Molecular structure of dynein and motility of a giant sperm axoneme provided with only the outer dynein arm

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is chara cterized by an extraordinarily high number of microtubule doublets (up to 2 ,500) arranged in double parallel spirals. Doublets of the inner row of eac h spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the out er arm, and no structure related to the central pair/radial spoke complex i s present. When analyzed by quick-freeze, deep-etch electron microscopy, th e structure of the dynein arms was shown to share the same organization des cribed in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus f lavus, since the foot region of the arms displays a globular extra-domain t hat is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. S DS-page suggested the presence of a sing e dynein high molecular weight ban d or, in the gels with the best electrophoretic resolution, of two very clo sely spaced bands. This polypeptide positively reacted with a polyclonal an tibody raised against a specific amino acid sequence located in the phospha te-binding loop of the dynein catalytic site. Dynein heavy chain-related DN A sequences corresponding to the catalytic phosphate-binding region were am plified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but th ey were unable to undergo processive movement in vitro. They propagated a t hree-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and mol ecular data are also discussed. (C) 2001 Wiley-Liss, Inc.