Decoration of spindle microtubules with Dynein: evidence for uniform polarity.

Abstract

Studies were conducted to determine whether the microtubules present within native spindles isolated from eggs of the surf clam, Spisula solidissima, could bind dynein obtained from axonemes of Tetrahymena thermophila . SDS gel electrophoresis revealed that the high molecular weight polypeptides that make up dynein cosedimented with the isolated spindles . Moreover, the ATPase activity of dynein bound to the spindle microtubules was stimulated approximately sevenfold . The birefringence retardation of spindles incubated without dynein decreased from 1 .4 nm to an undetectable level within 45 min, whereas that of spindles incubated for the same period of time with dynein was 1 .0 nm,^"70% of its initial value, thereby indicating that dynein stabilized spindle birefringence. Ultrastructural analysis revealed that each spindle microtubule was decorated with four to seven dynein arms attached by their "B" end, that which cross-bridges the B-subfiber within native axonemes . In addition, the polarity of the spindle microtubules could be determined by the orientation of the bound dynein arms . The results of these studies suggest that the half-spindle is composed of microtubules possessing the same polarity .Microtubules possess structural polarity, a property which may be involved in their ability to elicit directional, intracellular movements. Within the axoneme, microtubule doublets are of the same polarity, with their assembly or "plus" end located distal to the cell body (1, 5, 7) . The dynein arms on the Asubfiber of one microtubule doublet cyclically cross-bridge the B-subfiber of the adjacent doublet (15), causing it to slide toward the distal end of the axoneme (44) . Accordingly, the direction of microtubule sliding may be a manifestation of intrinsic microtubule polarity .It has been proposed that, during mitosis, sliding between microtubules of opposite polarity occurs and results in the separation of the mitotic poles (31,34,38) and in the procession of the chromosomes to the poles (34,38) . Recent studies involving serial sections and subsequent microtubule reconstruction of the central spindle of Diatoma vulgare during metaphase and anaphase demonstrate a change in microtubule profile, indicating that two groups of interdigitating, antiparallel microtubules may slide with respect to each other and result in spindle elongation (32,33,35) .Microtubule sliding within the mitotic apparatus would require the presence of a force-generating protein, and evidence exists for the participation of a dyneinlike ATPase in generating mitotic movements . For example, spindles isolated from sea urchin eggs contain both Mg"-stimulated ATPase activity and

Interaction of chlamydomonas dynein with tubulin

“…4a). B-end binding is characterized by an electron-transparent gap separating the dynein arm from the wall of the microtubule [Telzer and Haimo, 1981;Haimo and Fenton, 19841. In contrast, when dynein was added to a preparation of preassembled microtubules, the arms bound preferentially by their A ends (Fig.…”

“…4b), even with dynein present in excess to saturate the microtubules. A-end binding is characterized by the direct attachment of the arm to the wall of the microtubule [Telzer and Haimo, 1981;Haimo and Fenton, 19841. There are three possible explanations for the preferential attachment of the B end of the arm to microtu- Mass ratio values represent the mean f standard deviation for the ratio of the sum of the areas under the dynein heavy-chain peaks to the sum of the areas under the a and 0 tubulin peaks. ATPhanadate:…”

Substructure of the outer dynein arm

“…appear in traditional thin-section images (2). The "A end" (65) of the arm is depicted as a straight component that extends diagonally outward from the A microtubule, and the "B end" is represented as a larger hook-shaped component that extends towards the B microtubule of the adjacent doublet. The B end does not ordinarily appear to make contact with the B microtubule, leaving an interdoublet "gap.…”

Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles.

“…There have been numerous reports on the presence of dynein ATPase and dyneinlike ATPase activities in the mitotic apparatus of dividing ceils (see Pratt et al, reference 28, for example and brief review) as well as identification by histochemical methods of ATPase activity on microtubules in situ in nerve ceils (30). In addition, the finding that exogenous dynein from flagella binds to brain microtubules with the same periodicity found in axonemes (15) and that dynein from cilia binds to spindle microtubules (35) has suggested that dynein may function in association with cytoplasmic microtubules (16,23). On the basis of these observations it has been proposed that the force-generating molecules for motility may be endogenous, microtubule-based proteins such as dynein or other associated proteins with ATPase activity.…”