Biology

Kinetochore spindle fibres in flea-beetle metaphase primary spermatocytes have two regions with distinct morphologies. As seen after staining with antibodies against tubulin, the kinetochore microtubules are tightly bundled in the 5 microm closest to the kinetochore but they splay out in the region closest to the pole. This morphology persists throughout anaphase. This distinct morphology allows one to deduce the site where kinetochore microtubules depolymerise during anaphase. During poleward movement of the autosomes in anaphase, the bundled region shortens by about 0.25 microm for each 1 microm the chromosome moves poleward; this suggests that, during anaphase, 75% of the kinetochore microtubule shortening occurs at the pole end. Sex chromosomes in metaphase cells are separated from the autosomes and do not move poleward at the same time as the autosomes: they are reported to move poleward when the autosomes are in metaphase, to stop part way to the poles, and to move poleward ag...

We studied chromosome movement after kinetochore microtubules were severed. Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region. After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving. We examined actin and/or microtubules in irradiated cells by means of confocal fluorescence microscopy or serial-section reconstructions from electron microscopy. For each cell thus examined, chromosome movement had been recorded continuously until the moment of fixation. Kinetochore microtubules were completely severed by the ultraviolet microbeam in cells in which chromosomes continued to move poleward after the irradiation: none were seen in the irradiated regions. Similarly, actin filaments normally present in kinetochore fibres were severed by the...

We studied the distribution of acetylated α-tubulin in the microtubules of spermatogenic cells from the crane fly Nephrotoma suturalis (Loew) using a mono-clonal antibody specific for acetylated α-tubulin (6-11B-1). We found that cells in all stages of spermatogenesis contained ...

Areas of reduced birefringence (ARBs) produced by ultraviolet microbeam irradiation are areas of depolymerized microtubules. ARBs probably move poleward either by microtubule subunit addition at the kinetochore and loss at the pole, or by microtubule subunit addition at one edge of the ARB and loss from the other edge. In this paper we have used two approaches to try to distinguish between these two models. First, we determined whether the edges of the ARB move at the same rate; if ARB motion is due solely to addition at the kinetochore and loss at the pole, with the ARB edges unable to exchange subunits, then the two edges of each ARB should move at the same rate. On the other hand, if the exchange is at the ARB edges, then, from data from microtubules in vitro, the poleward edge should move much faster than the kinetochoreward edge. We found that the two edges of the ARB move at the same rate about half the time, but half the time they do not. Second, we studied the behaviour of t...

Marginal bands were isolated from newt red blood cells and, using monochromatic light from an ultraviolet microbeam, the marginal band microtubules were irradiated in vitro to produce areas of reduced birefringence (ARBs). The ARBs neither moved nor changed shape after they were formed, though the marginal bands sometimes changed shape during the irradiation. Marginal band ARBs were regions in which the microtubules were locally depolymerized, as determined by electron microscopy and immunofluorescence. The action spectrum for producing ARBs on marginal band microtubules in vitro matches very closely the action spectrum for producing ARBs on crane-fly spermatocyte chromosomal spindle fibres in vivo, which indicates that ARBs in vivo are produced by the ultraviolet light acting directly on the microtubules (as opposed to an intermediate component), and confirms, without complications inherent in the fixation of living cells, that ARBs on spindle fibres in vivo are regions in which mi...

Irradiation of birefringent chromosomal spindle fibres in cranefly spermatocytes in metaphase I or anaphase I produces an area of reduced birefringence (ARB) on the fibre. This ARB moves poleward and is lost at the pole. Ultrastructural and immunofluorescence analysis of ARBs obtained by irradiation with monochromatic ultraviolet light of wavelength 260 nm shows that the microtubules in the irradiated area are depolymerized, though the rest of the spindle appears unaffected. The area of microtubule depolymerization moves poleward with the ARB, and once the ARB reaches the pole the irradiated half-spindle appears normal. The motion of the ARB, therefore, appears to be due to the behaviour of the sheared microtubules in the chromosomal spindle fibre. The relative stability of the sheared microtubules shows that chromosomal fibre microtubules are not dynamically unstable, as are microtubules under certain conditions in vitro. However, ARB motion may be due to a moderated version of dyn...

Anaphase chromosome motion involves the disassembly of kinetochore microtubules. We wished to determine the site of kinetochore microtubule disassembly during anaphase in crane-fly spermatocytes. In crane-fly spermatocyte spindles, monoclonal antibody 6-11B-1 to acetylated α-tubulin labels kinetochore microtubules almost exclusively, with an area immediately adjacent to the kinetochore being weakly or not labelled. This ‘gap’ in acetylation at the kinetochore serves as a natural marker of kinetochore microtubules in the kinetochore fibre. We measured the length of the gap on kinetochore fibres in metaphase and anaphase in order to deduce the fate of the gap during anaphase; we used this information to determine where kinetochore microtubules disassemble in anaphase. Gap lengths were measured from confocal microscope images of fixed spermatocytes dual labelled with 6-11B-1 to acetylated α-tubulin and YL1/2 to tyrosinated α-tubulin, the latter being used to determine the positions of ...

Irradiation of birefringent chromosomal spindle fibres in crane-fly spermatocytes in metaphase I or anaphase I produces an area of reduced birefringence (ARB) on the fibre. This ARB moves poleward and is lost at the pole. Ultrastructural and immunofluorescence analysis of ARBs obtained by irradiation with monochromatic ultraviolet light of wavelength 260 nm shows that the microtubules in the irradiated area are depolymerized, though the rest of the spindle appears unaffected. The area of microtubule depolymerization moves poleward with the ARB, and once the ARB reaches the pole the irradiated half-spindle appears normal. The motion of the ARB, therefore, appears to be due to the behaviour of the sheared microtubules in the chromosomal spindle fibre. The relative stability of the sheared microtubules shows that chromosomal fibre microtubules are not dynamically unstable, as are microtubules under certain conditions in vitro. However, ARB motion may be due to a moderated version of dynamic instability. Possible models for ARB motion are discussed.

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