Structure and function of N- and O-glycosylated flagellar proteins in adhesion and gliding in Chlamydomonas reinhardtii

基于以前的工作，我们认为鞭毛蛋白的N-糖基化是将莱茵梭菌细胞附着到表面的关键(Xu等人，2020)。在下一步，我们研究了FMG1-B和FMG1-A蛋白在黏附和滑动中的功能。这些高度N-和O-糖基化的鞭毛蛋白可能参与形成围绕莱茵哈迪尔铁虫鞭毛的厚厚的糖萼。插入fmg1b和CRISPR/Cas9 fmg1a-fmg1b双突变体的实验挑战了FMG1-B单独对黏附和滑动是必需的(Shih等人。2013)。我们的数据表明，N-和O-糖基化鞭毛蛋白FMG1-B和FMG1-A对有效的黏附是必需的，但对滑行不是必需的。利用低温电子断层扫描，FMG1-A和FMG1-B的双重敲除导致剥离的糖蛋白密度丢失，留下弥漫的残留鞭毛。因此，FMG1-B和FMG1-A确实强烈地参与了糖萼的形成，这与它们在细胞表面黏附细胞的重要性一致。我们的目标是扩大这项工作，并在共同的cc125遗传背景下通过CRISPR/Cas9产生fmg1a和fmg1b基因的单敲除和双敲除突变。这对于允许对fmg1a和fmg1b单突变体以及双突变体的粘附性、滑移性和其他表型进行独立和比较分析是重要的。FMG1-B也将在野生型特异的fmg1b以及带有改变的N-和O-糖基化位点的基因的定点突变版本的表达中被挽救。这将允许确定特定的N-和O-糖基化位点对粘附性的重要性。粘附性将由TIRF显微镜使用无偏自动采集进行分析。此外，还将使用TIRF显微镜测量IFT和滑行速度。附着力将通过原子力显微镜(AFM)和微吸管力测量来测量。为了确定哪些鞭毛膜蛋白在没有FMG1-B和FMG1-A提供滑动能力的情况下与膜表面接触，我们将像Kamiya等人先前描述的那样，在微球移位过程中在fmg1a-fmg1b双突变体中鉴定鞭毛糖蛋白从膜表面分离。2018年)。候选基因将在wt和fmg1a-fmg1b双重突变背景中被敲除，并如上所述进行进一步分析。此外，糖蛋白突变体的鞭毛将通过低温电子断层扫描进行结构可视化。我们预计，这项工作将揭开莱茵哈迪尔乳杆菌黏附和滑动的结构和功能机制。

Based on previous work, we suggest that N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces (Xu et al., 2020). In a next step, we addressed the function of the FMG1-B and FMG1-A proteins in adhesion and gliding. These highly N- and O-glycosylated flagellar proteins are likely involved in forming the thick glycocalyx surrounding C. reinhardtii flagellar. Experiments with an insertional fmg1b and a CRISPR/Cas9 fmg1a-fmg1b double mutant challenged that FMG1-B alone is required for adhesion and gliding (Shih et al. 2013). Our data rather indicated that the N- and O-glycosylated flagellar proteins FMG1-B and FMG1-A are essential for efficient adhesion but not for gliding. Using cryogenic electron tomography, the dual knock-out of FMG1-A and FMG1-B resulted in a loss of the stripped glycoprotein density, leaving behind a diffuse remaining flagellar coat. Thus indicting, that FMG1-B and FMG1-A are indeed strongly involved in glycocalyx formation, consistent with their importance for adhering cells on surfaces. It is our aim to extent this work and generate single and double knock-out mutants of the genes fmg1a and fmg1b via CRISPR/Cas9 in a common cc125 genetic background. This is important to allow independent and comparative analysis of fmg1a and fmg1b single mutants and of the double mutant in regard of adhesion, gliding and other phenotypes. FMG1-B will be also rescued in expression with wildtype specific fmg1b as well as site-directed mutated versions of the gene with altered N- and O-glycosylated sites. This will allow determining the significance of specific N- and O-glycosylation sites for adhesion. Adhesion will be analyzed by TIRF microscopy using unbiased automatic acquisition. In addition, TIRF microscopy will be employed to measure IFT and gliding velocities. Adhesion forces will be measured via atomic force microscopy (AFM) and micropipette force measurements. To determine which flagella membrane proteins are in contact with the surface in absence of FMG1-B and FMG1-A providing gliding ability, we will identify flagellar glycoproteins detaching during microsphere translocation from the membrane surface in a fmg1a-fmg1b double mutant as previously described by (Kamiya et al. 2018). Candidate genes will be knocked out in a WT and fmg1a-fmg1b double mutant background and further analyzed as outlined above. Moreover, flagellar of glycoprotein mutants will be structurally visualized by cryogenic electron tomography. We envision that this work will unravel the structural and functional mechanism of adhesion and gliding in C. reinhardtii.