The expression, localisation and interactome of pigeon CRY2

Abstract

Cryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton.

Broadband 75–85 MHz radiofrequency fields disrupt magnetic compass orientation in night-migratory songbirds consistent with a flavin-based radical pair magnetoreceptor

“…Cryptochromes (Cry), currently the only group of vertebrate proteins known to form radical pairs upon photoexcitation (Ritz et al 2000 ; Liedvogel et al 2007 ; Maeda et al 2012 ; Zoltowski et al 2019 ; Xu et al 2021 ), have been proposed as the light-activated, radical-pair-forming, magnetosensory proteins in the avian retina (Ritz et al 2000 ; Mouritsen et al 2004 ; Maeda et al 2012 ; Nießner et al 2014 ; but see Bolte et al 2021 ; Hore and Mouritsen 2016 ; Günther et al 2018 ; Xu et al 2021 ; Wong et al 2021 ). At least six different cryptochrome variants are known to occur in retinal neurons of night-migratory songbirds: Cry1a (Mouritsen et al 2004 ; Möller et al 2004 ; Nießner et al 2011 ; Bolte et al 2021 ), Cry1b (Möller et al 2004 ; Bolte et al 2016 ; Nießner et al 2016 ), Cry2a (Mouritsen et al 2004 ; Möller et al 2004 ; Balay et al 2021 ; Einwich et al 2021 ), Cry2b (Hochstoeger et al 2020 ; Balay et al 2021 ), Cry4a (Liedvogel and Mouritsen 2010 ; Günther et al 2018 ; Wu et al 2020 ; Xu et al 2021 ), and Cry4b (Einwich et al 2020 ); a and b forms are alternative splicing variants.…”

Per1/Per2 Disruption Reduces Testosterone Synthesis and Impairs Fertility in Elderly Male Mice

“…GO analysis showed that differentially expressed (DE) mRNAs were mainly enriched in microtubule-related pathways, including microtubule-based movement (GO-BP), tubulin binding (GO-MF), and the microtubule (GO-CC; Figure 2). Many circadian molecules (PER2, CLOCK, ARTNL) and cell junction proteins (CTNNA1, CTNNA2) are associated with the microtubule motor protein, dynein (DYNC1H1, DCTN1) [19]. Correspondingly, two microtubule-related genes, Tubd1 and Pafah1b that are involved in spermatogenesis were significantly down-regulated (Figure 2D,F).…”

Dimerisation of European robin cryptochrome 4a

“…Crys can exist in the so-called dark-state, containing the fully oxidised form of the FAD, or in higher energy states formed by photo-excitation of the FAD followed by the formation of radical pairs, involving a triad or tetrad of tryptophan residues, which have been proposed as mediators of the mechanism of magnetoreception in migratory songbirds [15][16][17][18][19][20] . Three different Cry-genes, Cry1 [21][22][23][24][25][26] , Cry2 21,27 , and Cry4 [28][29][30][31][32] exist in most bird species, each with at least one splice-variant 22,33,34 . Several of these cryptochromes are found in the birds' retina, where they can easily be light-activated 21,24,25,27,29,32,34 .…”