Rigor and relaxed outer dynein arms in replicas of cryofixed motile flagella.
A method of image classification based on multivariate statistics has been developed and applied to freeze-etch images of outer dynein arms (ODAs) from cockerel sperm flagella. Demembranated flagella were cryofixed in three different nucleotide states: rigor (i.e. no added ATP); relaxed (i.e. 1 mM ATP plus vanadate); and active (i.e. 1 mM ATP). Freeze-etch replica fragments from them were coded to conceal their identities and sampled for flagella. From these a total of 6048 individual ODA images were successfully windowed and aligned, covering an angular range of 66 degrees. Each nucleotide condition produced a statistically significant ODA morphology. The relaxed and active morphologies differed only in the angulation of their heads, with the relaxed ODA favouring a more tilted position. The rigor morphology was more distinct and showed a conformational change involving a 12 nm distal shift relative to the A-tubule and highly variable heads and B-links, suggesting an ability to develop tension. Outer arms were classified by discriminant analysis as being either rigor-like, relaxed-like or active-like, and 80% of all ODAs were correctly classified. The misclassification of the remaining 20% indicated morphological heterogeneity in some of the groups. From the rigor group, 8.8% of the ODAs were misclassified as relaxed-like or active-like (i.e. non-rigor-like). It is speculated that this is a consequence of mechanochemical interactions between the B-tubules and the rigor ODAs and/or contaminating ATP remaining after demembranation. Active flagella were found to show all three morphologies, with 5.4% in the rigor conformation and 18.6% in the relaxed conformation. The finding of rigor-like and relaxed-like ODAs in flagella exposed to ATP is discussed in relation to the cross-bridge cycle.
Burgess
S A
SA
Department of Physiology, School of Medical Sciences, University of Bristol, UK.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001717:Birds; D004398:Dyneins / Q000737:chemistry / Q000648:ultrastructure*; D005613:Freeze Etching / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008297:Male; D015999:Multivariate Analysis; D011487:Protein Conformation; D013081:Sperm Motility / Q000187:drug effects; D013082:Sperm Tail / Q000187:drug effects / Q000648:ultrastructure*; D014638:Vanadates / Q000494:pharmacology
D014638:Vanadates; D000255:Adenosine Triphosphate; D004398:Dyneins