Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells.

Abstract

The role of the kinetochore in chromosome movement was studied by 532-nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 p,m and remain parallel to each other, not undergoing any poleward separation . Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it . Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of -3 ,um/ min . If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole . The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid . Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it .It is now generally accepted that mitotic spindle formation and chromosome movement involve several structures, including microtubules, centrioles, pericentriolar regions, and the centromere regions of the chromosomes . There have been several investigations into the ultrastructure and chemical composition of the kinetochore, or attachment site of microtubules to the centromeric region (14,(30)(31)(32)35) . Electron microscope studies have revealed a relatively uniform morphology of the kinetochore region in many different eukaryotes (including PtK 2 cells [34]) consisting of a trilaminar structure 0 .3-0 .6 ttm in diameter.Although the kinetochore has been demonstrated unequivocally to be a microtubule-organizing center in vitro (15,22,36,38), very little biochemical information is available on it . However, there are indications that ribonucleoprotein is a component of the kinetochore (9, 32) and it appears to be associated with the inner plate of the trilaminar kinetochore in PtK, cells .One of the earliest studies on the function of the centromere pertains to the mitotic behavior of x-ray-induced chromosome fragments lacking centromeres (11,12). Although these fragments did not join the metaphase plate, there was some sepa-THE JOURNAL OF CELL BIOLOGY " VOLUME 88 MARCH 1981 543-553 ©The Rockefeller University Press " 0021-9525/81/03/0543/11 $1 .00 ration of the chromatids at anaphase. However, they lagged behind the...

Holding Your Own: Establishing Sister Chromatid Cohesion

“…Cohesion between sister chromatids facilitates this orientation, most probably by physically constraining sister kinetochores to face in opposite directions (Rieder 1982;Waters et al 1996;Michaelis et al 1997;Tanaka et al 2000a). Fourth, kinetochores associated with spindle microtubules generate poleward-pulling forces that act to separate sister chromatids (McNeill and Berns 1981;Rieder et al 1986;Skibbens et al 1995). Cohesion opposes these kinetochore pulling forces, stretching the intervening chromatin and generating tension across the chromatid pair.…”

Drosophila S2 Cells as a Model System to Investigate Mitotic Spindle Dynamics, Architecture, and Function

“…5). Indeed, laser microsurgery has been seminal to elucidate the mechanistic basis of the spindle-assembly checkpoint (Rieder et al, 1995), the role of the centrosome in spindle assembly (Khodjakov et al, 2000b) and the role of KTs in chromosome movement McNeill and Berns, 1981). Drosophila culture cells offer the additional possibility of combining powerful live cell imaging and laser microsurgery with molecular tools such as RNAi.…”

Regulated Assembly of the Mitotic Spindle: A Perspective from Two Ends

“…In both cases, the predictions are borne out by experimentation. Mechanical or laser-induced severing between sister chromatids or ablating the kinetochore itself results in movement of the intact sister chromatid(s) toward its associated spindle pole (Nicklas and Staehly, 1967;McNeill and Berns, 1981;Rieder et al, 1986;Hays and Salmon, 1990;Skibbens et al, 1995;). Similarly, yeast cells harboring mutations in the cohesion factors such as Ctf7p/Eco1p, Mcd1p/Scc1p and Pds5p have partially elongated and often bent spindles with separated but unequal masses of DNA (Guacci et al, 1997;Skibbens et al, 1999;Hartman et al, 2000).…”