Signalling by a plant-like cryptochrome in diatoms

隐花色素是一种光感受器，存在于所有门中。虽然动植物隐色素的研究由来已久，但植物类隐色素的研究却是最近才发现的。它们可以在硅藻等稻草类动物中找到，也可以在绿藻和一些动物群体中找到，包括硬骨鱼和环节动物。三角褐指藻的CRYP就属于这一类，我们发现它是蓝光感受器。通常，FAD是结合的，伴随着一个天线发色团(亚甲基四氢叶酸)。当在大肠杆菌中过表达CRYP时，FAD处于半还原自由基态(FADH·)，在光谱的黄色和红色范围内显示吸收峰。与CRYP绑定的FADH·是异常稳定的。它可以可逆地还原为完全还原的FADH2，但氧化到完全氧化状态的速度非常慢。这导致了一种假设，即FADH·是CRYP的黑暗状态。植物隐花色素的常见蛋白质相互作用伙伴的基因在硅藻中缺失。因此，信号转导途径是不同的，必须从头开始研究。我们能够确定蛋白质相互作用伙伴，一个可能的转录因子BOLA和一个功能未知的蛋白质，只存在于硅藻和近亲中，ID42612。这些实验是在体外进行的，到目前为止，对体内相互作用和硅藻细胞内蛋白质的定位一无所知。此外，初步实验表明，CRYP以蓝光依赖的方式降解。为了阐明CRYP信号转导中的一些关键步骤，我们想要解决三个问题：(1)通过量化CRYP诱导的基因在蓝光、红光和黄光下的表达水平来证明CRYP在体内处于暗状态，并与WT和CRYP基因敲除突变体的暗状态进行比较。如果FADH·是暗状态，那么黄光和红光也能诱导WT的信号传递，但突变体不能。(Ii)CRYP与ID42612和BOLA的相互作用也应在体内得到证实，并确定蛋白质的定位。这将使用共聚焦显微镜中的BIFC来完成，即通过将GFP的部分分别连接到CRYP和BOLA或ID42612，当足够接近时，这些部分重新组成荧光团。(Iii)CRYP的降解终止了信号转导，ID4612的一个假定功能可能是直接或通过招募酶来降解CRYP，或保护其免受降解。这将使用CRYP-His在大肠杆菌中过表达，在不同的光颜色和黑暗中与三角假单胞菌的全细胞提取物孵育来检测。然后可以通过免疫印迹和抑制剂研究来跟踪降解过程，从而提供有关所涉及的蛋白酶种类的信息。总而言之，该项目将首次深入了解一种类似植物的隐花色素的特殊信号通路。

Cryptochromes are photoreceptors found in all phyla. Whereas animal and plant cryptochromes have been studied for long, plant-like cryptochromes were only discovered recently. They can be found in stramenopiles like diatoms, but also in chlorophytes and some animal groups, including teleosts and annelids. CryP of the diatom Phaeodactylum tricornutum belongs to this group and was shown by us to act as a blue-light photoreceptor. As common, FAD is bound, which is accompanied by an antenna chromophore (methenyltetrahydrofolate). When overexpressing CryP in E. coli, FAD is in its semireduced radical state (FADH•), showing absorbance peaks in the yellow and red range of the spectrum. The FADH• bound to CryP is unusually stable. It can be reversibly reduced to the fully reduced FADH2, but oxidation to the fully oxidised state is extremely slow. This led to the hypothesis that FADH• is the dark state of CryP. Genes for the usual protein interaction partners of plant cryptochromes are missing in diatoms. Thus, the signal transduction pathway is different and has to be examined from scratch. We were able to identify protein interaction partners, a putative transcription factor BolA and a protein of unknown function only present in diatoms and close relatives, ID42612. Those experiments were done in vitro and nothing is known so far about the in vivo interaction and the localisation of the proteins inside the diatom cells. In addition, preliminary experiments have shown that CryP is degraded in a blue-light dependent manner. To elucidate some crucial steps in the signal transduction of CryP we want to tackle three questions: (i) the dark state of CryP in vivo should be proven by quantifying the expression levels of CryP-induced genes under blue light, red light and yellow light in comparison to darkness in WT and knock-out mutants of CryP. If FADH• were the dark state, signalling should be inducible in WT by yellow and red light as well, but not in the mutants. (ii) the interaction of CryP and ID42612 and BolA should be proven in vivo as well, and the localisation of the proteins determined. This will be done using biFC in confocal microscopy, i.e. by coupling parts of GFP to CryP and BolA or ID42612, respectively, that reconstitute the fluorophore when coming close enough. (iii) degradation of CryP ends signalling and a putative function of ID4612 might be degradation of CryP either directly or by recruiting proteases, or protection from degradation. This will be examined using CryP-His overexpressed in E. coli incubated with whole cell extract of P. tricornutum under different light colours and in darkness. The degradation can then be followed by immunoblots and inhibitor studies will provide information on the kind of proteases involved. In summary the project will provide first insights into the peculiar signalling pathway of a plant-like cryptochrome.