Figure 2.
Poc1 localizes to the A–B and B–C inner junctions of triplet microtubules at the proximal end of the basal body. (A) Cross-sectional (top) and longitudinal (bottom) views of U-ExM-SIM expanded BBs from intact Tetrahymena cells, stained with anti-α-tubulin and anti-GFP antibodies to detect GFP:Poc1. The cells are poc1Δ rescued with GFP:Poc1. Scale bars are 500 nm, not corrected for expansion factor. The white arrowhead indicates the discontinuity of the Poc1 signal from the proximal to the core regions. (B) Focused refinement of the wild-type A-tubule and A–B inner junction (magenta arrowhead) at ∼13 Å shows a doughnut-shaped density (blue arrowheads) with 8 nm periodicity bridging protofilaments A01 and B10. (C) The A–B inner junction density is a WD40 domain. Left: Focused refinement of the A–B inner junction at 9.8 Å resolution, luminal side view. Middle and right show luminal side and top-down views, respectively, with ribbon diagrams of α- and β-tubulin (light green and cyan, PDB ID 8G2Z) modeled into protofilaments A01 and B10. An AlphaFold2 predicted structure of Poc1 (blue) is modeled into the A–B inner junction doughnut-shaped density. A portion of the C-terminal random coil of Poc1 is modeled into the horizontal densities that exit the WD40 domain. The N-terminus is not modeled. See also Video 2 and Materials and methods for fitting. (D) Focused refinement of the B–C inner junction at 10 Å, shows a WD40 density with 8 nm periodicity, interspersed with another protein density. Ribbon diagrams of α- and β-tubulin and Poc1 were modeled into the electron density as described above. (E) Individual structures and overlay of wild-type TMTs (gray) with poc1Δ complete TMTs (green) in cross-sectional view. Arrows indicate the positions of the A–B and B–C inner junctions and show a loss of density in poc1Δ TMTs. (F) Glancing cross sectional view on left, luminal side view on right. The WD40 densities described at the wild-type A–B and B–C inner junctions (blue asterisks) in C and D are missing in the poc1Δ complete TMT, while the interspersed density at the B–C inner junction (red asterisk) remains. (G) Schematic representation of the wild-type TMT structure from the proximal region of the Tetrahymena BB. Color scheme: gray, tubulin; dark green, A/C linker; cyan, pinhead; magenta, A–B inner junction; yellow, B–C inner junction; blue, Poc1. (H) Schematic representation of Poc1’s localization at the proximal region A–B and B–C inner junctions of Tetrahymena BBs. Poc1 is indicated in blue.

Poc1 localizes to the A – B and B – C inner junctions of triplet microtubules at the proximal end of the basal body. (A) Cross-sectional (top) and longitudinal (bottom) views of U-ExM-SIM expanded BBs from intact Tetrahymena cells, stained with anti-α-tubulin and anti-GFP antibodies to detect GFP:Poc1. The cells are poc1Δ rescued with GFP:Poc1. Scale bars are 500 nm, not corrected for expansion factor. The white arrowhead indicates the discontinuity of the Poc1 signal from the proximal to the core regions. (B) Focused refinement of the wild-type A-tubule and A–B inner junction (magenta arrowhead) at ∼13 Å shows a doughnut-shaped density (blue arrowheads) with 8 nm periodicity bridging protofilaments A01 and B10. (C) The A–B inner junction density is a WD40 domain. Left: Focused refinement of the A–B inner junction at 9.8 Å resolution, luminal side view. Middle and right show luminal side and top-down views, respectively, with ribbon diagrams of α- and β-tubulin (light green and cyan, PDB ID 8G2Z) modeled into protofilaments A01 and B10. An AlphaFold2 predicted structure of Poc1 (blue) is modeled into the A–B inner junction doughnut-shaped density. A portion of the C-terminal random coil of Poc1 is modeled into the horizontal densities that exit the WD40 domain. The N-terminus is not modeled. See also Video 2 and Materials and methods for fitting. (D) Focused refinement of the B–C inner junction at 10 Å, shows a WD40 density with 8 nm periodicity, interspersed with another protein density. Ribbon diagrams of α- and β-tubulin and Poc1 were modeled into the electron density as described above. (E) Individual structures and overlay of wild-type TMTs (gray) with poc1Δ complete TMTs (green) in cross-sectional view. Arrows indicate the positions of the A–B and B–C inner junctions and show a loss of density in poc1Δ TMTs. (F) Glancing cross sectional view on left, luminal side view on right. The WD40 densities described at the wild-type A–B and B–C inner junctions (blue asterisks) in C and D are missing in the poc1Δ complete TMT, while the interspersed density at the B–C inner junction (red asterisk) remains. (G) Schematic representation of the wild-type TMT structure from the proximal region of the Tetrahymena BB. Color scheme: gray, tubulin; dark green, A/C linker; cyan, pinhead; magenta, A–B inner junction; yellow, B–C inner junction; blue, Poc1. (H) Schematic representation of Poc1’s localization at the proximal region A–B and B–C inner junctions of Tetrahymena BBs. Poc1 is indicated in blue.

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