Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure

  1. Tylor R Lewis  Is a corresponding author
  2. Sebastien Phan
  3. Carson M Castillo
  4. Keun-Young Kim
  5. Kelsey Coppenrath
  6. William Thomas
  7. Ying Hao
  8. Nikolai P Skiba
  9. Marko E Horb
  10. Mark H Ellisman
  11. Vadim Y Arshavsky  Is a corresponding author
  1. Department of Ophthalmology, Duke University Medical Center, United States
  2. National Center for Microscopy and Imaging Research, School of Medicine, University of California, San Diego, United States
  3. Eugene Bell Center for Regenerative Biology and Tissue Engineering and National Xenopus Resource, United States
  4. Department of Pharmacology and Cancer Biology, Duke University Medical Center, United States
9 figures, 1 video, 3 tables and 1 additional file

Figures

Schematic illustration of incisure arrangements.

(A) Cartoon illustrating the structure of rod photoreceptors in mice and frogs. In each species, the outer segment contains hundreds of disc membranes in a stack. For simplicity, the connecting …

Incisures are observed only in fully enclosed discs of mouse rods.

(A–G) Representative TEM images of longitudinally sectioned WT mouse rods contrasted with a combination of tannic acid and uranyl acetate, which stains newly forming ‘open’ discs more intensely than …

Figure 3 with 1 supplement
Representative z-section from a STEM tomogram of a 750-nm-thick tangential section of a WT mouse retina.

The retina was contrasted with tannic acid/uranyl acetate. Full tomogram is shown in Figure 3—video 1. Because individual rods in the mouse retina are not perfectly aligned, adjacent cells are …

Figure 3—video 1
Tomogram associated with Figure 3.

Field of view: 12.0 µm x 12.0 µm.

Figure 4 with 4 supplements
Disc incisures are observed in mature but not newly forming discs.

(A) Representative z-sections at the depths of 0 (left),+168 (middle) and +244 nm (right) from a reconstructed electron tomogram of a 750 nm-thick WT mouse retinal section. Full tomogram is shown in …

Figure 4—video 1
Tomogram associated with Figure 4A.

Field of view: 1.91 µm x 1.91 µm.

Figure 4—video 2
Tomogram associated with Figure 4B.

Field of view: 1.91 µm x 1.91 µm.

Figure 4—video 3
Tomogram associated with Figure 4C.

Field of view: 1.91 µm x 1.91 µm.

Figure 4—video 4
Tomogram associated with Figure 4D.

Field of view: 1.91 µm x 1.91 µm.

Figure 5 with 1 supplement
Ultrastructure of mature disc incisures.

(A) Representative z-section from a reconstructed electron tomogram of a 750-nm-thick WT mouse retinal section. Full tomogram of the boxed area is shown in Figure 5—video 1. (B) Representative …

Figure 5—video 1
Tomogram associated with Figure 5.

Field of view: 0.63 µm x 0.84 µm.

Figure 6 with 1 supplement
Reduction in peripherin-2 level prevents incisure formation in mouse rods.

(A) Representative TEM images of tangentially sectioned WT outer segments and rds/+ outer segments preserving their cylindrical shape. Yellow asterisks indicate the ciliary axoneme; yellow …

Figure 6—figure supplement 1
Reduction in the level of peripherin-2 but not rhodopsin causes gross abnormalities in outer segment structure.

Representative TEM images of longitudinally sectioned WT, rds/+ and Rho+/- mouse retinas. Scale bar: +/- µm.

Increase in relative peripherin-2 level produces long incisures of varying shape.

(A–C) Representative TEM images of tangentially sectioned Rho+/- mouse retinas. Incisures can be relatively straight and extend nearly the entire disc diameter (A) or be bifurcated or twisted (B). …

Quantification of disc diameters in WT and Rho+/- mouse rods.

Each data point represents a single outer segment. For each genotype, three mice were analyzed (labeled as 1, 2, and 3), with 25 outer segments analyzed in each mouse. Error bars represent mean ± …

Figure 9 with 1 supplement
Loss of peripherin-2 in frogs (Xenopus tropicalis) prevents incisure formation.

(A–D) Representative TEM images of longitudinally sectioned WT and prph2-/- frog retinas. (E,F) Representative TEM images of tangentially sectioned WT and prph2-/- frog retinas. Magenta arrows point …

Figure 9—figure supplement 1
One incisure in frog rod discs is aligned with the ciliary axoneme.

Representative TEM image of a tangentially sectioned WT frog retina. Yellow asterisks indicate the ciliary axoneme; yellow arrowheads point to incisures. Scale bar: 1 µm.

Videos

Video 1
Reconstructed tomogram of a basal body nucleating the ciliary axoneme.

Shown is a 420 nm fragment of a 750-nm-thick retinal section. Tomogram pixel size is 0.7 nm. Field of view: 0.80 µm x 0.80 µm.

Tables

Table 1
Quantification of molar ratios between rhodopsin, peripherin-2 and ROM1 in mouse outer segments.
Protein molar ratio*WTrds/+Rho+/-
Rhodopsin: peripherin-218.2±0.630.9±6.08.5±1.5
Rhodopsin: ROM142.2±0.629.9±2.021.6±3.7
Peripherin-2: ROM12.3±0.11.0±0.22.6±0.6
Rhodopsin: (Peripherin-2 +ROM1)12.7±0.415.1±1.86.0±0.8
  1. *

    Values are shown as mean ± s.d. Three outer segment preparations from mice of each genotype were analyzed.

Table 1—source data 1

Quantification of molar ratios between rhodopsin, peripherin-2 and ROM1 in mouse outer segments – raw data.

https://cdn.elifesciences.org/articles/89160/elife-89160-table1-data1-v2.xlsx
Table 2
Summary of quantitative parameters determined for mouse discs.
Calculated parameterWTRho+/-
Disc surface area (both lamellae)3.53 µm21.80 µm2
Rhodopsin molecules per disc75,00038,200
Peripherin-2+ROM1 molecules per disc5,9106,360
Total rim length6.21 µmN/A*
Measured incisure length as % of disc diameter50%N/A*
Theoretical incisure length as % of disc diameter-155%
  1. *

    Incisure and total rim lengths in Rho +/- discs cannot be readily measured due to incisure complexity.

  2. The value is taken from the measurements shown in Figure 6.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
strain, strain background (Mus musculus)C57BL/6 JJackson LabsJax#:000664
genetic reagent (Mus musculus)RhoLem et al., 1999MGI:2680822
genetic reagent (Mus musculus)rdsvan Nie et al., 1978MGI:1856523
strain, strain background (Xenopus tropicalis)NigerianNational Xenopus ResourceRRID:NXR_1018
genetic reagent (Xenopus tropicalis)prph2This paperRRID:NXR_3003National Xenopus Resource

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