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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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MT initiation from kinetochores in the presence of centrosomes. (A–F) Selected frames from a combinational fluorescence/DIC time-lapse recording. Top part of each frame presents deconvolved and contrast-EGFP/α-tubulin fluorescence, whereas the bottom part of each frame is an overlay of fluorescence (green) over chromosome contours (red) from the corresponding DIC images. The cell is met with a mono-oriented chromosome, which is connected to one spindle pole via well-developed K-fiber, whereas the second kinetochore on this chromosome is completely devoid of MTs (A). This kinetochore remains unattached for several minutes (B). Then, however, formation of a small but discrete patch of GFP/α-tubulin fluorescence is seen in association with this kinetochore (C). The intensity of the patch gradually increases and eventually it begins to elongate forming a K-fiber (D–F). Note that the growing fiber is initially oriented away from the centrosome (E) but it then suddenly turns and becomes oriented toward the centrosome. Time is in min:s. Bar, 5 μm.
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fig3: MT initiation from kinetochores in the presence of centrosomes. (A–F) Selected frames from a combinational fluorescence/DIC time-lapse recording. Top part of each frame presents deconvolved and contrast-EGFP/α-tubulin fluorescence, whereas the bottom part of each frame is an overlay of fluorescence (green) over chromosome contours (red) from the corresponding DIC images. The cell is met with a mono-oriented chromosome, which is connected to one spindle pole via well-developed K-fiber, whereas the second kinetochore on this chromosome is completely devoid of MTs (A). This kinetochore remains unattached for several minutes (B). Then, however, formation of a small but discrete patch of GFP/α-tubulin fluorescence is seen in association with this kinetochore (C). The intensity of the patch gradually increases and eventually it begins to elongate forming a K-fiber (D–F). Note that the growing fiber is initially oriented away from the centrosome (E) but it then suddenly turns and becomes oriented toward the centrosome. Time is in min:s. Bar, 5 μm.

Mentions: Thus far, our data reveal that kinetochores in S2 cells can form K-fibers by a centrosome-independent mechanism. Does this kinetochore-driven K-fiber formation also occur on kinetochores that are oriented toward a centrosome? This question is difficult to address because in most cells the astral MT density makes it impossible to clearly follow how the kinetochore acquires its MTs. However, in highly flattened cells the density of astral MTs in the vicinity of the chromosomes is significantly decreased. This, in turn, decreases the chances of astral MT capture which allowed us to follow the formation of individual K-fibers on kinetochores that were oriented toward a centrosome. In these cells kinetochores remain free of MTs for several minutes (Fig. 3, A and B). At the end of this time a weak GFP signal, likely corresponding to several short MTs, appeared in the kinetochore region (Fig. 3, C and D). Soon thereafter, an MT bundle began to extend from the kinetochore outwards (Fig. 3, E and F). Because the initial growth direction of these forming MT bundles was not toward a centrosome (Fig. 3 E) they were not connected to a centrosome and thus could not have been formed via the classic search-and-capture mechanism. In the example shown in Fig. 3, the growing fiber exhibits a sudden turn, after which its free end orients toward a centrosome as it captures and is transported poleward on astral MTs (Fig. 3 F).


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

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

MT initiation from kinetochores in the presence of centrosomes. (A–F) Selected frames from a combinational fluorescence/DIC time-lapse recording. Top part of each frame presents deconvolved and contrast-EGFP/α-tubulin fluorescence, whereas the bottom part of each frame is an overlay of fluorescence (green) over chromosome contours (red) from the corresponding DIC images. The cell is met with a mono-oriented chromosome, which is connected to one spindle pole via well-developed K-fiber, whereas the second kinetochore on this chromosome is completely devoid of MTs (A). This kinetochore remains unattached for several minutes (B). Then, however, formation of a small but discrete patch of GFP/α-tubulin fluorescence is seen in association with this kinetochore (C). The intensity of the patch gradually increases and eventually it begins to elongate forming a K-fiber (D–F). Note that the growing fiber is initially oriented away from the centrosome (E) but it then suddenly turns and becomes oriented toward the centrosome. Time is in min:s. Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172442&req=5

fig3: MT initiation from kinetochores in the presence of centrosomes. (A–F) Selected frames from a combinational fluorescence/DIC time-lapse recording. Top part of each frame presents deconvolved and contrast-EGFP/α-tubulin fluorescence, whereas the bottom part of each frame is an overlay of fluorescence (green) over chromosome contours (red) from the corresponding DIC images. The cell is met with a mono-oriented chromosome, which is connected to one spindle pole via well-developed K-fiber, whereas the second kinetochore on this chromosome is completely devoid of MTs (A). This kinetochore remains unattached for several minutes (B). Then, however, formation of a small but discrete patch of GFP/α-tubulin fluorescence is seen in association with this kinetochore (C). The intensity of the patch gradually increases and eventually it begins to elongate forming a K-fiber (D–F). Note that the growing fiber is initially oriented away from the centrosome (E) but it then suddenly turns and becomes oriented toward the centrosome. Time is in min:s. Bar, 5 μm.
Mentions: Thus far, our data reveal that kinetochores in S2 cells can form K-fibers by a centrosome-independent mechanism. Does this kinetochore-driven K-fiber formation also occur on kinetochores that are oriented toward a centrosome? This question is difficult to address because in most cells the astral MT density makes it impossible to clearly follow how the kinetochore acquires its MTs. However, in highly flattened cells the density of astral MTs in the vicinity of the chromosomes is significantly decreased. This, in turn, decreases the chances of astral MT capture which allowed us to follow the formation of individual K-fibers on kinetochores that were oriented toward a centrosome. In these cells kinetochores remain free of MTs for several minutes (Fig. 3, A and B). At the end of this time a weak GFP signal, likely corresponding to several short MTs, appeared in the kinetochore region (Fig. 3, C and D). Soon thereafter, an MT bundle began to extend from the kinetochore outwards (Fig. 3, E and F). Because the initial growth direction of these forming MT bundles was not toward a centrosome (Fig. 3 E) they were not connected to a centrosome and thus could not have been formed via the classic search-and-capture mechanism. In the example shown in Fig. 3, the growing fiber exhibits a sudden turn, after which its free end orients toward a centrosome as it captures and is transported poleward on astral MTs (Fig. 3 F).

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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