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Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis.

Maiato H, Rieder CL, Khodjakov A - J. Cell Biol. (2004)

Bottom Line: This poleward transport results in chromosome bi-orientation and congression.Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores.However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany 12201, USA.

ABSTRACT
It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

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Spindle assembly in dynein-depleted cells. Selected frames from fluorescence time-lapse sequences of cells undergoing mitosis ∼72 h after dynein RNAi. (A) Spindle formation in a cell with centrosomes completely separated before NEB. The spindle forms between the separated centrosomes, and the poles are initially well focused. However, at a later time, the centrosomes detach from the poles and the K-fibers splay so that the spindle becomes barrel shaped. (B) Spindle formation in a cell with nonseparated centrosomes. Under this condition, the spindle initially forms as a fan-shaped structure, with MTs converging on the centrosome(s) only on one side of the chromosomes (2:00). However, within just a few minutes formation of prominent K-fibers is seen on the other side of the chromosomes (7:00). These K-fibers do not converge on a common point. Over time, the centrosomes detach from the spindle and then the K-fibers on the side of the spindle that was initially well focused begin to splay. As a result, the spindle eventually becomes barrel-shaped (62:00). Note that in this cell the centrosomes fused together (between 2:00 and 7:00), so that they appear as a single structure in later frames. Bars, 5 μm.
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fig6: Spindle assembly in dynein-depleted cells. Selected frames from fluorescence time-lapse sequences of cells undergoing mitosis ∼72 h after dynein RNAi. (A) Spindle formation in a cell with centrosomes completely separated before NEB. The spindle forms between the separated centrosomes, and the poles are initially well focused. However, at a later time, the centrosomes detach from the poles and the K-fibers splay so that the spindle becomes barrel shaped. (B) Spindle formation in a cell with nonseparated centrosomes. Under this condition, the spindle initially forms as a fan-shaped structure, with MTs converging on the centrosome(s) only on one side of the chromosomes (2:00). However, within just a few minutes formation of prominent K-fibers is seen on the other side of the chromosomes (7:00). These K-fibers do not converge on a common point. Over time, the centrosomes detach from the spindle and then the K-fibers on the side of the spindle that was initially well focused begin to splay. As a result, the spindle eventually becomes barrel-shaped (62:00). Note that in this cell the centrosomes fused together (between 2:00 and 7:00), so that they appear as a single structure in later frames. Bars, 5 μm.

Mentions: As reported by others, dynein knockdown induces S2 cells to accumulate in mitosis (Goshima and Vale, 2003; Fig. S2). In most of these cells the spindles were roughly bipolar and the chromosomes were organized into loose metaphase plates. However, compared with controls, under the experimental conditions we used, the poles were conspicuously less focused so that the spindle appeared barrel-shaped as in plants. Often the centrosomes were displaced from the spindle poles (Fig. 6 A), or had failed to separate (Fig. 6 B). These phenotypes are generally consistent with the effects of dynein inhibition reported previously in Drosophila (Robinson et al., 1999) and mammalian cells (Echeverri et al., 1996).


Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis.

Maiato H, Rieder CL, Khodjakov A - J. Cell Biol. (2004)

Spindle assembly in dynein-depleted cells. Selected frames from fluorescence time-lapse sequences of cells undergoing mitosis ∼72 h after dynein RNAi. (A) Spindle formation in a cell with centrosomes completely separated before NEB. The spindle forms between the separated centrosomes, and the poles are initially well focused. However, at a later time, the centrosomes detach from the poles and the K-fibers splay so that the spindle becomes barrel shaped. (B) Spindle formation in a cell with nonseparated centrosomes. Under this condition, the spindle initially forms as a fan-shaped structure, with MTs converging on the centrosome(s) only on one side of the chromosomes (2:00). However, within just a few minutes formation of prominent K-fibers is seen on the other side of the chromosomes (7:00). These K-fibers do not converge on a common point. Over time, the centrosomes detach from the spindle and then the K-fibers on the side of the spindle that was initially well focused begin to splay. As a result, the spindle eventually becomes barrel-shaped (62:00). Note that in this cell the centrosomes fused together (between 2:00 and 7:00), so that they appear as a single structure in later frames. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172442&req=5

fig6: Spindle assembly in dynein-depleted cells. Selected frames from fluorescence time-lapse sequences of cells undergoing mitosis ∼72 h after dynein RNAi. (A) Spindle formation in a cell with centrosomes completely separated before NEB. The spindle forms between the separated centrosomes, and the poles are initially well focused. However, at a later time, the centrosomes detach from the poles and the K-fibers splay so that the spindle becomes barrel shaped. (B) Spindle formation in a cell with nonseparated centrosomes. Under this condition, the spindle initially forms as a fan-shaped structure, with MTs converging on the centrosome(s) only on one side of the chromosomes (2:00). However, within just a few minutes formation of prominent K-fibers is seen on the other side of the chromosomes (7:00). These K-fibers do not converge on a common point. Over time, the centrosomes detach from the spindle and then the K-fibers on the side of the spindle that was initially well focused begin to splay. As a result, the spindle eventually becomes barrel-shaped (62:00). Note that in this cell the centrosomes fused together (between 2:00 and 7:00), so that they appear as a single structure in later frames. Bars, 5 μm.
Mentions: As reported by others, dynein knockdown induces S2 cells to accumulate in mitosis (Goshima and Vale, 2003; Fig. S2). In most of these cells the spindles were roughly bipolar and the chromosomes were organized into loose metaphase plates. However, compared with controls, under the experimental conditions we used, the poles were conspicuously less focused so that the spindle appeared barrel-shaped as in plants. Often the centrosomes were displaced from the spindle poles (Fig. 6 A), or had failed to separate (Fig. 6 B). These phenotypes are generally consistent with the effects of dynein inhibition reported previously in Drosophila (Robinson et al., 1999) and mammalian cells (Echeverri et al., 1996).

Bottom Line: This poleward transport results in chromosome bi-orientation and congression.Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores.However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany 12201, USA.

ABSTRACT
It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

Show MeSH