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. 2010 Jul 9;39(1):86-99.
doi: 10.1016/j.molcel.2010.06.012.

Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site

Rachel H Roberts-Galbraith  1 , Melanie D Ohi, Bryan A Ballif, Jun-Song Chen, Ian McLeod, W Hayes McDonald, Steven P Gygi, John R Yates 3rd, Kathleen L Gould
Affiliations

Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site

Rachel H Roberts-Galbraith et al. Mol Cell. .

Abstract

Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, the founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.

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Figures

Figure 1
Figure 1. Dephosphorylated Cdc15 binds Cdc12 and localizes to the contractile ring
A) Live cell imaging of the indicated strains grown asynchronously or after the indicated arrest. B) Denatured cell lysates were prepared from the indicated strains and probed with anti-Cdc15 serum. C) Cdc15 was immunoprecipitated from denatured cell lysates and subjected either to lambda phosphatase treatment or a buffer control prior to immunoblotting. D) Block and release of cdc25-22 cdc15-HA cells. At the indicated time points, cell cycle progression was monitored by septation index and percentage of binucleate cells. Cell pellets from each time point were subjected to native lysis. Lysates were probed with either bead-bound, recombinant MBP or MBP-Cdc12(aa1-764). Lysate samples and bound proteins were run on SDS-PAGE and Cdc15 was detected by an anti-HA antibody. E) Hyperphosphorylated Cdc15 was purified and subjected to lambda phosphatase treatment, phosphatase plus inhibitors, or buffer controls. Samples were run on SDS-PAGE and detected by silver staining. F) The Cdc15 purified in E was incubated with bead-bound MBP or MBP-Cdc12(aa1-764). Bound Cdc15 was detected by immunoblotting. G) Lysates of the indicated genotypes were subjected to membrane flotation. Fractions were taken for detection of proteins by immunoblotting. H) Quantitation of the amount of protein in G for the top three fractions of each sample. Bar: (A) 10 μm.
Figure 2
Figure 2. Dephosphorylation of Cdc15 induces conformational and oligomeric changes
A) Hyperphosphorylated Cdc15 was purified, treated with lambda phosphatase or a buffer control, and run on SDS-PAGE. B) Representative negative stain EM images of Cdc15 after treatment with buffer control. C) Representative negative stain EM images of Cdc15 after treatment with lambda phosphatase. Filaments were heterogeneous in length but always contained series of undulating curves. D) His6-Cdc15 F-BAR was purified and run on SDS-PAGE for detection by coomassie staining. E) Representative EM images of His6-Cdc15 F-BAR. F) Analytical ultracentrifugation traces of Cdc15 purified in its hyper- and hypophosphorylated forms. TEV contaminant is marked. G) TEV run alone for comparison to F. Bars: 100 nm.
Figure 3
Figure 3. Identification of Cdc15 phosphosites
A) Schematic of phosphorylation mutants. B) Cell lysates were prepared and run on SDS-PAGE. Cdc15 was detected by anti-Cdc15 serum and alpha-tubulin was detected with a DM1a antibody as a loading control. C) Wild type or mutant Cdc15 proteins were immunoprecipitated with anti-Cdc15 serum, subjected to lambda phosphatase treatment or buffer control, and detected by immunoblotting.
Figure 4
Figure 4. Cdc15 alanine mutants display precocious medial localization
A) Above, live cell imaging of asynchronous cells expressing endogenously-tagged wild type or mutant Cdc15. Arrows indicate medial localization and asterisks indicate filament-like GFP structures. Below, cdc25-22 cells with the indicated GFP-tagged cdc15 mutations were arrested and imaged live. B) Quantitation of images of cells of the indicated genotypes from A. C) A reconstruction of the cell middle of an arrested cdc25-22 cdc15(SP11A)-GFP cell (marked with the box) was rotated 90°. D) Wild type and mutant Cdc15-GFP intensity was measured and averaged for 15 cells per genotype. Bars are S.E.M. P<0.05 (*) or P<0.001 (**). Bars: (A) 10 μm, (C) 5 μm.
Figure 5
Figure 5. Effects of Cdc15 alanine mutants on other contractile ring components
A and B) cdc25-22 or cdc25-22 cdc15(SP11A) cells expressing the indicated GFP- or YFP-tagged proteins were arrested and imaged. C) Native lysates were probed with MBP or MBP-Cdc12(1-764) and Cdc15 was detected in cell lysates and pull down samples by immunoblotting. D) Native cell lysates were prepared from cells of the indicated genotypes/arrests. Co-immunoprecipitation of Cdc15 with Cyk3 or vice versa was determined by immunoblotting with anti-Cdc15 serum or an anti-FLAG antibody. E) Lysates were prepared for the indicated genotypes and arrests and used for membrane flotation as in Fig. 1G–H. F) cdc25-22 or cdc25-22 cdc15(SP11A) cells were arrested, fixed, and stained with Alexa Fluor-488 phalloidin for confocal imaging. G) cdc25-22 or cdc25-22 cdc15(SP11A) cells were grown to log phase, arrested, exposed to FM4-64 and immediately imaged live. Bars: 10 μm.
Figure 6
Figure 6. Altered dynamics in Cdc15 alanine mutants
A) cdc15-GFP, cdc15(27A)-GFP, or cdc15(27D)-GFP cells with sid4-RFP were grown to log phase, synchronized with a lactose gradient, and imaged by time lapse microscopy. Montages for each genotype were assembled from movies (images at 5 minute intervals). Ectopic spots are visible in the cdc15(SP27A)-GFP mutant. SPB separation is denoted in each movie with a carat. B) Quantitation of spot appearance before SPB separation in each indicated mutant (n>10 for each). Asterisks denote P ≤0.01 (*) or ≤0.001 (**). C and D) Quantitation of cytokinesis event timing for phosphorylation mutants from A (n>10 for each). Asterisks denote P ≤0.05 (*) or ≤0.001 (**). Error bars represent S.E.M. Bars: 5 μm.
Figure 7
Figure 7. Model for Cdc15 phosphoregulation
A) Model for alteration of Cdc15 conformation by phosphorylation and dephosphorylation. B) Depiction of hypophosphorylated Cdc15 in its active scaffolding role at the contractile ring.
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