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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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K-fibers regrow by MT plus-end polymerization after laser microsurgery. (A) Selected frames from fluorescence time-lapse recording depicting the behavior of K-fiber fragments generated by laser microsurgery. Note that the pole-connected fragment (P-fragment) rapidly depolymerizes, whereas the kinetochore-attached fragment (K-fragment) grows steadily. Green arrowheads point to the position of the free end of P-fragment, whereas red arrowheads mark the position of the K-fragment's free end. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. (B) Selected frames from a fluorescence time-lapse sequence of a combinational laser microsurgery/photobleaching experiment. In this cell, a short segment of an individual K-fiber is first photobleached with a low power 488-nm laser (compare −10 with −3 s frames). Then, the same fiber is cut with high power 532-nm laser pulses in the region immediately outside the bleached segment (compare −3 and 0 s frames). As the result, the K-fragment created by the operation now contains a fiduciary mark that allows us to determine where the elongation of the fragment is occurring via subunit incorporation into the kinetochore-associated (plus) or free (minus) ends of MTs. Red arrowheads indicate the position of the fragment's free end, whereas yellow arrowheads point at the bleached segment. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. As is evident from the preservation of the distance between the bleached segment and the free end of the fragment, elongation occurs via plus-end MT polymerization (in the kinetochore). It also reveals that the minus ends of MTs are stable under our conditions.
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fig5: K-fibers regrow by MT plus-end polymerization after laser microsurgery. (A) Selected frames from fluorescence time-lapse recording depicting the behavior of K-fiber fragments generated by laser microsurgery. Note that the pole-connected fragment (P-fragment) rapidly depolymerizes, whereas the kinetochore-attached fragment (K-fragment) grows steadily. Green arrowheads point to the position of the free end of P-fragment, whereas red arrowheads mark the position of the K-fragment's free end. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. (B) Selected frames from a fluorescence time-lapse sequence of a combinational laser microsurgery/photobleaching experiment. In this cell, a short segment of an individual K-fiber is first photobleached with a low power 488-nm laser (compare −10 with −3 s frames). Then, the same fiber is cut with high power 532-nm laser pulses in the region immediately outside the bleached segment (compare −3 and 0 s frames). As the result, the K-fragment created by the operation now contains a fiduciary mark that allows us to determine where the elongation of the fragment is occurring via subunit incorporation into the kinetochore-associated (plus) or free (minus) ends of MTs. Red arrowheads indicate the position of the fragment's free end, whereas yellow arrowheads point at the bleached segment. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. As is evident from the preservation of the distance between the bleached segment and the free end of the fragment, elongation occurs via plus-end MT polymerization (in the kinetochore). It also reveals that the minus ends of MTs are stable under our conditions.

Mentions: Our finding that some K-fibers grow from the kinetochores in S2 cells raises the important question of where tubulin subunits added to the forming K-fiber? MTs in K-fibers are organized in a parallel bundle with their minus ends terminating near the spindle pole and their plus ends ending within the kinetochore. In a mature K-fiber, tubulin heterodimers are constantly added in the kinetochore, and removed from the minus ends in the pole. As a result, even when the length of the fiber remains constant the subunits “flux” poleward through the fiber (Mitchison, 1989). Importantly, flux requires that MT plus- and minus-end dynamics within the K-fiber be precisely coordinated, so that during metaphase the length of the fiber remains constant. Obviously, during K-fiber growth from the kinetochore, the rate of MT polymerization must exceed the depolymerization rate. This implies that either the minus-end depolymerization is suppressed or that plus-end polymerization rate is increased in K-fibers before they connect to the pole. To differentiate between these two possibilities we severed individual K-fibers within the spindle with a focused laser beam and then followed them by fluorescence time-lapse microscopy (Fig. 4 and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200407090/DC1). Severing a K-fiber results in the formation of two fragments: one (the P-fragment) remains connected to the spindle pole by its MT minus ends, and it now has (new) free MT plus ends; the other (K-fragment) remains connected to the kinetochore via its MT plus ends, whereas its newly created MT minus ends are free. In agreement with similar K-fiber severing experiments of others (Inoue, 1964; Forer, 1965; Spurck et al., 1990; Czaban et al., 1993; Forer et al., 1997), we find that the behaviors of these fragments are strikingly different. In all cases (n = 15) P-fragments shorten rapidly and completely disappear in <10 s (Fig. 4, A–E). In contrast, K-fragments remain stable and then regrow (Fig. 5 A) to the length of the original K-fiber (Fig. 4, B–J). In five of these cells we were able to measure the shrinkage and regrowth rates, which were 21.8 ± 0.4 μm/min and 0.8 ± 0.2 μm/min, respectively. Notably, as the K-fragments begin to elongate they need not be oriented toward a centrosome (Fig. 4, F and G), but at some point the elongating MT bundle turns so that its minus end becomes oriented toward a centrosome, and growth then continues until the minus end reaches the spindle pole (Fig. 4, H–J). Thus, K-fragments exhibit both features of kinetochore-driven K-fiber formation: they elongate from the kinetochore toward the cell periphery, irrespective of the position of the centrosomes, and free ends are eventually captured and directed toward a centrosome (spindle pole) by astral MTs.


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

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

K-fibers regrow by MT plus-end polymerization after laser microsurgery. (A) Selected frames from fluorescence time-lapse recording depicting the behavior of K-fiber fragments generated by laser microsurgery. Note that the pole-connected fragment (P-fragment) rapidly depolymerizes, whereas the kinetochore-attached fragment (K-fragment) grows steadily. Green arrowheads point to the position of the free end of P-fragment, whereas red arrowheads mark the position of the K-fragment's free end. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. (B) Selected frames from a fluorescence time-lapse sequence of a combinational laser microsurgery/photobleaching experiment. In this cell, a short segment of an individual K-fiber is first photobleached with a low power 488-nm laser (compare −10 with −3 s frames). Then, the same fiber is cut with high power 532-nm laser pulses in the region immediately outside the bleached segment (compare −3 and 0 s frames). As the result, the K-fragment created by the operation now contains a fiduciary mark that allows us to determine where the elongation of the fragment is occurring via subunit incorporation into the kinetochore-associated (plus) or free (minus) ends of MTs. Red arrowheads indicate the position of the fragment's free end, whereas yellow arrowheads point at the bleached segment. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. As is evident from the preservation of the distance between the bleached segment and the free end of the fragment, elongation occurs via plus-end MT polymerization (in the kinetochore). It also reveals that the minus ends of MTs are stable under our conditions.
© Copyright Policy
Related In: Results  -  Collection

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fig5: K-fibers regrow by MT plus-end polymerization after laser microsurgery. (A) Selected frames from fluorescence time-lapse recording depicting the behavior of K-fiber fragments generated by laser microsurgery. Note that the pole-connected fragment (P-fragment) rapidly depolymerizes, whereas the kinetochore-attached fragment (K-fragment) grows steadily. Green arrowheads point to the position of the free end of P-fragment, whereas red arrowheads mark the position of the K-fragment's free end. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. (B) Selected frames from a fluorescence time-lapse sequence of a combinational laser microsurgery/photobleaching experiment. In this cell, a short segment of an individual K-fiber is first photobleached with a low power 488-nm laser (compare −10 with −3 s frames). Then, the same fiber is cut with high power 532-nm laser pulses in the region immediately outside the bleached segment (compare −3 and 0 s frames). As the result, the K-fragment created by the operation now contains a fiduciary mark that allows us to determine where the elongation of the fragment is occurring via subunit incorporation into the kinetochore-associated (plus) or free (minus) ends of MTs. Red arrowheads indicate the position of the fragment's free end, whereas yellow arrowheads point at the bleached segment. Blue arrowheads point at the end of the K-fiber terminating in the kinetochore. As is evident from the preservation of the distance between the bleached segment and the free end of the fragment, elongation occurs via plus-end MT polymerization (in the kinetochore). It also reveals that the minus ends of MTs are stable under our conditions.
Mentions: Our finding that some K-fibers grow from the kinetochores in S2 cells raises the important question of where tubulin subunits added to the forming K-fiber? MTs in K-fibers are organized in a parallel bundle with their minus ends terminating near the spindle pole and their plus ends ending within the kinetochore. In a mature K-fiber, tubulin heterodimers are constantly added in the kinetochore, and removed from the minus ends in the pole. As a result, even when the length of the fiber remains constant the subunits “flux” poleward through the fiber (Mitchison, 1989). Importantly, flux requires that MT plus- and minus-end dynamics within the K-fiber be precisely coordinated, so that during metaphase the length of the fiber remains constant. Obviously, during K-fiber growth from the kinetochore, the rate of MT polymerization must exceed the depolymerization rate. This implies that either the minus-end depolymerization is suppressed or that plus-end polymerization rate is increased in K-fibers before they connect to the pole. To differentiate between these two possibilities we severed individual K-fibers within the spindle with a focused laser beam and then followed them by fluorescence time-lapse microscopy (Fig. 4 and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200407090/DC1). Severing a K-fiber results in the formation of two fragments: one (the P-fragment) remains connected to the spindle pole by its MT minus ends, and it now has (new) free MT plus ends; the other (K-fragment) remains connected to the kinetochore via its MT plus ends, whereas its newly created MT minus ends are free. In agreement with similar K-fiber severing experiments of others (Inoue, 1964; Forer, 1965; Spurck et al., 1990; Czaban et al., 1993; Forer et al., 1997), we find that the behaviors of these fragments are strikingly different. In all cases (n = 15) P-fragments shorten rapidly and completely disappear in <10 s (Fig. 4, A–E). In contrast, K-fragments remain stable and then regrow (Fig. 5 A) to the length of the original K-fiber (Fig. 4, B–J). In five of these cells we were able to measure the shrinkage and regrowth rates, which were 21.8 ± 0.4 μm/min and 0.8 ± 0.2 μm/min, respectively. Notably, as the K-fragments begin to elongate they need not be oriented toward a centrosome (Fig. 4, F and G), but at some point the elongating MT bundle turns so that its minus end becomes oriented toward a centrosome, and growth then continues until the minus end reaches the spindle pole (Fig. 4, H–J). Thus, K-fragments exhibit both features of kinetochore-driven K-fiber formation: they elongate from the kinetochore toward the cell periphery, irrespective of the position of the centrosomes, and free ends are eventually captured and directed toward a centrosome (spindle pole) by astral MTs.

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