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

• Applicant: Professor Dr. Michael Hippler
• Subject Area: Plant Biochemistry and Biophysics
• Term: since 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 256628857

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.

• DFG Programme: Research Grants