Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres - PubMed (original) (raw)

Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres

Helder Maiato et al. Nat Cell Biol. 2005 Jan.

Abstract

The motion of a chromosome during mitosis is mediated by a bundle of microtubules, termed a kinetochore fibre (K-fibre), which connects the kinetochore of the chromosome to a spindle pole. Once formed, mature K-fibres maintain a steady state length because the continuous addition of microtubule subunits onto microtubule plus ends at the kinetochore is balanced by their removal at their minus ends within the pole. This condition is known as 'microtubule poleward flux'. Chromosome motion and changes in position are then driven by changes in K-fibre length, which in turn are controlled by changes in the rates at which microtubule subunits are added at the kinetochore and/or removed from the pole. A key to understanding the role of flux in mitosis is to identify the molecular factors that drive it. Here we use Drosophila melanogaster S2 cells expressing alpha-tubulin tagged with green fluorescent protein, RNA interference, laser microsurgery and photobleaching to show that the kinetochore protein MAST/Orbit - the single CLASP orthologue in Drosophila - is an essential component for microtubule subunit incorporation into fluxing K-fibres.

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Figures

Figure 1

Figure 1

CLASP is required for maintaining spindle and kinetochore fibre length in Drosophila S2T cells. (a) Mitotic progression in a control cell stably expressing GFP–α-tubulin. (b) Western blot showing the depletion of Drosophila CLASP by RNAi (−) compared with control S2T cells (+). Endogenous α-tubulin levels are shown as a loading control of protein extracts derived from 106 cells per lane. (c) Spindle collapse in a Drosophila S2T cell depleted of CLASP by RNAi. Note that several kinetochore fibres were generated by kinetochores (arrows) as the spindle collapsed. Asterisks indicate a centrosome not visible in some focal planes during the early recording stages. (d) Mature K-fibres, not attached to a centrosome/spindle pole (arrow), also shorten as the spindle collapses in cells depleted of CLASP. Time is shown in min:sec; scale bar represents 5 μm. (e) Spindle length over time for the control (a) and _CLASP-_RNAi-depleted (c) cells. ‘A’ indicates the onset of anaphase in the control cell in the chart. Movies of a and c can be found in the Supplementary Information.

Figure 2

Figure 2

CLASP is required for microtubule poleward flux. (a) Control cell showing the poleward motion (microtubule subunit flux) of a photobleached region (arrow) on a K-fibre. (b) In cells depleted of CLASP, photobleached marks on K-fibres (arrow) do not move poleward as the spindle gradually collapses. (c, d) Photobleached regions on K-fibres in dynein- or EB1-depleted cells, respectively, flux poleward (arrow) at normal rates. Time is shown in min:sec; scale bar represents 5 μm. (e) Kinetic traces of poleward flux, as assayed from the motion of photobleached regions on K-fibres, from two different cells per condition.

Figure 3

Figure 3

CLASP is required for subunit incorporation into K-fibre microtubules at their kinetochore-attached plus ends. (a) Control cell showing the re-growth (arrow) of a K-fibre after severing by laser microsurgery. (b) Similar to a but in a cell depleted of CLASP by RNAi. The severed K-fibre (arrow) does not re-grow even as the spindle gradually collapses. (c, d) K-fibres severed in dynein- or EB1-depleted cells, respectively, re-grow (arrow) at the same rates seen in control cells. Time is shown in min:sec; scale bar represents 5 μm. (e) K-fragment elongation curves after laser microsurgery for the cells shown in the previous panels. Movies of a–d can be found in the Supplementary Information.

Figure 4

Figure 4

Schematic depicting the role of CLASP at kinetochores. (a) CLASP (yellow) mediates the incorporation of tubulin subunits (green) into kinetochore microtubule plus-ends at kinetochores. When a mature K-fibre is photobleached (top orange arrow b–d) the subunits move, or ‘flux’, poleward through the fibre and are removed near the poles. When a K-fibre is photobleached (bottom orange arrow in b) and then severed (black arrow) closer to the pole, the fibre re-grows towards the pole (c–d) by microtubule subunit incorporation at the kinetochore but not at the newly created microtubule minus ends, which are stable. Once the re-growing fibre matures, factors are recruited to its microtubule minus ends (d) (most probably Kinesin-13 family members such as KLP10A/Kif2a; pink) that allow for (and maybe power) subunit flux. In the absence of CLASP (e), the incorporation of subunits into mature K-fibre microtubule plus-ends is shut down, which inhibits flux. However, as microtubule minus-end depolymerization still occurs near the poles, the K-fibres shorten and the spindle collapses.

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