Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome.

Primary mouse tracheal epithelial cell culture

Primary mouse tracheal epithelial cultures were derived as described 61 . In summary, tracheas from C57BL/6J mice aged 8 to 12 weeks were dissected. The tracheal cells were dissociated with protease. Dissociated cells were placed in a culture plate (Primaria) for several hours to remove fibroblasts. The non-adherent tracheal epithelial cells were seeded at a density of 0.33 × 10 5 cells per 24-well insert (Day 0). The cells were cultured for three days with mTEC/Plus medium in the basal and apical compartments of the transwell. After three days of incubation (Day 3), the medium was replaced with fresh mTEC/Plus medium. Air-liquid interface (ALI) differentiation was begun at day 5 by removing the medium in the apical transwell chamber and replacing the medium in the basal chamber with mTEC/NS. The medium was replaced every other day with mTEC/NS, and the filters were harvested at ALI day 14 for immunofluorescence experiments. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Francisco.

IMCD3 and fibroblast cell culture

IMCD3 cells were cultured in DMEM:F12 supplemented with 10% FBS. MEFs and human primary dermal fibroblasts were cultured in DMEM, Glutamax (Thermo Fisher), and 10% FBS. For imaging, IMCD3 cells, MEFs, and human primary dermal fibroblasts were seeded at 2.5 × 10 4 cells/cm 2 and grown to confluency. Cells were starved of serum in Opti-MEM reduced serum medium for 24 hours to induce ciliogenesis. Cell lines were not authenticated. No commonly misidentified cell lines were used. All growing cell lines were routinely tested for mycoplasma. Fixation and staining for immunofluorescence mTEC filters were harvested at ALI day 14 and the apical and basal compartments were washed with phosphate buffered saline (PBS) three times. The filters were fixed by adding 4% paraformaldehyde (PFA) in PBS to the apical compartment and incubating at room temperature for ten minutes. The PFA was removed by washing the filter with PBS six times with at least one minute of incubation between washes. The filters were then treated with a solution of 1% sodium dodecyl sulfate (SDS) for five minutes in PBS to reveal antigens. The SDS solution was removed by washing the filter with PBS. For SIM, filters were incubated in blocking buffer (PBS, 2.5 % BSA, 0.1 % Triton X-100, 0.02 % sodium azide) for ten minutes at room temperature. Primary antibodies were added to the filters in blocking buffer according as described in Supplementary Table S1 . The filters were incubated overnight (~16 hours) at 4 degrees on a rocker. The filters were washed with PBT (PBS, 0.2% Triton X-100) six times with at least one minute of incubation between washes. Secondary antibodies were added at a dilution of 1:1000 and incubated for two hours in blocking buffer on an orbital shaker. The secondary antibody was removed by six washes with PBT buffer. The filters were mounted on slides in Prolong Gold containing 4′,6-diamidino-2-phenylindole (DAPI). High performance coverslips were used, and the mounting medium was allowed to solidify for 16–24 hours before sealing the slides with nail polish and imaging. For STORM, filters were incubated in blocking buffer for ten minutes at room temperature. Primary antibodies were added to the filters incubating in blocking buffer according to the table of antibodies and concentrations (Table 1). The filters were incubated with the primary antibody overnight (~16 hours) at 4 °C on a rocker. The filter was washed with PBT. Secondary antibodies were incubated for two hours at room temperature or overnight (~16 hours) at 4 °C on the filters in blocking buffer. The secondary antibody was removed by six washes with PBT. Some samples for single color STORM were stained using labeled primary antibodies. In these cases, filters were incubated in blocking buffer for ten minutes at room temperature. Primary antibodies were added to the filters in blocking buffer at concentrations listed in Table 1. The filters were incubated with the primary antibody overnight (~16 hours) at 4 °C on a rocker. The filter was washed with PBT. For two-color STORM using primaries derived from the same species, filters were incubated in IF blocking buffer for ten minutes at room temperature. Primary antibody recognizing the experimental protein was added to the filters incubating in blocking buffer. The filters were incubated with the primary antibody overnight (~16 hours) at 4 °C on a laboratory rocker. The filter was washed with PBT. Secondary antibody labelled with Alexa 647 was added according to the table and incubated for two hours at room temperature on the filters in IF blocking buffer. The secondary antibody was removed by six washes with Wash buffer. Primary antibody labelled with Cy5.5 recognizing the reference protein was added to the filters incubating in blocking buffer according to the table of antibodies and concentrations. The filters were incubated with the primary antibody overnight (~16 hours) at 4 °C on a laboratory rocker. The filter was washed with PBT. All antibody information is available in Supplementary Table 1 Mounting filters in STORM buffer After immunofluorescence staining, mTEC filters were mounted on slides and high-performance coverslips in PBS with the addition of 100 mM mercaptoethylamine at pH 8.5, 5% glucose (wt/vol) and oxygen scavenging enzymes [0.5 mg/ml glucose oxidase (Sigma-Aldrich), and 40 mg/ml catalase (Roche Applied Science)]. The buffer remains suitable for imaging for two hours.

SIM image acquisition

SIM data were collected on a N-SIM microscope in 3D-SIM mode using an Apo TIRF 100x oil NA 1.49 objective (Nikon) following standard operation procedures. Image reconstruction was using the NIS Elements software (Nikon).

STORM instrumentation

STORM experiments were performed on a custom-built microscope based on a Nikon Ti-U inverted microscope. Three activation/imaging photodiode lasers (Coherent CUBE 405, OBIS 561 and CUBE 642) were combined using dichroic mirrors, aligned, expanded and focused to the back focal plane of the objective (Nikon Plan Apo 100x oil NA 1.45). The lasers were controlled directly by the computer. A quadband dichroic mirror (zt405/488/561/640rpc, Chroma) and a band-pass filter (ET705/70m, Chroma) separated the fluorescence emission from the excitation light. The focus drift of the microscope sample stage during image acquisition was stabilized by a closed-loop focus stabilization system that sends an infra-red laser beam through the edge of the microscope objective and detects the back reflection from the sample coverglass with a CCD camera 32 . After the emission light exits the microscope side port, we added a low-end piezoelectric deformable mirror (DM) (DMP40-P01, Thorlabs) at the conjugate plane of the objective pupil plane. This DM contains 40 actuators arranged in a circular keystone pattern. By first flattening the mirror and then manually adjusting key Zernike polynomials of the DM surface, we found that this DM has sufficient performance to correct aberrations induced by the optical system. We did not observe obvious depth-induced spherical aberrations caused by glass-water refractive index mismatch because transition zones we imaged were < 1 μm away from the coverglass surface. Moreover, after correcting the intrinsic aberrations, we increased the primary astigmatism Zernike coefficient of the DM surface to introduce astigmatism for 3D STORM. To experimentally generate a calibration curve of point spread function as a function of z, we immobilized Alexa 647–labeled antibodies on a coverglass and imaged individual molecules as the sample was scanned in z 32 . Compared to using a cylindrical lens 32 using the DM to introduce astigmatism did not cause magnification differences in the two lateral directions. It also allowed tractable optimization of the amount of astigmatism to achieve the best axial resolution. The Z localization precision (Z resolution) is estimated to be ~70nm full width at half maximum (FWHM) based on the reconstructed STORM images. The Z focal depth is ~800 nm. We measured the longitudinal distance between the transition zone and distal appendages by analyzing only transition zone structures with their axes close to the XY plane. Therefore, the precision was limited mostly by the XY resolution instead of Z resolution. The fluorescence was subsequently split by a 705nm beam splitter (T705lpxr, Chroma) to a long wavelength channel (ch1, >705 nm) and a short wavelength channel (ch2, 0.05. *, P ≤ 0.05. **, P ≤ 0.01. ***, P ≤ 0.001. ****, P ≤ 0.0001. Two sample comparisons were performed using an unpaired t test with a two tailed P value. Multiple comparisons were performed using an ordinary one-way ANOVA, along with Tukey’s multiple comparisons test to analyze pairwise differences. Fluorescence intensity measurements ( Figures 4 and 5 ) from multiple experiments were combined for statistical analysis. Negative normalized fluorescence intensity values were set to zero in graphs. Experiments were independently performed either twice ( Figures 4A, 4B , and 5C ) or thrice ( Figure 1G , 2E–H , 4C , 5A, and 5B ).

Data availability statement

Source data for experiments in figures 4 and 5 are provided in Supplementary Table 2 . All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Supplementary Material Supplemental Info Supplemental Table 1 Supplemental Table 2 Supplemental Table 3