2 PhD projects offered in the IPP summer call Molecular Mechanisms in Genome Stability & Gene Regulation

Scientific background

Many marine organisms time their reproduction according to the lunar cycle using an inner monthly calendar referred to as circalunar clock (Mat et al, 2023). Unfortunately, anthropogenic disturbances (e.g. nocturnal light pollution) are increasingly disturbing these moon-controlled reproduction cycles, imposing a threat to species survival and the marine ecosystem. Despite their high ecological relevance, the molecular bases of circalunar timing systems are still unknown. 
The marine bristle worm Platynereis dumerilii (Pdu) is an emerging genetically tractable model system for mass-spawning marine animals. We recently showed that  Pdu has a light-sensitive cryptochrome protein (L-Cry), that can discriminate between moonlight and sunlight and even between different moon phases, to enable the worm to synchronize its reproduction to the full moon phase (Pöhn, Krishnan et al, 2022). We found that L-Cry can form three distinct molecular states: 1. a dimeric, predominantly nuclear dark state with a fully oxidized light-sensing FAD cofactor, 2. a monomeric, predominantly cytosolic sunlight state with fully photoreduced FAD and 3. a half-reduced, predominantly nuclear moonlight state (Pöhn, Krishnan et al, 2022). Furthermore, we determined Cryo-EM structures of dark-state and light-activated L-Cry (Vu, Behrmann et al, 2023). To advance our molecular-mechanistic understanding of circalunar timing systems the prospective PhD students will investigate the molecular mechanisms underlying the differential responses of L-Cry to sunlight and moonlight, as well as L-Cry’s interactions with downstream ligands, using a combination of protein biochemical, spectroscopic, 3D-structural, molecular biology and cell-based approaches. 
 

PhD project 1: Molecular mechanisms underlying L-Cry’s responses to moonlight and sunlight

The aims of this project are to understand the molecular basis of the exceptionally high (moon)light sensitivity of L-Cry compared to its Drosophila CRY (dCRY) homolog (Zurl et al, 2022; Czarna et al, 2013; Berndt et al, 2007), to determine the molecular structure of the L-Cry moonlight state, to dissect conformational rearrangements, intermediate states and oligomeric state changes occurring during the transitions between the dark-, sunlight- and moonlight states of L-Cry, and to determine intensity thresholds separating moonlight- and sunlight state formation of L-Cry. These studies will elucidate molecular-mechanistic principles underlying moonlight receptor functions and help us predict the impact of nocturnal light pollution on L-Cry moonlight signaling.

The prospective PhD student will recombinantly express and purify L-Cry proteins (wildtype and mutants), analyse their light-response by time-resolved UV/VIS spectroscopy, analyse light-dependant changes of L-Cry’s oligomerisation state by SEC, native PAGE, MALS and native MS, determine L-Cry crystal structures and be involved in Cryo-EM studies of L-Cry. 

If you are interested in this project, please select Wolf (light) as your group preference in the IPP application platform.

PhD project 2: Molecular interactions and downstream signaling of L-Cry

To elucidate the molecular basis of circalunar oscillators, the PhD student will analyse L-Cry’s downstream signalling to the circalunar clock. To this end, she/he will search for new L-Cry interactors using pulldown/mass spectrometry (MS) under dark, sunlight and moonlight conditions, and mechanistically and functionally characterize their interactions with L-Cry in vitro and in vivo. Additionally, she/he will characterize L-Cry’s interactions with G3BP2 and YTHDF, which we previously identified as L-Cry ligands by pulldown/MS. YTHDF binds and decodes N6-methyladenosine (m6A) RNA modifications (Zaccara et al., 2019), whereas G3BP2 homologs are mostly involved in mRNA stability, RNA and protein transport as well as stress granule formation (Jin et al., 2022), indicating a link between L-Cry and RNA-dependant cellular signalling.

The prospective PhD student will recombinantly express and purify L-Cry proteins and L-Cry ligands, conduct pulldown/MS studies, test new ligands for direct binding to L-Cry in Y2H, SEC and pulldown experiments, and analyse the impact of the ligands on L-Cry’s light-response (time-resolved UV/VIS spectroscopy) and oligomerization (SEC, native PAGE, MALS, native MS). Stable L-Cry-ligand complexes will be subjected to structural studies using X-ray crystallography or Cryo-EM and their binding affinities will be determined by ITC, FP or SPR. Depending on the predicted roles of the L-Cry ligands, functional studies will be conducted, e.g. testing YTHDF binding to m6A methylated RNA.

If you are interested in this project, please select Wolf (int) as your group preference in the IPP application platform.

We encourage applications of highly motivated PhD candidates with a strong interest in structural biology, protein biochemistry, biophysics and structure-function relationship and a significant previous exposure to these research areas during their master education. 

Publications relevant to the project

Mat A, Vu HH, Wolf E, Tessmar-Raible K. (2023) All light, everywhere? Photoreceptors at non-conventional sites. Physiology (Bethesda). 2023 Oct 31. Link

Vu HH*, Behrmann H*, Hanić M, Jeyasankar G, Krishnan S, Dannecker D, Hammer C, Gunkel M, Solov’yov IA, Wolf E# and Behrmann E#. (2023) A marine cryptochrome with an inverse photo-oligomerization mechanism. Nat Commun 14, 6918 Link

Poehn B*, Krishnan S*, Zurl M, Coric A, Rokvic D, Häfker NS, Jaenicke E, Arboleda E, Orel L, Raible F, Wolf E # and Tessmar-Raible K # (2022). A Cryptochrome adopts distinct moon- and sunlight states and functions as sun- versus moonlight interpreter in monthly oscillator entrainment. Nat Commun 13: 5220; Link 

Zurl, M., Poehn, B., Rieger, D., Krishnan, S., Rokvic, D., Veedin Rajan, V.B., Gerrard, E., Schlichting, M., Orel, L., and Ćorić, A., et al. (2022). Two light sensors decode moonlight versus sunlight to adjust a plastic circadian/circalunidian clock to moon phase. PNAS 119, e2115725119. Link

Czarna, A., Berndt, A., Singh, H.R., Grudziecki, A., Ladurner, A.G., Timinszky, G., Kramer, A., and Wolf, E. (2013). Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function. Cell 153, 1394-1405. Link

Berndt, A., Kottke, T., Breitkreuz, H., Dvorsky, R., Hennig, S., Alexander, M., and Wolf, E. (2007). A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome. The Journal of biological chemistry 282, 13011-13021. Link

Zaccara, S., Ries, R.J., and Jaffrey, S.R. (2019). Reading, writing and erasing mRNA methylation. Nature reviews. Molecular cell biology 20, 608-624. Link

Jin, G., Zhang, Z., Wan, J., Wu, X., Liu, X., and Zhang, W. (2022). G3BP2: Structure and function. Pharmacological research 186, 106548. Link

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