Fig 1.
Mouse rod inner segments are nonpermissive to conventional Rho labeling strategies.
(A) Diagram of mouse rod photoreceptor layers adjacent to z-projection confocal fluorescent images of WT mouse retinal cryosections immunolabeled for Rho (magenta), STX3 (cyan)—which labels the IS and OPL—and DAPI nuclear staining (blue) in the ONL. Rho prominently labels rod OS. The STX3 channel is removed from part of the images for clarity. (B) Z-projection SIM images of thin (1 μm) plastic sections of WT retinas stained for immunofluorescence as whole retinas. The white arrow indicates the rod that is magnified in the Rho-N-GTX example, and the white asterisk indicates Rho-N-GTX labeling of the new OS discs in the magnified rod. Centrin (yellow) immunolabeling was used to localize the rod CC and BB. (C) Z-projection confocal image of an adult Rho-GFP/+ cryosection adjacent to z-projection SIM images of 2 μm Rho-GFP/+ cryosections. (D) Z-projection confocal image of a Rho-GFP-1D4/+ cryosection and z-projection SIM images of 2 μm Rho-GFP/+ cryosections. In both (C) and (D), a magnified region of a SIM image is shown with raised contrast and brightness (intensity) levels to depict faint IS GFP fluorescence in both heterozygous knock-in mouse lines (yellow arrows). Inverted images are shown to highlight this pattern. (E) Z-projection SIM images of Rho-GFP/+ thin plastic retina sections that were immunolabeled for NbGFP-A647 (magenta) and centrin (yellow) as whole retinas. Raised intensity images are shown to depict less intense NbGFP-A647 labeling in some proximal OSs and surrounding the CC. (F) SIM images of thin sections from Rho-GFP/+ and WT retinas that were stained as in (E) but with twice the amount of NbGFP-A647 (x2). Lack of staining in WT retinas demonstrates NbGFP-A647 specificity. BB, basal body; CC, connecting cilium; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; Rho, rhodopsin; SIM, structured illumination microscopy; STX3, syntaxin 3; WT, wild-type.
Fig 2.
OS peeling generates IS-enriched mouse retina samples.
(A) Diagram of the mouse retina OS peeling method. Mouse retina slices are iteratively peeled from filter paper 8 times to remove rod OSs (yellow) from the IS layer (magenta). (B) Western blot analysis comparing control full retina samples vs. retinas after OS peeling/IS-enriched retinas; both from Rho-GFP/+ mice. Approximately 2% of total volume from lysates from 1 retina (either control or peeled) were used from each condition for SDS-PAGE. Molecular weight marker sizes are indicated in kDa. Immunolabeled bands from antibodies targeting OS proteins, including NbGFP-A647, Rho-C-1D4, and ROM1 (black arrow points to the ROM1 monomer band), were reduced in peeled lysates, whereas IS proteins STX3 and Rab11a were roughly equal demonstrating IS enrichment. Tubulin immunoblotting was performed as a loading control. (C) Control full retina slices and (D) IS-enriched retinal slices from Rho-GFP/+ mice were fixed and stained for resin embedding, ultrathin sectioning, and TEM ultrastructure analysis. TEM images were pseudocolored to point out key rod structures as follows: OS = yellow, IS = magenta, CCs/BBs = blue. Single rod examples are magnified from both conditions to emphasize intact CC ultrastructure and the preservation of the IS plasma membrane (magenta arrows). The locations of the DAPs and rootlet are annotated in (D). (E) Z-projection SIM images of Rho-GFP/+ IS-enriched retinas immunolabeled with NbGFP-A647 (magenta) and centrin antibody (green). Fluorescence in the single rod example corresponding to the OS, IS, and CC are annotated. (F) Control SIM image of a WT IS-enriched retina immunolabeled and imaged as in (E) to demonstrate NbGFP-A647 labeling specificity. Scale bar values match adjacent panels when not labeled. BB, basal body; CC, connecting cilium; DAP, distal appendage; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; OS, outer segment; Rho, rhodopsin; STX3, syntaxin 3; SIM, structured illumination microscopy; TEM, transmission electron microscopy; WT, wild-type.
Fig 3.
A fraction of Rho-GFP molecules are localized at the rod IS plasma membrane.
(A) Confocal z-projection and (B) SIM z-projection images of an IS-enriched, Rho-GFP/+ retina section immunolabeled with NbGFP-A647 (magenta) and centrin antibody (green). Single rod examples are magnified in (B) to highlight Rho-GFP localization along the boundaries of the IS and CC. These same single rod images are also shown after 3D-deconvolution processing (SIM + 3D decon). Yellow arrows indicate a continuous line of Rho-GFP fluorescence between the IS and CC. Asterisks indicate strong Rho-GFP puncta staining in the proximal IS/myoid region. (C) STORM reconstruction of an IS-enriched Rho-GFP/+ retina section. In the magnified single rod examples, yellow arrows indicate Rho-GFP molecules located at the IS boundary. Magnified regions are indicated throughout with either a dashed box or a dashed white arrow. Some magnified images are rotated so that the OS end of the rod is at the top of the image. Scale bar values match adjacent panels when not labeled. CC, connecting cilium; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; OS, outer segment; Rho, rhodopsin; SIM, structured illumination microscopy; STORM, stochastic optical reconstruction microscopy.
Fig 4.
Rho colocalized with STX3 at the rod IS plasma membrane.
(A) Example SIM z-projection image (processed with 3D deconvolution) of a thin plastic section from a WT mouse retina immunolabeled with antibodies for anti-centrin (yellow), to label the CC and BB, and anti-Na,K ATPase B2 subunit (ATP1B2, cyan) to label the IS plasma membrane. (B) Example SIM z-projection image with 3D deconvolution of a thin plastic section from a WT mouse retina immunolabeled with anti-centrin and anti-STX3 (cyan) to label the IS plasma membrane. (C) SIM image of an IS-enriched Rho-GFP/+ retina immunolabeled for NbGFP-A647 (magenta), STX3 (cyan), and centrin (yellow). In the magnified single rod example, an arrowhead indicates a cytoplasmic IS region with Rho-GFP fluorescence and no STX3 fluorescence. To demonstrate Rho-GFP colocalization with STX3 at the IS plasma membrane, a row average intensity plot is shown for a portion of the IS from the magnified single rod example marked with a white dashed line. SIM images of (D) SNAP25 (magenta) and (E) PDC (magenta) immunolabeling in WT full retina sections that are each colabeled with STX3 (cyan) and centrin (yellow) antibodies. For both magnified single rod examples, row intensity plots are provided for the IS regions marked by white dashed lines. Fluorescence intensities are normalized on all plots for clarity. Throughout the figure, magnified regions are indicated with a dashed box. Some magnified images are rotated so that the OS end of the rod is at the top of the image. Scale bar values match adjacent panels when not labeled. Numerical values corresponding to graphical data are provided in Table A in S1 Data. BB, basal body; CC, connecting cilium; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; OS, outer segment; PDC, phosducin; Rho, rhodopsin; SIM, structured illumination microscopy; STX3, syntaxin 3; WT, wild-type.
Fig 5.
Endogenous IS mouse Rho is located near the cis-Golgi and at the plasma membrane.
(A) SIM z-projection images of a single rod example from an IS-enriched, WT retina section immunolabeled with Rho-C-1D4 (magenta) and centrin antibody (green). Rho-C-1D4 is localized along the boundaries of the IS and CC (yellow arrows). The OS is indicated. (B) SIM z-projection image of a WT full retina section immunolabeled with GM130 (magenta), STX3 (cyan), and centrin-2 (yellow) antibodies. Asterisks indicate strong GM130 puncta staining in the proximal IS/myoid region corresponding to the cis-Golgi in each rod IS. (C) SIM z-projection image of a Rho-GFP IS-enriched retina section immunolabeled with NbGFP-A647 (cyan), along with GM130 (magenta), STX3 (yellow), and centrin (yellow) antibodies. The white arrow indicates a magnified portion of the IS layer, in which an asterisk indicates a GM130 punctum located directly near a Rho-GFP punctum in the myoid of a rod. (D, E) SIM images of WT IS-enriched retinas immunolabeled with either (D) Rho-C-1D4 antibody (magenta) or (E) Rho-N-4D2 antibody (magenta); both are colabeled with STX3 (cyan) and centrin-2 (yellow) antibodies. Partial endogenous Rho colocalization with STX3 at the IS plasma membrane is indicated with white arrowheads, and row average intensity plots are from the magnified portion of the IS corresponding to the region in single rod example images marked with a dashed line. Fluorescence intensities are normalized on both plots for clarity. Throughout the figure, magnified regions are indicated with either a dashed box or a dashed white arrow. Some magnified images are rotated so that the OS end of the rod is at the top of the image. Scale bar values match adjacent panels when not labeled. Numerical values corresponding to all graphical data are provided in Table B in S1 Data. CC, connecting cilium; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; OS, outer segment; Rho, rhodopsin; SIM, structured illumination microscopy; STX3, syntaxin 3; WT, wild-type.
Fig 6.
STORM spatial analysis of Rho localization in rod ISs.
(A) STORM reconstruction of an IS-enriched Rho-GFP/+ retina immunolabeled with NbGFP-A647 (magenta) and STX3 (cyan) and centrin-2 (yellow) antibodies. OSs are indicated. The centrin-2+ widefield images are superimposed on STORM reconstruction images to mark the positions of rod CCs and BBs. In single rod examples, the IS region is indicated, and the IS hull—the manually defined STX3+ IS boundary—is outlined in cyan in a duplicate image with the STX3 STORM reconstruction removed. From the rod example in (A), the Rho-GFP and STX3 STORM molecule coordinates within the IS hull are plotted (Rho-GFP molecules = 4,239, STX3 molecules = 5,215). In the adjacent plot, a random distribution of coordinates within the IS hull matching the number of Rho-GFP molecules (4,239) are plotted in orange with the STX3 molecules. Nearest distance-to-hull measurements for Rho-GFP, STX3, and random molecules are plotted in a frequency graph and a CDF graph. Colors in the graphs match the molecule plots. (B-E) STORM reconstructions of IS-enriched WT retinas immunolabeled with either Rho-C-1D4 antibody (B) or Rho-N-4D2 (C), and full WT retinas immunolabeled with (D) SNAP25 or (E) PDC; all are co-immunolabeled with STX3 and centrin-2 antibodies. Single rod examples and IS hulls are indicated. Molecules coordinates are plotted along with corresponding randomly plotted molecules (orange) as in (A), and distance-to-hull measurements are plotted as frequency and CDF graphs. Molecule counts: (B) Rho-C-1D4 = 1,430, STX3 = 24,596, Random = 1,430; (C) Rho-N-4D2 = 3,199, STX3 = 24,044, Random = 3,199; (D) SNAP25 = 7,871, STX3 = 28,817, Random = 7,871; (E) PDC = 14,556, STX3 = 34,544, Random = 14,556. Dashed boxes indicate the single rod examples in magnified images. Scale bar values match adjacent panels when not labeled. (F) For each rod analyzed with STORM, the distance-to-hull frequency within 0.2 μm was divided by the distance-to-hull frequency within 0.2 μm of the corresponding random coordinates to acquire “normalized frequency <0.2 μm” values, which are compared as violin plots. STX3 n value (for number of rods analyzed) = 162, SNAP25 n = 31, PDC n = 31, Rho-GFP n = 40, Rho-GFP-1D4 n = 13, Rho-C-1D4 n = 21, and Rho-N-4D2 n = 25 conditions were tested for statistical significance using the Mann–Whitney U test. PDC vs. Rho-GFP ***P value < 0.001; PDC vs. Rho-GFP-1D4 ***P value < 0.001; PDC vs. 1D4 ***P < 0.001. (G) The same STORM data were used to perform Mosaic interaction analyses to test the colocalization between STX3 molecules and the other immunolabeled targets from the same rod ISs. Interaction strength values are compared as violin plots on a log scale. PDC vs. Rho-GFP, Rho-C-1D4, and Rho-N-4D2 (same n values as (F)) were tested for statistical significance using the Mann–Whitney U test. PDC vs. Rho-GFP *P = 0.026; PDC vs. Rho-GFP-1D4 ***P < 0.001. In violin plot graphs, circles = median values and dashed lines = mean values. Numerical values corresponding to all graphical data are provided in Table C in S1 Data. BB, basal body; CC, connecting cilium; CDF, cumulative distribution function; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; OS, outer segment; PDC, phosducin; Rho, rhodopsin; STORM, stochastic optical reconstruction microscopy; STX3, syntaxin 3; WT, wild-type.
Fig 7.
Super-resolution mapping of Rho IS surface labeling in mouse rods.
Z-projection SIM images of thin plastic sections of full (unpeeled) retinas from (A) WT, (B) Rho-GFP/+, and (C) RhoGFP-1D4 mice that were all labeled with WGA (cyan) and NbGFP-A647 (magenta) using normal, permeabilizing immunostaining conditions. Note that NbGFP-A647 only labels Rho-GFP/+ and Rho-GFP-1D4/+ rod OS tips as in Fig 1E and 1F. (D–F) Z-projection SIM images from full retinas labeled with WGA (cyan) and Rho-N-4D2 antibody (magenta) from WT (D), Rho-GFP/+ (E), or Rho-GFP-1D4/+ (F) mice using a surface labeling (no fixative, no detergent) protocol (see Materials and methods). Inverted WGA and Rho-N-4D2 single channel images are shown adjacent to the composite image. Red brackets indicate positive Rho-N-4D2 fluorescent signal in the IS. (G) Z-projection SIM image from a full WT retina labeled with WGA (cyan) and Rho-C-1D4 antibody (magenta). Lack of Rho-C-1D4 surface immunolabeling demonstrates Rho-N-4D2 extracellular labeling specificity and effectiveness. The OS, IS, and ONL layers, based on the WGA labeling pattern, are indicated for each SIM image. Scale bar values match adjacent panels when not labeled. (H–J) STORM reconstruction examples of rods from WT (H), Rho-GFP/+ (I), or Rho-GFP-1D4/+ (J) full retinas that were surface labeled with WGA (cyan) and Rho-N-4D2 antibody (magenta). The OS and IS regions are indicated. Adjacent to each STORM image is the Rho-N-4D2 molecular coordinates for the IS region. The number of Rho-N-4D2 molecules in (H) = 2,257, in (I) = 2,022, and in (J) = 2,764. Surface localization of Rho-N-4D2 molecules is indicated with orange arrows. Nearest distance-to-hull measurements for Rho-N-4D2 (magenta) and the corresponding set of random molecules (green) are plotted in a frequency graph above the molecule plots. For each rod analyzed with STORM, the distance to hull frequency within 0.2 μm was divided by the distance to hull frequency within 0.2 μm of the corresponding random coordinates to acquire “normalized frequency <0.2 μm” values, which are compared as violin plots grouped by genotype (K). WT n value (for number of rods analyze) = 23, “Rho-GFP” for Rho-GFP/+ rods n = 19, “Rho-GFP-1D4” for Rho-GFP-1D4/+ rods n = 22. No significant difference in these data were based on a one-way ANOVA test (P value = 0.144). In the violin plot graph, circles = median values and dashed lines = mean values. Numerical values corresponding to all graphical data are provided in Table D in S1 Data. IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; ONL, outer nuclear layer; OS, outer segment; Rho, rhodopsin; SIM, structured illumination microscopy; STORM, stochastic optical reconstruction microscopy; WGA, wheat germ agglutinin; WT, wild-type.
Fig 8.
STX3 coimmunoprecipitates with Rho in vivo.
Co-IP results from (A) Rho-GFP/+ IS-enriched retinal lysates incubated with GFP-Trap agarose beads, (B) Rho-GFP-1D4/+ IS-enriched retinal lysates incubated with GFP-Trap agarose beads, (C) Rho-GFP/+ IS-enriched retinal lysates incubated with anti-1D4 IgG agarose beads, and (D) WT IS-enriched retinal lysates incubated with anti-1D4 IgG agarose beads. In all western blots, input lanes correspond to 2% (% vol/vol) of the starting lysate volume, unbound lanes correspond to 2% (% vol/vol) of lysate volume post bead incubation, and bound lanes correspond to half the total eluate from each co-IP. Antibodies/nanobodies used for immunodetection are listed to the right of each corresponding western blot scan. Molecular weight marker sizes are indicated in kDa. Black arrows mark the correct size bands when other bands are present on the scan. Red asterisks indicate mouse IgG bands. In (B), the red arrow indicates the endogenous Rho band and the green arrow indicates the Rho-GFP-1D4 band. co-IP, coimmunoprecipitation; IgG, immunoglobulin G; Rho, rhodopsin; STX3, syntaxin 3; WT, wild-type.
Fig 9.
Rho localizes around the DAPs in mouse rods.
(A) SIM z-projection images of a WT full retina section immunolabeling with a centrin antibody that marks the location of rod CC and the DC of the BB in white, along with a CEP164 antibody that labels the DAPs in green. The SIM retina image is also shown after 3D-deconvolution processing (SIM + 3D decon.), and a single rod cilium example is magnified. (B) SIM image from peeled Rho-GFP/+ mouse labeled with NbGFP-A647 (magenta), anti-CEP164 antibody (green), and anti-centrin antibody (white). A single rod is magnified, and the DAPs region is further magnified. The remaining OSs and the IS region are indicated. (C) STORM reconstruction single rod examples and magnified DAPs regions from Rho-GFP/+ IS-enriched retina immunolabeled with NbGFP-A647 (magenta) and a CEP164 antibody (green). Centrin-2 antibody labeling marks the position of the CC (white), and residual NbGFP-A647 signal labels the OS; both are captured as widefield fluorescence images. White arrows indicate the regions that are magnified in the adjacent image. Scale bar values match adjacent panels when not labeled. BB, basal body; CC, connecting cilium; DAP, distal appendage; DC, daughter centriole; NbGFP-A647, GFP nanobody Alexa 647 conjugate; IS, inner segment; OS, outer segment; Rho, rhodopsin; SIM, structured illumination microscopy; STORM, stochastic optical reconstruction microscopy; WT, wild-type.
Fig 10.
Rho colocalizes with Rab11a in mouse rod ISs.
(A) Z-projection of a WT retinal cryosection co-immunolabeled with centrin-2 and Rab11a antibodies and counterstained with DAPI. (B) SIM z-projection image of a WT retina co-immunolabeled with centrin-2, STX3, and Rab11a antibodies. The SIM retina image is also shown after 3D-deconvolution processing (SIM + 3D decon.), and Rab11a+ puncta are localized in the IS layer. A single rod example is magnified, and a threshold image of the Rab11a channel is shown pseudocolored to depict puncta that are localized at the STX3+ IS membrane hull as magenta, and puncta that are localized internally or at the CC as white. (C) STORM reconstruction of a single rod from a WT retina immunolabeled as in (B). A magnified region is shown, and in the adjacent image, Rab11a+ clusters identified with Voronoi tessellation (see Materials and methods) are in white. (D) Z-projection of a WT retinal cryosection co-immunolabeled with centrin-2 and GMII antibodies and counterstained with DAPI. (E) SIM images, including 3D decon. processed images, of a WT retina co-immunolabeled with centrin-2, STX3, and GMII antibodies. As in (B), a single rod example is shown, and in the adjacent image, membrane-localized GMII+ puncta are pseudocolored magenta, and internal (and ciliary) puncta are white. (F) Frequency plots for FWHM measurements of individual Rab11a and GMII IS-localized puncta from the SIM data represented in (B) and (E). For Rab11a FWHM values, n (number of puncta) = 67. For GMII FWHM values, n (number of puncta) = 58. (G) Confocal z-projection image of a WT retina cryosection immunolabeled with centrin and DYNC1H1 antibodies, as well as DAPI counterstaining. DYNC1H1+ fluorescence fills the IS layer. In the adjacent panel, a STORM reconstruction of a WT retina immunolabeled with centrin-2, STX3, and DYNC1H1 antibodies is depicted. A single rod example is shown. (H) STORM reconstruction of a WT retina immunolabeled with centrin-2, STX3, and rootletin antibodies. A single rod example is shown, and the ciliary rootlet is indicated. (I-M) STORM images of a (I) Rho-GFP/+ IS-enriched retina or (J) Rho-GFP-1D4/+ IS-enriched retina, each co-immunolabeled with NbGFP-A647, and centrin, STX3, and Rab11a antibodies. STORM reconstruction channels—NbGFP-A647 (magenta) and Rab11a (cyan)—are superimposed with the matching widefield fluorescence image of centrin/STX3 immunolabeling (combined, yellow). IS regions are indicated. A single rod example is shown, and the CC is indicated. In the adjacent image, Rab11a+ clusters identified with Voronoi tessellation are in white, and the STX3+ IS hull is outlined in yellow. A white arrow indicates a further magnified region of Rho-GFP molecules localized around Rab11a clusters; however, there was a relatively low degree of colocalization between Rho and Rab11a. Next, Rho-GFP was co-immunolabeled with (K) DYNC1H1 antibody or (L) Rootletin antibody. In both, the locations of the CC and the IS outline are indicated, and in (K) areas where Rho-GFP and DYNC1H1 molecules overlap are indicated with white arrowheads. In (L), the location of the ciliary rootlet is indicated. (M) STORM data from (I-L) conditions were used to perform Mosaic interaction analyses to test the colocalization between Rho-GFP molecules and the other immunolabeled target from the same rod IS. Interaction strength values are compared as violin plots (circles = median values and dashed lines = mean values). N values, corresponding to the number of rods from each condition, are Rab11a vs. Rho-GFP, n = 15; Rab11a vs. Rho-GFP-1D4, n = 11; DYNC1H1 vs. Rho-GFP, n = 10; Rootletin vs. Rho-GFP, n = 15. In all panels, white arrows indicate regions that are magnified. Scale bar values match adjacent panels when not labeled. Numerical values corresponding to all graphical data are provided in Table E in S1 Data. CC, connecting cilium; FWHM, full width half maximum; GMII, Golgi alpha-mannosidase II; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; Rho, rhodopsin; SIM, structured illumination microscopy; STORM, stochastic optical reconstruction microscopy; STX3, syntaxin 3; WT, wild-type.