Figure 6. Poc1 stabilizes the inner... | Rockefeller University Press

Figure 6.

$Poc1 stabilizes the inner scaffold in the core region of the BB and promotes BB shape maintenance. (A) Fam161A and Poc16 localizations depend on Poc1 under high-force conditions. Wild-type and poc1Δ cells expressing Fam161A:mCherry (top left panels) and Poc16:mCherry (bottom left panels) at 30 and 38°C. Cells were fixed and stained with anti-mCherry antibody to visualize Fam161A or Poc16 (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 bars for full-cell images are 10 μm; scale bars for insets are 0.5 μm. Middle graphs show the quantification of Fam161A (top) and Poc16 (bottom) in wild-type versus poc1Δ BBs at 30°C using total cell fluorescence to account for differences in assortment and/or expression between cell lines. 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). BBs lacking Poc1 show defects in Fam161A, but not Poc16, binding under normal force conditions. Right graphs show the quantification of Fam161A (top) and Poc16 (bottom) at BBs in wild-type versus poc1Δ cells at 30oC and 38°C. Values were normalized to the signal for each strain at 30°C. Each point on the graph represents an independent experiment consisting of 150 BBs. Graphs show mean ± standard deviation. Both wild-type and poc1Δ BBs lose Fam161A upon shift to 38°C, whereas Poc16 is only lost in poc1Δ cells at 38°C. (B) Cross-sectional view of the complete TMT from the core region in poc1Δ alone (green) and overlaid with wild-type (gray). Arrowheads point to the inner scaffold attachment sites: A03 attachment site (teal), A–B inner junction attachment site (magenta), and B–C inner junction attachment site (yellow). Note the inner scaffold and A–B and B–C inner junction attachment sites are missing in poc1Δ TMTs, while the A03 attachment site is preserved albeit at a reduced level compared to wild-type. (C) Side view of the focused refinement of the A–B inner junction/stem attachment in wild-type (left) and poc1Δ (right) TMTs showing in wild-type BBs Poc1 binds directly to the A–B inner junction and coordinates the binding of an unknown protein directly to Poc1’s WD40 domain. In poc1Δ BBs, both the Poc1 density and the unidentified protein’s density are missing. (D) Representative cross-sectional views of the core region of whole BBs in ice with the subtomogram averages from wild-type and poc1Δ TMTs superimposed on the TMTs from the individual BBs. Note that wild-type BBs are circular, whereas poc1Δ BBs are flattened. Bottom, graph of circularity measurements from all wild-type (n = 61) and poc1Δ (n = 81) BBs used in this study shows that poc1Δ BBs are significantly less circular than wild-type BBs. Graph shows mean ± standard deviation. Scale bar is 50 nm. (E) U-ExM-SIM of wild-type and poc1Δ BBs at 38°C stained with anti-α-tubulin antibody in longitudinal views shows that wild-type BBs exhibit a gradual bend, whereas poc1Δ BBs display severe shape malformations that are likely to precede fracture and disassembly. The BB outline is marked with dashed white line. Scale bar is 500 nm. (F) U-ExM-SIM of wild-type and poc1Δ BBs at 38°C expressing Poc16:GFP and stained with anti-GFP (magenta) and anti-acetylated tubulin (green) antibody. Top panels show longitudinal views of individual BBs (with the distal end of BB at the top of the image) and bottom panels show cross sections of individual BBs. Poc16 is lost in poc1Δ BBs in places where tubulin defects are observable. Scale bars are 500 nm.$

Poc1 stabilizes the inner scaffold in the core region of the BB and promotes BB shape maintenance. (A) Fam161A and Poc16 localizations depend on Poc1 under high-force conditions. Wild-type and poc1Δ cells expressing Fam161A:mCherry (top left panels) and Poc16:mCherry (bottom left panels) at 30 and 38°C. Cells were fixed and stained with anti-mCherry antibody to visualize Fam161A or Poc16 (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 bars for full-cell images are 10 μm; scale bars for insets are 0.5 μm. Middle graphs show the quantification of Fam161A (top) and Poc16 (bottom) in wild-type versus poc1Δ BBs at 30°C using total cell fluorescence to account for differences in assortment and/or expression between cell lines. 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). BBs lacking Poc1 show defects in Fam161A, but not Poc16, binding under normal force conditions. Right graphs show the quantification of Fam161A (top) and Poc16 (bottom) at BBs in wild-type versus poc1Δ cells at 30^oC and 38°C. Values were normalized to the signal for each strain at 30°C. Each point on the graph represents an independent experiment consisting of 150 BBs. Graphs show mean ± standard deviation. Both wild-type and poc1Δ BBs lose Fam161A upon shift to 38°C, whereas Poc16 is only lost in poc1Δ cells at 38°C. (B) Cross-sectional view of the complete TMT from the core region in poc1Δ alone (green) and overlaid with wild-type (gray). Arrowheads point to the inner scaffold attachment sites: A03 attachment site (teal), A–B inner junction attachment site (magenta), and B–C inner junction attachment site (yellow). Note the inner scaffold and A–B and B–C inner junction attachment sites are missing in poc1Δ TMTs, while the A03 attachment site is preserved albeit at a reduced level compared to wild-type. (C) Side view of the focused refinement of the A–B inner junction/stem attachment in wild-type (left) and poc1Δ (right) TMTs showing in wild-type BBs Poc1 binds directly to the A–B inner junction and coordinates the binding of an unknown protein directly to Poc1’s WD40 domain. In poc1Δ BBs, both the Poc1 density and the unidentified protein’s density are missing. (D) Representative cross-sectional views of the core region of whole BBs in ice with the subtomogram averages from wild-type and poc1Δ TMTs superimposed on the TMTs from the individual BBs. Note that wild-type BBs are circular, whereas poc1Δ BBs are flattened. Bottom, graph of circularity measurements from all wild-type (n = 61) and poc1Δ (n = 81) BBs used in this study shows that poc1Δ BBs are significantly less circular than wild-type BBs. Graph shows mean ± standard deviation. Scale bar is 50 nm. (E) U-ExM-SIM of wild-type and poc1Δ BBs at 38°C stained with anti-α-tubulin antibody in longitudinal views shows that wild-type BBs exhibit a gradual bend, whereas poc1Δ BBs display severe shape malformations that are likely to precede fracture and disassembly. The BB outline is marked with dashed white line. Scale bar is 500 nm. (F) U-ExM-SIM of wild-type and poc1Δ BBs at 38°C expressing Poc16:GFP and stained with anti-GFP (magenta) and anti-acetylated tubulin (green) antibody. Top panels show longitudinal views of individual BBs (with the distal end of BB at the top of the image) and bottom panels show cross sections of individual BBs. Poc16 is lost in poc1Δ BBs in places where tubulin defects are observable. Scale bars are 500 nm.