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O-GlcNAcylation Levels Regulate Primary Ciliary Length by Promoting Axoneme Disassembly and Exploring Chlamydomonas reinhardtii as a Synthetic Template
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The sensory organelle primary cilium is involved in sensing and transducing relevant signaling cascades in almost all cells of our body. These ciliary-mediated pathways impact cellular homeostasis and metabolism profoundly. However, it is almost entirely unknown whether the cellular metabolic state affects the assembly of cilia. This study investigates how O-linked β-Nacetylglucosamine (O-GlcNAc), a sensor of cellular nutrients, regulates ciliary length. Pharmacological or genetic inhibition of O-GlcNAcylation led to longer length cilia and vice versa. Further biochemical assays revealed that both α-tubulin and HDAC6 (histone deacetylase 6) were O-GlcNAcylated in vivo. In vitro enzymatic assays showed that O-GlcNAcylation of either tubulin or HDAC6 promoted microtubule disassembly, which potentially caused ciliary shortening. Taken together, these results uncovered a negative regulatory role of O-GlcNAc in modulating the ciliary microtubule assembly. The crosstalk between O-GlcNAc and cilium is likely critical for fine-tuning a cellular response to nutrients, such as glucose, amino acids. Flagella of green algae, Chlamydomonas reinhardtii, have similar structure and function as mammalian primary cilia. Structurally, a flagellum consists of a microtubule-based axoneme surrounded by a specialized membrane extended from the cell membrane. An axoneme is composed of 9 doublet microtubules (DMTs) surrounded by two central singlet microtubules. The beating of the Chlamydomonas flagella relies on the force-generating motility appendages, including outer and inner dynein arms, radial spokes, and dynein regulatory complex (DRC) assembled onto DMTs in a highly specific and periodic manner. The highly organized and regularly arranged structures of DMTs make them an excellent natural template for synthesizing biomolecules by fusing target proteins with axoneme proteins. The concept was proven with successful transport and localization of tagged inner junction protein FAP20 and radial spoke protein RSP3 into the inner junction and radial spokes of flagella. This project tested whether Chlamydomonas could be utilized as a template for synthetic biology by inserting fusion proteins FAP20-TEV-Beta-lactamase (Bla), estrogen receptor alpha (ER) and RSP3-TEV-Bla, ER. These results confirmed that flagella are capable of assembling protein arrays. The RSP3-TEVBla and FAP20-TEV-Bla attached on the isolated flagella axoneme are enzymatically active; the flagella-bounded beta-lactamase was able to recycle for reuse; repeated deflagellation and regeneration can be used to increase the yield of tagged protein; RSP3-ER and FAP20-ER are able to bind and remove estrogen compounds. Altogether, the data indicate that flagella axoneme can serve as a template to synthesize biomolecules.
Tian, Jie (2019). O-GlcNAcylation Levels Regulate Primary Ciliary Length by Promoting Axoneme Disassembly and Exploring Chlamydomonas reinhardtii as a Synthetic Template. Doctoral dissertation, Texas A&M University. Available electronically from