Figure 3.

(A) Overproduction of Sid4ΔNp inhibits cytokinesis. Wild-type cells carrying a single integrated copy of sid4ΔN under control of the thiamin-repressible nmt1 promoter (KGY1347) were grown to mid-logarithmic phase at 32°C in medium containing thiamin (a and b). To induce overexpression of sid4ΔN, cells were then grown in medium lacking thiamin for 22 h at 32°C (c and d). Cells were fixed and stained with DAPI or with Calcofluor. (B) Sid4ΔNp overproduction prevents Sid4p SPB localization. sid4-HA cells carrying a single integrated copy of nmt1-sid4ΔN (KGY1742) were grown to mid-logarithmic phase at 32°C under repressing conditions (a and b) or were shifted to inducing conditions for 20 h at 32°C (c–e). Cells were stained with DAPI, anti-HA, and anti-Sad1p. (C) Full-length Sid4p rescues Sid4ΔNp overproduction phenotype. KGY1347 was transformed with plasmid pUR19 sid4⁺ carrying full-length Sid4p (sector 1) or control plasmid pUR19 (sector 2) and grown on minimal medium with or without thiamin to repress or induce production of Sid4ΔNp. (D) Sid4ΔNp overproduction prevents Spg1p SPB localization. spg1-HA cells carrying a single integrated copy of nmt1-sid4ΔN (KGY1741) were grown to mid-logarithmic phase at 32°C under repressing (a and b) or inducing conditions (c–h). Cells were stained with DAPI, anti-HA, and anti-Sad1p to detect SPBs. (Bar = 10 μm.)