1. Volume 27, Issue 4, Pages 534-542 (February 2017)
SIN-Dependent Dissociation of the SAD Kinase Cdr2 from the Cell Cortex Resets the Division Plane 
Sergio A. Rincon, Miguel Estravis, Florent Dingli, Damarys Loew, Phong T. Tran, Anne Paoletti 
Current Biology 
Volume 27, Issue 4, Pages (February 2017)
DOI: /j.cub
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2. Figure 1 S476 and S632 Are the Major Targets of the SIN on Cdr2
(A) Schematic representation of Cdr2 showing Cdr2 domains and the five Cdr2 RXXS sites found to be phosphorylated by mass spectrometry analysis in cdc mutant (see also Figure S1). The mutation of residues highlighted in red leads to defective dissociation of Cdr2 from the cortex during cell division. ++, Cdr2 basic domain.
(B) Time-lapse analysis of Cdr2-mEGFP or Cdr2RXXS-2A-mEGFP and Sid4-mCherry in wild-type (top), cdc7-24 (middle), and cdr2RXXS-2A cells (bottom) incubated at 36°C for 3 hr. Medial plane confocal images are shown. Scale bar, 5 μm.
(C) Quantification of Cdr2-mEGFP or Cdr2RXXS-2A-mEGFP amounts on the medial cortex or in the cytoplasm during cell division. Average GFP intensity was measured on maximum projections images in a circular region in the cell middle (cortical Cdr2) or close to a cell tip (cytosolic Cdr2), as represented on the scheme. Time 0 corresponds to mitotic entry (n = 10). Error bars, SD.
(D) Localization of wild-type or Cdr2RXXS-2A-mEGFP in a wild-type or cdc background, incubated for 3 hr at 36°C. Medial plane confocal images are shown. Scale bar, 5 μm.
(E) Quantification of Cdr2-mEGFP or Cdr2RXXS-2A-mEGFP cortical to cytosolic ratios in septating wild-type or cdc cells incubated for 3 hr at 36°C (n = 10). Error bars, SD.
See also Figures S1 and S2 and Tables S1 and S2.
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3. Figure 2
Rad24 Interacts with Cdr2 and Sequesters It in the Cytoplasm during Cell Division
(A) Co-immunoprecipitation assay between Cdr2-12myc and Rad24-GFP in wild-type or cdc cells incubated at 25°C or 36°C for 2 hr.
(B) Quantification of western blot signals from three independent experiments. Error bars, SD.
(C) Epifluorescence medial plane images showing Cdr2-mEGFP localization in nda3-KM311 cells synchronized in mitosis by incubation for 8 hr at 18°C and after block release at the indicated time points. Scale bar, 2 μm.
(D) Top: co-immunoprecipitation assays between Cdr2-12myc and Rad24-GFP in nda3-KM311 cells synchronized in mitosis by incubation for 8 hr at 18°C and after block release performed at the indicated time points. Bottom: percentage of septating cells at the indicated time points after block release.
(E) Co-immunoprecipitation assay between Cdr2-12myc or Cdr2RXXS-2A-12myc and Rad24-GFP in wild-type or cdc cells incubated at 36°C for 2 hr.
(F) Quantification of western blot signals from two independent experiments. Error bars, SD.
(G) Time-lapse analysis of Cdr2-mEGFP in rad24Δ cells expressing Sid4-mCherry. Medial plane confocal images are shown. Scale bar, 5 μm.
(H) Quantification of Cdr2-mEGFP amounts on the medial cortex or in the cytoplasm during cell division in rad24Δ cells. Measurement was performed like in Figure 1C. Time 0 corresponds to mitotic entry (n = 10). Error bars, SD.
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4. Figure 3
SIN-Dependent Dissociation of Cdr2 from the Cortex Resets the Division Plane
(A) Calcofluor staining of wild-type, cdr2RXXS-2A, or cdr2CAAX septating cells in a mid1+ or mid1nsm background. Scale bar, 5 μm.
(B) Quantification of asymmetric cell division in the same cells as in (A), measured by the ratio between the shorter and the longer daughter cells (cdr2+ mid1+, n = 117; cdr2RXXS-2A mid1+, n = 146; cdr2CAAX mid1+, n = 51; cdr2+ mid1nsm, n = 60; cdr2RXXS-2A mid1nsm, n = 110; cdr2CAAX mid1nsm, n = 138; ∗∗∗t test p value < 10−19).
(C) Maximum projections of confocal images showing Cdr2-mEGFP or Cdr2RXXS-2A-mEGFP distribution in cells also expressing Pom1-tdTomato and Rlc1-mCherry. Scale bar, 5 μm.
(D) Schematic representation of cell length (L) and of L1 and L2 corresponding to the shorter and longer distances between a cell tip and the border of the domain containing Cdr2 nodes, measured to define the position of Cdr2 domain along the cortex.
(E) Analysis of the localization of Cdr2 domain along the cortex measured by L1/L2 ratio (Cdr2-mEGFP, n = 325; Cdr2RXXS-2A-mEGFP, n = 381; ∗∗∗t test p value < 10−49).
(F) Analysis of the localization of Cdr2 domain along the cortex measured by L1/L2 ratio after binning according to cell length (L).
(G) Time-lapse analysis of Cdr2-mEGFP or Cdr2RXXS-2A-mEGFP localization in cells also expressing Pom1-tdTomato and Rlc1-mCherry. Time 0 corresponds to the end of acto-myosin ring constriction. Medial plane confocal images are shown. Scale bar, 5 μm.
See also Figure S3.
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5. Figure 4
cdr2RXXS-2A Aggravates the Division Plane-Positioning Defects of pom1Δ Cells
(A) Calcofluor staining of wild-type, cdr2RXXS-2A, cdr2bsc-3A, or cdr2bsc-3A + RXXS-2A cells in a mid1+ or mid1nsm background. Scale bar, 5 μm.
(B) Quantification of asymmetric cell division in wild-type, cdr2RXXS-2A, cdr2bsc-3A, or cdr2bsc-3A + RXXS-2A cells in a mid1+ or mid1nsm background, measured by analysis of the length ratio between the shorter and the longer daughter cells (cdr2+ mid1+, n = 238; cdr2RXXS-2A mid1+, n = 199; cdr2bsc-3A mid1+, n = 220; cdr2bsc-3A + RXXS-2A mid1+, n = 163; cdr2+ mid1nsm, n = 434; cdr2RXXS-2A mid1nsm, n = 406; cdr2bsc-3A mid1nsm, n = 325; cdr2bsc-3A + RXXS-2A mid1nsm, n = 302; ∗∗∗t test p values < 10−14).
(C) Calcofluor staining of cdr2+ pom1Δ or cdr2RXXS-2A pom1Δ septating cells. Scale bar, 5 μm.
(D) Quantification of asymmetric cell division in the same cells as in (C), measured as the length ratio between the shorter and the longer daughter cells (cdr2+ pom1Δ, n = 530; cdr2RXXS-2A pom1Δ, n = 468; ∗∗∗t test p value < 10−24).
(E) Epifluorescence medial plane images showing chromosome fate after cell division in pom1Δ cdr2-mEGFP or cdr2RXXS-2A-mEGFP cells expressing the histone Hht1 fused to RFP to visualize chromatin and stained for the cell wall and septum with calcofluor. Scale bar, 5 μm.
(F) Percentage of defective nuclear segregation or nuclear cut phenotype in the same cells as in (E). Average of two independent experiments (cdr2+ pom1Δ, n = 293; cdr2RXXS-2A pom1Δ, n = 415). Error bars, SD.
(G) Model recapitulating the SIN impact on division plane positioning. In wild-type cells (left), the SIN activity induces Cdr2 dissociation from the medial region of dividing cells, allowing de novo assembly of Cdr2 nodes in the middle of daughter cells by the end of cytokinesis. In contrast, in the cdr2RXXS-2A mutant (right), Cdr2 does not dissociate from the cortex during cell division, resulting in an asymmetric positioning of inherited Cdr2 nodes toward the new end of daughter cells. This asymmetry is not fully corrected at the next round of mitosis, leading to division plane-positioning defects in a mid1nsm background.
See also Figure S4 and Tables S1 and S2.
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