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Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

Goshima G, Nédélec F, Vale RD - J. Cell Biol. (2005)

Bottom Line: Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes.Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs.From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

View Article: PubMed Central - PubMed

Affiliation: The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

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Capture and transport of kinetochore fibers along centrosomal-nucleated microtubules. (A) A time-lapse image sequence of GFP-tubulin in an untreated (wild-type) S2 cell. Lower panels are enlarged images, and minus ends of K fibers are marked by colored dots. Kinetochore microtubule bundles (K fibers) are cross-linked near their minus ends (e.g., purple dots), followed by focusing to the spindle pole by transport along C-MTs (four dots are gradually focused to the pole during 99–234 s). See also Videos 1 and 2. Bars, 5 and 1 μm. (B) K fiber unfocusing upon C-MTs depolymerization. By replacing culture medium with serum-free medium (SFM), C-MTs are rapidly depolymerized. K fibers gradually shorten and become unfocused. Image acquisition began 30 s after SFM addition. See Fig. S1 for quantitation and see also Video 3 (left). Bar, 5 μm. (C) K fiber focusing upon C-MTs recovery. After 10 min of the serum-free medium (SFM) treatment, large amount of conditioned medium (CM) was added. C-MTs gradually reappeared and accordingly, K fibers became focused. Images acquisition began 60 s after conditioned medium addition (CM). See also Video 3 (right). Bar, 5 μm.
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fig1: Capture and transport of kinetochore fibers along centrosomal-nucleated microtubules. (A) A time-lapse image sequence of GFP-tubulin in an untreated (wild-type) S2 cell. Lower panels are enlarged images, and minus ends of K fibers are marked by colored dots. Kinetochore microtubule bundles (K fibers) are cross-linked near their minus ends (e.g., purple dots), followed by focusing to the spindle pole by transport along C-MTs (four dots are gradually focused to the pole during 99–234 s). See also Videos 1 and 2. Bars, 5 and 1 μm. (B) K fiber unfocusing upon C-MTs depolymerization. By replacing culture medium with serum-free medium (SFM), C-MTs are rapidly depolymerized. K fibers gradually shorten and become unfocused. Image acquisition began 30 s after SFM addition. See Fig. S1 for quantitation and see also Video 3 (left). Bar, 5 μm. (C) K fiber focusing upon C-MTs recovery. After 10 min of the serum-free medium (SFM) treatment, large amount of conditioned medium (CM) was added. C-MTs gradually reappeared and accordingly, K fibers became focused. Images acquisition began 60 s after conditioned medium addition (CM). See also Video 3 (right). Bar, 5 μm.

Mentions: To better understand the pole focusing process in Drosophila S2 cells, we performed time-lapse imaging of mitotic cells expressing GFP-tubulin. As shown in Fig. 1 A (and Video 1 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1), K fibers are generally focused at their minus end. However, this K fiber focusing appears to be a highly dynamic process, as live cell imaging shows continuous defocusing/refocusing of K fibers during metaphase (Video 2 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). This dynamic focusing process appears to involve the bundling of K fibers as well as cross-linking of microtubules between K fibers. Such K fiber focusing does not completely depend upon a functional centrosome, as it is observed in cells depleted of centrosomin, albeit often imperfectly (unpublished data). Live cell imaging also shows that K fibers can interact with and transport along C-MTs, resulting in a closer juxtaposition of centrosomes and the minus ends of K fibers and a tighter focus of the pole (Fig. 1 A and Video 1 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). Similar observations also have been reported by Maiato et al. (2004). These findings clearly indicate the separate and sequential processes of initial K fiber focusing and the subsequent transport of the K fibers toward the centrosome.


Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

Goshima G, Nédélec F, Vale RD - J. Cell Biol. (2005)

Capture and transport of kinetochore fibers along centrosomal-nucleated microtubules. (A) A time-lapse image sequence of GFP-tubulin in an untreated (wild-type) S2 cell. Lower panels are enlarged images, and minus ends of K fibers are marked by colored dots. Kinetochore microtubule bundles (K fibers) are cross-linked near their minus ends (e.g., purple dots), followed by focusing to the spindle pole by transport along C-MTs (four dots are gradually focused to the pole during 99–234 s). See also Videos 1 and 2. Bars, 5 and 1 μm. (B) K fiber unfocusing upon C-MTs depolymerization. By replacing culture medium with serum-free medium (SFM), C-MTs are rapidly depolymerized. K fibers gradually shorten and become unfocused. Image acquisition began 30 s after SFM addition. See Fig. S1 for quantitation and see also Video 3 (left). Bar, 5 μm. (C) K fiber focusing upon C-MTs recovery. After 10 min of the serum-free medium (SFM) treatment, large amount of conditioned medium (CM) was added. C-MTs gradually reappeared and accordingly, K fibers became focused. Images acquisition began 60 s after conditioned medium addition (CM). See also Video 3 (right). Bar, 5 μm.
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fig1: Capture and transport of kinetochore fibers along centrosomal-nucleated microtubules. (A) A time-lapse image sequence of GFP-tubulin in an untreated (wild-type) S2 cell. Lower panels are enlarged images, and minus ends of K fibers are marked by colored dots. Kinetochore microtubule bundles (K fibers) are cross-linked near their minus ends (e.g., purple dots), followed by focusing to the spindle pole by transport along C-MTs (four dots are gradually focused to the pole during 99–234 s). See also Videos 1 and 2. Bars, 5 and 1 μm. (B) K fiber unfocusing upon C-MTs depolymerization. By replacing culture medium with serum-free medium (SFM), C-MTs are rapidly depolymerized. K fibers gradually shorten and become unfocused. Image acquisition began 30 s after SFM addition. See Fig. S1 for quantitation and see also Video 3 (left). Bar, 5 μm. (C) K fiber focusing upon C-MTs recovery. After 10 min of the serum-free medium (SFM) treatment, large amount of conditioned medium (CM) was added. C-MTs gradually reappeared and accordingly, K fibers became focused. Images acquisition began 60 s after conditioned medium addition (CM). See also Video 3 (right). Bar, 5 μm.
Mentions: To better understand the pole focusing process in Drosophila S2 cells, we performed time-lapse imaging of mitotic cells expressing GFP-tubulin. As shown in Fig. 1 A (and Video 1 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1), K fibers are generally focused at their minus end. However, this K fiber focusing appears to be a highly dynamic process, as live cell imaging shows continuous defocusing/refocusing of K fibers during metaphase (Video 2 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). This dynamic focusing process appears to involve the bundling of K fibers as well as cross-linking of microtubules between K fibers. Such K fiber focusing does not completely depend upon a functional centrosome, as it is observed in cells depleted of centrosomin, albeit often imperfectly (unpublished data). Live cell imaging also shows that K fibers can interact with and transport along C-MTs, resulting in a closer juxtaposition of centrosomes and the minus ends of K fibers and a tighter focus of the pole (Fig. 1 A and Video 1 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). Similar observations also have been reported by Maiato et al. (2004). These findings clearly indicate the separate and sequential processes of initial K fiber focusing and the subsequent transport of the K fibers toward the centrosome.

Bottom Line: Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes.Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs.From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

View Article: PubMed Central - PubMed

Affiliation: The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

Show MeSH