Eukaryotic cilia and flagella are ancient cell organelles with both motile and sensory functions, essential for organismal development, reproduction and normal cell function. Unlike cytoplasmic microtubules (MTs), ciliary & flagellar doublet MTs are extremely stable and asymmetric, having complex periodic binding sites for associated dynein motors and components for regulation and signaling. This project focuses on doublet MTs, and specifically on a set of three protofilaments from A-tubules that can be isolated as long, hyper-stable "ribbons", composed of several proteins including tubulin and an integral filament of tektins. The location of tektin-ribbons in doublet MTs is unproven and controversial, and the solution could have profound implications. The most rapid, effective means to understanding the structure-function of tektin-ribbons is an integrated approach of biochemistry, state-of-the-art electron microscopy (EM) and cryo-electron tomography (cryo-ET) applied to sea urchin sperm flagella. The Objectives are: (1) To determine both the precise location of the ribbon in doublet MTs, and the position of the tektin filament within the ribbon. (2) To determine the high-resolution structure of the tektin filament. (3) To identify the tektin-binding domains in tubulin and tektin-bound inner dynein arms. The findings of this proposal will challenge current dogma and develop entirely new concepts for ciliary/flagellar motility and regulation. Based on the PI?s published research and preliminary studies, the PI?s hypothesis suggests the following original concepts: (i) That tektin-ribbons form a conserved core-skeleton of ciliary & flagellar axonemes. (ii) That tektins act as molecular rulers to determine the periodic binding sites of the effector molecules. (iii) That tektins interact with inner dynein arm motors, forming load-bearing cables that impact the mechanical and biophysical parameters crucial for ciliary & flagellar motility. (iv) That tektin filaments stabilize doublet MTs in their dynamic motions and during tubulin-turnover.
Broader Impacts of the Project This project provides excellent opportunities for the training of future educators, engineers, scientists and technicians. The PI has demonstrated success in recruiting and training under-graduate and high school students, as well as graduate students, including significant numbers of women and under-represented minorities. Future recruitment will come from the PI?s participation in the University of Minnesota Undergraduate Research Opportunities Program, the Multicultural Summer Research Opportunities Program, and local community colleges (a principal gateway to professional training for many minorities). This activity will introduce undergraduates to scientific research. They will learn standard and advanced techniques of protein chemistry and electron microscopy (EM) from the PI and through workshops given by the University's Characterization Facility. The PI will mentor their training, including the development of critical thinking, interpretive skills, writing and speaking. Moreover, the project will foster interactions and interdisciplinary training with the collaborator, Dr. Daniela Nicastro, an expert in cryo-ET and a role model for female scientists. An especially valuable benefit of this training for undergraduates is this collaborative training provided in modern EM techniques and image processing, which provide skills beyond biology. Outside the specific field of cilia & flagella this study will have broad impacts in other areas of cell biology (e.g., cytokinesis, intermediate filaments, Hedgehog signaling), on evolutionary biology (ancestral relationship of tektin to intermediate filament protein, and speciation of insects and other phyla), and on nano-technology and nano-fabrication. In terms of the latter point, the PI is involved at several levels with engineers and material scientists to foster interdisciplinary training and intellectual exchange among students of biology and the physical sciences.
