The Coordination of Cell Growth during Fission Yeast Mating Requires Ras1-GTP Hydrolysis

Abstract

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity.

Local Pheromone Release from Dynamic Polarity Sites Underlies Cell-Cell Pairing during Yeast Mating

“…Indeed, deletion of Gap1, the predicted GTPase activating protein (GAP) for Ras1 [39, 40], caused an important increase in zone lifetime: heterothallic gap1Δ M-cells lacking the P-factor protease Sxa2 exposed to homogeneous P-factor (0.01 to 1μM) displayed dynamic Scd2 zones, but these exhibited significantly longer lifetimes than gap1+ controls exposed to the same pheromone concentrations (Figure 4C). Some of these cells also lysed, as previously reported [40]. Thus, Ras inactivation promotes polarity zone disassembly.…”

Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase

“…However, while deletion of both Scd1 and Gef1 is lethal (Coll et al, 2003), we noticed that cells lacking scd2 and gef1 are viable ( Fig S1A) which suggested that other mechanisms for Scd1 recruitment and/or activation exist. Several pieces of data implicated the small GTPase Ras1 as positive regulator of Cdc42 acting upstream of Scd1 (Chang et al, 1994;Merlini et al, 2016;Weston et al, 2013). Indeed, we find that ras1 is required for viability of scd2Δ gef1Δ double mutant cells (Tables S1-S2), but not gef1Δ nor scd2Δ single mutants ( Fig S1B-C).…”

“…Notably, scd2 deletion causes cell rounding (Chang et al, 1994;Kelly and Nurse, 2011b) but does not severely affect Cdc42 activity, in contrast to scd1∆ , implying the existence of alternative mechanisms for Cdc42 activation. Several pieces of data implicate the small GTPase Ras1 as a Cdc42 regulator upstream of Scd1 (Chang et al, 1994;Merlini et al, 2018;Weston et al, 2013): Ras1 binds Scd1 directly (Chang et al, 1994), is localized to the plasma membrane and activated at the cell ends like Cdc42 , and its deletion causes partial cell rounding (Fukui et al, 1986).…”

Constitutively active RAS in S. pombe causes persistent Cdc42 signalling but only transient MAPK activation

“…Studies based on recombinant protein assays and yeast-2-hybrid analysis demonstrated 87 that Ras1 interacts with both MAPKKK Byr2 and Scd1, a GDP-GTP exchange factor (GEF) for Cdc42, 88 which regulates the actin cytoskeleton and cell morphology (Chang et al, 1994; Gronwald et al, 89 2001;Tu et al, 1997). These observations suggest that Ras1 simultaneously regulates both the 90 pheromone MAPK Spk1 and the Cdc42 pathways at the cell membrane (Weston et al, 2013). 91Indeed, a dynamic "Cdc42 zone" at the cell cortex prior to mating has been observed (Merlini et 92 al., 2013) and Ras1 and MAPK Spk1 cascade components are found there and are involved in the 93 process (Dudin et al, 2016;Merlini et al, 2013;Merlini et al, 2016;Merlini et al, 2018; Weston 94 et al, 2013).…”

“…Studies based on recombinant protein assays and yeast-2-hybrid analysis demonstrated 87 that Ras1 interacts with both MAPKKK Byr2 and Scd1, a GDP-GTP exchange factor (GEF) for Cdc42, 88 which regulates the actin cytoskeleton and cell morphology (Chang et al, 1994; Gronwald et al, 89 2001;Tu et al, 1997). These observations suggest that Ras1 simultaneously regulates both the 90 pheromone MAPK Spk1 and the Cdc42 pathways at the cell membrane (Weston et al, 2013). 91…”