Meiosis in Drosophila melanogaster. II. The prometaphase-I kinetochore microtubule bundle and kinetochore orientation in males.

Abstract

Fourteen prometaphase kinetochore microtubule bundles have been examined in electron micrographs of serial sections . The majority (54%) of the microtubules extend from the polar region towards the kinetochore but do not end in the kinetochore proper. Rather, they stop short of the kinetochore (21%), graze the kinetochore (19%), or pass through the kinetochore (9%), displaying a free end distal to the pole . Other microtubules that make up the kinetochore bundle include: kinetochore-to-pole microtubules (24%), chromosome-to-pole microtubules (5%), pieces with two free ends (14%), and those microtubules with one end in the kinetochore and a free end distal to the kinetochore (9%) . We conclude that the majority of the microtubules in the kinetochore bundle are most likely of polar origin rather than having been nucleated at the kinetochore.Prometaphase-I kinetochores can display any one of four patterns of microtubule connections with the poles, but the pattern of microtubule connections is not always correlated with kinetochore position . For instance, a kinetochore directly facing one pole may have microtubule connections with both poles while a kinetochore positioned 90°to the spindle axis may have microtubules running towards one pole only .Kinetochore orientation during prometaphase of meiosis can be very complex . The homologous kinetochores could theoretically participate in at least four orientation configurations: (a) unipolar orientation of homologous kinetochores ; (b) bipolar orientation of homologous kinetochores; (c) unipolar orientation of one pair of sister kinetochores and bipolar orientation of the homologous pair ; and (d) bipolar orientation of both pairs of sisters (1 cf. 8) .Bivalents, under normal conditions, usually achieve stable bipolar orientation before anaphase segregation . However, analyses of time-lapse ciné records of live cells in prometaphase demonstrate that brief unipolar orientation of homologous kinetochores is characteristic of some bivalents (1 cf. 18), as is unstable bipolar orientation (3 cf. 18). Mal-orientations are transient and reorientations of the kinetochores lead to stable bipolar orientation.Although prometaphase kinetochore orientations have been frequently described in fixed cells by light microscopy and analyzed in live cells by ciné analyses, little is known about the structural basis for such orientations . That homologous kinetochores are mechanically connected to the same pole in biva-

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

“…Those early structural studies described unattached Drosophila kinetochores as a "single bilaminar hemisphere" ~560 nm in diameter, with an inner layer ~280 nm thick and a more electron-dense outer layer ~60 nm thick (Goldstein 1981;Lin et al 1981;Church and Lin 1982). We also find the S2 kinetochore to be a bilaminar hemisphere, with a thin, darker-staining peripheral layer on the surface of a hemisphere.…”

“…In general, the ultrastructure of kinetochores in Drosophila somatic cells is similar to that reported for early prometaphase I bivalent kinetochores in Drosophila spermatocytes (Goldstein 1981;Church and Lin 1982). Those early structural studies described unattached Drosophila kinetochores as a "single bilaminar hemisphere" ~560 nm in diameter, with an inner layer ~280 nm thick and a more electron-dense outer layer ~60 nm thick (Goldstein 1981;Lin et al 1981;Church and Lin 1982).…”

“…3-Fig. 7 and Table 1 in Church and Lin 1982). Rather, in electron tomographic images we find that 81% of the kMTs terminate in a band of fibers at the kinetochore periphery that most likely corresponds to the peripheral layer seen in projection images.…”

“…We do not think that the difference in the number of MTs bound per kinetochore, between Drosophila S2 cells and spermatocytes, arises from differences in fixation protocols, because we used basically the same specimen preparation protocol, of glutaraldehyde fixation, osmium post fixation, and uranyl acetate en bloc staining, as had been employed in the previous studies (Lin et al 1981;Goldstein 1981;Church and Lin 1982). An interesting possibility is that the difference that we observe in numbers of attached kMTs arises from differences in kMT turnover between spermatocytes and somatic cells.…”

Merotelic kinetochore orientation occurs frequently during early mitosis in mammalian tissue cells and error correction is achieved by two different mechanisms

“…As shown in Fig. 11A, a curved kinetochore of a chromosome oriented with its intercentromere axis perpendicular to the interpolar spindle axis will be more likely to capture microtubules from opposite poles, as previously proposed for Drosophila meiotic cells (Church and Lin, 1982). Given available information, this mechanism seems the most likely explanation for the high frequency of merotelic kinetochore formation in early prometaphase in control cells.…”