Htl1 is important for SPB insertion into the nuclear envelope. (a) SPB phenotypes in large-budded wild-type and HTL1 shut-off cells expressing Spc110-GFP and Spc42-mCherry are shown. Images are maximum-intensity projections. Bar, 3 μm. (b) Wild-type and HTL1 shut-off strains were grown in rich medium containing glucose for 8 h. Cells were imaged each hour, and at least 100 large-budded cells were assessed for the phenotypes depicted in panel a. (c) The experiment outlined in panel b was repeated with HTL1 shut-off strains expressing Spc42-mCherry and Spc110-GFP and carrying either the empty vector or a high-copy plasmid expressing NBP1 from its native promoter. (d) TEM was performed on an HTL1 shut-off strain after 12 h of logarithmic growth in rich media containing glucose. All images are sections from the same large-budded cell. An abnormal nuclear protrusion (top), a dead SPB (red arrow; middle) and a mature SPB (red arrow; bottom) are shown. Bars: (large images) 0.5 μm; (insets) 0.1 μm.