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 characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each 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 outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified 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 they were unable to undergo processive movement in vitro. They propagated a three-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 molecular data are also discussed.
Mencarelli
C
C
Dipartimento di Biologia Evolutiva, Università di Siena, Siena, Italy.
Lupetti
P
P
Rosetto
M
M
Mercati
D
D
Heuser
J E
JE
Dallai
R
R
D000595:Amino Acid Sequence; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D004175:Diptera / Q000737:chemistry*; D004398:Dyneins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013081:Sperm Motility / Q000502:physiology*; D013082:Sperm Tail / Q000648:ultrastructure*; D013091:Spermatogenesis / Q000502:physiology; D013094:Spermatozoa / Q000648:ultrastructure
D017301:Ca(2+) Mg(2+)-ATPase; D004398:Dyneins