Kidd et al., 2005; Phillips et al., 2007; Sugioka et al., 2011; Robertson and Lin, 2012). The WRM-1 branch activates the nuclear export of ∼ 50% of the TCF/LEF DNA-binding protein, POP-1, via association with WRM-1 (Lo et al., 2004). POP-1 nuclear export enables the remaining nuclear POP-1 to complex with its coactivator β-catenin/SYS-1 (Kidd et al., 2005; Yang et al., 2015). The second branch of the WβA pathway regulates the stabilization and subsequent nuclear localization of SYS-1, which activates transcription of Wnt-target genes by complexing with POP-1 (Mizumoto and Sawa, 2007; Baldwin and Phillips, 2014).

SYS-1 is broadly expressed at the transcriptional level but expression, including asymmetric expression, is tightly regulated at the protein level (Huang et al., 2007; Phillips et al., 2007). One example of regulation of SYS-1 protein expression is via centrosomal localization and subsequent turnover. SYS-1 symmetrically localizes to the centrosomes during ACD, and that SYS-1 centrosomal localization is dependent on the centrosomal protein, RSA-2 (Huang et al., 2007; Phillips et al., 2007; Schlaitz et al., 2007; Christian, 2012; Vora and Phillips, 2015b; Vora and Phillips, 2016). After ACD in the absence of RSA-2, overall SYS-1 protein levels, including nuclear SYS-1, increase in both daughter cells (Vora and Phillips, 2015b; Vora and Phillips, 2016). These data suggest centrosomal regulation leads to the negative regulation and subsequent degradation of centrosomally localized SYS-1 and limits SYS-1 levels in daughter cells. Depletion of the proteasome core ATPase subunit of the 26S proteasome, RPT-4, results in elevated and stabilized centrosomal SYS-1 and decreased SYS-1 turnover compared with wild type (Vora and Phillips, 2015b; Vora and Phillips, 2016). These results are consistent with the hypothesis that the centrosomal proteasome plays a key role in regulating SYS-1 turnover at the centrosome via active degradation by the proteasome.

The microtubule motor dynein enhances the rapid accumulation of SYS-1 on mitotic centrosomes of large embryonic blastomeres via microtubule trafficking of SYS-1 (Thompson et al., 2022b), leading to SYS-1 mitotic turnover. Thus, dynein works as a negative regulator of SYS-1, by enhancing centrosomal SYS-1 levels and subsequent degradation by the centrosomal proteasome. Knockdown of single dynein subunits, including heavy chain DHC-1 or the light chain subunits DLC-1 and DYLT-1, lead to reduced SYS-1 centrosomal enrichment and elevated SYS-1 in both daughter cells. Additionally, loss of these dynein complex subunits results in the conversion of larval ACDs to symmetric divisions, increasing the Wnt-signaled cell fates (Thompson et al., 2022b). These data highlight the importance of microtubule trafficking for SYS-1 centrosomal localization and subsequent degradation. However, we previously showed that, when the worm homologue of mammalian proteasomal trafficking factor ECM29 (Leggett et al., 2002), ecps-1, is depleted, embryos with increased centrosomal SYS-1 were observed, in addition to a second substrate of the centrosomal proteasome, ZYG-1 (Peel et al., 2012; Thompson et al., 2022b). Thus, the current model suggests that SYS-1 and the proteasome are both actively trafficked via dynein to the centrosome during mitosis and, upon proper localization, result in SYS-1 degradation.

While the above model details SYS-1 negative regulation via centrosomal localization during mitosis in the mother cell, SYS-1 is also regulated after an ACD by the β-catenin destruction complex under the regulatory control of WβA in the daughter cells. In response to the Wnt ligand gradient, the positive Wnt regulators, Frizzled and Disheveled, are asymmetrically localized at the posterior cortex, resulting in their enrichment in Wnt-signaled daughter cell after cytokinesis (Park et al., 2004; Harrell and Goldstein, 2011; Sawa and Korswagen, 2013; Lam and Phillips, 2017). Members of the destruction complex, APC and Axin, are localized at the anterior pole of the mother cell and are asymmetrically enriched in the anterior Wnt-unsignaled daughter cell. The worm homologue of APC, APR-1, is essential for SYS-1 negative regulation during ACD (Rocheleau et al., 1997; Barth et al., 2008; Baldwin and Phillips, 2014). These data, coupled with the above centrosomal regulation data, suggest the current model of SYS-1 regulation occurs via centrosomal degradation of mother cell SYS-1 and asymmetric regulation of de novo synthesized SYS-1 by the destruction complex in the Wnt-signaled and -unsignaled daughter cells (Mizumoto and Sawa, 2007; Baldwin and Phillips, 2014;).

Lineage studies show that the Wnt-signaled daughters of Wnt-signaled cells respond more strongly to Wnt signaling in the subsequent division compared with their cousins, the Wnt-signaled daughters of Wnt-unsignaled cells (Zacharias et al., 2015; Zacharias and Murray, 2016). The heightened response can be visualized by both an enrichment of SYS-1 in the nucleus and increased target gene transcription (Zacharias et al., 2015; Zacharias and Murray, 2016). This nuclear SYS-1 enrichment suggests SYS-1 may be shielded from proteasomal degradation during nuclear localization, allowing for greater SYS-1 inheritance into the daughter cells of previously Wnt-signaled mother cells and progressively enriching the Wnt-signaled lineages for SYS-1 over time. Alternatively, enrichment of positive regulators of SYS-1 in Wnt-signaled lineages, such as Frizzled or Dishevelled (Sawa et al., 1996; Umbhauer et al., 2000), would also be predicted to increase downstream SYS-1 stabilization. In this latter case, inheritance of upstream Wnt pathway components would be expected to similarly increase WRM-1 and decrease nuclear POP-1 levels between cousins, which was indeed observed in this study (Lin et al., 1998; Zacharias et al., 2015; Zacharias and Murray, 2016). This suggests that increased SYS-1 inheritance from signaled mothers is unlikely to be the sole mechanism of cousin enrichment. However, the extent to which direct SYS-1 inheritance contributes to a Wnt pathway “memory” during normal ACD and how Wnt signaling regulates inherited and de novo SYS-1 remains unknown.

Here, we show centrosomal degradation of SYS-1 in the EMS mother cell is incomplete, leading to SYS-1 inheritance in the E and the MS daughter cells during wild-type ACDs. We developed a photoconvertible SYS-1 to observe the dynamics of preexisting and newly synthesized SYS-1 at the centrosome during mitosis. We found that preexisting and newly synthesized SYS-1 levels are trafficked to the mitotic centrosome via the dynein complex where they are both degraded. SYS-1 coupling, trafficking, and degradation at the mother cell centrosome are necessary for maintaining sister cell asymmetry and wild-type SYS-1 asymmetry in E and MS daughter cells during ACD. Surprisingly, de novo SYS-1 increases in both daughter cells after the overinheritance of mother cell SYS-1 during asymmetric cell division. Additionally, depletion of the negative regulator APR-1 increased both pools of SYS-1 in the E and MS nuclei, suggesting that, when inherited SYS-1 is affected, de novo SYS-1 is also misregulated and vice versa. Thus, we propose a model in which SYS-1 centrosomal degradation is a key regulator of SYS-1 inheritance that prevents the loss of daughter cell asymmetry. Further regulation of inherited and de novo SYS-1 in the E and MS daughter cells via Wnt signaling supports proper sister cell asymmetry due to a free exchange between the inherited and de novo SYS-1 pools.

MATERIALS AND METHODS

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Strains

Strains were maintained on OP50-inoculated nematode growth media (NGM) plates at 20°C (PHX5708 and N2) or 25°C (TX964) using typical C. elegans methods (Brenner, 1974). The genotypes of the investigated are as follows: N2 (wild type); TX964 (unc-119(ed3) III; him-3(e1147) IV; teIs98 [Ppie-1::GFP::SYS-1]); BTP254 (syb5708[Psys-1::DENDRA2::SYS-1]).

CRISPR/Cas9-mediated fluorescent tagging of endogenous SYS-1

An in-frame N-terminal DENDRA2-GAS tag was inserted in the endogenous sys-1 gene prior to the endogenous start codon (SunyBioTech, Fujian, China). Two synonymous nucleotide changes were generated in the donor sequence to prevent the donor from being recognized and cut by Cas9. Genome edited animals were sequence verified with DENDRA-2-sys-1 specific primers and backcrossed to wild-type N2 worms to reduce the chances of extraneous genetic variation or off target insertions impacting our experiments. The resulting strain, sys-1(syb5708[Psys-1::DENDRA2::SYS-1]), is designated by strain name BTP254.

RNAi

To perform RNAi knockdown of target genes, we used HT115 bacteria containing the pL4440 plasmid with a T7-flanked target gene insert. rsa-2, apr-1, and ecps-1 were obtained from a library supplied by Ahringer (Addgene) (Kamath et al., 2003). dlc-1 was obtained from the Phillips lab library and made as described in (Thompson et al., 2022a). Briefly, dlc-1 was amplified using target gene-flanking primers and C. elegans cDNA to generate a new insert. Bacteria with the desired insert were seeded onto isopropyl β-d-1-thiogalactopyranoside (IPTG)-containing plates to induce transcription from the T7 promotor as described previously (Conte et al., 2015). Worms were plated onto RNAi bacterial lawns after sodium hypochlorite synchronization as L1s, with the exception of worms plated on dlc-1 bacterial lawns which were washed from OP50 plates at 24 h before imaging (L3/L4) and plated on to the RNAi plate to avoid larval phenotypes (Timmons, 2006; Bekas and Phillips, 2020).

Confocal microscopy and FRAP

Confocal microscopy was performed with a Leica SP8 HyD detector system. The objective used was a 63 × HC PL APO CS2 objective with N.A. = 1.4, using type F immersion oil. Each analyzed image consisted of 35 summed z-images of the embryo, with each slice ∼ 0.6-µm thick. Fluorescence recovery after photobleaching (FRAP) was performed on the same system. Each embryo was imaged once then photobleached, at anaphase, telophase or cytokinesis. The entire EMS cell was selected as the region of interest (ROI) and bleached at an intermediate focal plane with 100% laser power for 200 iterations. Images were taken immediately after photobleaching at 4, 6, 8, and 10 min. Fluorescence intensity was measured on the sum of six Z-projection slices in FIJI using the mean fluorescence intensity of ROIs around the nuclei of the E and MS cells. Due to the time-intensive nature of these imaging sessions, data represented in our figures were collected over experiments separated in time, with appropriate controls in each session, to increase sample size.

Confocal microscopy and photoconversion of DENDRA2::SYS-1

Confocal microscopy was performed via a Leica SP8 HyD detector system using a 63 × HC PL APO CS2 objective with N.A. = 1.4 and type F immersion oil. Each image analyzed consisted of the sum of 35 z-images, ∼ 0.6-µm thick, across the embryo. Photoconversion of DENDRA2::SYS-1 was performed on the same system. For centrosome and cytoplasm photoconversion, the FRAP software was used to photoconvert the desired locale. Before photoconversion, one image was taken in early metaphase to observe and quantify preconversion green SYS-1 levels at the centrosome. The ROI was then photoconverted using the UV laser (405 nm diode laser) at an intermediate focal plane with 100% laser power for 200 iterations. Excitation light at 488 nm (490–550 nm) or 552 nm (580–670 nm) was provided by HyD lasers set at 30% laser power for both channels on standard mode. Images were taken immediately after photoconversion every 20 s through mitosis of the P1 cell with a frame and line average of 2. To obtain the CEI, the mean intensity of the embryonic cytoplasm was subtracted from the centrosomal mean (Thompson et al., 2022b).

For whole embryo photoconversion and quantification of DENDRA2::SYS-1 nuclear levels in the E and MS daughter cells, embryos were exposed to a UV laser (405 nm diode laser) at 100% laser power for 36 iterations (frame average: 6, line average: 6) using the normal imaging setting on the Leica SP8 HyD detector system. An image was taken of each embryo prior to photoconversion with the excitation light of 488 nm (490–550 nm) or 552 nm (580–670) HyD lasers set at 30% laser power for both channels on standard mode. Photoconversion was performed at the time of cytokinesis of the EMS cell, and 2 min after cytokinesis the time course was started, with 1 minute between each image taken for 7 min. DENDRA2::SYS-1 nuclear levels were normalized by N2 values utilizing the same settings described above.

RESULTS

Photobleaching reveals de novo and inherited SYS-1 pools in the E and MS cells and centrosomal SYS-1 dynamics over time

Alterations in centrosomal SYS-1 enrichment correlate with nuclear SYS-1 levels in WβA daughter cells (Vora and Phillips, 2015a; Thompson et al., 2022a). Disruption of SYS-1 centrosomal levels by knockdown of the centrosomal scaffold protein RSA-2, different dynein subunits (DHC-1, DLC-1, DYLT-1), or the proteasome trafficking adaptor protein ECPS-1 are sufficient to induce extra Wnt-signaled cell fates (Thompson et al., 2022a). These data suggest that impairing mother cell SYS-1 centrosomal localization or degradation increases SYS-1 inheritance in the daughter cells. However, it remains possible that degradation of mother cell SYS-1 is also incomplete in wild type and that inherited SYS-1 contributes to normal Wnt-dependent asymmetric cell fate specification. To better understand the dynamics of SYS-1 inheritance in wild type, we used FRAP in the E and MS daughter cells at different timepoints. sys-1 is broadly and highly expressed at the transcriptional and translational levels, but overall levels and asymmetric expression are controlled posttranslationally via proteasomal degradation (Phillips et al., 2007; Vora and Phillips, 2015a). Thus FRAP will identify newly translated, or folded and matured, GFP::SYS-1 (Chudakov et al., 2010). The EMS cell is polarized by Wnt ligand produced in the posterior P2 cell, resulting in E and MS daughter cells with high and low nuclear levels of SYS-1, respectively (Goldstein, 1992; Harrell and Goldstein, 2011; Robertson and Lin, 2012). EMS was photobleached at different times during mitosis, and nuclear SYS-1 levels in the E and MS daughter cells were measured 4 min after cytokinesis (Figure 1, A and B). If SYS-1 is cleared by the mother cell centrosomal processing, we expect no change between SYS-1 nuclear levels due to EMS photobleaching at different timepoints. Alternatively, if the mother cell SYS-1 protein is inherited by the daughter cells, we expect the largest decrease in SYS-1 levels to occur following a photobleach at cytokinesis. Anaphase photobleach gives slight, nonsignificant decrease (Figure 1B), but we speculated that this was due to late SYS-1 synthesis, giving rise to an unbleached population of mother cell SYS-1. To address this, we photobleached at telophase and at cytokinesis. Indeed, after the EMS telophase photobleach, we observed significant decreases in E and MS nuclear SYS-1, suggesting degradation of SYS-1 is incomplete in EMS mother cells (Figure 1B; Supplemental Figure S1). Finally, cytokinesis photobleaching, despite the late timing, resulted in visible GFP::SYS-1 in both daughter cell nuclei, revealing both a mother cell contribution and a de novo synthesized SYS-1 pool in the E and the MS daughter cells (Figure 1B). Regardless of the time of photobleaching, the asymmetry between E and MS SYS-1 nuclear levels is maintained (Figure 1B). These results suggest SYS-1 mother cell clearance is incomplete and that de novo SYS-1 is asymmetrically regulated during EMS ACD.

FIGURE 1:

FIGURE 1:

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Photobleaching reveals de novo and inherited SYS-1 pools in the E and MS cells and centrosomal SYS-1 dynamics over time. (A) Diagram of EMS cell FRAP assay. EMS cell is polarized by Wnt from the P2 cell. EMS mother cell is photobleached and SYS-1 nuclear levels are subsequently measured in the E and the MS daughters. (B) Quantification of nuclear SYS-1::GFP in E and MS cells without photobleaching (n = 8) and with photobleaching at anaphase (n = 12), telophase (n = 11), and cytokinesis (n = 10). Fluorescence recordings were measured 4 min after mother cell division is complete. Mean ± SEM. Student's two-tailed t test. (C) Photobleaching protocol for panel D and E. (D) Quantification of nuclear fluorescent SYS-1::GFP in the E and MS cells over time in nonbleached controls and EMS cell photobleached at cytokinesis. Mean ± SEM. Student's two-tailed t test. (E) Percentage of nuclear SYS-1 in daughter cells during a 2-min interval time course. N = denoted by gray figures in graph. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

To observe the dynamics of the de novo SYS-1 pool in the daughter cells, we photobleached the EMS mother cell at cytokinesis and measured de novo and total nuclear SYS-1 at different times during interphase (4, 6, 8, and 10 min after cytokinesis) (Figure 1C) with and without photobleaching. This timing was utilized to capture the effects of mother cell photobleaching on daughter cell SYS-1 nuclear levels, where SYS-1 functions in Wnt target gene expression, prior to centrosomal localization/degradation that begins again in daughter cell mitosis. Because de novo nuclear SYS-1 levels were reduced by 45–83% relative to total SYS-1 levels at each timepoint, our results confirm SYS-1 inheritance occurs in wild type, however, interestingly, the accumulation of de novo SYS-1 was not linear. Instead, we observed a significant increase in nuclear de novo SYS-1 levels in both daughter cells during minutes 4 through 6, as expected as total newly synthesized SYS-1 increases (Figure 1D). However, de novo SYS-1 levels plateau around 8 min and then decrease at 10 min, after cytokinesis of the mother cell (Figure 1D), suggesting de novo SYS-1 accumulation ceases or slows throughout the cell cycle. To quantify this change, we calculated the ratio of total to de novo nuclear SYS-1 levels over the time course in both daughter cells. In the E cell at 4 min, the de novo SYS-1 pool represents 28.9% of the total SYS level (Figure 1E) and increases to 54.2 and 51.5% by minutes 6–8, respectively, after mother cell division. Ten minutes after cytokinesis de novo SYS-1 decreases to 33%. Intriguingly, we observe a similar pattern in the MS cell (4 min = 17%; 6 min = 45%; 8 min = 50%; 10 min = 29%), though the absolute levels are higher in the Wnt-signaled E daughter (Figure 1D). This time course revealed variable rates of de novo SYS-1 synthesis and regulation in the nuclei of the E and MS daughter cells through the cell cycle, suggesting the role of Wnt signaling in SYS-1 stabilization is mostly complete by 8 min post-EMS division and that the “unsignaled” MS cell also stabilizes SYS-1, albeit at a lower level than E.

Photoconversion of DENDRA2::SYS-1 reveals turnover at the centrosome during mitosis

To visualize de novo and inherited SYS-1, we generated a photoconvertible sys-1 transgene, Psys–1::DENDRA2::SYS-1 that differentiates preexisting (red) and newly synthesized (green) SYS-1 centrosomal accumulation in the P1 cell, which has been previously utilized for SYS-1 centrosomal localization and processing studies (Vora and Phillips, 2015b; Thompson et al., 2022b). While observing P1 cellular division, in the green channel DENDRA2::SYS-1 is visible at the centrosome prior to photoconversion, consistent with previous studies (Vora and Phillips, 2015b; Thompson et al., 2022b) (Figure 2A), while the red channel detection shows background levels of centrosomal DENDRA2::SYS-1 (Figure 2B). To photoconvert DENDRA2::SYS-1 from green to red fluorescence, we exposed the entire P1 cell to a 405 nm UV diode laser (Chudakov et al., 2007) at 100% power for 36 iterations. After photoconversion, centrosomal DENDRA2::SYS-1 in the green channel sharply decreases (Figure 2C) and we clearly detect red fluorescent DENDRA2::SYS-1 at the centrosome (Figure 2D). After confirming successful photoconversion, we measured centrosomal DENDRA2::SYS-1 levels in 20-s increments through P1 mitosis (Figure 2E). To control possible photoconversion by the 488 nm laser (Woods et al., 2014), we followed the same imaging protocol without 405 nm photoconversion and followed both emissions over the time course. The data were normalized using N2 animals subjected to the same protocol to account for possible photobleaching or changes in development of the embryo induced by the imaging. Our data shows that there is no photoconversion induced by repetitive imaging with the 488 nm laser as no changes are observed in the red channel (Supplemental Figure S2A). The raw data revealed the preexisting, red centrosomal DENDRA2::SYS-1 decreased over time, likely due to centrosomal degradation. Interestingly, the newly synthesized, green centrosomal DENDRA2::SYS-1 decreased at a slower rate throughout the rest of P1 mitosis (Figure 2F). Additionally, to quantify photoconversion efficacy, we compared the level of postconversion red signal with the green signal after mock conversion, both normalized for green preconversion signal (Supplemental Figure S2, A and B). The results indicate ratios of approximately one and no significant difference between the two ratios, indicating successful photoconversion. Together, these data suggest that the rate of synthesis and trafficking is relatively equal to the rate of degradation of de novo SYS-1 at the centrosome, which is consistent with the previously published idea of a steady-state level of centrosomal GFP::SYS-1 that is stable during the P1 cell mitosis (Vora and Phillips, 2015a).

FIGURE 2:

FIGURE 2:

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Photoconversion of DENDRA2::SYS-1 reveals turnover at the centrosome during mitosis. (A) Preconversion image shows centrosomal localization of DENDRA2::SYS-1 in the green channel. (B) Centrosomal DENDRA2::SYS-1 is undetectable in the red channel prior to photoconversion by 405 nm blue laser exposure. (C) After photoconversion newly translated DENDRA2::SYS-1 is trafficked to the centrosome, and D shows photoconverted DENDRA2::SYS-1 at the centrosome. (E) Photoconversion protocol for centrosomal DENDRA2::SYS-1 time course. (F) Quantification of average centrosomal DENDRA2::SYS-1 in both channels over time. Mean ± SEM. N = 3. DENDRA2::SYS-1 fluorescent intensity was calculated by subtracting N2 autofluorescence in both channels at all timepoints. (G)  CEI of the time course shown in F. Mean ± SEM. N = 3. CEI was calculated in FIJI by subtracting the mean pixel intensity of a cytoplasmic ROI encompassing the posterior half of the cell with the dotted white line (A–D), from a circular centrosome ROI.

To specifically evaluate the enrichment of DENDRA2::SYS-1 at the centrosome, we used the centrosomal enrichment index (CEI) (Thompson et al., 2022b). CEI measures the degree to which the mother cell traffics cytoplasmic SYS-1 to the centrosome, thus enriching SYS-1 fluorescence at the centrosome, while also internally controlling for bleaching via repetitive imaging. CEI measurements provide a clearer observation of the initial increase in the red channel of centrosomal DENDRA2::SYS-1 after photoconversion (Figure 2G). CEI data show a similar rate of declining preexisting DENDRA2::SYS-1 centrosomal levels as the raw data but emphasizes the stability of newly translated centrosomal DENDRA2::SYS-1 enrichment, which is stable and low (Figure 2G). Photoconverted (older) SYS-1 accumulates at the centrosome at a greater rate than newly translated SYS-1. Because we would expect no centrosomal enrichment of preexisting DENDRA2::SYS-1 if SYS-1 were trafficked to a different locale to be degraded, our finding of older SYS-1 centrosomal enrichment is consistent with our model of SYS-1 enrichment from the cytoplasm and subsequent degradation at the centrosome. Altogether, these data suggest that newly synthesized and trafficked SYS-1, coupled with degradation of preexisting centrosomal SYS-1, lead to a stable steady state at the centrosomal locale, while indicating that the centrosome is the terminal locale of SYS-1. These data are consistent with a model where preexisting centrosomal SYS-1 is degraded prior to newly trafficked centrosomal SYS-1, which is stably replenished by cytoplasmic trafficking.

Photoconversion of the cytoplasm of the P1 cell unveils dynamics of dynein trafficking of DENDRA2::SYS-1 during mitosis

Previous data indicate that microtubule trafficking via the dynein complex is needed for SYS-1 centrosomal localization (Thompson et al., 2022b). By photoconverting subdomains of the posterior pole of the P1 cell cytoplasm, we tested the ability of the cell to traffic SYS-1 to the centrosomes. Following initial imaging during early mitosis, where preconversion levels of centrosomal DENDRA2::SYS-1 were measured with both green and red emission, a portion of the cytoplasm was photoconverted (Figure 3A). Embryos were imaged every 20 s during mitosis (Figure 3B) and DENDRA2::SYS-1 levels were measured in the proximal and the distal centrosomes in both the green and red channels. The photoconverted, red DENDRA2::SYS-1 represents newly trafficked SYS-1 from the anterior cytoplasmic region of the cell, while green DENDRA2::SYS-1 denotes older preexisting centrosomal SYS-1 and newly trafficked SYS-1 from the nonphotoconverted cytoplasmic SYS-1. Quantification of red newly trafficked SYS-1 showed centrosomal localization in both the proximal and distal centrosomes 20 s after photoconversion (Figure 3, C and D), suggesting that SYS-1 is highly mobile and quickly trafficked to both centrosomes in early mitosis. However, though photoconverted SYS-1 accumulates on both proximal and distal centrosomes, the rates of accumulation are not equal. When directly comparing red newly trafficked DENDRA2::SYS-1 levels in the proximal and distal centrosomes, we observe that proximal centrosomes have a higher enrichment of SYS-1 throughout mitosis when compared with distal centrosomes (Figure 3D). The difference in centrosomal DENDRA2::SYS-1 levels between the centrosomes suggest that cytoplasmic SYS-1 from any locale can be trafficked to both centrosomes but is more likely to be trapped and trafficked via dynein to the most proximal centrosome. We also noted a distinct pattern of newly translated DENDRA2::SYS-1 accumulation after partial cytoplasmic conversion (Figure 3C) compared with whole cell photoconversion (Figure 2F). Both cases see a drastic decline after photoconversion, but partial cytoplasm conversion (Figure 3C) results in a delay in this decrease compared with whole cell conversion that we attribute to initial SYS-1 accumulation from nonconverted regions of the cytoplasm followed by partial depletion of this pool.

FIGURE 3:

FIGURE 3:

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Photoconversion of the cytoplasm of the P1 cell unveils dynamics of dynein trafficking of DENDRA2::SYS-1 during mitosis. (A) Diagram of photoconversion of the P1 cell. Dotted square represents the photoconverted area of the cytoplasm at metaphase. (B) Photoconversion protocol for cytoplasm photoconversion in C, D, and E. (C) Quantification of raw DENDRA2::SYS-1 of the distal and proximal centrosome in green and red channels prephotoconversion and postphotoconversion of the cytoplasm, measured over a 2-min time course in 20 s intervals after photoconversion on control lacZ(RNAi). Mean ± SEM. N = 12. (D) Quantification of DENDRA2::SYS-1 of the distal and proximal centrosome in red channel prephotoconversion and postphotoconversion of the cytoplasm, measured over a 2-min time course in 20 s intervals after photoconversion on control lacZ(RNAi). Mean ±SEM. N = 12. (E) Time course depicting red, photoconverted DENDRA2::SYS-1 CEI on lacZ(RNAi) and dlc-1(RNAi). DENDRA2::SYS-1 fluorescent intensity was calculated by subtracting N2 autofluorescence in both channels at all timepoints. Mean ± SEM. N = 12 and 15, respectively.

To test the role of microtubule trafficking on DENDRA2::SYS-1, we depleted the light subunit of the dynein complex, DLC-1, via RNAi and measured the CEI of photoconverted DENDRA2::SYS-1 in the proximal centrosome. Previous results show DLC-1 is required for efficient centrosomal enrichment of SYS-1 (Thompson et al., 2022b). After dlc-1(RNAi), the red, newly trafficked DENDRA2::SYS-1 is stable throughout mitosis; however, DLC-1 knockdown hinders SYS-1 trafficking to the centrosome, resulting in a decrease of centrosomal SYS-1 enrichment throughout mitosis compared with controls (Figure 3E). This result is consistent with impaired SYS-1 centrosomal recruitment observed in cells lacking efficient dynein trafficking (Thompson et al., 2022b). These data, which are consistent with our model of SYS-1 capture and centrosomal trafficking, suggest microtubule trafficking via the dynein complex is key for centrosomal SYS-1 localization and subsequent degradation.

E and MS inheritance of mother cell SYS-1 is limited by EMS centrosomal processing

To determine the extent to which changes in SYS-1 centrosomal trafficking and degradation affect inheritance of mother cell SYS-1 into daughter cells, we photoconverted the entire EMS cell, the first ACD driven by the WβA pathway (Harrell and Goldstein, 2011), of DENDRA-2::SYS-1–expressing embryos at cytokinesis and measured inherited (red) SYS-1 in the E and MS daughter cell nuclei. Here, E is the Wnt-signaled cell that normally exhibits higher SYS-1 levels and MS is the unsignaled cell with lower SYS-1 levels (Huang et al., 2007; Phillips et al., 2007). For photoconversion of DENDRA2::SYS-1, we exposed EMS to UV (405 nm) (Chudakov et al., 2007) at 100% laser power for 36 iterations during EMS cytokinesis. Subsequently, we waited 2 min for the nuclear envelope of the E and MS daughters to reform and imaged every 60 s for 7 min (Figure 4A). Then, we normalized the nuclear fluorescence of DENDRA2::SYS-1 to the nuclear autofluorescence in wild-type (N2) embryos using identical measurements. Consistent with our FRAP studies, we observed inherited DENDRA2::SYS-1 present in both E and MS daughter cells (Figure 4B). Interestingly, the amount of DENDRA2::SYS-1 inherited by the E daughter was higher than that present in MS (Figure 4B), perhaps reflecting greater stability of inherited SYS-1 in the signaled E cell. We also noted that MS inheritance, while lower than E, was on average above autofluorescence. This result indicates SYS-1 inheritance persists even in the unsignaled MS cell. The time course also allowed us to observe changes in nuclear DENDRA2::SYS-1 over the cell cycle of the E and MS cells, up to 9 min after EMS division. For both the E and MS daughter cells, we found inherited nuclear DENDRA2::SYS-1 levels to remain stable throughout the time course (Figure 4B), indicating the asymmetry in inherited nuclear DENDRA2::SYS-1 occurs shortly after EMS division.

FIGURE 4:

FIGURE 4:

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E and MS inheritance of mother cell SYS-1 is limited by EMS centrosomal processing. (A) Photoconversion protocol for quantification of nuclear inherited DENDRA2::SYS-1 time course. (B) Quantification of inherited, red DENDRA2::SYS-1 nuclear levels of the E and MS cells at different timepoints starting 2 min after cytokinesis of the EMS mother cell in 60 s increments on the control lacZ(RNAi). Mean ± SEM. Two-way ANOVA followed by Tukey mixed effects analysis in multiple comparisons test. N = 12. (C) Quantification of inherited, red DENDRA2::SYS-1 nuclear levels of the E and MS cells time course on control versus dlc-1(RNAi) (N = 26 and N = 20, respectively). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (D) Quantification of inherited, red DENDRA2::SYS-1 nuclear levels of the E and MS daughter cells time course on control versus rsa-2(RNAi) (N = 26 and N = 20, respectively). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (E) Quantification of inherited, red DENDRA2::SYS-1 nuclear levels of the E and MS daughter cells time course on control versus ecps-1(RNAi) (n = 26 and n = 20, respectively). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (F) Quantification of fold change in asymmetry between the E and MS cells in lacZ, dlc-1, rsa-2 and ecps-1, (RNAi). DENDRA2::SYS-1 fluorescent intensity was calculated by subtracting N2 autofluorescence at all timepoints. * represent the P-value when comparing the averaged value of all timepoints of lacZ(RNAi) to experimental condition. Supplemental Table S1. P-values when comparing each timepoint. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Control data for this figure and Figures 5 and 6 have been combined since control data were collected in parallel with several matched experimental groups spanning multiple panels over several days as needed.

Next, we tested the requirement for dynein trafficking and centrosomal localization in SYS-1 inheritance. We predicted that impaired mother cell SYS-1 processing would lead to an increase in inherited SYS-1 levels, given the previously observed changes in cell fate under these conditions (DLC-1, RSA-2, and ECPS-1 knockdowns) (Thompson et al., 2022b). We observed that when microtubule trafficking is impaired by the knockdown of DLC-1 there is a significant increase in the amount of inherited DENDRA2::SYS-1 nuclear levels of both the E and MS cells (Figure 4C). Initial levels of inherited SYS-1 were 3- and 10-fold higher in the E and MS cells, respectively (Figure 4C), suggesting mother cell dynein trafficking is required to limit the amount of inherited SYS-1.

We further investigated the role of SYS-1 centrosomal localization in controlling SYS-1 inheritance by preventing centrosomal localization of SYS-1 via knockdown of the centrosomal scaffold protein RSA-2. Loss of centrosomal localization by rsa-2(RNAi) increased the amount of inherited nuclear DENDRA2::SYS-1 by 3.4- and 11-fold in E and MS daughter cells, respectively (Figure 4D). Last, we impaired the degradation of centrosomal SYS-1 by knocking down the adaptor protein ECPS-1, the worm homologue to EMC29, (a proteasome-dynein adaptor and scaffold) (Leggett et al., 2002; Lehmann et al., 2010). Previous studies showed that ECPS-1 knockdown increases SYS-1 levels at the centrosome, suggesting a role promoting SYS-1 centrosomal degradation (Thompson et al., 2022b). ecps-1(RNAi) lead to an increase of the amount of DENDRA2::SYS-1 inherited into the E and MS cells by 3- and 10.7-fold, respectively (Figure 4E). These data suggest that the various mechanisms required for mother cell centrosomal SYS-1 degradation are normally required to prevent SYS-1 overinheritance.

Finally, to assess the effect of the overinherited SYS-1 on loss of daughter cell SYS-1 asymmetry and further loss of the asymmetric cell division of EMS, we averaged the inherited DENDRA2::SYS-1 nuclear levels in each condition throughout the 7-min time course and measured the fold change between the E and the MS daughters. These data showed a loss of asymmetry of inherited SYS-1 in all the experimental conditions tested compared with control RNAi (Figure 4F). The data suggest that SYS-1 is normally inherited into both the E and the MS daughter cells and that, early in the cell cycle, the asymmetry between E and MS is established. For proper inheritance of SYS-1 during the EMS ACD the mother cell requires SYS-1 centrosomal trafficking, coupling, and degradation (also referred to as centrosomal processing) to prevent overinheritance of SYS-1 and loss of sister cell asymmetry.

Loss of centrosomal SYS-1 results in increased levels of nuclear de novo SYS-1 in EMS daughter cells

Given the fact that centrosomal processing functions in the mother cell limit SYS-1 inheritance, we predicted that mother cell, but not de novo translated DENDRA2::SYS-1 would be affected when mother cell centrosomal processing of SYS-1 is impaired. To test this, we photoconverted at EMS cytokinesis and measured green/de novo DENDRA2::SYS-1 nuclear levels in E and MS daughter cells starting 2 min after cytokinesis and in 60 s increments for 7 min (Figure 5, A and B). We observed that both the E cell, the Wnt-signaled cell, and MS, the unsignaled cell, were actively translating DENDRA2::SYS-1 (Figure 5A), consistent with our FRAP results (Figure 1C). The E cell also showed higher levels of de novo nuclear DENDRA2::SYS-1 levels than the MS cell (3.6-fold increase in E vs. MS), indicating robust asymmetry in synthesis or stability of newly translated DENDRA2::SYS-1 (Figure 5B). Additionally, the time course showed that the E and MS de novo nuclear DENDRA2::SYS-1 levels remained stable (Figure 5B). These data suggest that SYS-1 synthesis occurs in both the signaled E and unsignaled MS daughter cells and that the rate of synthesis:degradation is higher in E than MS.

FIGURE 5:

FIGURE 5:

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Loss of centrosomal SYS-1 results in increased levels of nuclear de novo SYS-1 in EMS daughter cells. (A) Photoconversion protocol for quantification of nuclear inherited DENDRA2::SYS-1 time course. (B) Quantification of green/de novo SYS-1 nuclear levels in the E and MS cells after photoconversion of the EMS cell at cytokinesis; the time course depicts SYS-1 nuclear levels at different timepoints starting 2 min after cytokinesis of the EMS cell in 60 s increments on the control lacZ(RNAi) (n = 26). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (C) Quantification of de novo SYS-1 nuclear levels of the E and MS cells time course on lacZ(RNAi) and dlc-1(RNAi) (n = 26 and n = 20, respectively). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (D) Quantification of de novo SYS-1 nuclear levels of the E and MS cells time course on lacZ(RNAi) and rsa-2(RNAi) (n = 26 and n = 20, respectively). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (E) Quantification of de novo SYS-1 nuclear levels of the E and MS cells time course on lacZ(RNAi) and ecps-1(RNAi) (n = 26 and n = 20, respectively). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (F) Quantification of fold change in asymmetry between the E and MS daughter cell in lacZ, dlc-1, rsa-2, and ecps-1 (RNAi). DENDRA2::SYS-1 fluorescent intensity was calculated by subtracting N2 fluorescence at all timepoints. * represent the P-value when comparing all timepoints and the average of control to experimental condition. Supplemental Table S1. P-values when comparing each timepoint. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Subsequently, we observed de novo nuclear DENDRA2::SYS-1 levels in the daughter cells after knocking down DLC-1, therefore impairing microtubule trafficking of SYS-1 to the centrosome in the mother cell (Thompson et al., 2022b). This surprisingly resulted in a significant increase in the amount of de novo nuclear SYS-1 levels in both daughter cells at all timepoints (Figure 5C). This increase in de novo SYS-1 was also observed in the other tested conditions that showed increased DENDRA2::SYS-1 inheritance, rsa-2 and ecps-1 (RNAi) (Figure 5, D and E). In all the tested conditions (dlc-1, rsa-2 and ecps-1 [RNAi]), we also observed a loss of asymmetry compared with the control (Figure 5F).

Thus, we find that impairing proper SYS-1 centrosomal processing during mitosis of the EMS cell showed increased levels of both inherited and de novo nuclear DENDRA2::SYS-1 in EMS daughter cells (Figures 4 and 5). Additionally, we observed that over time in the tested conditions, nuclear DENDRA2::SYS-1 consistently decreases in backgrounds with higher than normal SYS-1 compared with wild type, which stably maintains its SYS-1 levels in E and MS (Table 1). This pattern of increased rate of DENDRA2::SYS-1 clearance was observed in both DENDRA2::SYS-1 pools, suggesting that both de novo and inherited SYS-1 are processed similarly in both daughter cells. These data suggest that overinheritance of SYS-1 during asymmetric cell division affects the de novo SYS-1 that is either generated or matured in both daughter cells. This led us to speculate that these results may be due to a limited capacity of the β-catenin “destruction complex” activity in daughter cells to target newly synthesized SYS-1. If so, impairing the destruction complex, and resulting daughter cell de novo SYS-1 degradation, would also affect the inherited SYS-1 pool. We test this idea below.

TABLE 1:

Relative nuclear levels of DENDRA2::SYS-1 over time

Cell SYS-1 pool RNAi First to last image SYS-1 nuclear levels ratio SYS-1% decrease over time course
E inherited lacZ 0.4 7.4%
dlc-1 9.3 42.3%
rsa-2 11.3 47.1%
ecps-1 10.3 46%
de novo lacZ 1.1 22%
dlc-1 5.5 38.2%
rsa-2 3.3 21.3%
ecps-1 4.5 35.2%
MS inherited lacZ −0.2 −11.8%
dlc-1 9.4 50.5%
rsa-2 10.2 51%
ecps-1 9.8 49.2%
de novo lacZ −0.4 −44%
dlc-1 3.4 31.8%
rsa-2 3.4 28.3%
ecps-1 3.8 41.8%
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Depletion of the negative regulator of the Wnt signaling pathway, APR-1, affects both pools of nuclear SYS-1 in the E and MS cells

We targeted APR-1 a negative regulator of SYS-1 in the MS cell and elsewhere (Huang et al., 2007; Baldwin and Phillips, 2014) and a homologue of vertebrate APC, which is part of the β-catenin destruction complex and functions to limit β-catenin cytoplasmic accumulation in the Wnt-unsignaled state. APC also limits Wnt pathway activity after initial activation by targeting β-catenin for proteasomal degradation (Clevers and Nusse, 2012). As expected, we found that depletion of APR-1 via RNAi led to a significant increase in de novo DENDRA2::SYS-1 nuclear levels throughout the MS cell cycle (Figure 6A). We also observed a 2-fold increase in de novo DENDRA2::SYS-1 during the E cell cycle (Figure 6A), which has not been shown previously. This result was unexpected as a role of APR-1 as a negative regulator of SYS-1 had been primarily observed in the unsignaled MS cell, which displays higher APR-1 levels (Huang et al., 2007; Sugioka et al., 2011; Baldwin and Phillips, 2014; Baldwin et al., 2016), while these data suggest APR-1 regulates SYS-1 levels in both daughter cells.

FIGURE 6:

FIGURE 6:

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Partial loss of the negative regulator of the Wnt signaling pathway, APR-1, affects both pools of nuclear SYS-1 in the E and MS cells. (A) Quantification of de novo SYS-1 nuclear levels in the E and MS cells after photoconversion at cytokinesis over 7-min time course on lacZ(RNAi) and apr-1 (RNAi) (n = 26 and n = 20, respectively). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (B) Quantification of inherited SYS-1 nuclear levels in the E and MS cells after photoconversion at cytokinesis over 7-min timecourse on lacZ(RNAi) and apr-1 (RNAi). Mean ± SEM. Two-way ANOVA followed by Sidak mixed effects analysis in multiple comparisons test. (C) Graph representation of the fold change in de novo and inherited nuclear SYS-1 of APR-1 compared with the control in the E and MS cells. (D) Quantification of centrosomal DENDRA2::SYS-1 levels during telophase in the EMS cell on lacZ(RNAi) and apr-1 (RNAi) (n = 20 and n = 18, respectively). Mean ± SEM. Unpaired two-tailed t test. * represent the P-value when comparing all timepoints and the average of control to experimental condition. Supplemental Table S1. P-values when comparing each timepoint. DENDRA2::SYS-1 fluorescent intensity was calculated by subtracting N2 fluorescence at all timepoints. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Additionally, we observe different nuclear DENDRA2::SYS-1 dynamics when APR-1 is knocked down compared with other backgrounds with elevated de novo DENDRA2::SYS-1. The E and MS cells de novo DENDRA2::SYS-1 levels show a slower decrease compared with the DLC-1, RSA-2, and ECPS-1 knockdowns (Table 1), over the 7-min time course (Figure 6A). These results suggest that the more rapid SYS-1 decrease we observed after instances of elevated SYS-1 inheritance is due to the activity of the destruction complex. We conclude that de novo SYS-1 in both daughter cells is limited by the destruction complex in wild type and in situations with elevated inherited SYS-1.

We also observed increased levels of inherited nuclear SYS-1 in both E and MS daughter cells after APR-1 depletion (Figure 6B). Though the effect was visible in E, the highest fold change of de novo nuclear DENDRA2::SYS-1 after APR-1 depletion was in MS (Figure 6C). In MS, the de novo pool was more highly affected by loss of APR-1 than the inherited pool, suggesting that, in wild type, APR-1 is more critical for negatively regulation of de novo SYS-1 (Figure 6C). Though the effect of APR-1 depletion on inherited SYS-1 in MS leads to an average of 3.1-fold increase compared with wild type; this was less than the 7.9-fold increase we see in the MS de novo SYS-1 pool. By contrast, the difference between E inherited and E de novo SYS-1 after APR-1 depletion is not as striking (1.4-fold and 2.3-fold increase compared with wild type, respectively) (Figure 6C). Therefore, the major regulatory function of APR-1 on SYS-1 nuclear levels in EMS ACD is to limit accumulation of de novo SYS-1 in MS. In the E cell, APR-1 plays a minor role in SYS-1 regulation, likely due to APR-1 repression due to Wnt signaling.

Despite the decreased effect of APR-1 depletion on DENDRA2::SYS-1 nuclear levels in E, our results suggest that APR-1 is also functioning in this Wnt-signaled daughter cell. However, an alternative hypothesis explaining our increased SYS-1 levels in E after APR-1 depletion is that APR-1 negatively regulates SYS-1 earlier in the EMS lineage, such as in EMS itself. In this case, increased SYS-1 inheritance is would be expected to increase the total SYS-1 levels and possibly the stability of de novo SYS-1 in EMS daughter cells. To distinguish between these possibilities, we knocked down APR-1 and observed changes in total centrosomal DENDRA2::SYS-1 levels in the EMS cell during mitosis with no photoconversion. Centrosomal SYS-1 quantitation is a suitable readout of SYS-1 levels in the EMS cell and its progenitors because SYS-1 centrosomal accumulation reflects cytoplasmic SYS-1 levels (Vora and Phillips, 2015b) and any effect of SYS-1 levels can be clearly observed immediately before the cell divides, providing the latest timepoint possible before the birth of E and MS. We found that there is no significant change in centrosomal DENDRA2::SYS-1 levels during telophase of the EMS cell on apr-1(RNAi) compared with wild type (Figure 6D), suggesting SYS-1 is not negatively regulated by APR-1 in EMS or its progenitors. Thus, our observations suggest that when the inherited SYS-1 pool is affected in the EMS mother cell, the de novo SYS-1 pools are secondarily affected in the daughter cells. Additionally, here we report a role for APR-1 limiting the accumulation of both SYS-1 pools in both the signaled and unsignaled EMS daughter cells.

DISCUSSION

In C. elegans, successful asymmetric cell divisions throughout development depend on proper regulation of SYS-1 levels. Here we show that in wild-type conditions, proper SYS-1 centrosomal degradation in the EMS mother cell is key to regulate inheritance into the E and MS cells during ACD. It was previously established that SYS-1 centrosomal trafficking, coupling and degradation in the mother cell is key for regulation of limiting subsequent daughter cell SYS-1 levels and subsequent asymmetric cell fate specification (Vora and Phillips, 2015b; Thompson et al., 2022b). These data suggested SYS-1 inheritance was normally limited by centrosomal degradation and that de novo synthesized SYS-1, regulated by asymmetric Wnt pathway activity, was the source of nuclear SYS-1 important for ACD. However, SYS-1 inheritance (or the lack thereof) and the role of centrosomal degradation have not been demonstrated. Photobleaching experiments show that mother cell bleaching of GFP::SYS-1 decreases the total GFP::SYS-1 in the daughter cells, suggesting that inherited SYS-1 was present in daughter cell nuclei in wild-type conditions. DENDRA2::SYS-1 photoconversion at cytokinesis directly confirmed DENDRA2::SYS-1 inheritance to both the E and the MS cell and that loss of centrosomal SYS-1 degradation not only increases the inherited DENDRA2::SYS-1 pool but also unexpectedly increases/stabilizes the de novo synthesized, or de novo matured, pool. It should be noted that newly synthesized Dendra2 takes ∼40 min to mature (Wang et al., 2014), suggesting that the measured newly folded and fluorescent DENDRA2::SYS-1 ultimately derived from the early embryo and is only inherited and accumulated in a permissive cytoplasmic environment. We show that APR-1 constitutes an important part of this regulatory environment, as it negatively regulates both inherited and de novo SYS-1 in E and MS, and the rate of clearance of extra inherited SYS-1 is dependent on APR-1 function. Photoconvertible SYS-1 also allowed us to test the model of SYS-1 trafficking to its final destination in the cell, the mitotic centrosome. Photoconversion of DENDRA2::SYS-1 at the centrosome during metaphase demonstrated that newly synthesized DENDRA2::SYS-1 is continually trafficked to the centrosome during mitosis, resulting in stable steady state SYS-1 levels despite this localization leading to increased SYS-1 turnover (Vora and Phillips, 2015b). In contrast, preexisting SYS-1 is more enriched at the centrosome and decreases through mitosis. Thus, proper SYS-1 levels are dependent on both trafficking and degradation of SYS-1 in the mother cell along with negative regulation via the destruction complex in the daughter cells.

Though a linkage of apparently disparate cellular process in the daughter cells, including pericentriolar scaffolding, proteasomal transport, and dynein trafficking, cannot be currently ruled out as regulating SYS-1 nuclear localization in daughter cells, we propose the following model (Figure 7) that focuses on mother cell SYS-1 inheritance model since it builds on our previously observed mother cell SYS-1 phenotypes, such as SYS-1 centrosomal localization, while also incorporating data presented here. In wild-type conditions during ACD of the EMS cell, a portion of cytoplasmic SYS-1 is trafficked to the centrosome where the scaffold protein RSA-2 localizes SYS-1 to the centrosome. Centrosomal SYS-1 is degraded by the centrosomal proteosome, while the remaining cytoplasmic SYS-1 is inherited into the E and MS daughter cells. Inherited SYS-1 is then differentially regulated by APR-1, primarily (but not exclusively) in MS cell. De novo matured SYS-1 also accumulates in both daughter cells, with the E cell having higher nuclear SYS-1 levels than the MS cell, and the de novo pool is similarly regulated by APR-1 as the inherited pool (Figure 7A).

FIGURE 7:

FIGURE 7:

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Model for SYS-1 regulation during ACD. See text for details. Weight of the arrows and lines represent the relative changed effect, oval size represents size of the relevant SYS-1 pool. SYS-1i, inherited SYS-1; SYS-1dn, de novo SYS-1.

Loss of mother cell regulation of centrosomal SYS-1 levels via depletion of RSA-2, DLC-1, or ECPS-1 prevents proper degradation of mother cell SYS-1 leading to overinheritance of SYS-1 in both daughter cells. A higher rate of SYS-1 inheritance is correlated with higher de novo SYS-1 levels in both daughter cells, indicating an inability of the destruction complex to effectively regulate both SYS-1 pools due to dilution of the destruction complex activity. The overwhelming of the destruction complex allows SYS-1 nuclear levels to be higher in both the E and MS cell (Figure 7B).

Loss of the negative regulator APR-1 has a direct effect in the de novo levels in both the E and the MS cells as we see no effect on EMS mother cell SYS-1 levels (Figure 6D). Due to the similar regulation of de novo SYS-1 and inherited SYS-1 pools by APR-1, increased levels of both pools were observed after APR-1 depletion; however, this was likely due to increased stability of just the de novo pool. A loss of APR-1 therefore leads to increased total SYS-1 levels and a loss of daughter cell asymmetry (Figure 7C) (Huang et al., 2007).

Focusing on the EMS cell during cytokinesis, our model proposes that cell polarization by the Wnt gradient leads to differential distribution of APR-1. In the posterior region of the mother cell, APR-1 will not accumulate due to increased Wnt signaling (Figure 7D). In the anterior region of the cell, as documented previously (Sugioka et al., 2018), there will be a greater accumulation of APR-1. This pattern of APR-1 asymmetry will drive asymmetric stability of SYS-1 postdivision (Figure 7, E and F). In the first portion of the daughter cell cycle, from 2 to 6 min postcytokinesis (Figure 7E), the E cell increases its total nuclear SYS-1 levels, which encompasses the inherited and the de novo SYS-1 pools, due to decreased APR-1 levels and activity resulting from active Wnt signaling. During this same period, APR-1 in the MS cell decreases SYS-1 stability, leading to low total nuclear SYS-1 levels. Late during the cell cycle of the daughter cells, 8 to 10 min after cytokinesis (Figure 7F), we observe decreased total SYS-1 levels in the E cell due to increasing negative regulation via APR-1. In the MS cell, a more pronounced SYS-1 decrease is observed, suggesting a lack of Wnt signaling and the negative regulation of de novo and inherited SYS-1 via APR-1 (Figure 7F). Of note, Dendra2 maturation time is thought to be more rapid than original Dendra, but it remains likely that our quantitation of postconverted (green) DENDRA2::SYS-1 represents an underestimate of total (matured + immature) de novo protein.

Partial cytoplasmic photoconversions demonstrate that cytoplasmic SYS-1 can travel to either centrosome within 20 s, with a relatively minor difference between centrosomes proximal and distal (Figure 3, C and D). This minor difference was somewhat surprising given the essential role we previously noted for dynein trafficking (Thompson et al., 2022a), which would suggest a multistep process for efficient movement from the proximal to the distal centrosomal locale; namely: disengagement from proximal trafficking processes, diffusion to the distal cytoplasm, subsequent interaction with dynein components and then trafficking to the distal centrosome. A more parsimonious model may be that a rapid diffusion-capture mechanisms also plays a role at the centrosome directly and that dynein trafficking is required to elevate the steady state of centrosomal SYS-1 above baseline cytoplasmic levels. In this case, the trafficking mechanism depicted in Figure 7A would include passive diffusion and would only be partially decreased in the perturbations shown in Figure 7B.

Why is inherited SYS-1 asymmetric between E and MS?

Interestingly, the inherited DENDRA2::SYS-1 is not equal for both daughter cells; E shows higher levels of SYS-1 through the time course than its sister MS (Figure 4B). Previous studies show a strong asymmetry of APR-1 during the EMS cell division, accumulating in the anterior side (Sugioka et al., 2011; Sugioka et al., 2018). Our data show that APR-1 predominantly functions in the MS cell, though we do also see APR-1 depletion effects in E (Figure 6C). This suggests that SYS-1 inheritance may be symmetric (Vora and Phillips, 2015b), or at least correlates with the volume of the cytoplasm, but the stability of SYS-1 is asymmetric in the two daughter cells. In this model, inherited DENDRA2::SYS-1 in wild type is more stable and prevalent in the E cell (Figure 4B). However, it remains feasible that asymmetric inheritance of other Wnt signaling components, such as Frizzled or Dishevelled, contribute to the asymmetry observed. Indeed, asymmetric MOM-5/Frizzled has been reported in the early embryonic blastomeres (Park et al., 2004) and the seam cells (Mizumoto and Sawa, 2007), suggesting these components could be influencing the distribution of other Wnt signaling components during ACD. Together, these data suggest that, while SYS-1 is symmetrically inherited into the daughter cells at the time of cytokinesis, asymmetric daughter cell regulation and stability of the inherited SYS-1 via asymmetric inheritance of positive and negative regulators of Wnt signaling ultimately controls nuclear levels of inherited SYS-1.

Why does overinheritance of SYS-1 correlate with higher de novo SYS-1 levels?

While loss of microtubule trafficking, centrosomal coupling or degradation of SYS-1 in the mother cell led to an increase in the amount of inherited DENDRA2::SYS-1, we also observed increased levels of de novo DENDRA-2::SYS-1 in both daughter cells. These unexpected results led to further investigation of the role of negative regulators of SYS-1 in the Wnt signaling pathway. Because APR-1 depletion primarily affects de novo DENDRA2::SYS-1 rather than DENDRA2::SYS-1 levels in the mother cell (Figure 6, A and D), we used this background to test the effect of increased de novo DENDRA2::SYS-1 on maintenance of the inherited DENDRA2::SYS-1 pool. We observed increased inherited nuclear DENDRA2::SYS-1 levels in both daughter cells (Figure 6B). These results supported our hypothesis that when either of the pools is affected, this will influence the amount of nuclear SYS-1 in the other pool. These results could be explained by an overwhelming of the destruction complex that is equally targeting both de novo and inherited SYS-1 pools, thus leading to an accumulation of de novo SYS-1 as destruction complex activity is diluted by increased inherited SYS-1. Our data suggest that increased levels of SYS-1 in the daughter cells directly increases SYS-1 stability in the primarily affected pool (inherited DENDRA2::SYS-1 in dlc-1, rsa-2 and ecps-1[RNAi] or de novo DENDRA2::SYS-1 in apr-1[RNAi]), which in turn perturbs negative regulation of the other pool, leading to an increase in the total levels of nuclear SYS-1. Thus, we conclude that the SYS-1 pools are interchangeable, and both regulated by the destruction complex.

We also allowed tested the hypothesis that the rate of nuclear SYS-1 loss observed in cases of elevated inherited DENDRA2::SYS-1 levels (RSA-2, ECPS-1, or DLC-1 depletion) are due to the function of the destruction complex decreasing SYS-1 back to basal levels. APR-1 knockdown indeed led to a more moderate rate of decrease in de novo SYS-1 levels throughout the time course compared with the other conditions tested (dlc-1, rsa-2 and ecps-1[RNAi]), suggesting efficient clearance of extra SYS-1 requires APR-1 (Table 1) (Figure 6A).

APR-1 functions in the E cell?

Our data suggest that the destruction complex functions in the Wnt-signaled E cell. When knocking down APR-1, we found increased levels of de novo nuclear DENDRA2::SYS-1 in both the E and the MS, and while this result was expected for the MS cell, we did not expect to see significant changes in the E cell (Figure 6, A and B). Previous studies investigated the kinetic responses of β-catenin to Wnt signaling in mammalian cells and found that the destruction complex functions in both the absence and presence of Wnt signaling. However, when Wnt ligand is present, the destruction complex is only partially active (Hernández et al., 2012). While this has not yet been described in C. elegans, our results support those findings. FRAP experiments demonstrated that de novo GFP::SYS-1 plateaus 6 to 8 min after cytokinesis, suggesting its stabilization is temporally limited in E (Figure 1D), and this could be due to destruction complex function. Additionally, the partial depletion of APR-1 via RNAi in our experiments seems to lower the steady state of degradation when Wnt signaling is present, which supports the observed role for the destruction complex in the E cell.

While our studies focused on the EMS daughter cells, previous studies showed that in both the daughters of E (Ea and Ep) and MS (MSa and MSp), SYS-1 asymmetry is lost after apr-1(RNAi) (Huang et al., 2007). These results further support the idea that APR-1 regulation is key to maintain daughter cell asymmetry and its role in both E, the Wnt-signaled daughter cell and MS, the unsignaled cell. In addition, it has been previously shown that the observed asymmetry is not due to nuclear export or differential subcellular distribution of SYS-1, but to proteasomal degradation (Huang et al., 2007). These data suggest that SYS-1 asymmetry is tightly regulated by proteasomal degradation via mother cell centrosomal processing and the destruction complex function in the EMS daughters.

Our proposed mechanism of SYS-1 regulation and inheritance during ACD sheds light on the tight regulation of ACD processes and the several layers of negative regulation needed for proper asymmetric target gene activation. Indeed, trafficking and degradation of mother cell SYS-1 maintains SYS-1 levels in the daughters at a level that can be effectively regulated by the destruction complex.

Supplementary Material

mbc-36-ar25-s001.pdf (869.3KB, pdf)

Acknowledgments

We thank members of the Phillips lab for comments on the manuscript. Strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD01440). We also want to thank the Carver Center for Imaging at the University of Iowa and its director, Dr. Michael Dailey. This work was supported by NIH award RO1GM114007 (to B.T.P), and University of Iowa Graduate College Fellowships to M.V.

Abbreviations used:

ACD

Asymmetric Cell Division

APC

Adenomatous Polyposis Coli

CEI

Centrosomal Enrichment Index

CK1α

Casein Kinase 1α

FRAP

fluorescence recovery after photobleaching

GSK3β

Glycogen Synthase Kinase 3β

IPTG

isopropyl β-d-1-thiogalactopyranoside

NGM

Nematode Growth Media

NIH

National Institutes of Health

ROI

region of interest

WβA

Wnt/α-catenin Asymmetry

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E24-10-0441) on January 15, 2025.

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Supplementary Materials

mbc-36-ar25-s001.pdf (869.3KB, pdf)

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