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.

Cdc42 GTPase activating proteins Rga4 and Rga6 coordinate septum synthesis and membrane trafficking at the division plane during cytokinesis.

Fission yeast cytokinesis is driven by simultaneous septum synthesis, membrane furrowing and actomyosin ring constriction. The septum consists of a primary septum flanked by secondary septa. First, delivery of the glucan synthase Bgs1 and membrane vesicles initiate primary septum synthesis and furrowing. Next, Bgs4 is delivered for secondary septum formation. It is unclear how septum synthesis is coordinated with membrane furrowing. Cdc42 promotes delivery of Bgs1 but not Bgs4. We find that after primary septum initiation, Cdc42 inactivators Rga4 and Rga6 localize to the division site. In rga4Δrga6Δ mutants, Cdc42 activity is enhanced during late cytokinesis and cells take longer to separate. Electron micrographs of the division site in these mutants exhibit malformed septum with irregular membrane structures. These mutants have a larger division plane with enhanced Bgs1 delivery but fail to enhance accumulation of Bgs4 and several exocytic proteins. Additionally, these mutants show endocytic defects at the division site. This suggests that Cdc42 regulates primary septum formation and only certain membrane trafficking events. As cytokinesis progresses Rga4 and Rga6 localize to the division site to decrease Cdc42 activity to allow coupling of Cdc42-independent membrane trafficking events with septum formation for proper septum morphology.