Structural and molecular characterization of dynein in a gall-midge insect having motile sperm with only the outer arm.
The dipteran Monarthropalpus flavus possesses a peculiar sperm axoneme, characterized by multiple rows of microtubular doublets linked by the outer dynein arms only, lacking any equivalent of the central pair/radial spoke complex. The structure of these dynein molecules was studied by electron microscopy (EM). Using the quick-freeze, deep-etch method of EM, they were found to be similar to outer dynein arms described previously. Two globular "heads," each subdivided by a cleft, are clearly discernible. "Stalks" extend from proximal head to contact the B-tubule of the adjacent doublet. Unlike the situation in vertebrate sperm, the stalks sometimes branch into two thinner strands that contact the B-tubule at different sites. Treatment of demembranated sperm cells with ATP and vanadate induces conformational changes in the dynein outer arms. These are interpreted as the result of rotation of the dynein head with respect to what is observed in axonemes in rigor condition (after ATP depletion). SDS-PAGE indicates that the high-molecular-weight complement of this molecule comprises a single heavy chain. Specific dynein heavy chain-related DNA sequences corresponding to the catalytic-phosphate binding region were amplified by RT-PCR. Only one axonemal dynein sequence was identified among all amplified fragments. Southern blot analysis performed on genomic DNA using this sequence as a probe identified two hybridizing genes, only one of which is able to encode a functional product. Thus, genetic analysis indicates that this axonemal outer arm dynein is a homodymer of a single heavy chain subunit. In vivo, spermatozoa of this species are stored in a rolled configuration in female spermatheca, where they move rapidly with a wave-like motion. This movement could not be reproduced in vitro, except when spermatozoa were constrained in a bent configuration by some mechanical impediment. We propose that, in the absence of both the central pair/radial spoke complex and the inner arms, a curvature-dependent activation acts to trigger motility in these spermatozoa.
Lupetti
P
P
Dipartimento di Biologia Evolutiva, Università di Siena, Italy.
Mencarelli
C
C
Rosetto
M
M
Heuser
J E
JE
Dallai
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D048429:Cell Size / Q000235:genetics; D004175:Diptera; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D005614:Freeze Fracturing; D008297:Male; D008854:Microscopy, Electron / Q000379:methods; D008969:Molecular Sequence Data; D013081:Sperm Motility / Q000235:genetics / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure
D004398:Dyneins