Molecular Mechanisms of in vivo Channelrhodopsin Regulation in Chlamydomonas reinhardtii

在绿色衣原体模型中，视紫红质-1（ChR 1）和ChR 2作为趋光性的主要光感受器发挥作用，其中ChR 1通常在营养细胞中更丰富。虽然ChR广泛用于光遗传学，并且关于其结构和性质的越来越详细的知识来自于表达ChR的研究，但对它们在光遗传学中的调控和信号传导知之甚少。最近，多个ChR 1磷酸化，通过Ca 2+为基础的反馈回路调节，被证明是一个重要的组成部分，在适应趋光敏感性，以适应不同的生理条件。此外，光严格控制ChR水平。昼夜转录组数据显示，它们的转录水平在白天阶段的第一个小时内急剧下降。在平行的蛋白质水平上也发生快速的波长和光强度依赖性的减少，在存在和不存在放线菌酮。控制ChR 1水平的光感受器网络的某些部分已经出现，并指出光感受器在蓝色/紫外线范围内吸收。Phototropin（Phot）敲除突变体揭示Phot参与光强度依赖性ChR 1降解，而ChR 2降解不受影响。Phot缺失突变体的分析指出存在进一步的光依赖性组分。在这项提案中，我们的目标是通过使用CRISPR/Cas9产生的蓝光/UV光感受器的现有光感受器单/双缺失菌株，更详细地分析这种控制ChR水平的光网络。已知关键光信号传导因子的突变体也将包括在我们的分析中。ChR的性质和调节机制在一些重要的方面不同，例如Ca 2+依赖性，从不同的表达系统中观察到的。这可能是由于例如与其他蛋白质的相互作用。表达的功能性ChR是同源二聚体。我们的数据表明，在除ChRs形式也特定的高分子量复合物（HMMCs）。弱光刺激下的趋光性需要信号放大，其基础目前还完全未知。ChR与其他蛋白质的复合物形成被认为在这里发挥了中心功能，但从未在实验中显示。我们所描述的新型HMMC可能含有这些长期寻找的蛋白质。另一方面，HMMC的亚组可能与ChR降解有关。因此，在本项目中，我们也将分离这些HMMC，并通过质谱法鉴定其蛋白质组成。最后，将分析HMMC形成的条件。我们希望通过这一建议，更好地理解ChRs如何启动和控制趋光行为的分子机制，以及它们如何根据实际的光照条件进行调节。

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.