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

基于先前的工作，我们认为鞭毛蛋白的n -糖基化对于reinhardtii C.细胞粘附在表面上至关重要（Xu et al., 2020）。下一步，我们研究了FMG1-B和FMG1-A蛋白在粘附和滑动中的功能。这些高度N糖基化和o糖基化的鞭毛蛋白可能参与形成围绕着莱茵鞭毛的厚糖萼。插入fmg1b和CRISPR/Cas9 fmg1a-fmg1b双突变体的实验挑战了仅FMG1-B是粘附和滑动所必需的（Shih et al. 2013）。我们的数据表明，N-和o -糖基化鞭毛蛋白FMG1-B和FMG1-A对有效粘附至关重要，但对滑动却没有作用。使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损失，留下弥散的残余鞭毛被。因此，FMG1-B和FMG1-A确实强烈参与糖萼的形成，这与它们在表面粘附细胞的重要性是一致的。我们的目标是扩展这项工作，并通过CRISPR/Cas9在常见的cc125遗传背景下产生fmg1a和fmg1b基因的单敲除和双敲除突变体。这对于fmg1a和fmg1b单突变体以及双突变体在粘附、滑动和其他表型方面的独立和比较分析是很重要的。FMG1-B也将在野生型特异性FMG1-B和位点定向突变版本的基因表达中被拯救，这些基因的N和o糖基化位点发生了改变。这将允许确定特定的N-和o -糖基化位点对粘附的重要性。粘附性将通过无偏自动采集的TIRF显微镜进行分析。此外，TIRF显微镜将用于测量IFT和滑动速度。粘附力将通过原子力显微镜（AFM）和微移液管力测量来测量。为了确定在没有FMG1-B和FMG1-A提供滑行能力的情况下，哪些鞭毛膜蛋白与表面接触，我们将确定fmg1a-fmg1b双突变体在微球从膜表面易位过程中鞭毛糖蛋白的分离，如先前所述（Kamiya et al. 2018）。候选基因将在WT和fmg1a-fmg1b双突变背景下被敲除，并进一步分析如上所述。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。我们设想，这项工作将揭示C. reinhardtii粘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.