Figure 5.
Poc1 is an anchor for the inner scaffold to the TMTs in the core region. (A) Core region proteins Fam161A and Poc16 localize to BBs in Tetrahymena. Left, longitudinal view of Fam161A:mCherry (green) localization relative to Poc1:HaloTag (magenta) in wild-type cells. Right, longitudinal view of Poc16:mCherry (green) localization relative to Poc1:HaloTag (magenta) in wild-type cells. Longitudinal views are oriented with the cell exterior at the top of the image and the cell interior at the bottom. Diagrams of each protein’s localization are shown at right. Poc16 localizes to the core region only, whereas Fam161A has core and presumed transition zone localization. Images were acquired with a confocal microscope. Scale bars are 500 nm. (B) Structure of the wild-type TMT from the core region. Left, subclass of complete TMTs (see Fig. S4 A for overall average). Right, luminal side view of complete TMT. Arrowheads indicate the inner scaffold (lavender) and position of the inner scaffold attachment to the TMT at the A–B inner junction (magenta) and B–C inner junction (yellow). (C) Complete TMT subtomogram average mapped onto an individual BB in ice shows the inner scaffold (lavender) and cylindrical shape with 165 nm diameter. The A–B inner junction is indicated by blue dots in the images in the center and on the right. The longitudinal section image on the right shows that the scaffold is a structure of stacked rings. (D) Focused refinement on the A–B inner junction in the core region at 8.47 Å in luminal side view (left and middle) and top-down view (right). Ribbon diagrams for α- and β-tubulin and Poc1 are modeled into the density and colors are as described in Fig. 2 legend. An unknown protein density associates with the BB lumen-exposed side of Poc1 to form the A–B inner junction stem attachment to the inner scaffold. See also Video 4. (E) Zoomed-in view of the A–B inner junction from wild-type (gray) overlaid with poc1Δ (green) TMTs shows the WD40 density observed in the wild-type A–B inner junction (magenta arrowhead) is missing in the poc1Δ A–B inner junction. Both structures are from the subclasses of complete TMTs. (F) Schematic representation of the wild-type TMT in the core region of the BB. Color scheme is as follows: gray, tubulin; lavender, inner scaffold; teal, A03 inner scaffold attachment; pink shades, A–B inner junction stem; yellow shades, B–C inner junction inner scaffold attachment; green shades, B-tubule luminal densities; blue, Poc1. (G) Schematic representation of Poc1 (blue) localization in the BB core region with the inner scaffold represented by a lavender circle.

Poc1 is an anchor for the inner scaffold to the TMTs in the core region. (A) Core region proteins Fam161A and Poc16 localize to BBs in Tetrahymena. Left, longitudinal view of Fam161A:mCherry (green) localization relative to Poc1:HaloTag (magenta) in wild-type cells. Right, longitudinal view of Poc16:mCherry (green) localization relative to Poc1:HaloTag (magenta) in wild-type cells. Longitudinal views are oriented with the cell exterior at the top of the image and the cell interior at the bottom. Diagrams of each protein’s localization are shown at right. Poc16 localizes to the core region only, whereas Fam161A has core and presumed transition zone localization. Images were acquired with a confocal microscope. Scale bars are 500 nm. (B) Structure of the wild-type TMT from the core region. Left, subclass of complete TMTs (see Fig. S4 A for overall average). Right, luminal side view of complete TMT. Arrowheads indicate the inner scaffold (lavender) and position of the inner scaffold attachment to the TMT at the A–B inner junction (magenta) and B–C inner junction (yellow). (C) Complete TMT subtomogram average mapped onto an individual BB in ice shows the inner scaffold (lavender) and cylindrical shape with 165 nm diameter. The A–B inner junction is indicated by blue dots in the images in the center and on the right. The longitudinal section image on the right shows that the scaffold is a structure of stacked rings. (D) Focused refinement on the A–B inner junction in the core region at 8.47 Å in luminal side view (left and middle) and top-down view (right). Ribbon diagrams for α- and β-tubulin and Poc1 are modeled into the density and colors are as described in Fig. 2 legend. An unknown protein density associates with the BB lumen-exposed side of Poc1 to form the A–B inner junction stem attachment to the inner scaffold. See also Video 4. (E) Zoomed-in view of the A–B inner junction from wild-type (gray) overlaid with poc1Δ (green) TMTs shows the WD40 density observed in the wild-type A–B inner junction (magenta arrowhead) is missing in the poc1Δ A–B inner junction. Both structures are from the subclasses of complete TMTs. (F) Schematic representation of the wild-type TMT in the core region of the BB. Color scheme is as follows: gray, tubulin; lavender, inner scaffold; teal, A03 inner scaffold attachment; pink shades, A–B inner junction stem; yellow shades, B–C inner junction inner scaffold attachment; green shades, B-tubule luminal densities; blue, Poc1. (G) Schematic representation of Poc1 (blue) localization in the BB core region with the inner scaffold represented by a lavender circle.

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