Dynamic visits of cortical structures probe for cell size

Bhatia

Martin

2020

Preprint

Self Cite

The fission yeast cells Schizosaccharomyces pombe divide at constant cell size regulated by environmental stimuli. An important pathway of cell size control involves the membrane-associated DYRK-family kinase Pom1, which forms decreasing concentration gradients from cell poles and inhibits mitotic inducers at mid-cell. Here, we identify the phosphatase 2C Ptc1 as negative regulator of Pom1. Ptc1 localizes to cell poles in a manner dependent on polarity and cell-wall integrity factors. We show that Ptc1 directly binds Pom1 and can dephosphorylate it in vitro but modulates Pom1 localization indirectly upon growth in low glucose conditions by influencing microtubule stability. Thus, Ptc1 phosphatase plays both direct and indirect roles in the Pom1 cell size control pathway.

Section: Introductionmentioning

confidence: 99%

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

Bhatia

Martin

2020

Preprint

Self Cite

“…Because Pom1 clusters rather than individual molecules may shape the gradient (see Figure 1 above), and inspired by recent work showing visits of Cdr2 nodes by the downstream Wee1 kinase (Allard et al, 2018; Gerganova and Martin, 2018), we turned to live cell TIRF imaging. To test the method’s sensitivity and selectivity for cortical signals, we first compared the fluorescence levels of Pom1 WT , Pom1 1A , Pom1 3A and Pom1 5A phospho-mutants, which reproduced the increased Pom1 1A , Pom1 3A and Pom1 5A medial cortical localization seen by confocal microscopy (Figure 5C).…”

Section: Resultsmentioning

confidence: 99%

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

Floderer

Archetti

et al. 2019

eLife

Self Cite

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.

Section: Pom1 At the Mid-cell Cortex Controls Cell Length At Divisionmentioning

confidence: 99%

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

Floderer

Archetti

et al. 2019

Preprint

Self Cite

Protein concentration gradients convey information at a distance from the source to both pattern developing organisms and organize single cells. In the rod-shaped cells of Schizosaccharomyces pombe, the DYRK-family kinase Pom1 forms concentration gradients with maxima at the cell poles. Pom1 controls the timing of mitotic entry by inhibiting the SADfamily kinase 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. Whether the graded pattern directly alters Pom1 medial levels has been controversial. Here, using a combination of quantitative imaging approaches, including single particle tracking PALM and TIRF microscopy, we find that individual Pom1 molecules do not bind the membrane long enough to diffuse from cell pole to cell middle. Instead we propose they exchange within longer-lived clusters that form the functional gradient units. By creating an allelic series progressively blocking autophosphorylation, we show that multi-phosphorylation shapes and buffers the gradient to control the cortical mid-cell Pom1 levels, which represent the critical pool regulating Cdr2.Specific imaging of this cortical pool by TIRF microscopy demonstrates that more Pom1 overlaps with Cdr2 nodes in short than long cells, consistent with Pom1 inhibition of Cdr2 decreasing with cell growth. We conclude that Pom1 gradients modulate Pom1 mid-cell levels according to cell size.