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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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EB1-dependent, microtubule plus end tracking of Ncd. (A, top) A still image of Ncd-GFP in the spindle. (Bottom) Time-lapse imaging of the inset region shows tip association of Ncd-GFP on a growing astral microtubule (yellow arrowheads). Red arrowheads indicate the position of the punctate signals detected at time 0. See also Video 5. (B) Ncd-GFP in the spindle in a cell depleted of EB1 by RNAi. Fewer growing microtubules were observed and Ncd-GFP did not accumulate at the tips (arrowheads). See also Video 8. (C, top) A still image of a cell expressing Ncd-GFP-NES. (Bottom) Time-lapse sequences of the inset region. Enrichment of the signals at the growing tip is seen (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 6. (D) An Ncd-GFP-NES cell after EB1 RNAi (day 7). Accumulation at the tips of growing MTs of Ncd-GFP-NES is not detected (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 7. Bars of A–D are 10 μm (top images) or 1 μm (bottom images). (E) In vitro interaction of Ncd tail with COOH terminus EB1 fragment. Coomassie staining after GST pull-down experiment is shown. Binding reaction was performed in 200 μl volume with 12 μM dEB1C and 1 μM GST-Ncd-tail (lanes 2–6) or GST (lane 1). GFP-hAPC-C was preincubated with dEB1C at 1:10, 1:3, 1:1, and 2:1 molar ratio for competition (lanes 3–6). Bound fractions were 18-fold loaded compared with unbound fractions.
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fig4: EB1-dependent, microtubule plus end tracking of Ncd. (A, top) A still image of Ncd-GFP in the spindle. (Bottom) Time-lapse imaging of the inset region shows tip association of Ncd-GFP on a growing astral microtubule (yellow arrowheads). Red arrowheads indicate the position of the punctate signals detected at time 0. See also Video 5. (B) Ncd-GFP in the spindle in a cell depleted of EB1 by RNAi. Fewer growing microtubules were observed and Ncd-GFP did not accumulate at the tips (arrowheads). See also Video 8. (C, top) A still image of a cell expressing Ncd-GFP-NES. (Bottom) Time-lapse sequences of the inset region. Enrichment of the signals at the growing tip is seen (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 6. (D) An Ncd-GFP-NES cell after EB1 RNAi (day 7). Accumulation at the tips of growing MTs of Ncd-GFP-NES is not detected (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 7. Bars of A–D are 10 μm (top images) or 1 μm (bottom images). (E) In vitro interaction of Ncd tail with COOH terminus EB1 fragment. Coomassie staining after GST pull-down experiment is shown. Binding reaction was performed in 200 μl volume with 12 μM dEB1C and 1 μM GST-Ncd-tail (lanes 2–6) or GST (lane 1). GFP-hAPC-C was preincubated with dEB1C at 1:10, 1:3, 1:1, and 2:1 molar ratio for competition (lanes 3–6). Bound fractions were 18-fold loaded compared with unbound fractions.

Mentions: In addition to the localization of Ncd to K fibers, spinning-disk confocal microscopy revealed a previously unreported enrichment of Ncd-GFP at the tips of growing mitotic microtubules growing from centrosomes (Fig. 4 A, yellow arrowheads; and Video 5 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). Even though the microtubule shafts also have Ncd-GFP staining, our quantitation shows an at least threefold enrichment of the GFP signals at the microtubule tip (unpublished data). The tip localization was most evident in the cells expressing lower amounts of Ncd-GFP (i.e., levels shown in Fig. 3 A, 2; increasing expression led to more uniform staining of the spindle microtubules).


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

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

EB1-dependent, microtubule plus end tracking of Ncd. (A, top) A still image of Ncd-GFP in the spindle. (Bottom) Time-lapse imaging of the inset region shows tip association of Ncd-GFP on a growing astral microtubule (yellow arrowheads). Red arrowheads indicate the position of the punctate signals detected at time 0. See also Video 5. (B) Ncd-GFP in the spindle in a cell depleted of EB1 by RNAi. Fewer growing microtubules were observed and Ncd-GFP did not accumulate at the tips (arrowheads). See also Video 8. (C, top) A still image of a cell expressing Ncd-GFP-NES. (Bottom) Time-lapse sequences of the inset region. Enrichment of the signals at the growing tip is seen (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 6. (D) An Ncd-GFP-NES cell after EB1 RNAi (day 7). Accumulation at the tips of growing MTs of Ncd-GFP-NES is not detected (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 7. Bars of A–D are 10 μm (top images) or 1 μm (bottom images). (E) In vitro interaction of Ncd tail with COOH terminus EB1 fragment. Coomassie staining after GST pull-down experiment is shown. Binding reaction was performed in 200 μl volume with 12 μM dEB1C and 1 μM GST-Ncd-tail (lanes 2–6) or GST (lane 1). GFP-hAPC-C was preincubated with dEB1C at 1:10, 1:3, 1:1, and 2:1 molar ratio for competition (lanes 3–6). Bound fractions were 18-fold loaded compared with unbound fractions.
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Related In: Results  -  Collection

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fig4: EB1-dependent, microtubule plus end tracking of Ncd. (A, top) A still image of Ncd-GFP in the spindle. (Bottom) Time-lapse imaging of the inset region shows tip association of Ncd-GFP on a growing astral microtubule (yellow arrowheads). Red arrowheads indicate the position of the punctate signals detected at time 0. See also Video 5. (B) Ncd-GFP in the spindle in a cell depleted of EB1 by RNAi. Fewer growing microtubules were observed and Ncd-GFP did not accumulate at the tips (arrowheads). See also Video 8. (C, top) A still image of a cell expressing Ncd-GFP-NES. (Bottom) Time-lapse sequences of the inset region. Enrichment of the signals at the growing tip is seen (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 6. (D) An Ncd-GFP-NES cell after EB1 RNAi (day 7). Accumulation at the tips of growing MTs of Ncd-GFP-NES is not detected (yellow arrowheads). Red arrowheads indicate the position of the tip signals detected at time 0. See also Video 7. Bars of A–D are 10 μm (top images) or 1 μm (bottom images). (E) In vitro interaction of Ncd tail with COOH terminus EB1 fragment. Coomassie staining after GST pull-down experiment is shown. Binding reaction was performed in 200 μl volume with 12 μM dEB1C and 1 μM GST-Ncd-tail (lanes 2–6) or GST (lane 1). GFP-hAPC-C was preincubated with dEB1C at 1:10, 1:3, 1:1, and 2:1 molar ratio for competition (lanes 3–6). Bound fractions were 18-fold loaded compared with unbound fractions.
Mentions: In addition to the localization of Ncd to K fibers, spinning-disk confocal microscopy revealed a previously unreported enrichment of Ncd-GFP at the tips of growing mitotic microtubules growing from centrosomes (Fig. 4 A, yellow arrowheads; and Video 5 available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). Even though the microtubule shafts also have Ncd-GFP staining, our quantitation shows an at least threefold enrichment of the GFP signals at the microtubule tip (unpublished data). The tip localization was most evident in the cells expressing lower amounts of Ncd-GFP (i.e., levels shown in Fig. 3 A, 2; increasing expression led to more uniform staining of the spindle microtubules).

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