Fig 1.
Phylogenetic tree of AyNCXA (MG182344) and AyNCXB1-4 (MG182345, MG182346, MG182347, MG182348) (AyNCX sequences are highlighted blue) with other NCX and NCX-like proteins.
The following accession numbers were used for obtaining either predicted transcripts, mRNA, or protein sequences. Coral NCX proteins: Acropora digitifera (A.digitiferaNCXA: XP_015752015.1, A.digitiferaNCXB: XP_015772900.1)1, Acropora millepora NCX (A.milleporaNCXA: JT007757.1, A.milleporaNCXB: JT003571.1), Acropora cervicornis (A.cervicornisNCXA: GASU01080071.1, A.cervicornisNCXB: GASU01087165.1), Orbicella faveolata (O.faveolataNCXA: XM_020750226.1, O.faveolataNCXB: XP_020626605.1), Galaxea fascicularis (G.fascicularisNCXA: GFAZ01129628), Porites australiensis (P.australiensisNCXA: FX462417.1), Porites astreoides (P.astreoidesNCXA: GEHP01352486), Favia lizardensis (F.lizardensisNCXA: GDZU01041167), Pocillopora damicornis (P.damicornisNCXA: GEFF01028265), Stylophora pistillata (S.pistillataNCXA: GARY01000181.1 –this sequence was edited to fix frameshift errors that split the protein into three incomplete NCX proteins). All coral sequences were designated A or B based on their homology to AyNCX proteins. Invertebrate NCX proteins: Exaiptasia pallida (AnenomeNCX1: XP_020915137.1, AnenomeNCX2: XM_021049204.1, AnenomeNCX3: XP_020912295.1), Strongylocentrotus purpuratus (UrchinNCX2: XM_011685576.1, UrchinNCX3: XM_011663639.1- this sequence was originally annotated as an NCX1 protein in [36]), Crassostrea gigas (OysterNCX1: XP_011444293.1, OysterNCX2: XM_011445979.2, OysterNCX3: XM_020074533.1), Doryteuthis opalescens (SquidNCX: AAB52920.1)2, Drosophila melanogaster (FlyCalx: AAB63464.1)2, Chordate NCX proteins: Ciona intestinalis (TunicateNCX1: XM_002126723.4, TunicateNCX2: XM_002129316.4, TunicateNCX3: XM_002122937.3) Danio rerio (ZebrafishNCX1: NM_001037102.1, ZebrafishNCX3: XM_005156997.4), Callorhinchus milii (ElephantFishNCX1: XM_007893988.1, ElephantFishNCX3: XM_007893267.1), Squalus acanthias (DogfishNCX1: DQ068478.1)3, Gallus (ChickenNCX1: AJ012579.1, ChickenNCX3: AJ012580.1)4, Rattus norvegicus (RatNCX2: P48768.1, RatNCX3: P70549.1) 2, Mus musculus (MouseNCX1: AF004666.1, MouseNCX3: NM_080440.3)5, Canis sp. (DogNCX1: AAA62766.1) 2, Homo sapiens (HumanNCX1: NM_021097.2, HumanNCX2: NM_015063.2- the original accession number cited in paper, XM_0038970, no longer exists, HumanNCX3: NM_033262.4)6 NCKX proteins: Bos taurus (BullNCKX1: Q28139.2- was 108825 in reference but number has been updated)2, Rattus norvegicus (RatNCKX2: AAC19405.1)2 Other: Saccharomyces cerevisiae (YeastVX1: Q99385.1) 2, Homo sapiens (HumanNCLX: NP_079235.2). NCBI BLAST was used to identify most sequences. Others provided in papers are referenced as follows: 1 [20], 2 [35], 3 [36], 4 [37], 5 [17], 6 [38]. The scale bar represents an amount genetic change of 2.
Fig 2.
AyNCXA localizes in intracellular vesicles in sea urchin embryos.
A) Schematic of the fluorescent protein fusions used in these experiments. Protein colors match the fluorescence in micrographs B-D. B) The sea urchin embryo at ~20 hours post fertilization is a hollow, spherical, epithelial ball approximately 80 μm wide, and LCK is a cell plasma membrane marker. C) Two representative embryos expressing AyNCXA and LCK. Upper row: an equatorial cross section showing AyNCXA vesicles towards the apical surface of the cells. Lower row: Tangential section showing AyNCXA vesicles predominantly at the apical vertices between cells. D) Example of ABCC9 expressing embryo (surface projection) and a zoomed in cross-section with vesicles labeled with white arrows. E) ABCC9 localizes to vesicles, which colocalize with AyNCXA (white arrowhead).
Fig 3.
Validation of antibodies against AyNCXA.
A) The anti-AyNCXA antibodies recognize a ~100 kDa and ~75 kDa protein in homogenized A. yongei tissue. Both bands are eliminated when the antibody is pre-absorbed with the epitope peptide overnight, and neither band is present when the membrane is incubated with the pre-immune serum. All sample wells contain the same amount of protein and all three Western Blot images were taken at the same exposure. B) Immunofluorescence microscopy of A. yongei tissue reveals AyNCXA is present in all four tissue layers, including the calicodermis. C) Pre-absorption of antibodies with antigen peptide eliminates signal at the same exposure, confirming antibody specificity.
Fig 4.
Immunofluorescence microscopy of A. yongei tissue using structured illumination.
A) AyNCXA (red) is present in all four coral tissue layers, including near the apical membrane of the oral ectodermis (labeled 1), and in cytoplasmic structures of oral and aboral gastrodermis (arrows labeled 2), calcifying cells (labeled 3), and desmocytes (arrows labeled 4). B) The corresponding bright field image using differential interference contrast shows cell morphology. C) 400nm-thick cryosection indicates punctate AyNCXA signal in the calicoblastic cells (asterisk). D) 400nm-thick cryosection stained with antibodies against Na+/K+- ATPase (NKA) provides an example of basolateral staining/localization (arrowhead). E) Confocal microscopy confirms an immunostaining pattern consistent with vesicle localization in calicoblastic cells (asterisk). Nuclei are stained by Hoechst (blue). Abbreviations: SW- Seawater, Coel- Coelenteron, Sk- Skeleton.
Fig 5.
Transmission electron micrographs of the calicoblastic epithelium in A. yongei.
A-C) Vesicles of a variety of sizes are visible in calicoblastic cells. Larger vesicles are indicated by a black asterisk (*), smaller vesicles are indicated by black arrows. Scale bar is 500 nm. Abbreviations: AG- Aboral Gastroderm, mes- mesoglea, CE- Calicoblastic Epithelium, Sk- Skeleton.