Molecular Mechanisms of in vivo Channelrhodopsin Regulation in Chlamydomonas reinhardtii

• Applicant: Professor Georg Kreimer
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2021 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 468480235

Channelrhodopsin-1 (ChR1) and ChR2 function as major photoreceptors for phototaxis in the model green alga Chlamydomonas, with ChR1 being usually more abundant in vegetative cells. Although ChRs are widely used in optogenetics and an increasing detailed knowledge about their structure and properties is coming from studies with expressed ChRs, only little is known about their regulation and signaling in alga. Recently multiple ChR1 phosphorylation, regulated via a Ca2+-based feedback loop, was shown as one important component in adapting the phototactic sensitivity to varying physiological conditions. Further, light controls the ChR levels tightly. Diurnal transcriptome data show that their transcript levels dramatically drop within the first hour of the day phase. In parallel also on the protein level a rapid wavelength and light intensity-dependent decrease occurs, in both the absence and presence of cycloheximide. Some parts of a photoreceptor network controlling the ChR1 level are already emerging and point to photoreceptors absorbing in the blue/UV range. Phototropin (Phot) knock-out mutants revealed that Phot is involved in the light intensity-dependent ChR1 degradation, whereas ChR2 degradation is unaffected. Analysis of the Phot deletion mutants point to the presence of further light-dependent components. In this proposal, we aim to analyze this light-based network controlling the ChR levels in more detail by using the already existing photoreceptor single/double deletion strains of blue-light/UV photoreceptors generated by CRISPR/Cas9. Mutants in known key light signaling factors will also be included in our analyses. The ChR properties and regulatory mechanisms in alga differ in some important points, e.g. Ca2+-dependence, from those observed in different expression systems. This could be due to e.g. interactions with other proteins. Expressed functional ChRs are homodimers. Our data show that in alga in addition both ChRs form also specific high-molecular mass complexes (HMMCs). Phototaxis at low light stimuli requires signal amplification, the basis of which is currently still completely unknown. Complex formation of the ChRs with other proteins has been suggested to play here a central function, but has never been experimentally shown. The novel HMMCs described by us might contain these long sought-after proteins. On the other hand, a subgroup of the HMMCs might be related to ChR degradation. In this project, we therefore also will separate these HMMCs and identify their protein composition by mass spectrometry. Finally, the conditions under which the HMMCs are formed will be analyzed. We hope to contribute with this proposal to a better understanding of the molecular mechanisms how ChRs initiate and control phototactic behavior and how they are regulated depending on the actual light conditions.

• DFG Programme: Research Grants