Extraocular, rod-like photoreceptors in a flatworm express xenopsin photopigment
- Wellcome Sanger Institute, United Kingdom
- University of Cambridge, United Kingdom
- Marine Biological Laboratory, United States
- Université Côte D’Azur, CNRS, Institut de Biologie Valrose, France
- New York University School of Medicine, United States
- University of Manchester, United Kingdom
- University of Connecticut, United States
- University College London, United Kingdom
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- Dalhousie University, Canada
Figures
Figure 1 with 3 supplements
Phylogenetic analysis of metazoan opsins supports flatworm ciliary-like opsins as xenopsins and confirms a clade of flatworm rhabdomeric opsins.
Support for nodes is calculated using 1000 Ultrafast bootstrap replications as well as 1000 SH-aLRT replicates and approximate aBayes single Branch testing. Black dots indicate nodes with support …
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Figure 1—source data 1
Sequence data for opsins used in phylogenetic analyses for Figure 1.
- https://doi.org/10.7554/eLife.45465.007
Figure 1—figure supplement 1
Uncollapsed tree of IQ-TREE phylogenetic reconstruction of opsin relationships.
Node support values correspond to 1000 Ultrafast bootstrap replications, 1000 SH-aLRT replicates and approximate aBayes single Branch testing. Scale bar unit for branch length is the number of …
Figure 1—figure supplement 2
IQtree and RaxML trees showing the influence of the small opsin clades (i.e. chaopsins, bathyopsins, ctenophore and anthozoan opsins) on the position of xenopsins in relation to c-opsins and tetra-opsins (Neuropsin, Go-opsin and RRP); inclusion of these small opsin clades brings xenopsins close to tetraopsins (full dataset), their exclusion brings xenopsins close to c-opsins (reduced dataset).
https://doi.org/10.7554/eLife.45465.005
Figure 1—figure supplement 3
Alignment of major opsin clades showing conserved lysine in transmembrane domain VII, which binds to the retinal chromophore to form a photopigment.
Some xenopsins possess a tripeptide motif, NxQ, which is also found in ciliary opsins and known to be crucial for G-protein activation. A number of flatworm xenopsin sequences in clade A have …
Figure 2 with 1 supplement
In the larva (1 day post-hatching) xenopsin protein is localized to ciliary cells in the eyes.
(A) Apical view of larva showing xenopsin (red) in the epidermal eye (ee) (100% of individuals examined, n = 50) (OpenSPIM image, Syt gr = Sytox green, staining nuclei and bright-field image also …
Figure 2—figure supplement 1
Opsin localization and expression in Maritigrella embryos and larva.
(A and B) Xenopsin expression (red) in the epidermal eye (ee) during Maritigrella crozieri embryogenesis. The epidermal eye develops soon after gastrulation is complete and before development of the …
Figure 3
Acetylated tubulin staining identifies two dense clusters of extraocular cells, possible ciliary photoreceptors (CPR), either side of the adult brain.
(A) Live adult, red line shows plane of cross section in B-C. (B and C) Consecutive sections showing; (B) two clusters of acetylated tubulin+ cells and; (C) their distribution between the brain (br) …
Figure 4 with 4 supplements
The morphology of the ciliary phaosomal cells in adult Maritigrella crozieri.
(A) A dense cluster of intra-cellular vacuoles (phaosomes – highlighted in magenta) filled with cilia (n = 3 individuals). (B) Multiple cilia in the phaosome. (C) A possible unpigmented supporting …
Figure 4—video 1
Serial SEM images (101 × 500 nm sections = 50.5 μm total thickness) showing a cluster of ciliary phaosomes (intra-cellular vacuoles housing multiple cilia) that form the outer segment of the putative extraocular CPR cells in adult Maritigrella.
This cluster is located to the left of the brain.
Figure 4—video 2
Serial SEM images (47 × 400 nm sections = 18.8 μm total thickness) showing a complete phaosome.
At least 421 basal bodies were counted projecting cilia into the intra-cellular space that is formed by the cell itself. Note that in a nearby phaosome, the cilia appear to be arranged in a spirally …
Figure 4—video 3
Serial SEM images (72 x 100nm sections = 7.2μm total thickness) showing a third of a phaosome.
https://doi.org/10.7554/eLife.45465.014
Figure 4—video 4
A 3D reconstruction of series in Figure 4—video 3 shows that the cilia (pink) are unbranched and basal bodies (yellow) emerge all around the diameter of the cavity.
https://doi.org/10.7554/eLife.45465.015
Figure 5 with 1 supplement
Xenopsin is co-localised with acetylated tubulin and Gαi in extraocular ciliary phaosome cells and r-opsin is expressed with Gαq in eyes.
(A-E) Co-localization of acetylated tubulin and xenopsin is found throughout the cell (n, nucleus) (100% of acTub+ cells express xenopsin (in one cross section, n = 3 individual worms). (E) A cross …
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Figure 5—source data 1
Sequence data for G alpha subunits used in phylogenetic analyses for Figure 5.
- https://doi.org/10.7554/eLife.45465.018
Figure 5—figure supplement 1
G alpha subunit phylogeny and C terminal end alignment.
(A) IQ-TREE phylogenetic reconstruction of G alpha subunit relationships identifies those expressed in the Maritigrella crozieri larval transcriptome (red). Support for node is calculated using 1000 …
Figure 6 with 2 supplements
In human cells Maritigrella crozieri xenopsin forms a functional photopigment that predominantly couples to Gαi pathways.
(A,B) HEK293 cells were transfected with Glo22F and indicated opsins, + /- pertussis toxin, and exposed to light. In B, cells were treated with 2 µM forskolin prior to the light flash. (C) HEK293 …
Figure 6—figure supplement 1
Immunofluorescence to quantify opsin expression in HEK293 cells.
Cells were transfected with opsins, fixed, stained with 1D4 anti-rod opsin antibody and fluorescent secondary antibody, and imaged on a fluorescent microscope. (A) Representative images of cells …
Figure 6—figure supplement 2
Two further biological replicates of the secondary messenger assays show there was quantitative variation from day to day in the magnitude of responses to light and forskolin, but the qualitative response of each opsin was consistent (excluding one replicate in which rod opsin showed no activity, possibly due to a faulty preparation of plasmid DNA).
https://doi.org/10.7554/eLife.45465.021
Figure 7
An overview of metazoan ciliary photoreceptor morphology, opsin expression and Gα-protein coupling (determined from cellular assays), highlighting the distinct morphology of the ciliary phaosomes in flatworms and possible convergent evolution of enclosed ciliary membranes in invertebrate phaosomes and jawed vertebrate rods (*).
The colored lines under the phylum names represent the presence of the opsin sub-type in the taxonomic group; note the higher prevalence of xenopsins over ciliary opsins in Lophotrochozoa. (L) = larv…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-acetylated tubulin (monoclonal, mouse) | Sigma | T7451 RRID:AB_609894 | IF(1:500) |
| Antibody | Gα q/11 (C-19) (polyclonal, rabbit) | Santa Cruz Biotech | sc-392 RRID:AB_631537 | IF(1:300) |
| Antibody | Gαi-1 (R4) (monoclonal, mouse) | Santa Cruz Biotech | sc-13533 RRID:AB_2111358 | IF(1:300) |
| Antibody | Gαi-1/2/3 (35) (monoclonal, mouse) | Santa Cruz Biotech | sc-136478 RRID:AB_2722559 | IF(1:300) |
| Antibody | Gαo (A2) (monoclonal, mouse) | Santa Cruz Biotech | sc-13532 RRID:AB_2111645 | IF(1:300) |
| Antibody | Gαs/olf(A-5) (monoclonal, mouse) | Santa Cruz Biotech | sc-55545 RRID:AB_831819 | IF(1:300) |
| Antibody | Gαs/olf(C-18) (polyclonal, rabbit) | Santa Cruz Biotech | sc-383 RRID:AB_631539 | IF(1:300) |
| Antibody | Gα12 (E-12) (monoclonal, mouse) | Santa Cruz Biotech | sc-515445 | IF(1:300) |
| Cell line (Homo-sapiens) | Embryonic kidney cells | ATCC | CRL3216 RRID:CVCL_0063 | |
| Recombinant DNA reagent | pGlosensor 22 | Promega | E1290 | Live-Cell Biosensors |
| Recombinant DNA reagent | pcDNA3.1 | Invitrogen | V79020 | Mammalian Expression Vector |
| Recombinant DNA reagent | pcDNAFRT⁄TO vector | Thermo | V652020 | Expression vector |
| Chemical compound | 9-cis retinal | Sigma | R5754 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.45465.023
