carried out the lab work and analysed the data

carried out the lab work and analysed the data. the outer segments of UV cones, but restricted to the tip of the segments. CRY1 was found in all UV cones across the entire retina, with the highest densities near the fovea. Pre-exposure of birds to different wavelengths of light did not result in any difference in CRY1 detection, suggesting that CRY1 did not undergo any detectable functional changes as result of light activation. Considering that CRY1 is likely not involved in magnetoreception, our findings open the possibility for an involvement in different, yet undetermined functions in the avian UV/V cones. and cryptochromes, light activation of FAD has been shown to result in a conformational switch of the C-terminal end of the cryptochrome protein39C43. Since Niessner and colleagues observed CRY1a labelling only after exposures to UV to yellow light, but not after exposure in darkness or under reddish light, and their antibody was directed against the C-terminal segment of the cryptochrome, they proposed that their antibody only detected the protein after a light-triggered conformational switch25C28. It is worth mentioning that during the revision of this manuscript, Bolte and genes, unlike genes, exhibits a clear circadian expression profile, suggesting a role in the circadian regulation of physiological processes rather than in magnetoreception23. Here, we examined the cellular localisation and distribution of CRY1 protein across the zebra finch retina and tested whether the detection of CRY1 protein was wavelength dependent by examining the large quantity of CRY1 after exposure to monochromatic lights. Results CRY1 antibody To detect the presence of CRY1 in the zebra finch retina we used a commercial polyclonal antibody designed to target a peptide unique to CRY1 (Fig.?1). The target sequence is almost identical, albeit shorter, to the sequence used by Niessner26,27,56, and more recently by Bolte44, to detect CRY1 in retinas of other bird and mammal species. Even though the western blot analysis on FLAG tag Peptide total protein extracted from your retinas of the zebra finch with our antibody revealed a single band at a lower molecular FLAG tag Peptide weight than the expected size (observe supplemental information), the immunofluorescent transmission location and pattern coincides with that independently reported by Niessner26,27,56, and Bolte44 and colleagues, strongly supporting that this antibody used in this paper is likely detecting CRY1. Open in a separate window Physique 1 Alignment of the C-terminal amino acids of mouse ( em Mus musculus /em ) CRY1 with chicken ( em Gallus gallus /em ) CRY1 and several zebra finch ( FLAG tag Peptide em Taeniopygia guttata /em ) cryptochromes. The green box highlights the target sequence detected by the ABCAM CRY1 antibody used in this FLAG tag Peptide paper, as well as the target sequence used by Niessner26,27,56, and Bolte44. Accession figures: mouse CRY1 (“type”:”entrez-protein”,”attrs”:”text”:”NP_031797.1″,”term_id”:”6681031″,”term_text”:”NP_031797.1″NP_031797.1), chicken CRY1 (“type”:”entrez-protein”,”attrs”:”text”:”NP_989576.1″,”term_id”:”45383636″,”term_text”:”NP_989576.1″NP_989576.1), zebra finch CRY1a (“type”:”entrez-protein”,”attrs”:”text”:”XP_030118992.2″,”term_id”:”1820590200″,”term_text”:”XP_030118992.2″XP_030118992.2), zebra finch CRY1b (“type”:”entrez-protein”,”attrs”:”text”:”XP_030118993.2″,”term_id”:”1820590202″,”term_text”:”XP_030118993.2″XP_030118993.2), zebra finch CRY2a (“type”:”entrez-protein”,”attrs”:”text”:”XP_030130159.1″,”term_id”:”1720776538″,”term_text”:”XP_030130159.1″XP_030130159.1), zebra finch CRY2b (“type”:”entrez-protein”,”attrs”:”text”:”XP_012429630.1″,”term_id”:”823462197″,”term_text”:”XP_012429630.1″XP_012429630.1), zebra finch CRY4 (“type”:”entrez-protein”,”attrs”:”text”:”XP_002198533.1″,”term_id”:”224085023″,”term_text”:”XP_002198533.1″XP_002198533.1). It is important to note that this alignment shown in Fig.?1 includes different isoforms for CRY1 and CRY2. When we started the current study, no isoforms of CRY1 were known for the zebra finch, which is why we make no differentiation between CRY1 and CRY1a throughout the text. Nevertheless, the alignment clearly shows that the detected epitope corresponds to the CRY1a isoform, as is also the case for the protein detected by Niessner26,27,56, and Bolte44. CRY1 expression in UV cones Evaluation of cross sections of the zebra FLAG tag Peptide finch retina revealed CRY1 immunolabelled cells exclusively in the photoreceptor layer (Fig.?2A, B, third panel). Some non-specific transmission in the inner nuclear layer and inner plexiform layer did not seem to be associated with any other retinal cells. We believe it to be background noise present in that channel, or faint autofluorescence since it is also apparent in the unfavorable control without main antibody (Fig. S2C). The strong signal visible in the photoreceptor layer in the DAPI channel is most likely due to the collapse of the pigment epithelium layer Rabbit polyclonal to APPBP2 during dissection and sectioning (also apparent from your flattened appearance of the outer segments). The retinal pigment epithelium contains lipofuscin, a known source of autofluorescence in the vertebrate retina57,58, and perhaps broken oil droplets (Fig. S2C). Open in a separate window Figure.