Molecular Mechanisms of in vivo Channelrhodopsin Regulation in Chlamydomonas reinhardtii

莱茵衣藻体内通道视紫红质调节的分子机制

• 批准号: 468480235
• 负责人: Professor Georg Kreimer
• 金额: --
• 依托单位: Lehrstuhl für Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/468480235?language=en
• 关键词: Molecular; Mechanisms; vivo; Channelrhodopsin; Regulation

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.

通道视紫红质-1(ChR1)和ChR2是模式绿藻衣藻趋光性的主要光感受器，其中ChR1通常在营养细胞中含量更高。尽管CHRs在光遗传学中得到了广泛的应用，通过对表达的CHRs的研究，人们对它们的结构和性质有了越来越详细的了解，但对它们在藻类中的调控和信号转导知之甚少。最近，通过基于钙离子的反馈环调节的多个ChR1磷酸化被证明是适应不同生理条件的趋光敏感度的重要组成部分。此外，光严格控制着CHR的水平。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，在没有和存在放线菌亚胺的情况下，都会发生快速的波长和光强度依赖性的下降。控制ChR1水平的光感受器网络的一些部分已经出现，并指向在蓝/紫外光范围内吸收光感受器。PTOT基因敲除突变体表明，PTOT参与了光强度依赖的ChR1的降解，而ChR2的降解不受影响。对PhotT缺失突变体的分析表明，还存在更多依赖于光的成分。在这项建议中，我们的目标是利用CRISPR/Cas9产生的蓝光/紫外光感受器的已有光感受器单/双缺失菌株，更详细地分析这个基于光的网络控制CHR水平。已知的关键光信号因子的突变也将包括在我们的分析中。藻类中的ChR特性和调控机制在一些重要方面与在不同表达系统中观察到的不同，如钙依赖。这可能是由于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，在藻类中，除了这两种CHRs外，还形成了特异性的高分子质量复合体(HMMCs)。在弱光刺激下的趋光性需要信号放大，其基础目前仍完全不清楚。CHRS与其他蛋白质的复杂形成被认为在这里发挥着中心功能，但从未被实验证明。我们所描述的新型HMMCs可能含有这些长期被追捧的蛋白质。另一方面，HMMC的一个亚群可能与ChR降解有关。因此，在这个项目中，我们还将分离这些HMMC，并通过质谱学鉴定它们的蛋白质组成。最后，分析了HMMC形成的条件。我们希望通过这一提议有助于更好地理解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条件进行调控。