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

Abstract

Studies at the cellular and molecular level of magnetoreception - sensing and responding to magnetic fields - is a relatively new research area. As it appears that different mechanisms of magnetoreception in animals evolved from different origins, many questions about the 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 (PI-PLC) we show that EPG is associated to the membrane via glycosylphosphatidylinositol (GPI) anchor. EPGs 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 homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues in order 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 field.

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

“…Data accessibility. DNA sequences for all constructs utilized in this paper can be found in the associated electronic supplementary material [29].…”

A Genetic Programming Approach to Engineering MRI Reporter Genes

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“…The optimal concentration and labeling duration for each dye depend on many different conditions, like cell type, protein tagged, SLE used, ligand and dye itself. Previous studies used comparable dyes for LM applications at concentrations between 10 and 500 nM, and stained for 10 – 30 min (Beatty, 2019; Broadbent et al, 2023; Grimm, Brown, et al, 2017; Grimm et al, 2020; Joshua et al, 2022; Ricker et al, 2022), in case up to 1 µM (Binns et al, 2020; Braselmann et al, 2018), to label proteins conjugated to HaloTag. We used a concentration of 100 nM for 30 min for all dyes to keep conditions comparable.…”

“…Our tests for in-resin fluorescence of the green JF479, the red fluorogenic JF585 and the far-red dyes JF646, JFX646 and JFX650, included higher concentrations of 1 µM for each of these dyes, and even 10 µM for JF479 were tested. Previous studies used these JF dyes for LM of labeled proteins conjugated to HaloTag at concentrations between 100-500 nM (Beatty, 2019; Broadbent et al, 2023; Joshua et al, 2022; Klump et al, 2023; Ricker et al, 2022), sometimes up to 1 µM (Binns et al, 2020; Braselmann et al, 2018). However, in our application the increased concentrations did not improve the results on EPON sections.…”