Vora, Setu et al. (2015) International Worm Meeting "Centrosome-associated degradation limits SYS-1/beta-catenin inheritance after asymmetric cell division."

Vora, Setu, Phillips, Bryan

[International Worm Meeting, 2015]

Cells of the C. elegans embryo navigate through invariant lineages in serial asymmetric cell divisions (ACDs), many of which are controlled by a modified Wnt/beta-catenin signaling strategy. Daughter cells derived from a Wnt-dependent ACD exhibit nuclear asymmetry of the transcriptional coactivator SYS-1/beta-catenin, resulting in differential activation of Wnt-responsive target genes and distinct cell fates. We investigated how dynamic localization of SYS-1 to mitotic centrosomes influenced SYS-1 inheritance and cell fate outcomes in daughter cells after division of the endomesodermal (EMS) blastomere. Through yeast two-hybrid screening, we identified the centrosomal protein RSA-2 as a SYS-1 binding partner and showed that localization of SYS-1 to mitotic centrosomes was dependent on RSA-2. Uncoupling SYS-1 from the centrosome by RSA-2 depletion increased SYS-1 inheritance after ACD and promoted Wnt-dependent cell fate. Photobleaching experiments revealed that the entire fraction of centrosome-bound SYS-1 turns over rapidly during mitosis. Interestingly, disruption of the proteasome led to an increased accumulation of SYS-1 at the centrosome but decreased the mobile fraction and turnover rate. We conclude that centrosomal targeting of SYS-1 promotes its degradation during asymmetric cell division. We propose a model whereby centrosome-associated SYS-1 degradation couples negative regulation with cell division timing to facilitate SYS-1 clearance from the mother cell at the time of asymmetric division. These results suggest that proper spatial organization of signaling effectors such as SYS-1/beta-catenin is necessary for permitting their efficient regulation during dynamic processes such as ACD. .

Vora, Setu M. et al. (2013) International Worm Meeting "Centrosomal localization of SYS-1/beta-catenin is required for proper expression patterns during asymmetric cell division."

Vora, Setu M., Phillips, Bryan T.

[International Worm Meeting, 2013]

The Wnt/b-catenin asymmetry pathway is responsible for carrying out a number of different asymmetric cell divisions throughout C. elegans development. These divisions give rise to daughter cells with differential activity of Wnt signaling components. SYS-1/b-catenin is a principal transcriptional effector in this pathway and allows daughter cells from an asymmetric division to adopt distinct transcriptional profiles and according cellular fates. Before division, SYS-1 symmetrically localizes to the centrosomes and is asymmetrically expressed between daughter cells after division. Interestingly, vertebrate b-catenin also displays centrosomal localization during division, but it is unknown what role centrosomal localization plays in any b-catenin regulation. Through yeast two-hybrid screening, we have identified the centrosomal scaffold protein RSA-2 (regulator of spindle assembly) as a strong positive interacting partner of SYS-1. RNAi knockdown of RSA-2 results in decreased centrosomal targeting of SYS-1. We are using rsa-2 (RNAi) to examine the fate of centrosomally uncoupled SYS-1 during asymmetric divisions. Our research shows that in rsa-2 (RNAi), SYS-1 relocalizes near kinetochore microtubules during division, suggesting a trafficking mechanism for targeting of nuclear SYS-1 to the centrosomes. Furthermore, uncoupling SYS-1 from the centrosome promotes Wnt-signaled cell fate and disrupts patterns of SYS-1 asymmetry after division. Taken together, these results suggest that SYS-1 localization to the centrosome results in SYS-1 clearance from the mother cell before cytokinesis to allow faithful regulation of de novo SYS-1 in the daughter cells. These analyses will allow us to understand dynamic expression patterns of SYS-1 during asymmetric division and explore how subcellular localization relates to regulation of cell fate determinants during development.

Phillips, Bryan et al. (2021) International Worm Meeting "Beta-catenin centrosomal localization regulates Wnt signaling in C. elegans development and human cells"

Vora, Setu, Phillips, Bryan

[International Worm Meeting, 2021]

The Wnt/beta-catenin signaling pathway is central to metazoan development as well as adult tissue homeostasis and Wnt signaling is routinely dysregulated in cancer and congenital disorders. Caenorhabditis elegans embryos serially employ a modified Wnt/beta-catenin pathway embryos to drive asymmetric cell division (ACD) and rapidly diversify cell fate. Wnt-dependent ACDs rely on nuclear asymmetry of the transcriptional coactivator SYS-1/beta-catenin between daughter cells to differentially activate Wnt-responsive target genes. Interestingly, SYS-1 dynamically localizes to mitotic centrosomes in a symmetrical fashion during many embryonic ACDs. This centrosomal localization pattern enables proteasomal processing and limits SYS-1 levels in daughter cells; uncoupling SYS-1 from the centrosome leads to increased nuclear SYS-1 in both daughter cells and loss of robust control of Wnt-dependent cell fate. Photobleaching experiments reveal that centrosome-bound SYS-1 turns over rapidly, a process that requires the proteasome. These data demonstrate that centrosomal processing of SYS-1 represents a novel form of beta-catenin negative regulation specifically at mitosis that reinforces SYS-1 regulation by Wnt signaling. Intriguingly, beta-catenin centrosomal localization is well-conserved between nematodes and mammals so we examined the functional contribution of centrosomes to Wnt signaling, beta-catenin regulation, and posttranslational modifications using HEK293 cells. After pharmacological depletion of centrosomes, the cellular response to Wnt signaling is attenuated despite normal beta-catenin synthesis and degradation rates. The attenuated Wnt response is due to accumulation of a novel high-molecular-weight form of phosphorylated beta-catenin. Wnt signaling normally operates by inhibiting a destruction complex that phosphorylates and degrades beta-catenin, but the high-molecular-weight form of phosphorylated beta-catenin is unexpectedly increased by Wnt signaling. Furthermore, gel-shift analyses and mass spectrometry suggest the high-molecular weight form of phospho-beta-catenin results from mono-ubiquitination. These studies have thus identified a pool of beta-catenin effectively shielded from regulation by Wnt. We present a model whereby centrosomes across species regulate beta-catenin stability: centrosomes actively degrade beta-catenin during ACD and prevent inappropriate beta-catenin modifications that antagonize normal stabilization by Wnt signals.

Thompson, Josh et al. (2015) International Worm Meeting "Mechanisms of SYS-1/beta-catenin centrosomal localization in early embryonic blastomeres."

Thompson, Josh, Vora, Setu, Phillips, Bryan

[International Worm Meeting, 2015]

Asymmetric cell division, the unequal distribution of cell fate determinants between daughter cells, is a critical system underlying the development and maintenance of the varied tissue types of a mature organism. Despite widespread utilization of such divisions, the mechanisms behind induction of asymmetry are less understood due to its complex, pleiotropic, and often functionally redundant effects. Throughout C. elegans development, Wnt/beta-catenin signaling induces asymmetric distribution of Wnt signaling components to polarize a mother cell, differentially regulating Wnt target genes in its daughter cells. For instance, a proper Wnt signaling response results in accumulation of cytoplasmic SYS-1/beta-catenin in the proximal 'signaled' cell, which, along with co-activator POP-1/TCF, activates genes to induce the corresponding cell fate. Despite its asymmetric regulation immediately post-division, cytoplasmic SYS-1 localizes symmetrically to mitotic centrosomes. Recent data from our lab indicate that centrosomal localization of SYS-1 serves as a clearance mechanism to increase the robustness of Wnt-mediated polarity. However, the method of SYS-1 trafficking to and accumulation at the centrosome is unknown. In the early embryo, centrosomal localization of the b-catenin SYS-1 is dependent on the centrosomal scaffolding protein RSA-2. When SYS-1 is prevented from loading to the centrosome, by loss of RSA-2, it co-localizes to the region of kinetochore microtubules. This localization suggests a directed transport of SYS-1 to centrosomes along microtubules via the action of molecular motors. We conducted an RNAi and chemical screen specifically aimed at depleting microtubules and microtubule transport proteins and quantified resulting SYS-1 centrosomal accumulation. We focused on dynein and dynactin components as the primary motor machinery for the observed minus end trafficking. We identified several dynein subunits by RNAi knockdown that are required for proper centrosomal localization of embryonic SYS-1/beta-catenin. Further, the preliminary data from microtubule-destabilizing and ATP-depleting chemical treatment indicate that both ATP and microtubules are important for SYS-1 centrosomal localization. Together, these results begin to characterize a regulatory role for the multimeric dynein/dynactin cargo binding complex in SYS-1/beta-catenin localization.

Thompson, Joshua W. et al. (2017) International Worm Meeting "Mechanisms of SYS-1/beta-catenin centrosomal localization in early embryonic blastomeres."

Thompson, Joshua W., Vora, Setu M., Phillips, Bryan T.

[International Worm Meeting, 2017]

Asymmetric cell division, the unequal distribution of cell fate determinants between daughter cells, is a critical system underlying the development and maintenance of varied tissue types in a multicellular organism. Despite widespread utilization of such divisions, the mechanisms responsible for induction of asymmetry are less understood due to the complex, pleiotropic, and often functionally redundant signaling systems involved. Throughout C. elegans development, the Wnt/beta-catenin asymmetry pathway induces the asymmetric distribution of Wnt signaling components to polarize a mother cell and therefore differentially regulate Wnt target genes in its daughter cells. Despite this asymmetry in its regulation immediately post-division, cytoplasmic SYS-1 localizes symmetrically to mitotic centrosomes in a manner consistent with well conserved beta-catenin localization. The subfunctionalization of C. elegans' 4 beta-catenin homologs allows us to investigate the role of this phenomenon with particular focus on the transcriptional regulation of the Wnt/ beta -catenin asymmetry pathway. Recent data indicate that centrosomal localization of SYS-1 serves as a clearance mechanism to increase the robustness of Wnt-mediated polarity. However, the method and regulation of SYS-1 trafficking to and accumulation at the centrosome is unknown. In the early embryo, this centrosomal localization of the SYS-1 is dependent on the centrosomal scaffolding protein RSA-2. Loss of RSA-2 by RNAi prevents SYS-1 centrosomal loading, forcing it to accumulate in the approximate region of kinetochore microtubules. This localization suggests that SYS-1 may be directly transported to centrosomes along microtubules via the action of molecular motors. Given the minus-end directed movement implied by the localization at a microtubule organizing center, we focused on dynein and dynactin components as the primary minus-end directed motor. Preliminary data from microtubule-destabilizing and ATP-depleting chemical treatment indicate that both ATP and microtubules are important for SYS-1 centrosomal localization. A temperature sensitive allele of dynein heavy chain show a disproportionate effect on SYS-1 localization. Additionally, an RNAi screen aimed at depleting these microtubules and microtubule transport proteins has identified several dynein light chain subunits that promote proper centrosomal localization of embryonic SYS-1/beta-catenin. Together, these results suggest an active role for the multimeric dynein/dynactin cargo binding complex in SYS-1/beta-catenin regulation and signaling status.