A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis in fission yeast.

Cdc42, a conserved regulator of cell polarity, is activated by two GEFs, Gef1 and Scd1, in fission yeast. Why the cell needs two GEFs is unclear, given that they are partially redundant and activate the same GTPase. Using the GEF localization pattern during cytokinesis as a paradigm, we report a novel interplay between Gef1 and Scd1 that spatially modulates Cdc42. We find that Gef1 promotes Scd1 localization to the division site during cytokinesis through recruitment of the scaffold protein Scd2, via a Cdc42 feedforward pathway. Similarly, during interphase Gef1 promotes Scd1 recruitment at the new end to enable the transition from monopolar to bipolar growth. Reciprocally, Scd1 restricts Gef1 localization to prevent ectopic Cdc42 activation during cytokinesis to promote cell separation, and to maintain cell shape during interphase. Our findings reveal an elegant regulatory pattern in which Gef1 primes Cdc42 activation at new sites to initiate Scd1-dependent polarized growth, while Scd1 restricts Gef1 to sites of polarization. We propose that crosstalk between GEFs is a conserved mechanism that orchestrates Cdc42 activation during complex cellular processes.This article has an associated First Person interview with the first author of the paper.

Arp2/3-dependent endocytosis ensures Cdc42 oscillations by removing Pak1-mediated negative feedback.

The GTPase Cdc42 regulates polarized growth in most eukaryotes. In the bipolar yeast Schizosaccharomyces pombe, Cdc42 activation cycles periodically at sites of polarized growth. These periodic cycles are caused by alternating positive feedback and time-delayed negative feedback loops. At each polarized end, negative feedback is established when active Cdc42 recruits the Pak1 kinase to prevent further Cdc42 activation. It is unclear how Cdc42 activation returns to each end after Pak1-dependent negative feedback. We find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. Using experimental and mathematical approaches, 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. 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.

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.