Figures and data in The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

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Videos
Tables
Additional files

7 figures, 1 video, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement

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The IJ structures of *Chlamydomonas* and *Tetrahymena* doublet.

(**A–D**) Surface renderings and schematics of the 48 nm repeat cryo-EM maps of *Chlamydomonas* (**A, B**) and *Tetrahymena* (**C, D**) doublets viewed from the tip of the cilia. Black arrow indicates longitudinal view in (E), (F) and (G). (**E–F**) The longitudinal section of the *Chlamydomonas* doublet at the IJ complex from the inside (E) and outside (F). (G) The longitudinal section of *Tetrahymena* doublet viewed from the inside. Color scheme: FAP20: dark green; PACRG: gray; FAP52: light green, Y-shaped density: purple; FAP45: yellow green; fMIP-B8B9: orange; Tubulin: light gray; Unknown density: yellow; Rest of MIPs: white; Tether density 1 and 2: red; Tether density 3, pink. Plus and minus ends are indicated by + and - signs. (H) Cross sectional views of the different Tether densities from *Chlamydomonas* (left) and *Tetrahymena* (right). In *Chlamydomonas*, there is a Y-shaped density (purple) that cradles the FAP52 density. The Y-shaped density is absent in *Tetrahymena*. In *Tetrahymena*, we observed Tether density 3, which is absent in *Chlamydomonas*.

Figure 1—figure supplement 1

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Data related to the doublet structures.

(A) Schematics of fractionation of the axoneme in this study. Doublets were split from axoneme, and outside proteins were removed to obtain a simpler sample for cryo-EM. (B) A typical cryo-EM image of *Chlamydomonas* doublets. (C) Gold-standard Fourier Shell Correlation of the 48 nm repeat and 16 nm repeat doublet maps of *Chlamydomonas* and *Tetrahymena*. (D) Local resolution estimation of the 16 nm repeat maps from *Chlamydomonas* and *Tetrahymena* using MonoRes. The resolution of the B-tubule in *Tetrahymena* is lower due to the loss of the IJ PF. (E) The remaining PACRG- and FAP20-like densities in the *Tetrahymena* doublet structure. (F) Superimposition of the tomographic structure of the intact doublet (EMD-2132) with the 48 nm structure of the *Chlamydomonas* doublet in this study. The DRC is colored green. (G) Enlarged view of the missing PACRG unit at the IJ PF, where the DRC binds. Bars in E and H denote a length of 5 nm.

Figure 2 with 1 supplement

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16 nm structure of *Chlamydomonas* doublet.

(**A, B**) 16 nm repeat structure of *Chlamydomonas* doublet and model at the IJ region. Tubulin densities are depicted as transparent gray, except in A in which tubulins are shown in dark blue. (C) Atomic model of the IJ complex, consisting of PACRG, FAP20, FAP52 and FAP276. Color scheme: FAP20: dark green; PACRG: gray; FAP52: light green, FAP276: purple; tubulin: transparent gray density. (**D, E**) Maps and models of PF A1 and B10, and IJ PF illustrating how the IJ PF interacts with tubulins. The views are indicated in the schematics. Dashed boxes indicate the views in (F), (G) and (H). Color scheme: α-tubulin: green; β-tubulin: blue; PACRG: gray; FAP20: dark green; FAP276: purple. (F) Electrostatic surface charge of PACRG, FAP20 and α- and β-tubulins of PF A1. Tubulin surface is negatively charged while the interacting interface of PACRG and FAP20 are positively charged. (G) The interaction of the PACRG with the inter-dimer interface of tubulins from PF B10 is shown. H136 from PACRG is conserved and is likely to take part in the interaction with D127 and C129 from α-tubulin of PF B10. (H) The C-terminus of β-tubulin of PF A1 interacts with PACRG and FAP20. Potential residues involved in the interaction of C-tail of β-tubulin and PACRG and FAP20 are shown. (I) *Chlamydomonas* PACRG has a long N-terminus 1–117. The N-terminus of *Chlamydomonas* PACRG (red) forms a stable triple helix arrangement with the core of the protein. This is not observed in the human PACRG (PDB: 6NDU) shown in yellow. In addition, the N-terminus of PACRG going into the wedge between PF A13 and A1.

Figure 2—figure supplement 1

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Atomic models of PACRG, FAP20, FAP52 and FAP276.

Atomic model of (A) PACRG, (C) FAP20, (E) FAP52 and (G) FAP276. Illustration of the cryo-EM density quality at selected regions of (B) PACRG, (D) FAP20, (F) FAP52 and (H) FAP276.

Figure 3 with 1 supplement

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Interaction between PACRG and FAP20.

(A) Consecutive molecules of PACRG and FAP20 in the IJ PF. PACRG and FAP20 form a heterodimer as indicated by brackets. (B) Magnified view of the interacting region of PACRG and FAP20. Residues Q264 and D258 of PACRG form hydrogen bonds with residue T38 and K20 of FAP20, respectively. (**C, D**) Electrostatic interactions between PACRG and FAP20 illustrated by their surface charge. The dashed boxes in (**A, C, D**) highlight the interacting loops between PACRG and FAP20 (B). (E) Multiple sequence alignment of PACRG in the regions of FAP20-binding loop. Asterisks indicate residues that are involved in FAP20 binding.

Figure 3—figure supplement 1

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Multiple sequence alignment of FAP20 shows that it is highly conserved.

Figure 4 with 1 supplement

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Structure of FAP52 and its interaction with tubulins and PACRG.

(A) Structure of FAP52 in a top view from the outside of the B-tubule looking down on the A-tubule. Black arrow indicates the direction of view in (B). (B) Three-point contacts of FAP52 with α-tubulins from PF B9 and B10 and PACRG, indicated by the black dashed boxes. The α-K40 loops are colored red. (**C, D**) The structure of the α-K40 loop from PF B10. Red dashed boxes indicate the α-K40 loop. (E) Interaction of α-K40 loop of PF B10 with FAP52. FAP52’s K229 is within favorable distance to form hydrogen bonds with the backbone of D39 and T41. Hydrophobic interactions between L226 and I42 and interactions involving R225 are also possible.

Figure 4—figure supplement 1

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Data related to FAP52.

(A) Atomic model of *Chlamydomonas* FAP52 from inside the *Chlamydomonas* density map. (B) Atomic model of *Chlamydomonas* FAP52 fitted inside the *Tetrahymena* map highlights the longer loop (red) from *Chlamydomonas*. (C) Alignment of FAP52 from several species shows that *Chlamydomonas* has a longer loop in one beta propeller blade. The long loop is responsible for the interaction with PACRG. (**D, E**) α-K40 loop from PF B9 in *Chlamydomonas*. (**F, G**) Superimposition of the acetylated α-K40 loops from *Chlamydomonas* PF B9, B10 and *Tetrahymena* A12.

Figure 5 with 1 supplement

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Structure of the Tether densities.

(**A–B**) At higher resolution, Tether densities 1 and 2 appear to be a single polypeptide chain in both *Chlamydomonas* (A) and *Tetrahymena* (B). Color scheme: tubulin: transparency gray; Tether densities 1 and 2 (Tether loop/FAP106): red; FAP276: purple; PACRG: light gray; Densities between PF A13 and A12 (turquoise). The dashed regions indicate the location of FAP52, which has been digitally removed to show the Tether densities underneath. (C) Model of FAP106 fitted inside the segmented Tether loop from *Chlamydomonas*. (D) Model of FAP106 tethering the B-tubule and A-tubule. Dashed box indicates view in (E). (E) Helix H3 and H4 of FAP106 insert into the gap formed by four tubulin dimers of PF B9 and B10. (F) Structure of Tether density 3 from *Tetrahymena*, which binds on top of the wedge between PF A13 and A1. (G) Overlay of the PACRG from *Chlamydomonas* onto the structure of *Tetrahymena* shows a hypothetical steric clash of a long *Tetrahymena* PACRG N-terminus with Tether density 3.

Figure 5—figure supplement 1

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Data related to the Tether densities.

(A) The density of the search motif [FHY]xWxxKxx[FHY used for identifying FAP106 (left panel) and the fitted model segment (right panel). (B) The model of the N-terminus (left) and C-terminus (right) of FAP106 inside the electron density. (**C, D**) The small helix (H2) (indicated by the dashed box) from *Chlamydomonas* (A) and *Tetrahymena* homolog for Tether loop appear to interact with α-tubulin. (E) Multiple sequence alignment of FAP106 from a few organisms. (F) Secondary structure prediction of FAP106. The big cylinder represents helical prediction. Low confidence predictions are omitted.

Figure 6 with 1 supplement

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Structure of FAP126 and its interaction.

(A) View from the top of the PF A12 and A13 showing the density of FAP126 (dark turquoise). Dashed box indicates view in (B). (B) Closeup view of the interactions of FAP126 with the Tether loop, FAP106. T76 and Q77 of FAP126 form hydrogen bonds with Q126 and Q129 of FAP106, respectively. (C) Complimentary electrostatic surface charges of tubulins and FAP126. (D) Electrostatic charge of FAP126 on the tubulin interacting surface. (E) The N-terminus of PACRG and the hook density go into the wedges between PF A12 and A13, and PF A13 and A1, respectively. This likely contributes to the curvature of this region. (F) Inter-PF angles of the A- and B-tubules from *Chlamydomonas* and *Tetrahymena* (Ichikawa et al., 2019) showing very similar angle distributions. (G and H) Correlation graphs of consensus normalized expression levels for two selected pairs of genes (ENKURIN(FAP106)/FAP126 and FAP52/FAP126). Tissues showing high levels of expression of one or both genes are labeled. Correlation coefficients (r) are indicated. By excluding testis, corpus callosum, pons and medulla and fallopian tube, the correlation coefficients between ENKUR and FAP126 and between FAP52 and FAP126 are 0.66 and 0.84, respectively.

Figure 6—figure supplement 1

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Data related to FAP126.

(A) Multiple sequence alignment of FAP126 from a few organisms. FAP126 of *Chlamydomonas* still have the SH3 binding domain while lacks the proline-rich region compared to other species. (B) Atomic model of FAP126 fitted inside its segmented density. The dotted box indicated the view in (C). (C) The density of the search motif WPxxxxxW used for identifying FAP126. (D) Table of pairwise correlation coefficients between tissue mRNA expression levels, color-coded from low (red) to high (blue) values. ENKUR is the homolog of FAP106 in human. DCX is a microtubule associated protein in neuron, picked as a control. (**E, F**) Correlation graphs of consensus normalized expression levels for two selected pairs of genes (PACRG/FAP20 and PACRG/FAP52). Tissues showing high levels of expression of one or both genes are labeled. Correlation coefficients (r) are indicated.

Figure 7

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Inner junction structure and proposed model of IJ formation.

(A) Model of the IJ complex including PACRG, FAP20, FAP52, FAP126, FAP276 and FAP106. FAP45 is not depicted here. Tubulin is depicted as transparent. (B) The B-tubule starts growing laterally from the outer junction side as shown in Schmidt-Cernohorska et al. (2019). PACRG and FAP20 form a heterodimer, which binds onto the outside surface of PF A1. Following this, multiple alternative hypotheses are possible. One hypothesis is that FAP52, FAP276 and FAP106 would bind onto PF A12 and A13. FAP45 and other fMIP proteins would then be incorporated inside the B-tubule, which fixes the proper curvature so that PF B9 and B10 can interact with other IJ proteins. FAP52 binds both PF B9 and B10 through their K40 loops and finally, PACRG and FAP20 interact with the lateral side of PF B10 allowing for B-tubule closure.

Videos

Video 1

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posterframe for video — The IJ complex of the *Chlamydomonas reinhardtii* flagella.

Tables

Table 1

Proteins completely missing (no peptide detected) in FAP52 knockout mutant

Names	Mol. weight (kDa)	p-values (WT vs FAP52)	Exclusive unique peptide counts in WT (quantitative values after normalization)
ARL3	20	0.0013	2, 2, 1 (2, 1, 2)
CHLREDRAFT_171815	57	0.035	2, 6, 1 (2, 5, 2)
CHLREDRAFT_156073	11	0.024	1, 1, 1 (2, 1, 2)
FAP276	10	0.015	3, 3, 2 (8, 7, 14)
FAP52	66	0.0046	27, 21, 15 (59, 73, 105)
FAP36	41	0.0023	3, 3, 1 (3, 3, 2)
CrCDPK1	54	<0.0001	3, 5, 2 (3, 4, 4)
CHLREDRAFT_176830	110	0.024	2, 1, 2 (2, 1, 1)
FAP173	33	0.012	3, 3, 1 (5, 3, 2)
FAP29	112	0.00045	2, 3, 2 (3, 3, 4)
CHLREDRAFT_181390	41	0.028	1, 1, 1 (1, 1, 2)
ANK2	60	0,0041	1, 2, 1 (1, 1, 2)

Table 2

Normalized spectral count of proteins detected by mass spectrometry.

Name	Molecular Weight in kDa (MW)	Average quantitative Value (AQV)	Rough Stoichiometric Peptide Abundance (RSPA)*	T. thermophila Homologs**	Human homologs***	Localization in C. reinhardtii
TUA1	50	1077.97	215.59	TBA_TETTH	TUBA1C	Doublet
TUB1	50	625.46	125.09	TBB_TETTH	TUBB4B	Doublet
RIB72	72	116.72	16.21	TTHERM_00143690	EFHC1	MIP
PACRG	25	38.39	15.35	TTHERM_00446290	PACRG	IJ
PF16	50	74.09	14.82	TTHERM_000157929	SPAG6	Central Pair
RSP9	30	41.79	13.93	TTHERM_00430020	RSPH9	Radial Spoke
FAP86	30	36.11	12.04	-	-	Doublet
FAP1	22	26.46	12.03	-	-	Doublet
FAP52	66	79.12	11.99	TTHERM_01094880	CFAP52	MIP
FAP20	22	26.08	11.86	TTHERM_00418580	CFAP20	IJ
FAP126	15	16.89	11.26	-	CFAP126	MIP
RSP1	88	98.73	11.22	TTHERM_00047490	RSPH1	Radial Spoke
FAP115	27	29.98	11.10	TTHERM_00193760	-	Doublet
FAP106	27	29.81	11.04	TTHERM_00137550	ENKUR	IJ?
Tektin	53	57.61	10.87	-	TEKT5	IJ?
RSP3	57	60.55	10.62	TTHERM_00566810	RSPH3	Radial Spoke
FAP252	39	39.97	10.25	TTHERM_00899430	CETN3	Axonemal
RSP2	77	77.95	10.12	-	CALM2	Radial Spoke
FAP161	43	43.50	10.12	TTHERM_00155380	CFAP161	Axonemal
IDA4	29	27.29	9.41	TTHERM_00841210	DNALI1	Dynein
FAP107	26	23.66	9.10	-	FLG2	Axonemal
DHC2	457	414.12	9.06	TTHERM_01027670	DNAH1	Dynein
FAP12	54	48.85	9.05	-	DAGLB	Cytoplasmic
RSP7	34	30.62	9.01	TTHERM_00194419	CALML5	Radial Spoke
RSP5	56	49.32	8.81	-	-	Radial Spoke
FAP230	45	39.59	8.80	-	-	Axonemal
FAP77	29	23.88	8.24	TTHERM_00974270	CFAP77	Axonemal
FAP55	111	90.47	8.15	-	MYH14	Axonemal
FAP90	28	22.35	7.98	-	WBP11	Axonemal
RSP10	24	19.10	7.96	TTHERM_00378600	RSPH1	Radial Spoke
FAP71	32	24.86	7.77	TTHERM_00077710	EWSR1	Axonemal
EEF1	51	39.04	7.66	TTHERM_00655820	Multiple	Axonemal
FAP182	49	36.69	7.49	TTHERM_01049330	C9orf116	Axonemal
Rib43a	43	32.06	7.46	TTHERM_00624660 TTHERM_00641119	RIBC2	MIP
FAP45	59	43.20	7.32	TTHERM_001164064	CFAP45	MIP

^*RSPA was calculated by (AQV)/(MW)*10.

^** T. thermophila homologs were BLAST searched using the Uniprot database.
^***Human homologs were taken from the ChlamyFP project (Pazour et al., 2005).