The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography

Abstract

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryotomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner Junctions, underscoring its precise spatial regulation.

Keywords: assembly; basal body; centriole; cilium; electron cryo-tomography.

Figure 1.

Electron cryo-tomography structures of cilium inner Junctions. (A) A schematic diagram of a cilium in Tetrahymena, including BB and axoneme. The three regions in the BB, the proximal, central core, and distal, are highlighted in different green colors. Their approximate longitudinal spans are indicated. The three grey-colored cross-sections indicate the location of the structures presented in this work. (B) Left, schematic diagrams of the cross-section of the proximal, the central core region of the BB, and the axoneme. Right, representation of the triplet MT or doublet MT from the three regions. Distinct structures attached to the microtubule wall in each region, such as the cartwheel and A-C linker in the proximal region, the inner scaffold in the central core region, and the Dynein complexes (outer Dynein arm, ODA; inner Dynein arm, IDA; Dynein regulatory complex, DRC) and radial spokes (RS) in the axoneme, are indicated. The red dashed-line boxes highlight the A-B inner Junctions (IJ). (C) Three representative subtomograms-averaged structures presented in this work. From bottom to top are the proximal, the central core region of the BB, and the axoneme, as indicated in the cartoons in (B).

Figure 2.

Structure of the inner Junctions in the proximal region of the BB. (A) The A-B inner junction and the MIPs are identified in the BB proximal region. A schematic diagram of TMT is on the right. The pfs shown in the structure are highlighted in yellow. A dash line indicates the cutting plane and an arrow indicates the direction of view. (B) Comparison of A-B inner Junctions in the BB’s proximal and central core regions. FAP52 binds to B-tubule at different locations in the two structures. The proximal region structures are on the left, and the central core region structures are on the right. (C) Upper: a composite map showing the proximal region’s A-B and B-C inner Junctions. The A-B and B-C inner ladders crosslinking pfs A13-B10 and B7-C10 are highlighted green and indicated by red arrowhead. A schematic diagram of TMT is on the right. The pfs shown in the structure are highlighted in yellow. Arrows indicate the directions of view in the bottom panels. Bottom: longitudinal cross-section views of the A-B and B-C inner Junctions. The A-B inner ladder and B-C inner ladder are colored green.

Figure 2.

Structure of the inner Junctions in the proximal region of the BB. (A) The A-B inner junction and the MIPs are identified in the BB proximal region. A schematic diagram of TMT is on the right. The pfs shown in the structure are highlighted in yellow. A dash line indicates the cutting plane and an arrow indicates the direction of view. (B) Comparison of A-B inner Junctions in the BB’s proximal and central core regions. FAP52 binds to B-tubule at different locations in the two structures. The proximal region structures are on the left, and the central core region structures are on the right. (C) Upper: a composite map showing the proximal region’s A-B and B-C inner Junctions. The A-B and B-C inner ladders crosslinking pfs A13-B10 and B7-C10 are highlighted green and indicated by red arrowhead. A schematic diagram of TMT is on the right. The pfs shown in the structure are highlighted in yellow. Arrows indicate the directions of view in the bottom panels. Bottom: longitudinal cross-section views of the A-B and B-C inner Junctions. The A-B inner ladder and B-C inner ladder are colored green.

Figure 3.

Structural changes at the transition from the proximal to the central core region of the BB. (A) Focused classification on the subtomograms from the proximal region identifies two structures where FAP52 binds at different locations (indicated by red arrows). (B) The longitudinal distribution of the subtomograms from the proximal region. The 150 nm long proximal region is divided into 14 bins. Based on the two classes identified in (A), for each class, the number of subtomograms (y-axis) found in each bin is plotted along the length (x-axis), showing their longitudinal distribution. Right: A schematic diagram indicates the weighted average longitudinal length for Class 2 is 130 nm. The weighted average length is L = ∑ j*Nj / ∑ Nj (Nj: number of subtomograms found in bin j). (C) Left: The Class 2 average in a cross-section view. A red arrowhead indicates the A-B inner junction. A red dashed line and a black arrow indicate the cross-section and viewing direction of the structure on the right. Right: The Class 2 average longitudinally expanded to 70 nm shows FAP52 (red arrowheads) shifting binding site, indicated by a yellow arrowhead. The shift coincides with the termination of the unidentified ladder-like protein in the A-B inner junction (red arrow). (D) Similar to (B), the longitudinal distribution of the subtomograms from the central core region. The 300 nm long central core region is divided into 28 bins. Based on the classification result shown in (Figure S3C), the longitudinal distribution of subtomograms in Class 6 and the other five classes are plotted. The weighted average longitudinal position of Class 6 is at 170 nm from the very proximal end of the BB. This is illustrated in a schematic diagram on the right. (E) The averaged structure in Class 6 (Figure S3C) shows the changes of TMT transitioning from the proximal to the central core region. A red arrowhead indicates the inner scaffold. A red arrow indicates FAP52. A yellow arrowhead indicates the A-C linker’s termination and the inner scaffold’s emergence.

Figure 3.

Structural changes at the transition from the proximal to the central core region of the BB. (A) Focused classification on the subtomograms from the proximal region identifies two structures where FAP52 binds at different locations (indicated by red arrows). (B) The longitudinal distribution of the subtomograms from the proximal region. The 150 nm long proximal region is divided into 14 bins. Based on the two classes identified in (A), for each class, the number of subtomograms (y-axis) found in each bin is plotted along the length (x-axis), showing their longitudinal distribution. Right: A schematic diagram indicates the weighted average longitudinal length for Class 2 is 130 nm. The weighted average length is L = ∑ j*Nj / ∑ Nj (Nj: number of subtomograms found in bin j). (C) Left: The Class 2 average in a cross-section view. A red arrowhead indicates the A-B inner junction. A red dashed line and a black arrow indicate the cross-section and viewing direction of the structure on the right. Right: The Class 2 average longitudinally expanded to 70 nm shows FAP52 (red arrowheads) shifting binding site, indicated by a yellow arrowhead. The shift coincides with the termination of the unidentified ladder-like protein in the A-B inner junction (red arrow). (D) Similar to (B), the longitudinal distribution of the subtomograms from the central core region. The 300 nm long central core region is divided into 28 bins. Based on the classification result shown in (Figure S3C), the longitudinal distribution of subtomograms in Class 6 and the other five classes are plotted. The weighted average longitudinal position of Class 6 is at 170 nm from the very proximal end of the BB. This is illustrated in a schematic diagram on the right. (E) The averaged structure in Class 6 (Figure S3C) shows the changes of TMT transitioning from the proximal to the central core region. A red arrowhead indicates the inner scaffold. A red arrow indicates FAP52. A yellow arrowhead indicates the A-C linker’s termination and the inner scaffold’s emergence.

Figure 4.

Structure of the inner Junctions in the central core region of the BB. (A) Two orthogonal views of the 48-nm repeat structure at the A-B inner junction. The pfs shown in the structure are highlighted in yellow in a schematic diagram of the TMT on the right. A dash line indicates the cutting plane and an arrow indicates the direction of the view. (B) Comparing the A-B inner junction from the BB central core region to the axoneme. (C) A MIP with a leucine-rich repeat (LRR) motif in the A-B inner junction. An LRR model predicted by AlphaFold2 from the protein (UniProt: Q22N53) fits into the density map. The LRR motif makes potential interactions with pfs B10, A13, and A1 of the TMT wall, Poc1, and IJ34. (D) An AlphaFold3 predicted model of a right-handed 4-helix bundle fits into the density map. It is formed by two NTDs from the neighboring LRR-containing proteins as an antiparallel dimer. Each monomer is colored in either dark or light blue. (E) Localization of Poc39 to the Tetrahymena BB by fluorescence microscopy. Left: fluorescence signal of GFP-Poc39 expressed in Tetrahymena cell. Center: immunofluorescence signal of a BB protein Centrin in the same cell. Right: merge of the two images on the left. The inset is an enlarged view of a local area showing the colocalization of the GFP-Poc39 (green) and the Centrin (red) signals. Scale bar: 10 μm. (F) Comparing the inner Junctions between the central core region and the axoneme. Left: the models of the inner junction in the central core of BB and the axoneme. Center: superposition of the two models using pf A1 as a reference. Pf B10 (α/β tubulin) in the BB central core are in light green and blue. Pf B10 (α/β tubulin) in the axoneme are dark green and blue. Right: schematic illustration of the change. The solid circles represent the pf B10 and FAP52 from the central core. The dashed circles represent the pf B10 and FAP52 in the axoneme. The two curved arrows indicate the movement of pf B10 and FAP52 from the central core to the axoneme. (G) Comparing the structures of IJ34 in the central core region and the axoneme. The left and center show that IJ34 and FAP52 fit into the density maps. Right: superposition of the two IJ34 using their main domains as a reference. Their C-terminal helices are 10 Å apart.

Figure 4.

Structure of the inner Junctions in the central core region of the BB. (A) Two orthogonal views of the 48-nm repeat structure at the A-B inner junction. The pfs shown in the structure are highlighted in yellow in a schematic diagram of the TMT on the right. A dash line indicates the cutting plane and an arrow indicates the direction of the view. (B) Comparing the A-B inner junction from the BB central core region to the axoneme. (C) A MIP with a leucine-rich repeat (LRR) motif in the A-B inner junction. An LRR model predicted by AlphaFold2 from the protein (UniProt: Q22N53) fits into the density map. The LRR motif makes potential interactions with pfs B10, A13, and A1 of the TMT wall, Poc1, and IJ34. (D) An AlphaFold3 predicted model of a right-handed 4-helix bundle fits into the density map. It is formed by two NTDs from the neighboring LRR-containing proteins as an antiparallel dimer. Each monomer is colored in either dark or light blue. (E) Localization of Poc39 to the Tetrahymena BB by fluorescence microscopy. Left: fluorescence signal of GFP-Poc39 expressed in Tetrahymena cell. Center: immunofluorescence signal of a BB protein Centrin in the same cell. Right: merge of the two images on the left. The inset is an enlarged view of a local area showing the colocalization of the GFP-Poc39 (green) and the Centrin (red) signals. Scale bar: 10 μm. (F) Comparing the inner Junctions between the central core region and the axoneme. Left: the models of the inner junction in the central core of BB and the axoneme. Center: superposition of the two models using pf A1 as a reference. Pf B10 (α/β tubulin) in the BB central core are in light green and blue. Pf B10 (α/β tubulin) in the axoneme are dark green and blue. Right: schematic illustration of the change. The solid circles represent the pf B10 and FAP52 from the central core. The dashed circles represent the pf B10 and FAP52 in the axoneme. The two curved arrows indicate the movement of pf B10 and FAP52 from the central core to the axoneme. (G) Comparing the structures of IJ34 in the central core region and the axoneme. The left and center show that IJ34 and FAP52 fit into the density maps. Right: superposition of the two IJ34 using their main domains as a reference. Their C-terminal helices are 10 Å apart.

Figure 4.

Structure of the inner Junctions in the central core region of the BB. (A) Two orthogonal views of the 48-nm repeat structure at the A-B inner junction. The pfs shown in the structure are highlighted in yellow in a schematic diagram of the TMT on the right. A dash line indicates the cutting plane and an arrow indicates the direction of the view. (B) Comparing the A-B inner junction from the BB central core region to the axoneme. (C) A MIP with a leucine-rich repeat (LRR) motif in the A-B inner junction. An LRR model predicted by AlphaFold2 from the protein (UniProt: Q22N53) fits into the density map. The LRR motif makes potential interactions with pfs B10, A13, and A1 of the TMT wall, Poc1, and IJ34. (D) An AlphaFold3 predicted model of a right-handed 4-helix bundle fits into the density map. It is formed by two NTDs from the neighboring LRR-containing proteins as an antiparallel dimer. Each monomer is colored in either dark or light blue. (E) Localization of Poc39 to the Tetrahymena BB by fluorescence microscopy. Left: fluorescence signal of GFP-Poc39 expressed in Tetrahymena cell. Center: immunofluorescence signal of a BB protein Centrin in the same cell. Right: merge of the two images on the left. The inset is an enlarged view of a local area showing the colocalization of the GFP-Poc39 (green) and the Centrin (red) signals. Scale bar: 10 μm. (F) Comparing the inner Junctions between the central core region and the axoneme. Left: the models of the inner junction in the central core of BB and the axoneme. Center: superposition of the two models using pf A1 as a reference. Pf B10 (α/β tubulin) in the BB central core are in light green and blue. Pf B10 (α/β tubulin) in the axoneme are dark green and blue. Right: schematic illustration of the change. The solid circles represent the pf B10 and FAP52 from the central core. The dashed circles represent the pf B10 and FAP52 in the axoneme. The two curved arrows indicate the movement of pf B10 and FAP52 from the central core to the axoneme. (G) Comparing the structures of IJ34 in the central core region and the axoneme. The left and center show that IJ34 and FAP52 fit into the density maps. Right: superposition of the two IJ34 using their main domains as a reference. Their C-terminal helices are 10 Å apart.

Figure 5.

poc1Δ mutants destabilize the A-B inner junction and allow axonemal components to incorporate into the assembly. (A) Upper: representative tomogram slices from poc1Δ BB show the TMTs split at the A-B inner junction at various longitudinal locations. Bottom: the blue lines highlight the split TMTs in the upper panels. The yellow arrows indicate the splits. The minus ends of TMT are at the bottom, and the plus ends are at the top. Scale bar: 100 nm. (B) 3D classification of subtomograms from poc1Δ BB, focusing on the A-B inner junction. The resulting three subsets show structure variations at the A-B inner junction. In Class 1, the B-tubule is incomplete and detached from the A-tubule at the inner junction. In Class 2, the B-tubule is complete and remains attached to the A-tubule, though the Poc1 position is empty. In Class 3, the B-tubule is complete and connected to the A-tubule, while other proteins occupy the Poc1 site. Yellow dashed circles and arrows outline the position of Poc1 in the wild-type. An image from the wild-type and a schematic diagram is shown for comparison. (C) Further refinement of Class 3 at 8.00 Å shows the FAP20/PACRG filament at the mutant’s inner junction gap. (D) Comparing the A-B inner junction in the central core region between wild-type and poc1Δ Class 3 shows an increased gap in the junction. Pf A1 is used as a reference for superposition. For Pf B10 (α/β tubulin), the wild-type is in light green and blue, and the poc1Δ is in dark green and blue. (E) Focused classification of subtomograms from poc1Δ BB (Class 3) identifies additional axoneme MIP. Left, the mutant structure is shown in green. Right, the inner junction from the wild-type axoneme DMT. The corresponding MIP is colored in lavender. (F) In poc1Δ BB, Poc39 partially remains in the Class 2 average but is absent in Class 3, where the inner junction is occupied by FAP20/PACRG. For comparison, the wild-type structure is shown in grey. (G) Mapping the location of subtomogram from 3 subsets identified in the central core region of poc1Δ BBs. 8 representative BBs are shown. The green dots represent the Class 1 subset, an incomplete B-tubule detached from the A-tubule at the inner junction. The blue dots represent the Class 2 subset, complete B-tubule without Poc1. The red dots represent the Class 3 subset, the axoneme-like inner junction where FAP20/PACRG fills in the space left by Poc1. The arrows point in the direction from the proximal to the distal end of the BBs.

Figure 5.

poc1Δ mutants destabilize the A-B inner junction and allow axonemal components to incorporate into the assembly. (A) Upper: representative tomogram slices from poc1Δ BB show the TMTs split at the A-B inner junction at various longitudinal locations. Bottom: the blue lines highlight the split TMTs in the upper panels. The yellow arrows indicate the splits. The minus ends of TMT are at the bottom, and the plus ends are at the top. Scale bar: 100 nm. (B) 3D classification of subtomograms from poc1Δ BB, focusing on the A-B inner junction. The resulting three subsets show structure variations at the A-B inner junction. In Class 1, the B-tubule is incomplete and detached from the A-tubule at the inner junction. In Class 2, the B-tubule is complete and remains attached to the A-tubule, though the Poc1 position is empty. In Class 3, the B-tubule is complete and connected to the A-tubule, while other proteins occupy the Poc1 site. Yellow dashed circles and arrows outline the position of Poc1 in the wild-type. An image from the wild-type and a schematic diagram is shown for comparison. (C) Further refinement of Class 3 at 8.00 Å shows the FAP20/PACRG filament at the mutant’s inner junction gap. (D) Comparing the A-B inner junction in the central core region between wild-type and poc1Δ Class 3 shows an increased gap in the junction. Pf A1 is used as a reference for superposition. For Pf B10 (α/β tubulin), the wild-type is in light green and blue, and the poc1Δ is in dark green and blue. (E) Focused classification of subtomograms from poc1Δ BB (Class 3) identifies additional axoneme MIP. Left, the mutant structure is shown in green. Right, the inner junction from the wild-type axoneme DMT. The corresponding MIP is colored in lavender. (F) In poc1Δ BB, Poc39 partially remains in the Class 2 average but is absent in Class 3, where the inner junction is occupied by FAP20/PACRG. For comparison, the wild-type structure is shown in grey. (G) Mapping the location of subtomogram from 3 subsets identified in the central core region of poc1Δ BBs. 8 representative BBs are shown. The green dots represent the Class 1 subset, an incomplete B-tubule detached from the A-tubule at the inner junction. The blue dots represent the Class 2 subset, complete B-tubule without Poc1. The red dots represent the Class 3 subset, the axoneme-like inner junction where FAP20/PACRG fills in the space left by Poc1. The arrows point in the direction from the proximal to the distal end of the BBs.

Figure 5.

poc1Δ mutants destabilize the A-B inner junction and allow axonemal components to incorporate into the assembly. (A) Upper: representative tomogram slices from poc1Δ BB show the TMTs split at the A-B inner junction at various longitudinal locations. Bottom: the blue lines highlight the split TMTs in the upper panels. The yellow arrows indicate the splits. The minus ends of TMT are at the bottom, and the plus ends are at the top. Scale bar: 100 nm. (B) 3D classification of subtomograms from poc1Δ BB, focusing on the A-B inner junction. The resulting three subsets show structure variations at the A-B inner junction. In Class 1, the B-tubule is incomplete and detached from the A-tubule at the inner junction. In Class 2, the B-tubule is complete and remains attached to the A-tubule, though the Poc1 position is empty. In Class 3, the B-tubule is complete and connected to the A-tubule, while other proteins occupy the Poc1 site. Yellow dashed circles and arrows outline the position of Poc1 in the wild-type. An image from the wild-type and a schematic diagram is shown for comparison. (C) Further refinement of Class 3 at 8.00 Å shows the FAP20/PACRG filament at the mutant’s inner junction gap. (D) Comparing the A-B inner junction in the central core region between wild-type and poc1Δ Class 3 shows an increased gap in the junction. Pf A1 is used as a reference for superposition. For Pf B10 (α/β tubulin), the wild-type is in light green and blue, and the poc1Δ is in dark green and blue. (E) Focused classification of subtomograms from poc1Δ BB (Class 3) identifies additional axoneme MIP. Left, the mutant structure is shown in green. Right, the inner junction from the wild-type axoneme DMT. The corresponding MIP is colored in lavender. (F) In poc1Δ BB, Poc39 partially remains in the Class 2 average but is absent in Class 3, where the inner junction is occupied by FAP20/PACRG. For comparison, the wild-type structure is shown in grey. (G) Mapping the location of subtomogram from 3 subsets identified in the central core region of poc1Δ BBs. 8 representative BBs are shown. The green dots represent the Class 1 subset, an incomplete B-tubule detached from the A-tubule at the inner junction. The blue dots represent the Class 2 subset, complete B-tubule without Poc1. The red dots represent the Class 3 subset, the axoneme-like inner junction where FAP20/PACRG fills in the space left by Poc1. The arrows point in the direction from the proximal to the distal end of the BBs.

Figure 6.

(A) Schematic illustration summarizing the inner junction components in three regions of the cilium. In poc1Δ, the axoneme-specific MIPs are in red; note that many BB components are partially bound in the mutant as the inner junction structure is disrupted without Poc1. (B) A model illustrates the poc1Δ BB A-B inner junction partially morphing into an axoneme-like architecture. The double-ended arrows indicate expansion of the inner junction gap as PACRG and FAP20 are incorporated in. This causes the disassociation of Poc39 and facilitates the binding of axonemal components, such as CCDC81b and BMIP1.

Figure 6.

(A) Schematic illustration summarizing the inner junction components in three regions of the cilium. In poc1Δ, the axoneme-specific MIPs are in red; note that many BB components are partially bound in the mutant as the inner junction structure is disrupted without Poc1. (B) A model illustrates the poc1Δ BB A-B inner junction partially morphing into an axoneme-like architecture. The double-ended arrows indicate expansion of the inner junction gap as PACRG and FAP20 are incorporated in. This causes the disassociation of Poc39 and facilitates the binding of axonemal components, such as CCDC81b and BMIP1.