Lupetti P, Mencarelli C, Rosetto M, Heuser J E, Dallai R
Dipartimento di Biologia Evolutiva, Università di Siena, Italy.
Cell Motil Cytoskeleton. 1998;39(4):303-17. doi: 10.1002/(SICI)1097-0169(1998)39:4<303::AID-CM5>3.0.CO;2-3.
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.
双翅目昆虫黄单节螨拥有一种独特的精子轴丝，其特征是有多排微管双联体，仅由外侧动力蛋白臂相连，缺乏任何相当于中央微管对/辐条复合体的结构。通过电子显微镜（EM）研究了这些动力蛋白分子的结构。使用EM的快速冷冻、深度蚀刻方法，发现它们与先前描述的外侧动力蛋白臂相似。可以清楚地看到两个球状“头部”，每个头部都有一条裂缝将其细分。“柄”从近端头部延伸，与相邻双联体的B微管接触。与脊椎动物精子的情况不同，柄有时会分支成两条较细的链，在不同位置与B微管接触。用ATP和钒酸盐处理去膜精子细胞会诱导动力蛋白外侧臂的构象变化。这些变化被解释为动力蛋白头部相对于在僵直状态（ATP耗尽后）轴丝中观察到的情况发生旋转的结果。SDS-PAGE表明，该分子的高分子量成分包含一条重链。通过RT-PCR扩增了与催化磷酸结合区域相对应的特定动力蛋白重链相关DNA序列。在所有扩增片段中仅鉴定出一个轴丝动力蛋白序列。使用该序列作为探针在基因组DNA上进行的Southern印迹分析鉴定出两个杂交基因，其中只有一个能够编码功能性产物。因此，遗传分析表明，这种轴丝外侧臂动力蛋白是单个重链亚基的同型二聚体。在体内，该物种的精子以卷曲的形态储存在雌性受精囊中，在那里它们以波浪状运动快速移动。这种运动在体外无法重现，除非精子受到某种机械阻碍而处于弯曲形态。我们提出，在缺乏中央微管对/辐条复合体和内侧臂的情况下，一种曲率依赖性激活作用会触发这些精子的运动。
