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Raw data for the article: A Cryptochrome adopts distinct moon- and sunlight states and functions as sun- versus moonlight interpreter in monthly oscillator entrainment

Citation

Poehn, Birgit et al. (2022), Raw data for the article: A Cryptochrome adopts distinct moon- and sunlight states and functions as sun- versus moonlight interpreter in monthly oscillator entrainment, Dryad, Dataset, https://doi.org/10.5061/dryad.wm37pvmq8

Abstract

The moon’s monthly cycle synchronizes reproduction in countless marine organisms. The mass-spawning bristle worm Platynereis dumerilii  uses an endogenous monthly oscillator to phase reproduction to specific days. Classical work showed that this oscillator is set by full moon. But how do organisms recognize such a specific moon phase? We uncover that the light receptor L-Cryptochrome (L-Cry) is able to discriminate between different moonlight durations, as well as between sun- and moonlight. Consistent with L-Cry’s function as light valence interpreter, its genetic loss leads to a faster re-entrainment under artificially strong nocturnal light. This suggests that L-Cry blocks “wrong” light from impacting on the monthly oscillator. A biochemical characterization of purified L-Cry protein, exposed to naturalistic sun- or moonlight, reveals the formation of distinct sun- and moonlight states characterized by different photoreduction- and recovery kinetics of L-Cry’s co-factor Flavin Adenine Dinucleotide. In vivo, L-Cry’s sun- versus moonlight states correlate with distinct sub-cellular localizations, indicating different signalling. In contrast, r-Opsin1, the most abundant ocular opsin, is not required for monthly oscillator entrainment. Our work reveals a new concept for correct moonlight interpretation involving a “valence interpreter” that provides entraining photoreceptor(s) with light source and moon phase information. These findings advance our mechanistic understanding of a fundamental biological phenomenon: moon-controlled monthly timing. Such level of understanding is also an essential prerequisite to tackle anthropogenic threats on marine ecology.

Methods

This dataset consists of

1. Western blot data
2. Records of spawning behaviour
3. Immunohistochemistry data
4. Light spectra measurement
5. SEC/MALS data
6. UV/VIS spectroscopy data

1. Western blot
Worm head protein lysates were analysed using SDS-PAGE (10% acrylamide) and subsequent transfer to a 0.45µm nitrocellulose membrane. The membrane was probed with two primary antibodies against the Platynereis dumerilii L-Cry protein (raised in mouse) and a primary antibody against beta-actin (raised in rabbit). The primary antibodies were specifically bound with the corresponding secondary antibodies: anti-mouse IgG-peroxidase-linked and anti-rabbit IgG-peroxidase-linked or fluorescently labelled antibody. Detection was performed accordingly, either using a peroxidase substrate or detection of fluorescence. This dataset comprises all membranes for L-Cry and/or beta-actin detection for every biological replicate (if BRs are applicable).

2. Records of spawning behaviour
Mature male and female worms were recorded on a daily basis, taking into consideration days of the lunar month.

3. Immunohistochemistry data
Immunohistochemistry samples were imaged using Zeiss LSM 700 confocal microscope with a 40x oil objective. In order to determine fluorescence in the nuclei, images of the 405 nm channel (Hoechst staining) were segmented using the deep learning-based algorithm Cellpose. The resulting regions were overlayed as Regions Of Interest (ROI) on images of the 555nm channel (L-Cry antibody staining) in Fiji/ImageJ. In addition, the entire image was selected as a ROI as well. As background regions, nuclei of the cells that do not express L-Cry and/or some non-nuclear regions of the image were used (at least 3 background ROIs per image).Fiji/ImageJ was used to determine the fluorescence signal intensity (by measuring the Area and Mean Gray Value of each ROI). Corrected Total Cell Fluorescence (CTCF) was calculated the following way: CTCF =Area (ROI_1 )*Mean (ROI_1 )-Area (ROI_1 )*Mean(ROI_(background ROIs) ). Total nuclear CTCF was calculated by adding all nuclear ROI together. To obtain the cytoplasmic fluorescence intensity, the summed nuclear values were subtracted from the CTCF of the ROI for the entire image. In order to compare results across experiments, total nuclear CTCF and total cytoplasmic CTCF values were normalized to the highest value within each experiment. The ratio between nuclear and cytoplasmic signal intensity was calculated as well. In some experiments, the analysis was performed in a "blinded" manner, i.e. the names of the images were enciphered. Experiment 1 and 3 contained all three conditions, while the experiments 2 and 4 contained only two conditions. Experiments 3 and 4 contained the l-cry -/- mutant samples that were used as threshold control.

4. Light spectra measurement
Under water measurements of natural sun- and moonlight at the habitat of Platynereis were acquired using a RAMSES-ACC-VIS hyperspectral radiometer (TriOS GmbH) for UV to IR spectral range (see V. B. Veedin Rajan et al., Nat. Ecol. Evol. 5, 204–218 (2021) for details). Radiometers were placed at 4m and 5m water depth close to Posidonia oceanica meadows, which are a natural habitat for P. dumerilii. Measurements were recorded automatically every 15min across several weeks in the winter 2011/2012 (at 5m depth) and during spring 2011 (at a 4m depth).
To obtain an exemplary sunlight spectrum, the sunlight measurements taken at 5m depth between 10 am-4 pm on 25.11.2011 were averaged.  To obtain a fullmoon spectrum (FM) for the 5m depth location measurements taken from 10pm to 1am on a clear fullmoon night (10-11.11.2011) were averaged. To subtract baseline noise from this measurement, a new moon (NM) spectrum was obtained by averaging measurements between 7:15pm to 5am on a new moon night on 24.11.2011, and subtracted from the fullmoon spectrum. To validate that this spectrum is representative of a typical full moon spectrum at the habitat of Platynereis, we averaged moonlight measured at 4m depth between 10:15 pm to 2am during a full moon night in April (17.-18.04.2012) and subtracted a new moon spectrum measured two weeks earlier. To benchmark these moonlight spectra measured under water with moonlight measured on land, we compared the underwater spectra to a publicly available full moon spectrum measured on land on 14.04.2014 in the Netherlands (http://www.olino.org/blog/us/articles/2015/10/05/spectrum-of-moon-light). The resulting light spectra are plotted in the Supplementary Figure 1.
For primary data, see Zurl et al. 2022, Data accompanying article: Two light sensors decode moonlight versus sunlight to adjust a plastic circadian/circalunidian clock to moon phase, Dryad, Dataset, https://doi.org/10.5061/dryad.2v6wwpzkr

5. SEC/MALS data
Purified L-Cry protein was analyzed for its oligomeric state by SEC and SEC-MALS.

6. UV/VIS spectroscopy data
The L-Cry light responses to blue light, sunlight and moonlight and recovery kinetics were analyzed by UV/VIS spectroscopy, observing the light-induced photoreduction and reoxidation (recovery) of L-Cry’s cofactor flavin adenine dinucleotide (FAD).

Usage Notes

To open the data files included with this submission, the following programs and/or software are required:

1. GraphPadPrism 8.4.2

2. MS Excel 2010

3. Origin version 7.5/10.5 (trial)

4. a .PDF reader, such as Adobe Acrobat

Funding

Austrian Science Fund, Award: F78

Johannes Gutenberg-Universität Mainz

Deutsche Forschungsgemeinschaft, Award: GSC 266

IMB Mainz