Determining the Location of Tektins within a Vital Molecular Machine

Additional contributors: Christna Ouche; Dr. Richard W. Linck (faculty mentor)Flagella and cilia play crucial roles in keeping life moving, one of the most easily recognizable flagella being a spermazoon’s tail. These remarkable molecular machines contain a ring of 9 outer doublet microtubules (hollow tubes, 30 nm in diameter) each composed of protofilaments (PFs). Within each microtubule is a hyper-stable, 3-PF “ribbon” containing the integral, filamentous protein tektin. Two hypotheses exist regarding the location of tektin: (i) the dynein-spoke-tektin filament and (ii) the partition model: (i) suggests that tektin forms one of the three PFs. (ii) suggests that a tektin filament lies on top of the ribbon and is not one of the PFs. In this project ribbons were first isolated from Strongylocentrotus purpuratus (sea urchins). Secondly, they were partially digested by several different enzymes and sized (showing degree of digestion) by gel electrophoresis. Electron microscopy verified that the ribbons were still partially intact. Finally, flanking tubulin fragments still loosely associated with the tektin filament were to be identified by mass spectrometry. By comparing these short fragments of tubulin to their known PF structure, we would be able to deduce whether the tektin filament binds to the sides of the flanking tubulin PFs (as in model i) or binds along the inside surface of the ribbon (as in model ii). The optimal digestion has yet to be achieved; future studies aim to optimize these conditions, through use of different digestive enzymes and digestion times, and subsequently distinguish which model is indeed a more accurate representation of tektin location