Tektins from echinoderm flagella were analyzed for microheterogeneity, self-associations and association with tubulin, resulting in a general model of tektin filament structure and function applicable to most eukaryotic cilia and flagella. Using a new antibody to tektin consensus peptide RPNVELCRD, well-characterized chain-specific antibodies and quantitative gel densitometry, tektins A, B and C were found to be present in equimolar amounts in Sarkosyl-urea-stable filaments. In addition, two isoforms of tektin A are present in half-molar ratios to tektins B and C. Cross-linking of AB filaments indicates in situ nearest neighbor associations of tektin A1B and A2B heterodimers, -trimers, -tetramers and higher oligomers. Soluble purified tektin C is cross-linked as homodimers, trimers and tetramers, but not higher oligomers. Tektin filaments associate with both loosely bound and tightly bound tubulin, and with the latter in a 1:1 molar ratio, implying a specific, periodic association of tightly bound tubulin along the tektin axis. Similarly, in tektin-containing Sarkosyl-stable protofilament ribbons, two polypeptides (∼67/73 kDa, homologues of rib72, efhc1 and efhc2) are present in equimolar ratios to each other and to individual tektins, co-fractionating with loosely bound tubulin. These results suggest a super-coiled arrangement of tektin filaments, the organization of which has important implications for the evolution, assembly and functions of cilia and flagella.
Bibliographical noteFunding Information:
This work was supported by grants GM 20644 (NIH) to R.E.S., GM 35648 (NIH) and DIR9602237/BIR9113444 (NSF) to R.W.L., and a UROP Fellowship (University of Minnesota) to P.W.S. We thank Dr. Michael Hadfield, Director of the Kewalo Marine Laboratory, University of Hawaii, Dr. Ian Gibbons and the late Dr. Robert E. Kane, former director of that institution, for their accommodations, hospitality and advice during periods of this work. We thank Richard Chock for collection of sea urchins, Dr. Andre Thierault (Division of Medical Technology, University of Hawaii School of Medicine) for use of his laboratory facilities, Gwen Prior and Nicole Lemieux for technical assistances and Drs. David Hamilton, Paul Lefebvre, Mary Porter and Carolyn Silflow for stimulating scientific discussions. Finally, we thank the reviewers of this work for their valuable comments.
- Coiled coils