Molecular Mechanisms of in vivo Channelrhodopsin Regulation in Chlamydomonas reinhardtii

通道视紫红质-1 （ChR1）和ChR2是模式绿藻衣藻的主要光感受器，其中ChR1在营养细胞中含量较高。尽管ChRs在光遗传学中得到了广泛的应用，并且对其结构和性质的详细了解也越来越多地来自于对表达ChRs的研究，但对其在藻类中的调控和信号传导知之甚少。最近，通过Ca2+为基础的反馈回路调节的多个ChR1磷酸化被证明是适应不同生理条件的光致敏感性的一个重要组成部分。此外，光严格控制ChR水平。每日转录组数据显示，它们的转录水平在一天的第一个小时内急剧下降。同时，在蛋白质水平上，无论环己亚胺存在与否，都会发生波长和光强度依赖性的快速下降。控制ChR1水平的光感受器网络的某些部分已经出现，并指向蓝光/紫外线范围内的光感受器吸收。光致蛋白（Phot）敲除突变体表明，Phot参与光强度依赖性的ChR1降解，而ChR2降解不受影响。对光缺失突变体的分析指出存在进一步的光依赖成分。在本提案中，我们旨在利用CRISPR/Cas9产生的蓝光/紫外线光感受器单/双缺失菌株，更详细地分析控制ChR水平的光基网络。已知关键光信号因子的突变也将包括在我们的分析中。藻类的ChR特性和调控机制在一些重要的方面有所不同，例如Ca2+依赖性，与在不同的表达系统中观察到的不同。这可能是由于与其他蛋白质的相互作用。表达的功能性ChRs为同型二聚体。我们的数据表明，在藻类中，这两种chr也形成特定的高分子质量复合物（hmcs）。弱光刺激下的趋光性需要信号放大，其基础目前仍完全未知。ChRs与其他蛋白质的复杂形成被认为在这里起着核心作用，但从未被实验证明。我们所描述的新型hmcs可能包含这些长期寻求的蛋白质。另一方面，hmcs的一个亚群可能与ChR降解有关。因此，在本项目中，我们也将分离这些hmcs并通过质谱法鉴定它们的蛋白质组成。最后，分析了hmc形成的条件。我们希望通过这一建议来更好地理解ChRs如何启动和控制光致行为以及它们如何根据实际光照条件进行调节的分子机制。

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.