sid4.T584E compromises anchorage of Sid4 and Cdc11 to the SPB. (A) Representative fluorescence images showing how the SPB affinity of the T584 phosphomimetic Sid4.T584EGFP fusion protein is reduced at 36°C. The cell walls of wild-type sid4.GFP cells were stained by resuspension in red lectin before being mixed with unstained sid4.T584EGFP cells and mounting the mixture for capture of a series of slices in the z axis that were merged to give the maximum projection shown. Red arrowheads indicate signals at SPBs of wild-type cells, and white arrows indicate signals at SPBs of sid4.T584E cells. n = 3. BF, brightfield. (B) Western blots of mid-log phase cultures of the indicated strains in which the transcription of an ectopic copy of sid4⁺ at the hph.171 locus was either repressed (Sid4⁻) by the inclusion of 20 µM thiamine or de-repressed by the removal of thiamine 24 h before sampling (Sid4⁺). n = 3. (C) Representative fluorescence images of Cdc11.GFP in the indicated strains at the temperatures shown. Both fields show a mixture of sid4⁺ cdc11.GFP and sid4.T584E cdc11.GFP cells. The sid4⁺ cdc11.GFP cells were stained by transient resuspension in red fluorescent lectin to identify them in the mixed field of view to highlight the reduction in fluorescence intensity arising from the sid4.T584E mutation. n = 3. (D) The scheme for the anchorage of Sid4.T584EGFP to the SPB for the spot tests in E that show that sid4.T584E sin⁻ lethality arises from Sid4 departure from the SPB. (E) Spot tests in which serial dilution of the indicated strains were placed onto agar plates and incubated at 36°C.