Chinsen, O. | bioRxiv Science

bioRxiv2025

Scd1 diffuses end to end along the cytoplasm to facilitate Cdc42 activation and bipolar growth

The conserved GTPase Cdc42 is a major regulator of polarized growth in most eukaryotes. In Schizosaccharomyces pombe, Cdc42 activity displays anticorrelated oscillatory dynamics between the growing ends enabling bipolarity. Cdc42 at each end is activated only when the opposite end loses activity. This suggests that a regulator of Cdc42 likely travels end-to-end to activate Cdc42. The oscillatory dynamics between the growing ends have also been observed in Cdc42 activator Scd1, its scaffold Scd2. It is unclear how these proteins move between the ends to facilitate bipolarity. We find that Scd1 does not travel between the cell ends via actin-mediated delivery. Instead, we show that Scd1 is mostly cytoplasmic and diffuses between the cell ends. The rate of diffusion is not entirely proportional to increasing the mass of Scd1 and cells lacking the inhibitor Pak1 kinase show decreased diffusion. Moreover, we show that Scd1 diffuses at a much faster rate compared to its scaffold Scd2. These findings suggest that Scd1 diffusion is not random and is regulated by Pak1 kinase. We find that decreasing the rate of diffusion disrupts Cdc42 oscillatory dynamics and results in monopolarity. Our results show that end-to-end Scd1 diffusion drives Cdc42 oscillatory dynamics and regulates cell polarity. SIGNIFICANCE STATEMENTO_LICdc42 activation shows oscillatory dynamics between the sites of growth C_LIO_LIThe Cdc42 GEF Scd1 diffuses from site of activation to the opposite end to facilitate these oscillatory dynamics C_LIO_LIThis diffusion is not random and likely depends on intrinsic properties of the Scd1 protein. C_LI

cell biology↗

bioRxiv2023

The Arp2/3 complex promotes periodic removal of Pak1-mediated negative feedback to facilitate anticorrelated Cdc42 oscillations.

The conserved GTPase Cdc42 is a major regulator of polarized growth in most eukaryotes. Cdc42 periodically cycles between active and inactive states at sites of polarized growth. These periodic cycles are caused by positive feedback and time-delayed negative feedback loops. In the bipolar yeast S. pombe, both growing ends must regulate Cdc42 activity. At each cell end, Cdc42 activity recruits the Pak1 kinase which prevents further Cdc42 activation thus establishing negative feedback. It is unclear how Cdc42 activation returns to the end after Pak1-dependent negative feedback. Using genetic and chemical perturbations, we find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. With our experimental data and mathematical models, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. In agreement with these observations, our model and experimental data show that in each oscillatory cycle, Cdc42 activation increases followed by an increase in Pak1 recruitment at that end. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity.

cell biology↗