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. 2023 Nov;13(11):230019.
doi: 10.1098/rsob.230019. Epub 2023 Nov 22.

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells

Brianna Ricker  1 , Sunayana Mitra  1 , E Alejandro Castellanos  2 , Connor J Grady  2 , Daniel Woldring  1 , Galit Pelled  3   4 , Assaf A Gilad  1   3
Affiliations

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells

Brianna Ricker et al. Open Biol. 2023 Nov.

Abstract

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

Keywords: GCaMP6m; HaloTag; Kryptopterus vitreolus; glycosylphosphatidylinositol (GPI) anchor; magnetoreception; phosphatidylinositol-specific phospholipase C (PI-PLC).

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Conflict of interest statement

We declare we have no competing interests.

Figures

Figure 1.
Figure 1.
Fluorescently labelled HaloTag indicates EPG's cellular localization. (a–d) HeLa cells expressing Halo-N-EPG (a,b) and Halo-C-EPG (c,d) labelled with fluorescent HaloTag ligands: (a,c) membrane permeable JFX650, and (b,d) membrane impermeable AF488. (e,f) Illustrations to demonstrate the interaction of HaloTag-EPG fusion proteins with AF488. (e) As reflected in (b), Halo-N-EPG expressing cells were labelled with AF488 indicating its presence as a membrane associated protein with its N terminus exposed to the extracellular space. (f) As reflected in (d), Halo-C-EPG expressing cells were not labelled with AF488, indicating this construct does not localize to the membrane. Scale bars represent 50 µm.
Figure 2.
Figure 2.
Lysate analysis and PI-PLC digestion signify EPG undergoes posttranslational modification to receive a glycosylphosphatidylinositol anchor. (a) Lysate of HeLa cells expressing HaloTag fusion proteins labelled with JFX650 run on an SDS-PAGE gel visualized with Far-Red excitation and 715/30 filter emission (white bands), and Stain-Free imaging (black bands). The expected size of the HaloTag fusion proteins is approximately 46 kDa. Halo-N-EPG presents as a series of bands between 50 and 75 kDa. Halo-C-EPG exhibits a band at 46 kDa. Halo-N-CD59 presents a band just above 50 kDa. Mock transfected cells do not present JFX650 associated bands. (b,c) HeLa cells expressing HaloTag fusion proteins labelled with JFX650 were treated with PI-PLC, effectively releasing any GPI anchored protein from the membrane as illustrated by (b). Media from treated (+) and untreated (−) cells visualized by SDS-PAGE with Far-Red excitation and 715/30 filter emission (white bands) and Stain-Free imaging (black bands) show a band at approximately 61.5 kDa in the Halo-N-EPG (+) group that is not present in the untreated (−) group—indicative of the presence of a GPI anchor. Halo-C-EPG did not present any JFX650 associated bands. The positive control, Halo-N-CD59, presented bands at approximately 51.5 kDa. Mock transfected cells did not present any JFX650 associated bands.
Figure 3.
Figure 3.
Developing an assay using GCaMP6m to determine if EPG is still functional after the addition of HaloTag. (a–f) HeLa cells expressing GCaMP6m and various EPG-HaloTag fusion constructs visualized before (a,c,e) and after (b,d,f) electromagnetic stimulation. (a,b) Cells expressing EPG-IRES-tdT appear more intense after stimulation. (c,d) Cells expressing Halo-N-EPG appear more intense after stimulation. (e,f) Cells expressing Halo-C-EPG appear relatively unchanged after stimulation. Scale bars represent 200 μm. (g–i) Average intensity of GCaMP6m over time with various electromagnetic stimuli. Error bars are representative of 95% CI. Significant increases in intensity were observed between the no stimulus/sham and active groups in both (g) (p < 0.0001, unpaired t test) and (h) (p < 0.0001, unpaired t test). No significant difference was observed between any groups in (i). (j–l) Percentage of individual cells that produced a signal greater than 3×SD+mean of the corresponding no stimulus group. Significant differences were observed between sham and active groups in both (j) and (k). No significant difference was observed between sham and active groups in (l). The EPG-IRES-tdT group included n = 177, n = 134, and n = 163 cells over four experiments for no stimulus, sham, and active groups respectively. The Halo-N-EPG group included n = 282, n = 269, and n = 279 cells over four experiments for no stimulus, sham, and active groups respectively. The Halo-C-EPG group included n = 160, n = 182, and n = 189 cells over three experiments for the no stimulus, sham, and active groups respectively.
Figure 4.
Figure 4.
Observing the effect of electromagnetic stimulation on homologues of EPG from different species. (a) Alignment of amino acid sequences for EPG and homologues B.g., BNCR, and CD59. (b,c) HeLa cells expressing Halo-N-B.g. and labelled with AF488 (b) and JFX650 (c) demonstrating this protein is membrane-associated. (d) Average intensity of GCaMP6m in HeLa cells expressing Halo-N-B.g. with various stimuli. (e,f) HeLa cells expressing Halo-N-BNCR and labelled with AF488 (e) and JFX650 (f) showing the protein, unexpectedly, does not localize to the membrane. (g) Average intensity of GCaMP6m in HeLa cells also expressing Halo-N-BNCR with various stimuli. (h,i) HeLa cells expressing Halo-N-CD59 labelled with AF488 (h) and JFX650 (i) demonstrating this protein is membrane-associated. (j) Average intensity of GCaMP6m in HeLa cells also expressing Halo-N-CD59 with various stimuli. (d,g,j) Error bars are representative of 95% CI. All scale bars indicate 50 μm. The Halo-N-B.g. group included n = 84, n = 97, and n = 91 cells over 3 experiments for no stimulus, sham, and active groups respectively. The Halo-N-BNCR group included n = 118, n = 120, and n = 105 cells over 3 experiments for no stimulus, sham, and active groups respectively. The Halo-N-CD59 group included n = 130, n = 124, and n = 120 cells over 4 experiments for the no stimulus, sham, and active groups respectively.
Figure 5.
Figure 5.
Site directed mutagenesis of the three-phenylalanine region results in change of function. (a) Predicted structure of EPG with the three phenylalanine residues highlighted in red. (b) Stability of amino acid swaps for EPG in a heatmap; red is a stabilizing swap, yellow is neutral, and blue is a destabilizing swap. (c–g) Average intensity of GCaMP6m in HeLa cells expressing various 3F mutants over time with various stimuli. Error bars are representative of 95% CI. (c–f) Amino acids were swapped with the most stabilizing residue indicated in (b). (c) First F in 3F region knocked out. (d) Second F in 3F region knocked out. (e) Third F in 3F region knocked out. (f) All three F residues in 3F region knocked out. (g) 3F motif inserted into CD59. The Halo-N-EPG3Fm-MFF group included n = 77, n = 90, and n = 89 cells over 3 experiments for no stimulus, sham, and active groups respectively. The Halo-N-EPG3Fm-FWF group included n = 87, n = 96, and n = 86 cells over 3 experiments for the no stimulus, sham, and active groups respectively. The Halo-N-EPG3Fm-FFW group included n = 87, n = 85, and n = 88 cells over 3 experiments for the no stimulus, sham, and active groups respectively. The Halo-N-EPG3Fm-MWW group included n = 84, n = 83, and n = 89 cells over 3 experiments for the no stimulus, sham, and active groups respectively. The Halo-N-3FCD59 group included n = 86, n = 92, and n = 95 cells over 3 experiments for no stimulus, sham, and active groups respectively.
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