PAK-family kinases promote cell fusion irreversibility by preventing cell-wall repair.

Abstract

Cell-cell fusion is critical for the sexual life cycle, as it drives the unidirectional transition between haploid and diploid phases. In fungi, fusion requires not only plasma membrane merging but also local cell-wall removal. However, the integrity of the cell wall, which resists internal turgor pressure and is essential for survival, is normally monitored by a cell-wall integrity pathway (CIP) involving Rho1 GTPase, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) signaling, which promotes damage repair. How the CIP allows localized cell-wall degradation is not known. In the fission yeast Schizosaccharomyces pombe, we previously identified pak2Δ mutants that exhibit transient fusion: cells briefly fuse and exchange cytoplasmic contents, which signal catastrophic haploid meiosis, but then reseal their fusion pore. Here, we show that the kinase activity of p21-activated kinases (PAK) is essential for cell-cell fusion, with major and minor contributions from Pak2 and the essential Pak1, respectively. Strikingly, osmotic stabilization largely suppresses all PAK-associated fusion defects, pointing to a role in cell-wall remodeling. Pak2 accumulates at the fusion site, where it promotes fusion pore opening and expansion. In its absence, Rho1-GTP, PKC, and beta-glucan synthase Bgs4 show elevated levels at the fusion site, and their partial loss of function rescues fusion. Pak1 mutants have elevated MAPK activity. Thus, Pak2 and Pak1 both antagonize the CIP. Furthermore, correlative light and cryo-soft-X-ray tomography reveals that resealed pores in PAK mutants rebuilt an intact cell wall. We conclude that PAKs enforce fusion directionality by antagonizing cell-wall repair mechanisms that otherwise restore separation between mating partners.