Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy

Abstract

Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions.

FIGURE 1:

INVS, ANKS6, NEK8, and NPHP3 colocalize in the INVc. (A) Subcellular localization of the INVc in confluent, serum-starved WT RPE1 cells. Endogenous INVS (green) was detected by IF with a polyclonal anti-INVS primary and an AF488-conjugated secondary antibody measured by DLM. Cell nuclei (“Nuc.”) were labeled with DAPI (shown in gray). Cilia were detected by IF labeling of acetylated tubulin in the ciliary axoneme (AcTub, AF647 secondary, blue) and CEP170 at the basal body (AF568 secondary, red). The merge image of the inset (magenta border) contains only the INVS, AcTub, and CEP170 channels. The schematic of an RPE1 cell (top left, not drawn to scale) illustrates the positioning of the cilium relative to the nucleus in a typical RPE1 cell, as well as the positioning of the cilium relative to the coverslip and microscope objective (“Optics”). Most RPE1 cilia are positioned roughly parallel to the coverslip, as depicted. (B, C) DLM images of cilia with INVcs detected by fluorescence. For each INVc protein (pINVc), cilia from six individual cells have been oriented basal body down, cilium tip up, aligned side by side. (B, top two panels) DLM images of cilia in WT RPE1 cells stably expressing GFP-NEK8 or GFP-INVS. GFP was detected directly by fluorescence of the GFP protein. GFP signal is shown in green and shifted 600 nm to the right of the ciliary markers that were detected by IF: ARL13B (AF647 secondary, blue) and CEP170 (AF568 secondary, red). (B, bottom panel) Schematic illustrating the coloring and relative positioning of fluorescent markers shown in the images in B and C. (C) DLM of WT RPE1 cilia with inversin compartments detected by IF with primary antibodies to endogenous INVc proteins (pINVc): anti-INVS, anti-ANKS6, or anti-NPHP3, all detected with AF488 secondary antibodies and all shown here in green, shifted 600 nm to the right of the axoneme (AcTub, AF647 secondary, blue) and basal body (CEP170, AF568 secondary, red). (D) Pairwise colocalization of all four INVc proteins measured by SIM. GFP-NEK8 was detected by immunolabeling with anti-GFP primary and AF488-conjugated secondary antibodies (green). Endogenous ANKS6, NPHP3, and INVS were detected with specific primary antibodies and AF568-conjugated secondary antibodies (red). Samples were also stained with anti-acetylated tubulin (AcTub) primary detected with AF647-conjugated secondary (blue). Each of the three cilia is shown in a three-color merged image (top row of images) and with the red and blue channels shifted relative to the merge to show each channel individually (bottom row of images). (D, bottom panel) Schematic illustrating the coloring and relative positioning of fluorescent markers in the merged and shifted images. (E) Distribution of absolute INVc lengths measured in µm by DLM of endogenous INVS in WT cells (black, mean = 2.19, stdev = 0.61, n = 154) or GFP-INVS in WT cell lines stably expressing GFP fusion proteins (green, mean = 2.87, stdev = 0.70, n = 126) in one representative experiment. Only INVS-positive cilia are included. INVc lengths are significantly different between the two cell lines (p = 5.94 × 10^–16). (F) Distribution of relative INVc lengths. INVc length was normalized relative to cilium length: [length INVc]/[length AcTub+ARL13B]. Endogenous INVS: mean = 0.46, stdev = 0.15, n = 154. GFP-INVS: mean = 0.55, stdev = 0.14, n = 126. Only INVS-positive cilia are included. Lengths are significantly different between the two cell lines (p = 4.00 × 10^–7). (G) Distribution of INVS densities (see Supplemental Figure S2 for definition) within the INVc, measured as mean pixel intensity in arbitrary units (A.U.) for a manually masked INVS-positive region for the same 126 cilia as measured in E and F. Only INVS-positive cilia are included. Representative images of dim (low density, I), average (medium density, II), and bright (high density, III) compartments are shown.

FIGURE 2:

3D SM SR reconstructions of the INVc and other ciliary markers. All localizations in the reconstructions are represented by 3D Gaussians with diameter 25 nm and an opacity set to show the localization density. Cilia are generally oriented tip up. Asterisks (*) indicate reconstructions for which supplemental movies and reconstruction statistics are provided (Supplemental Movies 1–4 and Supplemental Figures S18 and S19). (A) Two-color 3D SM SR reconstruction of a cilium immunolabeled with anti-NPHP3 to detect the INVc (AF647 secondary, green) and anti-acetylated tubulin to detect the axoneme (AcTub, CF568 secondary, magenta). Multiple views of the same cilium are shown, including diffraction-limited (D.L.) images of each channel separately and a merge (top left), SM SR reconstructions of each channel and a merge (top middle), rotated and tilted views of the merge (bottom), and cross-sections of three different regions of the INVc (top right). Cross-sections: the position and length of each region are annotated to the left of its corresponding cross-section. Rotated and tilted views: the XYZ-axes accompanying each reconstruction are defined by the position of the sample relative to the microscope, where the Z-axis represents sample depth as illustrated in the schematic in panel B. The “tilted view” is a tilted perspective viewing along the ciliary axis from ciliary tip (foremost) to base. The other three views (bottom right) are rotations of the reference image (connected by white arrows) around the Y-axis only. The degree of rotation is noted at the ciliary tip and is illustrated by the axes pinned to the ciliary base. The INVc (NPHP3) in this cilium appears to contain two linear substructures, “fibrilloids,” which are traced on a duplication of the 180° Y-rotation view (bottom right corner). (B–F) Single-color 3D SM SR reconstructions of ciliary proteins immunolabeled with specific primary antibodies and AF647-conjugated secondary antibodies. The color of each localization in the reconstruction maps to its Z-position in the sample. The depth range of the color scale is unique to each reconstruction. The 0 position (purple) is the edge of the reconstruction volume closest to the coverslip, not a fixed position relative to the sample stage. (B) Top: schematic defining the XYZ-axes of the sample, the coverage of the depth color scale used in single-color 3D SM SR reconstructions, and the relative orientations of the microscope objective (“Optics”), the coverslip, and an RPE1 cell with a cilium and nucleus (“Nuc.”) Objects are not drawn to scale. Bottom: 3D SM SR reconstruction of a cilium stained with anti-ARL13B to detect a ciliary protein independent of the INVc. Multiple views of the same cilium are shown (as in A): a D.L. image, cross-sections, and Y-axis rotations. (C) 3D SM SR reconstruction of a cilium stained with anti-INVS to detect the inversin compartment. Multiple views of the same cilium are shown (as in A and B): a D.L. image, cross-sections, and Y-axis rotations. The compartment in this reconstruction has four fibrilloids. (D–F) 3D SM SR reconstructions of multiple INVcs detected with either anti-INVS (D), anti-ANKS6 (E), or anti-NPHP3 (F). A single view of each cilium is shown (four cilia per panel, total 12 unique cilia in D–F). The number of fibrilloids counted in each reconstruction is noted at the bottom. (G) Distribution of the number of fibrilloids per cilium observed in 3D SM SR reconstructions of INVcs detected by immunolabeling for INVS, ANKS6, or NPHP3. Reconstructions of these INVcs are shown in panels C–F and Supplemental Figure S21, F–H. (H) Distribution of the lengths of individual fibrilloids measured in INVcs detected by INVS, ANKS6, or NPHP3 (as in G). From reconstructions of 18 compartments, a total of 58 fibrilloids were measured. The afibrilloid compartment in Supplemental Figure S21G is excluded from this graph.

FIGURE 3:

INVS is the primary organizer of the INVc. (A) Schematic illustrating the coloring and relative positioning of fluorescent markers in the images in B. (B) DLM images of cilia in which endogenous INVS, ANKS6, and NPHP3 were detected by IF (green, left three columns) in WT RPE1 cells and INVS-KO, ANKS6-KO, NEK8-KO, and NPHP3-KO cell lines. Each genotype is represented by a single row. GFP fluorescence measured by DLM in WT RPE1 cells and INVS-KO, ANKS6-KO, NEK8-KO, and NPHP3-KO cell lines stably expressing GFP-NEK8 or GFP-INVS (green, right two columns). In all samples, ARL13B (red) and AcTub (blue) were detected by IF to mark cilia. Six representative cilia are shown in each subpanel. Additional images are provided in Supplemental Figures S23–S25, S30, and S31. (C) Quantification of ciliary density (arbitrary units, A.U.) and INVc frequency for fluorescent INVc markers (anti-INVS, anti-ANKS6, anti-NPHP3, GFP-NEK8, and GFP-INVS, all shown in green in B) measured in WT and gINVc-KO cell lines. Ciliary area was masked using the ARL13B and AcTub channels. Ciliary densities measured for each sample are plotted as a box plot (bottom axis) on top of a gray bar representing the percentage of cilia that contained an INVc (top axis). The number of cilia (n) measured for each condition (sum of two replicates) is reported to the right of each box/bar. See Materials and Methods for a description of the statistics represented by these box plots. Supplemental Figure S28 reports the results of pairwise t tests performed for each sample represented in this panel.

FIGURE 4:

ANKS6 and NEK8 link NPHP3 to INVS. (A) Schematic illustrating the coloring and relative positioning of fluorescent markers in the images in B. (B) DLM of ANKS6 detected by IF (AF568 secondary, red) in WT, INVS-KO, ANKS6-KO, NEK8-KO, and NPHP3-KO cell lines stably expressing GFP-INVS. GFP-INVS was detected by native GFP fluorescence (green). ARL13B (AF647 secondary, blue) marks the whole cilium. Additional images are provided in Supplemental Figure S32. (C) Mean ANKS6 density, measured as mean pixel intensity of the red channel in the ciliary region defined by ARL13B (arbitrary units, A.U.) in WT, INVS-KO, ANKS6-KO, NEK8-KO, and NPHP3-KO cell lines either not transduced (NT,-) or stably expressing GFP-INVS (+). Number of cilia (n) measured for each condition is noted to the right. See Supplemental Figure S34A for results of pairwise t tests performed for each sample represented in these plots. Supplemental Figure S32F provides INVc-specific density measurements. (D) Schematic illustrating the coloring and relative positioning of fluorescent markers in the images in E. (E) DLM of NPHP3 detected by IF (AF568 secondary, red) in WT, INVS-KO, ANKS6-KO, NEK8-KO, and NPHP3-KO cell lines stably expressing GFP-INVS. GFP-INVS detected by native GFP fluorescence (green). ARL13B (AF647 secondary, blue) marks the whole cilium. Additional images are provided in Supplemental Figure S33. (F) Mean ciliary intensity of NPHP3 in WT, INVS-KO, ANKS6-KO, NEK8-KO, and NPHP3-KO cell lines stably expressing GFP-INVS. Number of cilia (n) measured for each sample noted. See Supplemental Figure S34B for results of pairwise t tests performed for each sample represented in these plots. Supplemental Figure S32F provides INVc-specific density measurements. (G–J) Scatterplots and pairwise correlations of GFP-NEK8 or GFP-INVS (X-axis) with ANKS6 and NPHP3 (Y-axis) in WT RPE1 cells stably expressing GFP-NEK8 or GFP-INVS (see Supplemental Figure S36 for similar measurements for NEK8-KO cells rescued with GFP-NEK8 and INVS-KO cells rescued with GFP-INVS). GFP-NEK8 and GFP-INVS were detected directly by native GFP fluorescence and endogenous ANKS6 and NPHP3 were detected by indirect IF with AF568 secondary (as in B and E). The densities of the GFP-alleles and endogenous proteins within the INVc were measured as the mean pixel intensity in arbitrary units (A.U.) within a user-defined subcompartment mask drawn around the GFP and/or AF568-positive region of each cilium. (G) Correlation between ANKS6 and GFP-NEK8, n = 79, Pearson’s r value: r = 0.804 (p = 4.81 × 10^–19). (H) Correlation between NPHP3 and GFP-NEK8, n = 79, Pearson’s r value: r = 0.726 (p = 2.62 × 10^–15). (I) Correlation between ANKS6 and GFP-INVS, n = 86, Pearson’s r value: r = -0.0743 (p = 0.497). (J) Correlation between NPHP3 and GFP-INVS, n = 78, Pearson’s r value: r = -0.0139 (p = 0.904). (K) Diagram of hierarchical order of dependence for the localization of INVS, ANKS6, NEK8, and NPHP3 in the INVc, determined by IF of each INVc protein in gINVc-KO cells and gINVc-KO cells rescued with GFP-INVS.

FIGURE 5:

SIM of two ciliary localization patterns of NPHP3. (A) SIM IF of NPHP3 (red, AF568 secondary) in a RPE1 cell lines of relevant genotypes. One cilium per genotype is shown in each column (left to right): WT and INVS-KO cell lines; WT, INVS-KO, ANKS6-KO, and NEK8-KO cell lines stably expressing GFP-INVS (green, detected with anti-GFP primary antibody, AF488 secondary). Acetylated tubulin (AcTub, AF647 secondary, blue) marks the axoneme. Top row: three-color merges of NPHP3, GFP-INVS, and AcTub. Bottom row: each color is shifted to show each channel individually, left to right: AcTub, GFP-INVS, and NPHP3. Pound symbols (#) mark cilia in which NPHP3 is not localized in the INVc. Additional reconstructions are provided in Supplemental Figures S38–S44. (B) Schematic illustrating the coloring and relative positioning of fluorescent markers in the images in A. (C) Frequency of cilia in which NPHP3 is localized within the INVc for each cell line, assessed by SIM reconstructions as shown in A and Supplemental Figures S38–S44. Only NPHP3-positive cilia are included. For GFP-INVS cell lines, only cilia which were positive for both NPHP3 and GFP are included.

FIGURE 6:

A new model for the structure and composition of the INVc. (A) 3D SM SR reconstructions of INVS (left) suggest that the INVc is composed of fibrilloid substructures. Based on these data, a schematic of a cilium with an INVc, which is composed in this example of three fibrilloids (green) that are aligned roughly parallel along the axoneme (magenta), is shown. (B–G) Illustrations of the structure and composition of the INVc at various levels of detail and in different genetic contexts (C–F). Each INVc protein is shown in following colors: INVS (red), ANKS6 (blue), NEK8 (green), and NPHP3 (yellow). In the highest-resolution illustrations, each INVc protein is illustrated with some structural details pertaining to predicted protein domains within each molecule (defined in G). (B) A possible model for fibrilloid composition based on the order of dependence for the assembly of the four INVc proteins in the compartment. The 1:1:1:1 stoichiometry for all INVc proteins shown here is symbolic, and the relative positions of the circular molecules in the middle-resolution illustration are intentionally ambiguous as to whether each fibrilloid is a heteropolymer of all or some INVc proteins or a homopolymer, consisting for example of INVS only, on which ANKS6-NEK8 and NPHP3 then assemble (see Supplemental Figure S59 for candidate polymer models). In the highest-resolution illustrations (bottom), NPHP3 associates directly with the ciliary membrane via its N-terminal myristoylation, and ANKS6–NEK8 complexes link NPHP3 to INVS. (C) Illustrations of a cilium in a WT cell in which the INVc is a fibrilloid structure composed of INVS, ANKS6, NEK8, and NPHP3. Three fibrilloids are shown in the more detailed illustration (top left). Two fibrilloids are shown in the simplified illustration (top right), where ANKS6 and NEK8 are together represented in cyan. INVS is the core organizer and is illustrated here as being more directly associated with the axoneme. (D) A cilium in an NPHP3-deficient cell. The INVc is still present and structurally unaffected by loss of NPHP3. (E) A cilium in an ANKS6- or NEK8-deficient cell in which GFP-INVS expression has rescued compartment formation. The INVc contains INVS, which we expect still forms fibrilloids. Ciliary NPHP3 density is reduced, and NPHP3 is not sequestered in the INVc, but rather relocalized throughout the ciliary volume. The failure of INVS to sequester NPHP3 is due to the loss of the ANKS6–NEK8 complex that is predicted to link these two proteins physically, based on our measurements of NPHP3 localization in ANKS6-KO or NEK8-KO cells stably expressing GFP-INVS. (F) A cilium in an INVS knockout cell. No INVc is present. NPHP3 is reduced and found throughout the cilium, albeit at a low density, similar to that observed for ANKS6- or NEK8-deficient cells. (G) Schematic of the protein domains and motifs present in the four INVc proteins as cartooned in panels B–F. All four proteins are oriented left to right from N- to C-terminus. The INVS protein contains an ankyrin repeat domain (ARD) consisting of 16 ankyrin repeats, two D-boxes (known to bind APC; Morgan et al., 2002), a bipartite NLS (Otto et al., 2003), two IQ domains (known to bind calmodulin; Yasuhiko et al., 2001), and a C-terminal ninein-like domain (NLD) that is important for centriolar targeting of INVS (Shiba et al., 2009) ANKS6 contains an N-terminal ARD with 11 ankyrin repeats, followed by a serine-rich patch and a C-terminal SAM domain. The ANKS6 ARD likely mediates an interaction with NEK8 through the N-terminal NEK8 kinase domain (KD; Czarnecki et al., 2015). NEK8 contains a C-terminal RCC (regulator of chromatin condensation) domain, which is predicted to fold into a propeller structure and is important for mediating an interaction of NEK8 and INVS (Zalli et al., 2012; Czarnecki et al., 2015). The N-terminus of NPHP3 is myristoylated (Myr.), which could insert in the ciliary membrane (Wright et al., 2011; Nakata et al., 2012; as illustrated in panel B). NPHP3 also contains a coiled-coil domain (amino acids 83–207), which may be important for targeting NPHP3 to the basal body (Nakata et al., 2012). The C-terminus of the NPHP3 contains at least 11 tetratricopeptide repeats (TPR). The first 200 amino acids of NPHP3 localize to the cilium (Wright et al., 2011) but fail to be sequestered in the INVc (Supplemental Figure S37), suggesting that the interaction between NPHP3 and ANKS6 or NEK8 is either through the TPR repeats or through some other domain present in the C-terminus of the protein (amino acids 201–1330).