A Cryptochrome adopts distinct moon- and sunlight states and functions as sun- versus moonlight interpreter in monthly oscillator entrainment

Abstract

The moon's monthly cycle synchronizes reproduction in countless marine organisms. The mass-spawning bristle worm Platynereis dumerilii uses an endogenous monthly oscillator set by full moon to phase reproduction to specific days. But how do organisms recognize specific moon phases? We uncover that the light receptor L-Cryptochrome (L-Cry) discriminates between different moonlight durations, as well as between sun- and moonlight. 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 Platynereis, L-Cry's sun- versus moonlight states correlate with distinct subcellular localizations, indicating different signaling. In contrast, r-Opsin1, the most abundant ocular opsin, is not required for monthly oscillator entrainment. Our work reveals a photo-ecological concept for natural light interpretation involving a "valence interpreter" that provides entraining photoreceptor(s) with light source and moon phase information.

Fig. 1. l-cry^–/– mutants are loss-of-function alleles.

a Overview of the l-cry genomic locus for wt and mutants. Both mutant alleles result in an early frameshift and premature stop codons. The Δ34 allele has an additional 9 bp deletion in exon 3. b Western Blots of P. dumerilii heads probed with anti-L-Cry antibody. In the context of further investigations such Western blots of mutant versus wild types have been performed more than 10 times with highly consistent results. Also see further analyses in this manuscript and ref. . c overview of P. dumerilii. d whole mount in situ hybridization against l-cry mRNA on worm head. ae, anterior eye; pe, posterior eye. e–j Immunohistochemistry of premature wild-type (e–h) and mutant (i, j) worm heads sampled at zt19/20 using anti-L-Cry antibody (green) and Hoechst staining (magenta), dorsal views, anterior up. e, f: z-stack images (maximal projections of 50 layers, 1.28 µm each) in the area highlighted by the rectangle in (d), whereas (g–j) are single layer images of the area highlighted by the white rectangles in (e, f). In the context of further investigations such stainings of mutant versus wild types have been performed more than 10 times with highly consistent results. Also see further analyses in this manuscript and ref. .

Fig. 2. L-Cry shields the circalunar clock from light that is not naturalistic moonlight.

a–d, j Spawning of l-cry ^+/+ (a), l-cry ^{+/– (Δ34)} (b) and l-cry ^{−/−(Δ34/ Δ34)} (c) animals over the lunar month in the lab with 8 nights of artificial moonlight (a–c), under natural conditions in the sea (d, replotted from ref. ,,) and in the lab using naturalistic sun- and moonlight (j, 8 nights moonlight). e–h, k Data as in (a–d, j) as circular plot. 360° correspond to 30 days of the lunar month. The arrow represents the mean vector, characterized by the direction angle µ and r (length of µ). r indicates phase coherence (measure of population synchrony). p-values inside the plots: result of Rayleigh Tests. Significance indicates non-random distribution of data points. The inner circle represents the Rayleigh critical value (p = 0.05). i–l Results of two-sided multisample statistics on spawning data shown in (a–h, j, k). The phase differences in days can be calculated from the angle between the two mean vectors (i.e. 12°= 1 day).

Fig. 3. l-cry−/− mutants entrain the circalunar clock faster than wt to a high-intensity artificial moonlight stimulus.

a Nocturnal moonlight exposure protocol of lunar phase shift (entrained by 8 nights, phased shifted by 6 nights of artificial culture moon, light green). b, c Number of mature animals (percent per month, rolling mean with a window of 3 days) of l-cry wild-type (b) and homozygous mutant (c) animals. p-values indicate results of Kolomogorov–Smirnov tests. Dark blue arrowheads- old FM phase: wt show a spawning minimum, indicative that the worms are not properly phase shifted. Mutants spawn in high numbers, but don’t spawn at the old NM indicated by light blue arrowhead. Also compare to initial FM and NM in months 1,2. d, e Circular plots of the data shown in (b) and (c). Each circle represents one lunar month. Each dot represents one mature worm. The arrow represents the mean vector characterized by the direction angle µ and r. r (length of µ) indicates phase coherence (measure of population synchrony). The inner circle represents the Rayleigh critical value (p = 0.05). f, g Results of two-sided multisample statistics of data in (d, e). Phase differences in days can be calculated from the angle between the two mean vectors (i.e. 12°= 1 day).

Fig. 4. l-cry has a minor contribution as entraining photoreceptor to circalunar clock entrainment.

a Nocturnal moonlight exposure protocol of lunar phase shift with 8 nights of naturalistic moonlight (dark green). Number of mature animals (percent per month, rolling mean with a window of 3 days) of l-cry wild-type (b) and mutant (c) animals. p-values: Kolomogorov–Smirnov tests. Black arrowheads indicate spawning-free intervals of the wildtype, which shifted to the position of the new FM (under free-running conditions: FR-FM). d, e Data as in (b, c) plotted as circular data. 360° correspond to 30 days of the lunar month. The arrow represents the mean vector characterized by the direction angle µ and r. r (length of µ) indicates phase coherence (measure of population synchrony). p values are results of Rayleigh Tests: Significance indicates non-random distribution of data points. The inner circle represents the Rayleigh critical value (p = 0.05). f, g Results of two-sided multisample statistics on spawning data shown in (a–e). Phase differences in days can be calculated from the angle between the two mean vectors (i.e. 12°= 1 day).

Fig. 5. L-Cry forms differently photoreduced sunlight- and moonlight states.

a Multi-Angle Light Scattering (MALS) analyses of dark-state L-Cry fractionated by size exclusion chromatography (SEC). Black dashed line: normalized UV absorbance, solid line: normalized scattering signal. The molar mass of about 130 kDa derived from MALS (mass signal shown in red) corresponds to an L-Cry homodimer. b Absorption spectrum of L-Cry in darkness (black) and after sunlight exposure (orange). Additional timepoints: Supplementary Fig. 6a. c Dark recovery of L-Cry after 20 min sunlight on ice. Absorbance at 450 nm in Supplementary Fig. 6b. d, e Absorption spectra of L-Cry after exposure to naturalistic moonlight for different durations. f Full spectra of dark recovery after 6 h moonlight. Absorbance at 450 nm: Supplementary Fig. 6d. g Absorption spectrum of L-Cry after 6 h of moonlight followed by 20 min of sunlight. h Absorption spectrum of L-Cry after 20 min sunlight followed by moonlight first results in dark-state recovery. Absorbance at 450 nm: Supplementary Fig. 6e. i Absorption spectrum of L-Cry after 20 min sunlight followed by 4 h and 6 h moonlight builds up the moonlight state. j Model of L-Cry responses to sunlight (orange), moonlight (green) and darkness (black). Only transitions between stably accumulating states are shown. Absorbances in (b–i) were normalized when a shift in the baseline occurred between different measurements of the same measurement set, which is then indicated on the Y-axis as “normalized absorbance”.

Fig. 6. Naturalistic moon- and sunlight impact differently on L-Cry localization and levels.

a,a′,a″ Overview of sampling timepoints. 16h day (light) and 8h night (dark or moonlight) per 24 h, with 8 nights of moonlight per month. NM and FM-1 have the same light regime, but are named differently for accuracy as they refer to different days relative to the lunar month. b relative L-Cry levels at indicated timepoints, as determined by western Blot. Individual data points as well as mean ± SEM are shown. Ordinary one-way ANOVA: p < 0.0001; adjusted p-values of Tukey’s multiple comparison test: FM-1 zt0–10 min vs FM-1 zt 8: p < 0.0001, FM7 zt0–10 min vs FM7 zt8: p < 0.0001, FM-1 zt0–10 min vs FM7 zt 0–10 min: p = 0.9141. c Representative Western Blot used for quantification in (b), see Supplementary Fig. 7 for all other. In addition, in the context of further investigations such Western blots of identical or similar conditions were performed completely independently meanwhile more than three times with highly consistent results. d P. dumerilii head. Dashed ovals designate the oval-shaped posterior domains between the posterior eyes. Green dots: L-Cry+ cells. ae, anterior eye; pe, posterior eye. e–g Confocal single layer (1.28 µm) images of worm heads stained with anti-L-Cry antibody (green) and HOECHST (magenta: nuclei). White rectangles: areas of the zoom-ins presented below. e′–g‴ zoomed pictures of the areas depicted in e–g. Arrows: predominant nuclear L-Cry, arrowheads: predominant cytoplasmic L-Cry. Scale bars: 10 µm. Overview images with nuclear stain: Supplementary Fig. 8a–c. For a complete set of examples from a randomly chosen experimental repetition see Supplementary Fig. 10. In addition, in the context of further investigations such immunohistochemical stainings of worm heads from similar conditions were performed completely independently meanwhile more than three times with highly consistent results. h quantification of subcellular localization of L-Cry as nuclear/cytoplasmic ratio at indicated timepoints. Individual data points as well as mean ± SEM are shown. p values: two-tailed t-test. NM zt0 –10 min vs NM zt0 + 10 min: p = 0.0019, NM zt0 –10 min vs FM7 zt0–10 min: p = 0.3837, NM zt0 + 10 min vs FM7 zt0 −10 min: p < 0.0001. Biological replicates: NM zt0 −10 min n = 22; NM zt0 + 10 min n = 18; FM7 zt0 −10 min n = 16. For quantification as categorical data, see Supplementary Fig. 8a′–f.

Fig. 7. The entrainment of the monthly oscillator requires the detection of a specific moon phase.

a Representation of the presence of the moon on the sky depending on moon phase. As a full cycle of the moon around the earth takes on average 24.8 h the presence of the moon relative to the sun shifts every night by ~49 min, indicated by the green diagonal. Worms need to specifically detect the full moon phase for circalunar oscillator entrainment, which requires that they can realize when a specific light (moonlight) starts and ends. b L-Cry’s function as moonlight duration and intensity detector for circalunar clock entrainment. L-Cry’s biochemical property to only reach the full moonlight state after extended periods of (naturalistic) moonlight illumination allows for a discrimination of moonlight duration. As moon phases are characterized by the duration (and intensity) of the moon on the night sky, moonlight exposure duration translates into moon phase detection. When L-Cry is in its moonlight state it permits (thick green arrow) efficient entrainment of the circalunar oscillator via a yet unidentified photoreceptor X, while L-Cry (partly) blocks (dotted line) the light for circalunar entrainment when it is in its sunlight state. L-Cry might also provide minor information as entraining photoreceptor to the circalunar oscillator (thin green arrow).