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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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Model for initiation of K-fiber formation by kinetochores during normal spindle formation. (A) Chromosomes/centromeres provide a favorable environment for MT nucleation, e.g., by generating a RanGTP gradient (green circles represent α/β-tubulin heterodimers, and white squares represent a putative initiation factor). This leads to formation of short MTs in the vicinity of kinetochores, where these MTs can be easily captured. When an MT is captured by its plus end, it begins to polymerize steadily within the kinetochore. As the result, MT minus ends are pushed away from the kinetochores. Additional factors are likely responsible for cross-linking individual MTs to form a K-fiber. (B) Two routes leading to K-fiber formation and spindle morphogenesis. When unattached kinetochores capture astral MTs (top kinetochore in the schematics) they immediately establish a connection to the pole and move poleward. Then, a mature K-fiber can form either by acquiring additional astral MTs or through kinetochore-mediated MT polymerization (shown). Importantly, in this scenario the growth of the K-fiber from the kinetochore will be directed to the attached pole. An alternative route of K-fiber formation (bottom kinetochore in the schematics) is used when the connection between the kinetochore and the pole is delayed (e.g., kinetochores facing away from the pole). Under this condition, the kinetochores themselves initiate formation of K-fibers, via the mechanism presented in A. The initial growth of these K-fibers is not directed toward spindle poles, but eventually the minus ends of growing K-fibers interact with astral MTs and are subsequently incorporated into the spindle.
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fig7: Model for initiation of K-fiber formation by kinetochores during normal spindle formation. (A) Chromosomes/centromeres provide a favorable environment for MT nucleation, e.g., by generating a RanGTP gradient (green circles represent α/β-tubulin heterodimers, and white squares represent a putative initiation factor). This leads to formation of short MTs in the vicinity of kinetochores, where these MTs can be easily captured. When an MT is captured by its plus end, it begins to polymerize steadily within the kinetochore. As the result, MT minus ends are pushed away from the kinetochores. Additional factors are likely responsible for cross-linking individual MTs to form a K-fiber. (B) Two routes leading to K-fiber formation and spindle morphogenesis. When unattached kinetochores capture astral MTs (top kinetochore in the schematics) they immediately establish a connection to the pole and move poleward. Then, a mature K-fiber can form either by acquiring additional astral MTs or through kinetochore-mediated MT polymerization (shown). Importantly, in this scenario the growth of the K-fiber from the kinetochore will be directed to the attached pole. An alternative route of K-fiber formation (bottom kinetochore in the schematics) is used when the connection between the kinetochore and the pole is delayed (e.g., kinetochores facing away from the pole). Under this condition, the kinetochores themselves initiate formation of K-fibers, via the mechanism presented in A. The initial growth of these K-fibers is not directed toward spindle poles, but eventually the minus ends of growing K-fibers interact with astral MTs and are subsequently incorporated into the spindle.

Mentions: However, our observations combined with demonstrations that MT nucleation is stimulated in the vicinity of chromosomes, offer a straightforward explanation for how kinetochores can generate K-fibers with the correct polarity (Fig. 7 A). We believe that kinetochores do not nucleate but rather capture short MTs that form in their vicinity as described by Witt and colleagues (Witt et al., 1980). Nucleation of these short MTs is likely to be promoted by a Ran-GTP gradient (for review see Karsenti and Vernos, 2001), although we have yet no experimental proof for this hypothesis. It has been shown, however, that RCC1 (the GTP exchange factor for Ran) is concentrated in the centromere (Bischoff et al., 1990). In fact, RCC1 was independently identified as the centromere-associated protein CENP-D (Kingwell and Rattner, 1987). Furthermore, other proteins involved in the Ran GTP/GDP cycle are also enriched in the centromere/kinetochore region (Joseph et al., 2004). Thus, it is likely that the favorable environment in the vicinity of kinetochores promotes MT nucleation.


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

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

Model for initiation of K-fiber formation by kinetochores during normal spindle formation. (A) Chromosomes/centromeres provide a favorable environment for MT nucleation, e.g., by generating a RanGTP gradient (green circles represent α/β-tubulin heterodimers, and white squares represent a putative initiation factor). This leads to formation of short MTs in the vicinity of kinetochores, where these MTs can be easily captured. When an MT is captured by its plus end, it begins to polymerize steadily within the kinetochore. As the result, MT minus ends are pushed away from the kinetochores. Additional factors are likely responsible for cross-linking individual MTs to form a K-fiber. (B) Two routes leading to K-fiber formation and spindle morphogenesis. When unattached kinetochores capture astral MTs (top kinetochore in the schematics) they immediately establish a connection to the pole and move poleward. Then, a mature K-fiber can form either by acquiring additional astral MTs or through kinetochore-mediated MT polymerization (shown). Importantly, in this scenario the growth of the K-fiber from the kinetochore will be directed to the attached pole. An alternative route of K-fiber formation (bottom kinetochore in the schematics) is used when the connection between the kinetochore and the pole is delayed (e.g., kinetochores facing away from the pole). Under this condition, the kinetochores themselves initiate formation of K-fibers, via the mechanism presented in A. The initial growth of these K-fibers is not directed toward spindle poles, but eventually the minus ends of growing K-fibers interact with astral MTs and are subsequently incorporated into the spindle.
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Related In: Results  -  Collection

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

fig7: Model for initiation of K-fiber formation by kinetochores during normal spindle formation. (A) Chromosomes/centromeres provide a favorable environment for MT nucleation, e.g., by generating a RanGTP gradient (green circles represent α/β-tubulin heterodimers, and white squares represent a putative initiation factor). This leads to formation of short MTs in the vicinity of kinetochores, where these MTs can be easily captured. When an MT is captured by its plus end, it begins to polymerize steadily within the kinetochore. As the result, MT minus ends are pushed away from the kinetochores. Additional factors are likely responsible for cross-linking individual MTs to form a K-fiber. (B) Two routes leading to K-fiber formation and spindle morphogenesis. When unattached kinetochores capture astral MTs (top kinetochore in the schematics) they immediately establish a connection to the pole and move poleward. Then, a mature K-fiber can form either by acquiring additional astral MTs or through kinetochore-mediated MT polymerization (shown). Importantly, in this scenario the growth of the K-fiber from the kinetochore will be directed to the attached pole. An alternative route of K-fiber formation (bottom kinetochore in the schematics) is used when the connection between the kinetochore and the pole is delayed (e.g., kinetochores facing away from the pole). Under this condition, the kinetochores themselves initiate formation of K-fibers, via the mechanism presented in A. The initial growth of these K-fibers is not directed toward spindle poles, but eventually the minus ends of growing K-fibers interact with astral MTs and are subsequently incorporated into the spindle.
Mentions: However, our observations combined with demonstrations that MT nucleation is stimulated in the vicinity of chromosomes, offer a straightforward explanation for how kinetochores can generate K-fibers with the correct polarity (Fig. 7 A). We believe that kinetochores do not nucleate but rather capture short MTs that form in their vicinity as described by Witt and colleagues (Witt et al., 1980). Nucleation of these short MTs is likely to be promoted by a Ran-GTP gradient (for review see Karsenti and Vernos, 2001), although we have yet no experimental proof for this hypothesis. It has been shown, however, that RCC1 (the GTP exchange factor for Ran) is concentrated in the centromere (Bischoff et al., 1990). In fact, RCC1 was independently identified as the centromere-associated protein CENP-D (Kingwell and Rattner, 1987). Furthermore, other proteins involved in the Ran GTP/GDP cycle are also enriched in the centromere/kinetochore region (Joseph et al., 2004). Thus, it is likely that the favorable environment in the vicinity of kinetochores promotes MT nucleation.

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