New aspects of dynein arm structure and positional relationships

Abstract

The dynein arm is the major ATPase of ciliary and eukaryotic flagellar axonemes. It is responsible for the force transduction that powers sliding of adjacent doublet microtubules, thus leading to the generation of active bends along the ciliary shaft. Using negative stain and thin section electron microscopy, I have been studying various structural aspects of the arm of ciliary axonemes obtained from Tetrahymena thermophila SB 255 (a mucocyst lacking mutant).;Dynein arms observed in the presence of 50 {dollar}\mu{dollar}M AMP-PCP (a non-hydrolyzable ATP analog) are composed of head, body, and cape subunits. The cape and body subunits permanently attach to A subfiber of doublet no. N. The head subunit articulates with the body and transiently attaches to the B subfiber of doublet no. N+1 during cross-bridge formation. Two distinct forms of dynein arm-microtubule attachments are observed: the TRANS and CIS orientations. The TRANS orientation supports a critical step in the mechanochemical cycle of the arm. The CIS orientation is critical in interpreting negative stain images of dynein arms decorating both axonemal and cytoplasmic microtubules.;In the absence of trypsin treatment, membraneless axonemes obtained from Tetrahymena actively extrude their doublet microtubules when treated with 50 {dollar}\mu{dollar}M ATP. Negative stain stereo images of these doublet preparations reveal the positional relationship of outer and inner arm rows of dynein arms to the other structures appended to the doublet microtubule (i.e., radial spoke groups and the interdoublet links).;A computer model of the ciliary doublet has been constructed based on findings concerning dynein arm morphology and positional relationships of the arms to the other attached structures of the microtubule. The model establishes that the 'bouquet' form of dynein observed in solution requires compacting in order to function in the interdoublet space. Confirming observations have been made using inner (pf-23) and outer arm (pf-13A) mutants of Chlamydomonas reinhardtii.