Poc1 stabilizes the pinhead at the proximal end of the BB. (A) Bld10 is lost in high force conditions from poc1Δ BBs. Left images: Wild-type and poc1Δ cells expressing Bld10:mCherry at 30 and 38°C. Cells were fixed and stained with anti-mCherry antibody to visualize Bld10 (grayscale, red) and anti-centrin antibody to visualize all BBs (green). Cells were starved for 12 h to attenuate new BB assembly and temperature shifted for 12 h before fixation. Scale bar for full-cell images is 5 μm; scale bar for insets is 0.5 μm. Middle: Comparison of BB Bld10:mCherry levels at 30°C between wild-type and poc1Δ cells. Each point on the graph represents the average of 15 BBs, normalized to the total cell levels of mCherry determined for that cell (see Materials and methods). There is no statistical difference in amounts of Bld10 at BBs between wild-type and poc1Δ cells. Graph shows mean ± standard deviation. Right: Comparison of BB Bld10:mCherry levels in wild-type and poc1Δ cells at 30 and 38°C. Bld10mCh levels at 30°C for each cell line were normalized to 1. Each point on the graph represents an independent experiment consisting of 150 BBs. Graphs show mean ± standard deviation. BBs lose Bld10 at 38°C in poc1Δ cells, but not in wild-type cells. (B) Cross-sectional view of poc1Δ complete TMTs (green) alone and overlaid with wild-type TMTs (gray) shows loss of electron density at the pinhead. Right, zoomed in view of overlaid structure in cross-sectional and luminal side views. The pinhead is lost in poc1Δ TMTs even though the A-tubule structure where it should attach is intact. Cyan arrowhead, pinhead; magenta arrowhead, A–B inner junction.