Role for intraflagellar transport in building a functional transition zone

Abstract

Genetic disorders caused by cilia dysfunction, termed ciliopathies, frequently involve the intraflagellar transport (IFT) system. Mutations in IFT subunits-including IFT-dynein motor DYNC2H1-impair ciliary structures and Hedgehog signalling, typically leading to "skeletal" ciliopathies such as Jeune asphyxiating thoracic dystrophy. Intriguingly, IFT gene mutations also cause eye, kidney and brain ciliopathies often linked to defects in the transition zone (TZ), a ciliary gate implicated in Hedgehog signalling. Here, we identify a C. elegans temperature-sensitive (ts) IFT-dynein mutant (che-3; human DYNC2H1) and use it to show a role for retrograde IFT in anterograde transport and ciliary maintenance. Unexpectedly, correct TZ assembly and gating function for periciliary proteins also require IFT-dynein. Using the reversibility of the novel ts-IFT-dynein, we show that restoring IFT in adults (post-developmentally) reverses defects in ciliary structure, TZ protein localisation and ciliary gating. Notably, this ability to reverse TZ defects declines as animals age. Together, our findings reveal a previously unknown role for IFT in TZ assembly in metazoans, providing new insights into the pathomechanism and potential phenotypic overlap between IFT- and TZ-associated ciliopathies.

Keywords: C. elegans; IFT; cilia; dynein; transition zone.

Figure 1. Screen for retrograde IFT defects identifies a temperature‐sensitive allele of the dynein heavy chain CHE‐3, the orthologue of mammalian DYNC2H1

1. Genetic screen for retrograde IFT defects uncovers a temperature‐sensitive (ts) mutation in C. elegans CHE‐3 (dynein‐2 heavy chain). Paraquat‐resistant strains were first screened for dye‐filling defects (Dyf phenotype) typically indicative of cilia structure anomalies (examples of normal and defective DiI dye‐filling are shown). Three different IFT reporters were then introduced into the dyf mutants (CHE‐11::GFP shown as an example) to reveal candidate retrograde IFT mutants showing strong IFT protein accumulation at cilia tips. Whole‐genome sequencing revealed the likely causative mutation in one of the strains, namely in the retrograde IFT‐dynein motor CHE‐3 (DYNC2H1 human orthologue). A variable Dyf phenotype at 20°C suggested the possibility of a ts mutation. Scale bar, 20 μm.

2. The Dyf phenotype of the ts‐che‐3 mutant was confirmed, with animals grown at the permissive temperature (15°C) showing the ability to uptake dye, while those raised at the restrictive temperature (25°C) are Dyf. Scale bar, 20 μm.

3. Using the homodimeric kinesin OSM‐3 as a marker for IFT, proper localisation is seen in the ts‐che‐3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM‐3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 μm.

4. The ts‐che‐3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature, indicating defective chemosensation. The osm‐9 mutant is included as a positive control. n = 50 animals; error bars are standard error.

Figure EV1. The temperature‐sensitive mutation in C. elegans CHE‐3 (orthologue of human DYNC2H1) is in a glycine residue next to the ATP binding site and immediately adjacent to an evolutionarily conserved aspartic acid (Asp; D) likely required for ATP binding/hydrolysis in the AAA2 domain

1. Multiple sequence alignment showing a portion of the AAA2 domain containing the glycine (G1997) residue mutated to glutamic acid (Glu; E) in CHE‐3 (G2056 residue in human DYNC2H1).

2. Close‐up of Gly and Asp amino acids in relation to ATP‐Mg²⁺ within the AAA2 domain of human DYNC2H1 (crystal structure PDB accession number 4RH7).

Figure 2. Loss of IFT‐dynein function at restrictive temperature results in TZ protein localisation defects that are reversed at permissive temperature

1. Three TZ markers (NPHP‐4, CEP‐290 and MKS‐6) are shown at both the permissive (15°C) and restrictive (25°C) temperatures in the ts‐che‐3 mutant. At the permissive temperature, all three markers localise to the TZ, while at the restrictive temperature exhibit ectopic localisation (leakage) distally into the ciliary axoneme. While NPHP‐4 and MKS‐6 show significant accumulations, CEP‐290 is grossly normal. At the permissive temperature, both IFT co‐markers (CHE‐13 and XBX‐1) display strong localisation to the basal body (bb) and along the axoneme. At the restrictive temperature, the IFT markers show accumulation in the axoneme. Accumulations emphasised using asterisks.

2. To determine whether ciliary compartmentalisation could be restored, young ts‐che‐3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature. The TZ proteins reassembled at the TZ, with loss of the ectopic accumulations, and IFT protein localisation was also restored—strongest at the basal body, similar to animals grown at the permissive temperature.

3. After 24 h at the restrictive temperature, TZ proteins appear to begin to accumulate distally within the axoneme in ts‐che‐3 mutants. This is seen, in small amounts, for NPHP‐4 and CEP‐290 but with stronger ectopic localisation for MKS‐6. The IFT proteins CHE‐13 and XBX‐1 also show accumulations in the axoneme after 24 h at the restrictive temperature.

Data information: Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4–7 animals), Kruskal–Wallis test, *P < 0.05, **P < 0.01. tz, transition zone; bb, basal body; scale bars are 4 μm.

Figure EV2. The null and ts‐che‐3 mutant, but not the heterozygous ts mutant show ectopic TZ protein localisation, which recovers after FRAP in the ts mutant

1. In the null allele che‐3(e1124), five transition zone (TZ) proteins show distal ectopic accumulation (marked by asterisks) in the bulbous axonemes of the mutant cilia, something not observed in wild‐type animals. TZs are indicated with an arrowhead. Dotted lines and arrows indicate direction and location of either cilia or dendrites (den). Scale bar is 4 μm.

2. When grown at the restrictive temperature the cilia of animals heterozygous for the ts‐che‐3 allele resemble those found in wild‐type, with no apparent ectopic TZ (MKS‐6) or IFT (XBX‐1) protein accumulations seen in the homozygous mutant. Scale bar is 4 μm.

3. After photobleaching the distal end of a truncated axoneme in a ts‐che‐3 mutant grown at the restrictive temperature, MKS‐6 shows fluorescence recovery within seconds. White boxes indicate pixels analysed for the graph, which represents a ratio of fluorescence intensity between the area photobleached (indicated by the red box) to an area not photobleached. Scale bar is 0.5 μm.

Figure 3. Abrogating CHE‐3 function causes defects in transition zone gate function and ultrastructure

1. In the ts‐che‐3 mutant grown at the permissive temperature (15°C), both RPI‐2/RP2 and TRAM‐1/TRAM1 are localised to the PCMC, just proximal to the ciliary axoneme. At the restrictive temperature, TRAM‐1 shows leakage into the axoneme with its strongest localisation at the PCMC. Intriguingly, RPI‐2 now displays strong localisation to the axoneme, indicating that in the absence of IFT, RPI‐2 may be targeted into the cilium. When ts‐che‐3 mutant animals grown at the restrictive temperature (25°C) are shifted at young adult to the permissive (15°C) for 24 h, both TRAM‐1 and RPI‐2 exhibit restoration of their PCMC localisation. Consistent with other TZ markers, both MKS‐2 and MKSR‐1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature. Also consistent with what was observed for other markers, after 24 h at the permissive temperature, animals grown at the restrictive temperature show loss of the ectopic localisation. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (MKS‐2 and MKSR‐1) or accumulation (RPI‐2 and TRAM‐1) for statistical analyses. n = 10 cilia (4–6 animals), Kruskal–Wallis test, *P < 0.05, **P < 0.01; scale bars are 4 μm.

2. Loss of CHE‐3 function results in short, bulbous cilia containing electron‐dense accumulations and abnormal membrane–microtubule connections. Shown are transmission electron micrograph (TEM) cross sections of an amphid channel cilium in wild‐type and che‐3(e1124) null mutant animals. Representative images of wild‐type cilia show intact middle segment (containing doublet microtubules) and transition zone (with Y‐shaped links) (left top and bottom panels) regions. Representative images of che‐3(e1124) cilia reveal apparently intact transition zones, with visible Y‐link structures, but enlarged ciliary ends filled with electron‐dense accumulations, which often appear vesicular. The bulbous structures reveal doublet microtubules associated with the membrane, and occasionally ectopic microtubule‐to‐membrane connections, which sometimes appear similar to transition zone Y‐links in the region just distal to the seemingly “normal” TZ structure. Schematics show longitudinal (left images) and cross section (right images) representations of wild‐type and che‐3(e1124) cilia. tz, transition zone; pcmc, periciliary membrane compartment; scale bars are as indicated in (nm) for TEM images.

Figure EV3. Ultrastructural defects observed in cilia of che‐3 null and temperature sensitive mutants

At the restrictive temperature of 25°C, the temperature‐sensitive che‐3 mutant, che‐3(nx159ts), has ultrastructural features similar to those of the che‐3(e1124) null mutant at 20°C. Left panels show transmission electron micrograph (TEM) cross sections of amphid channel cilia. Schematics show the amphid channel in longitudinal and cross‐section orientations (only 3 of 10 cilia shown), indicate the section positions (in microns, relative to position “0”), and denote the phenotypes observed (summarised in point form). Scale bars are 500 nm (all images identically scaled). Some images placed on a grey background. PCMC, periciliary membrane compartment. Arrowheads indicate transition zones, and asterisks show middle and distal ciliary segments.

Figure 4. Ciliary maintenance and IFT function upon shift to the restrictive temperature (25°C)

1. Using the IFT marker BBS‐7, cilia lengths were measured for wild‐type and the ts‐che‐3 mutant animals raised at the permissive (15°C) and shifted to the restrictive temperature (25°C) for three time points. After 12 h, some accumulations (indicated by asterisks in the fluorescence images) are observed within the cilia, but there is not a significant reduction in cilium length. After 18 and 24 h, significant reduction in ciliary length is observed and large accumulations of BBS‐7 is seen within short bulbous cilia, similar to what is observed in ts‐che‐3 animals raised at the restrictive temperature. n = 30–34 cilia, one per animal; Kruskal–Wallis test, **P < 0.01; error bars are mean absolute deviation; scale bars are 4 μm.

2. IFT velocities were measured for wild‐type or ts‐che‐3 mutant animals (grown at permissive temperature) and imaged at the permissive temperature, restrictive temperature or after 3 h at the restrictive temperature. Compared to wild‐type, anterograde velocities in the ts‐che‐3 dynein mutant are similar, while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments. Interestingly, the ts‐che‐3 IFT‐dynein mutant appears to not only have slow retrograde IFT trains, but the trains also appear much larger and less frequent. Initially upon the temperature shift (< 15 min), IFT rates are much faster for both wild‐type and ts‐che‐3 dynein mutant, although retrograde IFT remains much slower in the mutant. After 3 h at the restrictive temperature, IFT velocities are intermediate between the rates seen at 15°C and those observed at the initial shift to 25°C. Error bars are 95% CI. Wild‐type (10–15 animals): anterograde n = 141–326, retrograde n = 100–271; for che‐3(nx159ts) (18–25 animals): anterograde n = 234–312, retrograde n = 64–103; y‐axis scale bar is 2 s; x‐axis scale bar is 1 μm.

Figure EV4. IFT behaviour in ts‐che‐3 mutant

1. Anterograde IFT velocities of CHE‐11/IFT140 are quite similar for wild‐type and che‐3(nx159ts) when animals raised at the permissive temperature are imaged at the permissive or restrictive temperatures (< 30 min). However, retrograde velocities are much reduced in che‐3(nx159ts) mutants compared to wild‐type animals. All velocities observed were markedly increased when the imaging was performed at 25°C compared to cilia imaged at 15°C, indicating a temperature dependence on IFT rates. Error bars are 95% CI. For wild‐type (13–15 animals): anterograde n = 301–493, retrograde n = 309–440; for che‐3(nx159ts) (18–48 animals): anterograde n = 727–776, retrograde n = 401–407. Y‐axis scale bars are 2 s; x‐axis scale bars are 1 μm.

2. After 3 h at the restrictive temperature, some ts dynein mutants raised at the permissive temperature begin to show very slow retrograde IFT, whereas others exhibit no IFT in either direction. This suggests that in the ts‐che‐3 mutant, IFT stochastically shuts down over time after the shift to the restrictive temperature. Y‐axis scale bars are 2 s; x‐axis scale bars are 1 μm.

Figure 5. Transition zone repair potential decreases with age while older adults show reduced TZ protein localisation defects

1. A, B

   To assess the effects of IFT restoration on TZ assembly, ts‐che‐3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development (days 1, 4 and 8 of adulthood). The TZ markers MKS‐6 (A) and NPHP‐4 (B) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood. After a shift to the permissive temperature for 24 h, day 2 adults show “repaired” TZ protein localisation resembling that of wild‐type animals. For both markers, day 4 adults (maintained at the restrictive temperature) show less ectopic TZ protein localisation than day 1 adults. The correction of ectopic localisation following shift to the permissive temperature for 24 h was reduced in day 4 (to day 5) of adulthood compared to that observed in day 1 (to day 2) adults. For MKS‐6, but not NPHP‐4, day 8 adults (maintained at the restrictive temperature) also display less ectopic accumulation than day 1 adults. The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers, indicating that this repair is most effective in day 1 adults. In summary, as the adults age, ectopic TZ protein localisation is reduced, while the ability to correct this phenotype by restoring IFT function also reduces. tz, transition zone; scale bars are 4 μm. Heat map graphs corresponding to the bar graphs are located in Fig EV5. n = 10 cilia (4–7 animals), Kruskal–Wallis test, *P < 0.05, **P < 0.01, error bars are mean absolute deviation.

Figure EV5. TZ and IFT repair potential decreases with age while older adults show reduced localisation defects

Shown are animals grown at restrictive temperature and shifted at days 1, 4 or 8 of adulthood to the permissive for 24 h. For both TZ markers—MKS‐6 and NPHP‐4—there is significant reduction in ectopic protein localisation (marked by asterisks) after a 24‐h shift to the permissive temperature except for NPHP‐4 at day 4, which shows a reduction but is not statistically significant. For both IFT markers (XBX‐1 and CHE‐13), the same trend is observed. After a 24‐h shift to the permissive temperature, there is significant reduction in ectopic accumulation except at day 4 for CHE‐13, which shows a reduction but is not statistically significant. Despite a reduction in ectopic localisation for all markers, as the animals age, the “repair” seen after 24 h at the permissive temperature (i.e., IFT function is restored) is reduced as the animals age. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4–7 animals), Kruskal–Wallis test, *P < 0.05, **P < 0.01. tz, transition zone; bb, basal body; scale bars are 4 μm.

Figure 6. Model for the role of intraflagellar transport (IFT) in the correct assembly and function of the ciliary transition zone (TZ), and relationship to ciliopathies

1. Kinesin‐ and dynein‐dependent anterograde and retrograde particles (1 and 2, respectively) are shown harbouring IFT‐A, IFT‐B and BBS subcomplexes involved in ciliary cargo transport. Under normal conditions, any misassembly or partial disassembly of proteins from the TZ (3) can be recovered by the retrograde IFT machinery (4). Disruption of retrograde transport (and consequently, anterograde transport) leads to an accumulation of TZ proteins in a short, bulbous cilium as well as impaired TZ assembly and function (as observed in our temperature‐sensitive IFT‐dynein mutant at the restrictive temperature), phenotypes that are reversible when IFT is restored at the permissive temperature.

2. Cross‐talk between different ciliary modules and distinct classes of ciliopathies. Most ciliary modules are associated with a specific class of ciliopathies; for example, IFT‐A and IFT‐dynein are linked to skeletal ciliopathies, BBS proteins are associated with Bardet–Biedl syndrome, and transition zone proteins are associated with Joubert syndrome and related disorders (JSRDs), MKS, NPHP and eye‐related disorders, but not skeletal ciliopathies. However, a subset of proteins are associated with ciliopathies generally typical of a different module, indicating potential overlap in function. The module–disease connections are shown as solid lines, with the width correlating to the number of causative genes currently identified (1 to 10+, as indicated). MKS, Meckel syndrome; JBTS, Joubert syndrome; COACH, cerebellar vermis hypoplasia/oligophrenia/ataxia/coloboma/hepatic fibrosis; OFD, oral‐facial‐digital syndrome; SLSN, Senior‐Løken syndrome; NPHP, nephronophthisis; RP, retinitis pigmentosa; CRD, cone‐rod dystrophy; LCA, Leber congenital amaurosis; BBS, Bardet–Biedl syndrome; CED, cranioectodermal dysplasia; SRTD, short‐rib thoracic dysplasia; JATD, Jeune asphyxiating thoracic dystrophy.