Research Projects

Ubiquitination is a post translational mechanisms wherein ubiquitin molecules are attached to lysine residue in a target protein which can regulate cellular trafficking, protein degradation or signal transduction. As no proteosome is present in the flagella, the proteins that get ubiquitinated are generally trafficked out of the cilia in the cell body. The ubiquitin-proteasome system (UPS) maintains cellular homeostasis by regulating protein turnover, trafficking, and quality control. Ubiquitination, a reversible post-translational modification, involves the covalent attachment of ubiquitin to lysine residues via a cascade of E1, E2, and E3 enzymes. Distinct ubiquitin chain linkages determine functional outcomes K48-linked chains typically signal proteasomal degradation, while K63-linked chains mediate non-proteolytic roles such as intracellular trafficking, DNA repair, and signalling. Flagella are dynamic microtubule-based organelles essential for motility and sensory functions. In Chlamydomonas reinhardtii, flagellar assembly and disassembly are tightly regulated processes involving precise control of protein synthesis, transport, and degradation. Recent evidence implicates ubiquitination in flagellar dynamics, especially during disassembly and quality control, yet mechanistic details remain poorly understood. We hypothesize that ubiquitination regulates flagellar proteome turnover and protein quality control, particularly through specific ubiquitin linkages and interactions with trafficking machinery. Our objectives are to: (1) investigate how ubiquitination governs flagellar protein turnover and quality control; (2) define the types of ubiquitin linkages involved (e.g., K48 vs. K63); (3) identify key ubiquitinated proteins during flagellar disassembly; (4) assess how trafficking mutants (e.g., fla10, fla8, and BBSome mutants) alter ubiquitination patterns To test this, we will induce flagellar disassembly using sodium pyrophosphate (NaPPi), followed by isolation of flagella and detection of ubiquitinated proteins via immunofluorescence and western blotting. Tandem Ubiquitin Binding Entities (TUBEs) with specificity for K48 and K63 linkages will enrich ubiquitinated proteins for mass spectrometry, enabling identification of ubiquitination sites, chain types, and interaction networks. Comparative analysis of wild-type, IFT-defective (fla10, fla8), and BBSome mutant strains will reveal how disrupted trafficking affects ubiquitin-mediated protein turnover. Proteomic data will be integrated to build a regulatory map of ubiquitination in flagellar maintenance. This research will provide fundamental insights into how the UPS regulates ciliary structure and function. Understanding ubiquitin-mediated regulation of flagella may offer novel perspectives on the cellular basis of human ciliopathies and inform therapeutic strategies targeting ubiquitin pathways.