ABSTRACT

The kinetochores of isolated HeLa cell chromosomes attached to an electron microscope specimen grid, fixed in formaldehyde, and stained with alcoholic phosphotungstic acid are visible as dark, preferentially stained structures distinct from the chromatin with which they are associated. When unfixed chromosomes are immobilized by attachment to grids and incubated with chick brain tubulin, microtubules are observed to assemble onto the kinetochores. This demonstrates the competence of kinetochores in isolated chromosomes to act in vitro as microtubule assembly sites and suggests that they also possess this capacity in vivo. In addition, the results provide a possible means for isolating and characterizing kinetochores. The kinetochore is the site of attachment for the chromosomal microtubules of the mitotic apparatus. While the kinetochore may not be recognizable as a distinct, morphologically identifiable structure in some organisms (1) or may exist as a diffuse region along the chromosome in others (2, 3), in mammalian cells the kinetochore is a localized, welldefined area from which microtubules arise. Considerable detail of the fine structure of the mammalian kinetochore is known from the study of thin sections in the electron microscope (4-6); characteristically, the organelle consists of a tripartite, disc-shaped structure localized at the primary constriction of the chromosome. However, it is only recently that techniques have been defined which permit electron microscopic visualization of the kinetochore in whole mount chromosomes (7). By application of these techniques to surface spreads of the meiotic chromosomes of the locust (7), hamster (8), and man (9) as well as spreads of mitotic chromosomes of HeLa cells (10) the kinetochore has been revealed to be a preferentially stained structure distinct from the chromatin with which it is associated.There are various sites for the attachment and presumptive assembly of microtubules in eukaryotic cells; among these are the basal bodies of cilia and flagella, centrioles of the mitotic apparatus, and the kinetochores of chromosomes.With the development of methods for the in vitro assembly of brain tubulin (11), it has become possible to study the assembly of microtubules onto these sites. Isolated flagellar axonemes of Chiamydonas (12,13) and sea urchin sperm (12) as well as isolated basal bodies of Chlamydomonas (14) will act as initiating sites for the in vitro assembly of tubulin.Moreover, it has been demonstrated that brain tubulin will reversibly assemble and disassemble onto the isolated mitotic apparatuses of marine eggs (15) and rat kangaroo cells (16). The poles of marine egg mitotic apparatuses have been separated from the rest of the structure and have been shown to be capable of acting as sites for initiation of microtubule assembly (microtubule organizing centers; 17). However, it has not been demonstrated whether the kinetochores of chromosomes are also capable of initiating microtubule assembly, and an answer to this question would have s...

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

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.

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