Sun- versus moonlight responses and molecular interactions of a marine Cryptochrome

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Many marine organisms time their maturation and reproduction according to the lunar cycle using an inner monthly calendar, also referred to as circalunar clock or circalunar oscillator. The marine bristle worm Platynereis dumerilii (Pdu) has a light-sensitive cryptochrome (Cry) protein, called L-Cry, that can discriminate between moonlight and sunlight and between different moon phases. This enables the worm to synchronize its reproduction to the full moon phase via its circalunar clock. We previously showed that L-Cry can form three distinct molecular states with unique biochemical and functional properties: 1. a dimeric, predominantly nuclear, dark state with its light-sensing cofactor flavine adenine dinucleotide (FAD) in the fully oxidized form, 2. a monomeric, predominantly cytosolic, photoactivated sunlight state with a fully reduced anionic FAD°- radical and 3. a half-reduced, predominantly nuclear, moonlight state. Here, we will further elucidate the molecular mechanisms underlying the differential responses of L-Cry to sunlight and moonlight, using a combination of protein biochemical, spectroscopic, 3D-structural and cell-based approaches. Our specific aims are to understand the molecular basis of the exceptionally high light sensitivity of L-Cry that enables it to sense very dim moonlight, to uncover the molecular structure of the enigmatic L-Cry moonlight state, and to dissect conformational rearrangements and intermediate states occurring during the transitions between the dark-, sunlight- and moonlight states of L-Cry. Furthermore, we will determine the upper and lower limits of the light intensity ranges, that lead to the formation of functional moonlight- or sunlight states of L-Cry. This will enable us to predict the impact of anthropogenic artificial nocturnal light pollution on L-Cry moonlight signalling and on the circalunar behaviour of the worms. To elucidate the hitherto unknown molecular basis of circalunar oscillators, we will also analyse L-Cry’s downstream signalling to the circalunar clock. To this end, we will search for new L-Cry interactors using pulldown/mass spectrometry, and mechanistically and functionally characterize their interactions with L-Cry in vitro and in vivo.