The Meckel syndrome- associated protein MKS1 functionally interacts with components of the BBSome and IFT complexes to mediate ciliary trafficking and hedgehog signaling

<div><p>The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT) proteins. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS) complex protein MKS1 and the BBSome protein BBS4. We find that <i>Mks1; Bbs4</i> double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh) dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between <i>Mks1</i> and a hypomorphic allele of the IFTB component <i>Ift172</i>. Despite each single mutant surviving until around birth, <i>Mks1; Ift172</i>^<i>avc1</i> double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that <i>Mks1</i> interacts genetically with an allele of <i>Dync2h1</i>, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of <i>Mks1</i> with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure.</p></div

Summary of embryonic phenotypes in double mutant crosses.

<p>Summary of embryonic phenotypes in double mutant crosses.</p

Ciliary trafficking defects in <i>Mks1</i>^{<i>krc/krc</i>}<i>;Bbs4</i>^<i>-/-</i> double mutant cells.

<p><b>(A-H)</b> MEFs derived from embryos of the indicated genotypes were cultured under serum-free conditions for 48 hours to induce cilia formation and immunostained for γ-Tubulin (red), acetylated α–Tubulin (green) and the ciliary membrane proteins ARL13B (A-D, magenta) or INPP5E (E-H, magenta). Two representative images for each condition are included. <b>(I-P)</b> Serum starved MEFs derived from embryos of each genotype were treated for the final 24 hours with either DMSO vehicle (I,K,M,O) or SMO agonist (SAG) to activate the Hh pathway (J,L,N,P). Cells were stained for γ-Tubulin (red), acetylated α–Tubulin (green), or SMO (magenta). Two representative images for each condition are included. <b>(Q-X)</b> MEFs of each genotype were treated as described for (I-P) and immunostained for γ-Tubulin (red), acetylated α–Tubulin (green), or GPR161. Due to the low intensity of GPR161 relative to acetylated α–Tubulin, each of these channels is shown separately next to the corresponding merged channel image. Two representative images for each condition are included. <b>(Y-F’)</b> MEFs of each genotype were treated as described in I-X and immunostained for α–Tubulin (red), acetylated α-Tubulin (green), or GLI2 (magenta). Two representative images for each condition are included. Scale bar, 5μm.</p

<i>Mks1</i>^{<i>krc/krc</i>}<i>;Bbs4</i>^<i>-/-</i> double mutants exhibit enhanced defects in ciliary localization of Arl13b, but not acetylated Tubulin.

<p><b>(A-H)</b> Mouse embryonic fibroblasts (MEFs) were derived from embryos of the indicated genotype, and immunostained for γ-Tubulin (red) and either acetylated α–Tubulin (A-D, green) or ARL13B (E-H, green) to label cilia. Scale bar, 20μm. <b>(I-L)</b> Tissue sections from the embryonic neural tube of double heterozygous (A) <i>Bbs4</i>^<i>-/-</i> (B), <i>Mks1</i>^{<i>krc/krc</i>} (C), or <i>Mks1</i>^{<i>krc/krc</i>}<i>;Bbs4</i>^<i>-/-</i> double mutant (D) at E10.5. Sections were immunostained for ARL13B to label ciliary membranes (red) and for γ-Tubulin to label centrioles (green). Scale bar, 50μm. <b>(M-N)</b> Quantitation of the percentage of ciliated cells in each genotype positive for either acetylated α-Tubulin (M) or ARL13B (N) following 48 hours of serum starvation. The percentage of cells with cilia stained for acetylated α–Tubulin was not different between double mutants and <i>Mks1</i>^{<i>krc/krc</i>} single mutant cells (M, p = 0.3493). By contrast, significantly fewer double mutant cells had cilia stained with ARL13B compared with <i>Mks1</i>^{<i>krc/krc</i>} single mutant cells (N, *** p = 0.0001).</p

Model summarizing the trafficking defects in <i>Mks1</i>^{<i>krc/krc</i>} mutants as well as double mutants with <i>Bbs</i> and <i>Ift</i>.

<p>All conditions are depicted in the state of HH pathway activation upon stimulation with SAG. <b>(A)</b> WT and double heterozygous cells. Red outline depicts the normal ciliary membrane integrity (based on ARL13b and INPP5E localization) SMO is present in the ciliary membrane, GPR161 is reduced within the ciliary membrane due to pathway activation. GLI2 and KIF7 are enriched at the ciliary tip. IFT components are enriched at the ciliary tip and the transition zone near the basal body (shown in purple). <b>(B)</b> <i>Mks1</i>^{<i>krc/krc</i>} cells exhibit defects in the localization of ciliary membranes (dashed red line)- ARL13b is reduced and INPP5E is absent. SMO is reduced, and GLI2, KIF7, and IFT81 are no longer restricted to the ciliary tip. <b>(C)</b> <i>Mks1</i>^{<i>krc/krc</i>} cells that also lack BBS4 exhibit additional defects in the trafficking of ciliary membrane proteins. ARL13B is absent, however SMO is restored in the cilia by the loss of BBS4. GLI2 is not restricted to the ciliary tip, as with <i>Mks1</i>^{<i>krc/krc</i>} single mutant cells, however a lower percentage of cells have ciliary GLI2. <b>(D)</b> <i>Mks1</i>^{<i>krc/krc</i>} cells that also have impaired IFT function due to a homozygous hypomorphic mutation in <i>Ift172</i> have severe ciliogenesis defects. Less than 10% of <i>Mks1</i>^{<i>krc/krc</i>}<i>;Ift172</i>^{<i>avc1/avc1</i>} cells are ciliated. Those cilia that are present are shorter than normal. IFT88 is absent from the transition zone or the cilium, while IFT81 and KIF7 are no longer restricted to the tip, as with <i>Mks1</i>^{<i>krc/krc</i>} single mutant cells.</p

<i>Mks1</i>^{<i>krc/krc</i>}<i>;Ift172</i>^{<i>avc1/avc1</i>} double mutants exhibit phenotypes consistent with severely reduced Hh signaling.

<p><b>(A-D)</b> Double heterozygous (A), <i>Ift172</i>^{<i>avc1/avc1</i>} (B), <i>Mks1</i>^{<i>krc/krc</i>} (C), or <i>Mks1</i>^{<i>krc/krc</i>}<i>;Ift172</i>^{<i>avc1/avc1</i>} double mutant (D) embryos at E10.5. In contrast to <i>Ift172</i>^{<i>avc1/avc1</i>} or <i>Mks1</i>^{<i>krc/krc</i>} single mutant embryos, double mutants exhibit a pointed midbrain flexure, enlarged branchial arches, and a twisted body axis reminiscent of other mutants in which cilia are absent or ciliogenesis is severely perturbed. <b>(E-L)</b> Transverse sections through the neural tube of embryos of the indicated genotype at E10.5. Sections were taken at the level of the forelimbs and immunostained with antibodies against FOXA2 (red) and NKX2.2 (green) in E-H, or OLIG2 (red) and ISL1 (green) in I-L. Scale bar, 100μm.</p

Loss of <i>Bbs4</i> enhances Hh signaling phenotypes in <i>Mks1</i>^<i>krc</i> mutant embryos.

<p><b>(A-D)</b> Double heterozygous (A), <i>Bbs4</i>^<i>-/-</i> (B), <i>Mks1</i>^{<i>krc/krc</i>} (C), or <i>Mks1</i>^{<i>krc/krc</i>}<i>;Bbs4</i>^<i>-/-</i> double mutant (D) embryos at E13.5. Note that double mutant embryos exhibit microopthalmia, polydactyly, and general edema. <b>(E-H)</b> Higher magnification images of the hindlimbs of the embryos indicated above. Each digit is denoted by *. Approximately 25% of <i>Mks1</i>^{<i>krc/krc</i>} exhibit polydactyly on at least one limb as in (G). Double mutant embryos exhibit fully penetrant polydactyly on all four limbs (H). <b>(I-L)</b> Transverse sections through the neural tube of embryos of the indicated genotype at E10.5. Sections were taken at the level of the forelimbs and immunostained with antibodies against OLIG2 (red) and NKX2.2 (green). Scale bar, 100μm.</p

Basal body localization of IFT88 is impaired in <i>Mks1</i>^{<i>krc/krc</i>}<i>;Ift172</i>^{<i>avc1/avc1</i>} double mutant cells.

<p><b>(A-L)</b> MEFs derived from embryos of the indicated genotype were immunostained for IFT88 (A-D, green) IFT81 (E-H, green), or KIF7 (I-L, green) together with γ-Tubulin (red) and acetylated α-Tubulin (magenta) following 48 hours of serum starvation. Two representative images for each condition are shown. Scale bar, 2μm. <b>(M-O)</b> Quantitation of the percentage of cells with IFT88 or IFT81 localized to either the basal body or ciliary axoneme. Compared with <i>Mks1</i>^{<i>krc/krc</i>} or <i>Ift172</i>^{<i>avc1/avc1</i>} single mutant cells, significantly fewer double mutant cilia were positive for IFT88 localized to the basal body (M, **** p< 0.0001). Localization of IFT81 was not significantly disrupted in double mutants compared with either single mutant, however all of the mutants exhibited reduced localization compared with double heterozygous cells (N, p = 0.0015). The percentage of ciliated cells with KIF7 within the axoneme was also significantly reduced in <i>Mks1</i>^{<i>krc/krc</i>}<i>;Ift172</i>^{<i>avc1/avc1</i>} double mutants compared with single mutant or double heterozygous cells (O, p< 0.001). Quantification for each genotype was based on imaging of 4 separate fields on 3 coverslips per condition. <b>(P-Q)</b> Quantitation of the percentage of cells positive for IFT81 or KIF7 for each genotype in which localization was not restricted to the ciliary tip. Compared with double heterozygous cells, significantly more ciliated <i>Mks1</i>^{<i>krc/krc</i>} cells exhibited localization of IFT81 (p = 0.0005) or KIF7 (p< 0.0001) that was not restricted to the ciliary tip. Quantification for each genotype was based on imaging of 4 separate fields on 3 coverslips per condition.</p