Multi-phosphorylation reaction and clustering tune Pom1 gradient mid-cell levels according to cell size

Abstract

Protein concentration gradients pattern developing organisms and single cells. In Schizosaccharomyces pombe rod-shaped cells, Pom1 kinase forms gradients with maxima at cell poles. Pom1 controls the timing of mitotic entry by inhibiting Cdr2, which forms stable membrane-associated nodes at mid-cell. Pom1 gradients rely on membrane association regulated by a phosphorylation-dephosphorylation cycle and lateral diffusion modulated by clustering. Using quantitative PALM imaging, we find individual Pom1 molecules bind the membrane too transiently to diffuse from pole to mid-cell. Instead, we propose they exchange within longer lived clusters forming the functional gradient unit. An allelic series blocking auto-phosphorylation shows that multi-phosphorylation shapes and buffers the gradient to control mid-cell levels, which represent the critical Cdr2-regulating pool. TIRF imaging of this cortical pool demonstrates more Pom1 overlaps with Cdr2 in short than long cells, consistent with Pom1 inhibition of Cdr2 decreasing with cell growth. Thus, the gradients modulate Pom1 mid-cell levels according to cell size.

Direct and indirect regulation of Pom1 cell size pathway by the protein phosphatase 2C Ptc1

“…At the plasma membrane, Pom1 auto-phosphorylates to promote its return to the cytosol. It also forms cortical clusters that support its graded distribution from cell poles to mid-cell (Allard et al, 2019;Gerganova et al, 2019). These clusters, together with autophosphorylation-mediated detachment, promote robustness of the gradient (Hersch et al, 2015;Saunders et al, 2012).…”

“…Recent work proposed that this feature is cell surface (Facchetti et al, 2019;Pan et al, 2014), consistent with the membrane-associated localization of all components of the pathway. As Pom1 levels at mid-cell diminish and Wee1 visits increase as cells grow, the pathway is well positioned to act as cell size sensor by linking Wee1 inhibition with cell dimensions for size homeostasis (Allard et al, 2018;Gerganova et al, 2019;Gerganova and Martin, 2018). However, as both pom1 and cdr2 mutants retain homeostatic capacity, this pathway may function in parallel to other homeostatic systems (Facchetti et al, 2019;Wood and Nurse, 2013).…”

“…Dephosphorylation of a series of autophosphorylation sites in the Pom1 corticalbinding domain by the Tea4-associated PP1 Dis2 phosphatase promotes association of Pom1 at the plasma membrane (Gerganova et al, 2019). This dephosphorylation normally takes place at cell poles, where Tea4 and its binding partner Tea1 are deposited by microtubules (Chiou et al, 2017;Martin and Arkowitz, 2014).…”

A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1

“…However, the functional role of this region within kinase domains suggests that other kinases with insertions at the GHI subdomain may be regulated by mechanisms similar to the one we describe here. Phosphorylation of these residues in Wee1 explains the cell size defects of cdr1∆ mutants, and likely represent the functional output of the well-studied Pom1-Cdr2-Cdr1-Wee1 pathway (Martin and Berthelot-Grosjean, 2009;Moseley et al, 2009;Pan et al, 2014;Allard et al, 2018Allard et al, , 2019Gerganova et al, 2019). Efficient signaling in this pathway requires organization within oligomeric clustered structures at the cell cortex.…”

A toolbox of Stable Integration Vectors (SIV) in the fission yeastSchizosaccharomyces pombe

“…However, we refrain from using different constructs to compare between conditions as genomic position effects may become evident only in certain backgrounds (Allshire and Ekwall, 2015). While we present the full vector series for the first time here, some plasmids have been used in published studies (Billault-Chaumartin and Martin, 2019;Gerganova et al, 2019;Lamas et al, 2019;Marek et al, 2019;Vještica et al, 2018) which illustrates their application in fission yeast research.…”