Cryptochrome 1a localisation in light- and dark-adapted retinae of several migratory and non-migratory bird species: no signs of light-dependent activation

Abstract

The magnetic compass of birds seems to be based on light-dependent radical-pair processes in the eyes. Cryptochromes are currently the only candidate proteins known in vertebrates that may serve as the primary radical-pair-based magnetoreceptor molecules. Previous immunohistochemical studies have suggested that cryptochrome 1a (Cry1a) is localised in the photoreceptor outer segments of the ultraviolet/violet (UV/V) cones, and it has been claimed that differences in Cry1a antibody staining intensities show that Cry1a is activated by light and that this should make Cry1a the most likely magnetoreceptive candidate molecule. Here, we present an independent study of Cry1a distribution within retinae of several bird species, ranging from non-migratory domestic chicken and rock pigeon to night-migratory passerines, using both the previously used antibody and two newly generated antibodies, one against the same epitope as the originally used antibody and one against a different epitope of Cry1a. We confirm the UV/V cone outer segment localisation of Cry1a in all the tested bird species. In some stainings, we found Cry1a immunoreactivity as a distinct punctate pattern throughout the whole length of the UV/V cone outer segments. These dots with a diameter of around 170 nm might suggest that many Cry1a molecules accumulate in distinct spots in the UV/V cone outer segments. However, we did not see any notable difference in Cry1a immunoreactivity between lightand dark-adapted retinae. We find no evidence whatsoever that a C-terminal antibody against Cry1a labels only a light-activated form of the Cry1a protein.

Cryptochrome expression in avian UV cones: revisiting the role of CRY1 as magnetoreceptor

“…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 Niessner 26 , 27 , 56 , and Bolte 44 .…”

“…It is worth mentioning that during the revision of this manuscript, Bolte el at . 44 confirmed that CRY1a is present in the outer segments of UV cones in European robins, Eurasian blackcaps ( Sylvia atricapilla ) and domestic chickens, using an antibody directed against the same target as Niessner et al 26 . However, contrary to Niessner et al .…”

“…However, contrary to Niessner et al . ’s results, the detection of the CRY1a signal did not differ between light- and dark-adapted retinas, arguing against the involvement of a conformational change of CRY1a 44 .…”

“…The target sequence is almost identical, albeit shorter, to the sequence used by Niessner 26 , 27 , 56 , and more recently by Bolte 44 , to detect CRY1 in retinas of other bird and mammal species. Even though the western blot analysis on total protein extracted from the retinas of the zebra finch with our antibody revealed a single band at a lower molecular weight than the expected size (see supplemental information), the immunofluorescent signal location and pattern coincides with that independently reported by Niessner 26 , 27 , 56 , and Bolte 44 and colleagues, strongly supporting that the antibody used in this paper is likely detecting CRY1.

Figure 1 Alignment of the C-terminal amino acids of mouse ( Mus musculus ) CRY1 with chicken ( Gallus gallus ) CRY1 and several zebra finch ( Taeniopygia guttata ) cryptochromes.

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Figure 1 Alignment of the C-terminal amino acids of mouse ( Mus musculus ) CRY1 with chicken ( Gallus gallus ) CRY1 and several zebra finch ( Taeniopygia guttata ) cryptochromes. The green box highlights the target sequence detected by the ABCAM CRY1 antibody used in this paper, as well as the target sequence used by Niessner 26 , 27 , 56 , and Bolte 44 . Accession numbers: mouse CRY1 (NP_031797.1), chicken CRY1 (NP_989576.1), zebra finch CRY1a (XP_030118992.2), zebra finch CRY1b (XP_030118993.2), zebra finch CRY2a (XP_030130159.1), zebra finch CRY2b (XP_012429630.1), zebra finch CRY4 (XP_002198533.1).

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