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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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Formation of a K-fiber on the distal kinetochore of a mono-oriented chromosome leads to chromosome congression. (A–E) Selected frames from a combinational fluorescence (top)/DIC (bottom) time-lapse sequence. This cell contains a mono-oriented chromosome (A, bottom, arrow), which is positioned near the top spindle pole. A K-fiber forms in association with the unattached kinetochore that faces away from centrosome-generated astral MTs (top, arrows). This fiber initially grows from the kinetochore toward the cell's periphery (B and C), but it then suddenly turns toward the bottom spindle pole and begins to glide poleward (compare C with D). As the result of the gliding, the fiber becomes incorporated into the spindle, and the chromosome congresses onto the metaphase plate (E). Note that poleward sliding of the fiber is initiated when it interacts with an astral MT emanating from the bottom pole (arrowheads in C and D). Time is in min:s. Bar, 5 μm.
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fig2: Formation of a K-fiber on the distal kinetochore of a mono-oriented chromosome leads to chromosome congression. (A–E) Selected frames from a combinational fluorescence (top)/DIC (bottom) time-lapse sequence. This cell contains a mono-oriented chromosome (A, bottom, arrow), which is positioned near the top spindle pole. A K-fiber forms in association with the unattached kinetochore that faces away from centrosome-generated astral MTs (top, arrows). This fiber initially grows from the kinetochore toward the cell's periphery (B and C), but it then suddenly turns toward the bottom spindle pole and begins to glide poleward (compare C with D). As the result of the gliding, the fiber becomes incorporated into the spindle, and the chromosome congresses onto the metaphase plate (E). Note that poleward sliding of the fiber is initiated when it interacts with an astral MT emanating from the bottom pole (arrowheads in C and D). Time is in min:s. Bar, 5 μm.

Mentions: In those cells where centrosome separation was incomplete at the time of NEB, many chromosomes were positioned at NEB outside of the region saturated with astral MTs. Nevertheless, these chromosomes consistently developed K-fibers on those kinetochores that faced away from the centrosomes, and were thus shielded from astral MTs by the chromosome body (Fig. 1 B and Video 2). These “distal” K-fibers were observed in all eight cells, and they first appeared in the vicinity of the kinetochore (Fig. 2 and Video 3) and then extended outward away from the centromere region. It was not unusual in cells that contained multiple distal K-fibers, for the ends of the fibers to join to form an additional spindle pole lacking a centrosome (Fig. 1 B). This configuration was usually transient as, one by one the chromosomes became incorporated into a single bipolar spindle as the ends of their distal kinetochore K-fibers moved toward one of the centrosomal spindle poles. After all K-fibers were incorporated into a bipolar spindle (Fig. 1 B) the cell entered and completed a normal anaphase (not depicted). Although the duration of prometaphase in these cells was highly variable, it was usually significantly longer (up to 60 min) than in those cells in which NEB occurred in the presence of completely separated centrosomes.


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

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

Formation of a K-fiber on the distal kinetochore of a mono-oriented chromosome leads to chromosome congression. (A–E) Selected frames from a combinational fluorescence (top)/DIC (bottom) time-lapse sequence. This cell contains a mono-oriented chromosome (A, bottom, arrow), which is positioned near the top spindle pole. A K-fiber forms in association with the unattached kinetochore that faces away from centrosome-generated astral MTs (top, arrows). This fiber initially grows from the kinetochore toward the cell's periphery (B and C), but it then suddenly turns toward the bottom spindle pole and begins to glide poleward (compare C with D). As the result of the gliding, the fiber becomes incorporated into the spindle, and the chromosome congresses onto the metaphase plate (E). Note that poleward sliding of the fiber is initiated when it interacts with an astral MT emanating from the bottom pole (arrowheads in C and D). Time is in min:s. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Formation of a K-fiber on the distal kinetochore of a mono-oriented chromosome leads to chromosome congression. (A–E) Selected frames from a combinational fluorescence (top)/DIC (bottom) time-lapse sequence. This cell contains a mono-oriented chromosome (A, bottom, arrow), which is positioned near the top spindle pole. A K-fiber forms in association with the unattached kinetochore that faces away from centrosome-generated astral MTs (top, arrows). This fiber initially grows from the kinetochore toward the cell's periphery (B and C), but it then suddenly turns toward the bottom spindle pole and begins to glide poleward (compare C with D). As the result of the gliding, the fiber becomes incorporated into the spindle, and the chromosome congresses onto the metaphase plate (E). Note that poleward sliding of the fiber is initiated when it interacts with an astral MT emanating from the bottom pole (arrowheads in C and D). Time is in min:s. Bar, 5 μm.
Mentions: In those cells where centrosome separation was incomplete at the time of NEB, many chromosomes were positioned at NEB outside of the region saturated with astral MTs. Nevertheless, these chromosomes consistently developed K-fibers on those kinetochores that faced away from the centrosomes, and were thus shielded from astral MTs by the chromosome body (Fig. 1 B and Video 2). These “distal” K-fibers were observed in all eight cells, and they first appeared in the vicinity of the kinetochore (Fig. 2 and Video 3) and then extended outward away from the centromere region. It was not unusual in cells that contained multiple distal K-fibers, for the ends of the fibers to join to form an additional spindle pole lacking a centrosome (Fig. 1 B). This configuration was usually transient as, one by one the chromosomes became incorporated into a single bipolar spindle as the ends of their distal kinetochore K-fibers moved toward one of the centrosomal spindle poles. After all K-fibers were incorporated into a bipolar spindle (Fig. 1 B) the cell entered and completed a normal anaphase (not depicted). Although the duration of prometaphase in these cells was highly variable, it was usually significantly longer (up to 60 min) than in those cells in which NEB occurred in the presence of completely separated centrosomes.

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