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-糖基化对于C. reinhardtii细胞粘附到表面上（Xu等，2020年）。下一步，我们研究了FMG 1-B和FMG 1-A蛋白在粘附和滑动中的功能。这些高度N-和O-糖基化的鞭毛蛋白可能参与形成C.莱氏鞭毛虫使用插入fmg 1 B和CRISPR/Cas9 fmg 1 a-fmg 1 B双突变体的实验挑战了粘附和滑动仅需要FMG 1-B的事实（Shih等人，2013）。我们的数据表明，N-和O-糖基化鞭毛蛋白FMG 1-B和FMG 1-A是必要的有效的粘附，但不是滑行。使用低温电子断层扫描，FMG 1-A和FMG 1-B的双重敲除导致剥离的糖蛋白密度的损失，留下弥漫的剩余鞭毛外套。因此，表明FMG 1-B和FMG 1-A确实强烈参与糖萼形成，这与它们在表面上粘附细胞的重要性一致。我们的目标是扩展这项工作，并在共同的cc 125遗传背景下通过CRISPR/Cas9产生基因fmg 1a和fmg 1b的单和双敲除突变体。这对于允许fmg 1a和fmg 1b单突变体和双突变体在粘附、滑动和其他表型方面的独立和比较分析是重要的。FMG 1-B也将在野生型特异性fmg 1 B以及具有改变的N-和O-糖基化位点的基因的定点突变形式的表达中被拯救。这将允许确定粘附的特定N-和O-糖基化位点的重要性。使用无偏自动采集，通过TIRF显微镜分析粘附。此外，TIRF显微镜将用于测量IFT和滑动速度。将通过原子力显微镜（AFM）和微量移液器力测量来测量粘附力。为了确定在不存在提供滑动能力的FMG 1-B和FMG 1-A的情况下哪些鞭毛膜蛋白与表面接触，我们将鉴定在微球易位期间从fmg 1 a-fmg 1 B双突变体的膜表面分离的鞭毛糖蛋白，如先前所述（Kamiya et al. 2018）。将在WT和fmg 1a-fmg 1b双突变体背景中敲除候选基因，并如上所述进一步分析。此外，鞭毛的糖蛋白突变体将结构可视化的低温电子断层扫描。我们设想，这项工作将揭示的结构和功能机制的粘附和滑翔在C。莱因哈德氏菌

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.