Figure 4. The Cdc15 F-BAR Domain Coordinates Other Binding Partners Required for Proper CR Architecture.

(A) Distance from the PM (d_center) of the indicated CR components calculated from fPALM. Components of the CR membrane-proximal layer are in blue, intermediate in green, and distal in red. Boxplots depict first and third quartiles and median; whiskers, minimum and maximum; notches, 95% confidence intervals. See also Table S4.

(B and C) Representative images of GFP-Pxl1 (B) and Ppb1-GFP (C). Sum projections are shown, and scale bars = 5 µm. Arrows indicate cells without CR signal.

(D) Left: representative time-lapse montages. Minutes elapsed since spindle pole body separation are indicated; deconvolved max projections are shown. Scale bar = 2 µm. Right: quantification of the mean duration of CR events, ****p < 0.0001, Student’s t test. Error bars represent SEM, and number of cells analyzed (n) is indicated.

(E) Coomassie-stained SDS-PAGE of proteins PD with amylose resin after in vitro binding assay. Both F-BAR constructs also contain E30K E152K mutations. Experiment was repeated with similar results.

(F) Immunoblot (IB) of Cdc15 after immunoprecipitation (IP) from asynchronous cells and treatment with either a control or recombinant calcineurin (CN) phosphatase reaction. Numbers indicate position of molecular weight markers in kDa. Experiment was repeated with similar results.

(G) Distance from the PM (d_center) of the indicated CR components in cells treated with DMSO or FK506, as in (A). See also Table S4.

(H) Model for the role of the Cdc15 F-BAR domain in driving nanoscale architecture of the CR. Left: the Cdc15 F-BAR domain recruits the formin Cdc12, but also Pxl1, which is required for CN localization. CN dephosphorylates Cdc15 to promote its open conformation. Right: in cdc15-3A, the protein network established by the Cdc15 F-BAR domain is disrupted, resulting in a shallower CR. Model not drawn to scale.