ABS 047: Investigating pathways regulating size-dependent accumulation of mitotic activator Cdc25

Dylan Hernandez , Rachel E. Burke, Benjamin Kuran, Logan Anglemyer, and Kristi E. Miller

Van Wickle (2025) Volume 1, ABS047

Introduction: Cells maintain a characteristic size to function properly, often by delaying cell cycle transitions until reaching a threshold size. In fission yeast, mitotic entry occurs at a defined cell surface area, regulated in part by the size-dependent nuclear accumulation of the phosphatase Cdc25. Recent studies show that Cdc25 accumulates specifically with cell volume, but the mechanisms underlying this regulation remain unknown.

Here, we investigate pathways influencing Cdc25 nuclear accumulation in response to cell volume changes. We focused on fission yeast mutants known to divide at smaller sizes, hypothesizing that these deletions disrupt negative regulators of Cdc25. Using live-cell fluorescence microscopy and an ImageJ/MATLAB pipeline, we quantified mNeonGreen-tagged Cdc25 in both cytoplasm and nucleus of wild-type and small-size mutants.

We identified six mutants that exhibit increased nuclear Cdc25 accumulation relative to wild type. Four are involved in glucose-sensing/cAMP signaling (Pka1, Git3, Git5, Gpa2), one encodes a SWI/SNF complex subunit (Sol1), and one encodes a nuclear poly(A) binding protein (Pab2). Given the enrichment of glucose-signaling components, we investigated glucose-dependent regulation of Cdc25. Prior studies suggest that in glucose-rich conditions, Cdc25 is targeted for degradation by the E3 ubiquitin ligase Pub1.

We show that pub1 deletion mutants also accumulate elevated nuclear Cdc25 in a size-dependent manner, similar to cAMP pathway mutants. This implies a potential mechanistic link between Pub1-mediated degradation and PKA signaling in restricting Cdc25 accumulation in glucose-rich conditions.
Ongoing experiments use genetic interaction and protein stability assays to define how Pka1 and Pub1 interact to regulate Cdc25 levels. Together, our findings reveal a conserved regulatory network connecting nutrient sensing and protein degradation to cell size control, providing insight into how cells integrate growth signals to maintain size homeostasis.

Methods: To measure the concentration of Cdc25 in our wild type and small-sized mutant cells, we tagged Cdc25 with the green fluorescent marker mNeonGreen. Using a spinning disk confocal microscope, we were able to take three separate images: Cdc25-mNG (for measuring Cdc25 concentration), brightfield (to visualize cell outlines), and BFP-NLS (to visualize/locate the nucleus. Binary masks were made for the brightfield and BFP-NLS images and then laid over the mNG images, which we ran through a semi-automated pipeline in MATLAB that was able to measure the Cdc25-mNG in the nuclei and cytoplasm. This MATLAB protocol also allowed us to measure the cell surface area and volume.

Results: We found that Cdc25 concentration increased in git5Δ, git3Δ, sol1Δ, pab2Δ, pka1Δ, gpa2Δ mutant cells. We have proven that cells grown in low glucose also accumulate higher concentrations of Cdc25 compared to nutrient rich controls. We have also found that pub1 deletion cells also accumulate more Cdc25 in the nucleus as a function of size compared to wild type cells.

Discussion: We found that Cdc25 concentration increased in git5Δ, git3Δ, sol1Δ, pab2Δ, pka1Δ, gpa2Δ mutant cells, suggesting that these genes may encode negative regulators of Cdc25. Pka may negatively regulate Cdc25 directly. Cdc25 has been shown to be a target of Pka in Xenopus, mouse-fertilized egg, and mammalian oocytes (Duckworth et al., 2002; Cui et al., 2008; Pirino et al., 2009). Cdc25 has been shown to be a target of Pub1-mediated protein delegation (Nefsky and Beach, 1996; Kishimoto and Yamashita, 2000). Future studies will reveal if Pub1 is regulated by Pka1 to control Cdc25 levels for cell adaptation to low glucose.