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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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Distinct effects of Ncd and cytoplasmic dynein on pole coalescence. (A) Representative spindle morphology after RNAi of indicated genes. Green, tubulin; blue, DNA. Bar, 10 μm. (B) Immunoblot showing knockdowns of indicated proteins. Samples were collected at day 7 after RNAi (dsRNA was treated at day 0 and day 4). Note that single and double RNAi result in similar level of reduction of Ncd or Dhc64C. (C) Quantitation of K fiber unfocusing. Relative mean width of the K fiber minus ends (green line) are shown with standard errors bars (control no RNAi cells; 2.2 ± 1.2 μm [mean ± SD; n = 94]). Experiments were performed twice and combined data is presented. Statistically significant increase was seen after Ncd (n = 57), EB1 (n = 91) and Ncd/Dhc64C (n = 59) double RNAi (asterisk; P < 0.0001 for each experiment), whereas Dhc64C single RNAi (n = 73) did not produce significant effects at this sample size (P > 0.05, t test). The frequency of metaphase spindles with multiple poles or unfocused K fiber(s) was also scored and is described at the bottom (n >50 for each treatment). EB1, Ncd, and double Ncd/Dhc64C RNAi significantly increased the spindles with multiple poles or unfocused K fibers. (D) Quantitation of centrosome detachment in the metaphase spindle. The gap distance between centrosome (stained by γ-tubulin) and the minus end of K fiber that is most closely located to the centrosome was measured in three independent experiments (blue line), and relative average distance to accompanying control sample (average 1.2 ± 0.7 μm [mean ± SD; n = 307]) is shown with standard error bars after single Ncd (n = 63), Dhc64C (n = 188), EB1 RNAi (n = 109), or double Ncd/Dhc64C RNAi (n =128). In each spindle, the pole with the wider gap distance was chosen for measurement. We confirmed that the severe detachment seen after Dhc64C RNAi was not due to prolonged metaphase (Goshima and Vale, 2003), as RNAi of APC/cyclosome component Cdc16 or double Cdc16/Ncd also accumulated in metaphase but such a defect was not observed. Reduction of Ncd often leads to formation of the spindles with multiple asters (Goshima and Vale, 2003). However, we selected the cells whose spindle had overall bipolar structure (i.e., chromosomes are aligned and visible in a single line) and single aster on either side.
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fig2: Distinct effects of Ncd and cytoplasmic dynein on pole coalescence. (A) Representative spindle morphology after RNAi of indicated genes. Green, tubulin; blue, DNA. Bar, 10 μm. (B) Immunoblot showing knockdowns of indicated proteins. Samples were collected at day 7 after RNAi (dsRNA was treated at day 0 and day 4). Note that single and double RNAi result in similar level of reduction of Ncd or Dhc64C. (C) Quantitation of K fiber unfocusing. Relative mean width of the K fiber minus ends (green line) are shown with standard errors bars (control no RNAi cells; 2.2 ± 1.2 μm [mean ± SD; n = 94]). Experiments were performed twice and combined data is presented. Statistically significant increase was seen after Ncd (n = 57), EB1 (n = 91) and Ncd/Dhc64C (n = 59) double RNAi (asterisk; P < 0.0001 for each experiment), whereas Dhc64C single RNAi (n = 73) did not produce significant effects at this sample size (P > 0.05, t test). The frequency of metaphase spindles with multiple poles or unfocused K fiber(s) was also scored and is described at the bottom (n >50 for each treatment). EB1, Ncd, and double Ncd/Dhc64C RNAi significantly increased the spindles with multiple poles or unfocused K fibers. (D) Quantitation of centrosome detachment in the metaphase spindle. The gap distance between centrosome (stained by γ-tubulin) and the minus end of K fiber that is most closely located to the centrosome was measured in three independent experiments (blue line), and relative average distance to accompanying control sample (average 1.2 ± 0.7 μm [mean ± SD; n = 307]) is shown with standard error bars after single Ncd (n = 63), Dhc64C (n = 188), EB1 RNAi (n = 109), or double Ncd/Dhc64C RNAi (n =128). In each spindle, the pole with the wider gap distance was chosen for measurement. We confirmed that the severe detachment seen after Dhc64C RNAi was not due to prolonged metaphase (Goshima and Vale, 2003), as RNAi of APC/cyclosome component Cdc16 or double Cdc16/Ncd also accumulated in metaphase but such a defect was not observed. Reduction of Ncd often leads to formation of the spindles with multiple asters (Goshima and Vale, 2003). However, we selected the cells whose spindle had overall bipolar structure (i.e., chromosomes are aligned and visible in a single line) and single aster on either side.

Mentions: We next performed RNAi of the dynein heavy chain (Dhc64C), Ncd, or double RNAi of both motors and then quantitatively examined pole focusing of metaphase mitotic spindles by fixed cell immunofluorescence (Fig. 2 A). Significant reduction of motor protein levels after a 7-d RNAi treatment was confirmed by immunoblot analysis (Fig. 2 B). We quantitated the two phenomena involved in pole focusing that our live cell imaging suggested as being separable to some extent: the lateral spread of the kinetochore minus ends (herein termed K fiber distance; Fig. 2 C) and the distance between the centrosome and the base of the K fibers (centrosome to K fiber distance; Fig. 2 D). We confirmed the results of Maiato et al. (2004) and Morales-Mulia and Scholey (2005) of centrosome detachment (an increase in the centrosome to K fiber distance) after Dhc64C RNAi (Fig. 2, A and D). A similar phenotype was observed after RNAi of two dynactin subunits (p50/dynamitin and p150/Glued; unpublished data). Interestingly, Ncd depletion by RNAi did not alter the centrosome to K fiber distance (P > 0.1). However, a weak but statistically significant synergistic effect was apparent upon double RNAi of Ncd and Dhc64C (Fig. 2 D; P < 0.001, compared with Dhc64C alone). In contrast, RNAi of Ncd showed a strong phenotype on K fiber focusing, as was qualitatively described previously (Goshima and Vale, 2003). Maiato et al. (2004) and Morales-Mulia and Scholey (2005) reported that dynein RNAi also caused K fiber unfocusing, but the phenotype was not quantitatively analyzed. We found more K fiber unfocusing in dynein RNAi than control cells, although the effect was much smaller than Ncd RNAi (Fig. 2 C). Again, K fiber unfocusing was much more dramatic after simultaneous knockdowns of Dhc64C and Ncd than each single RNAi (Fig. 2, A and C; P < 0.001). One possible cause of the dramatic synthetic phenotype upon double knockdown could be the presence of polyploidy due to massive failure in previous cell division. However, we did not observe an increase in the DNA content per cell after 7 d Ncd/Dhc64c RNAi treatment (Fig. S2 A available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). Moreover, a synthetic pole focusing effect of double RNAi treatment was even observed after a 2-d RNAi treatment, although fewer cells in the population exhibited spindle defects (Fig. S2 B). These results indicate that the synthetic phenotype of double Ncd/Dhc64c RNAi treatment is not due to polyploidy but rather derives from dysfunction of the motors for spindle microtubules.


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

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

Distinct effects of Ncd and cytoplasmic dynein on pole coalescence. (A) Representative spindle morphology after RNAi of indicated genes. Green, tubulin; blue, DNA. Bar, 10 μm. (B) Immunoblot showing knockdowns of indicated proteins. Samples were collected at day 7 after RNAi (dsRNA was treated at day 0 and day 4). Note that single and double RNAi result in similar level of reduction of Ncd or Dhc64C. (C) Quantitation of K fiber unfocusing. Relative mean width of the K fiber minus ends (green line) are shown with standard errors bars (control no RNAi cells; 2.2 ± 1.2 μm [mean ± SD; n = 94]). Experiments were performed twice and combined data is presented. Statistically significant increase was seen after Ncd (n = 57), EB1 (n = 91) and Ncd/Dhc64C (n = 59) double RNAi (asterisk; P < 0.0001 for each experiment), whereas Dhc64C single RNAi (n = 73) did not produce significant effects at this sample size (P > 0.05, t test). The frequency of metaphase spindles with multiple poles or unfocused K fiber(s) was also scored and is described at the bottom (n >50 for each treatment). EB1, Ncd, and double Ncd/Dhc64C RNAi significantly increased the spindles with multiple poles or unfocused K fibers. (D) Quantitation of centrosome detachment in the metaphase spindle. The gap distance between centrosome (stained by γ-tubulin) and the minus end of K fiber that is most closely located to the centrosome was measured in three independent experiments (blue line), and relative average distance to accompanying control sample (average 1.2 ± 0.7 μm [mean ± SD; n = 307]) is shown with standard error bars after single Ncd (n = 63), Dhc64C (n = 188), EB1 RNAi (n = 109), or double Ncd/Dhc64C RNAi (n =128). In each spindle, the pole with the wider gap distance was chosen for measurement. We confirmed that the severe detachment seen after Dhc64C RNAi was not due to prolonged metaphase (Goshima and Vale, 2003), as RNAi of APC/cyclosome component Cdc16 or double Cdc16/Ncd also accumulated in metaphase but such a defect was not observed. Reduction of Ncd often leads to formation of the spindles with multiple asters (Goshima and Vale, 2003). However, we selected the cells whose spindle had overall bipolar structure (i.e., chromosomes are aligned and visible in a single line) and single aster on either side.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171195&req=5

fig2: Distinct effects of Ncd and cytoplasmic dynein on pole coalescence. (A) Representative spindle morphology after RNAi of indicated genes. Green, tubulin; blue, DNA. Bar, 10 μm. (B) Immunoblot showing knockdowns of indicated proteins. Samples were collected at day 7 after RNAi (dsRNA was treated at day 0 and day 4). Note that single and double RNAi result in similar level of reduction of Ncd or Dhc64C. (C) Quantitation of K fiber unfocusing. Relative mean width of the K fiber minus ends (green line) are shown with standard errors bars (control no RNAi cells; 2.2 ± 1.2 μm [mean ± SD; n = 94]). Experiments were performed twice and combined data is presented. Statistically significant increase was seen after Ncd (n = 57), EB1 (n = 91) and Ncd/Dhc64C (n = 59) double RNAi (asterisk; P < 0.0001 for each experiment), whereas Dhc64C single RNAi (n = 73) did not produce significant effects at this sample size (P > 0.05, t test). The frequency of metaphase spindles with multiple poles or unfocused K fiber(s) was also scored and is described at the bottom (n >50 for each treatment). EB1, Ncd, and double Ncd/Dhc64C RNAi significantly increased the spindles with multiple poles or unfocused K fibers. (D) Quantitation of centrosome detachment in the metaphase spindle. The gap distance between centrosome (stained by γ-tubulin) and the minus end of K fiber that is most closely located to the centrosome was measured in three independent experiments (blue line), and relative average distance to accompanying control sample (average 1.2 ± 0.7 μm [mean ± SD; n = 307]) is shown with standard error bars after single Ncd (n = 63), Dhc64C (n = 188), EB1 RNAi (n = 109), or double Ncd/Dhc64C RNAi (n =128). In each spindle, the pole with the wider gap distance was chosen for measurement. We confirmed that the severe detachment seen after Dhc64C RNAi was not due to prolonged metaphase (Goshima and Vale, 2003), as RNAi of APC/cyclosome component Cdc16 or double Cdc16/Ncd also accumulated in metaphase but such a defect was not observed. Reduction of Ncd often leads to formation of the spindles with multiple asters (Goshima and Vale, 2003). However, we selected the cells whose spindle had overall bipolar structure (i.e., chromosomes are aligned and visible in a single line) and single aster on either side.
Mentions: We next performed RNAi of the dynein heavy chain (Dhc64C), Ncd, or double RNAi of both motors and then quantitatively examined pole focusing of metaphase mitotic spindles by fixed cell immunofluorescence (Fig. 2 A). Significant reduction of motor protein levels after a 7-d RNAi treatment was confirmed by immunoblot analysis (Fig. 2 B). We quantitated the two phenomena involved in pole focusing that our live cell imaging suggested as being separable to some extent: the lateral spread of the kinetochore minus ends (herein termed K fiber distance; Fig. 2 C) and the distance between the centrosome and the base of the K fibers (centrosome to K fiber distance; Fig. 2 D). We confirmed the results of Maiato et al. (2004) and Morales-Mulia and Scholey (2005) of centrosome detachment (an increase in the centrosome to K fiber distance) after Dhc64C RNAi (Fig. 2, A and D). A similar phenotype was observed after RNAi of two dynactin subunits (p50/dynamitin and p150/Glued; unpublished data). Interestingly, Ncd depletion by RNAi did not alter the centrosome to K fiber distance (P > 0.1). However, a weak but statistically significant synergistic effect was apparent upon double RNAi of Ncd and Dhc64C (Fig. 2 D; P < 0.001, compared with Dhc64C alone). In contrast, RNAi of Ncd showed a strong phenotype on K fiber focusing, as was qualitatively described previously (Goshima and Vale, 2003). Maiato et al. (2004) and Morales-Mulia and Scholey (2005) reported that dynein RNAi also caused K fiber unfocusing, but the phenotype was not quantitatively analyzed. We found more K fiber unfocusing in dynein RNAi than control cells, although the effect was much smaller than Ncd RNAi (Fig. 2 C). Again, K fiber unfocusing was much more dramatic after simultaneous knockdowns of Dhc64C and Ncd than each single RNAi (Fig. 2, A and C; P < 0.001). One possible cause of the dramatic synthetic phenotype upon double knockdown could be the presence of polyploidy due to massive failure in previous cell division. However, we did not observe an increase in the DNA content per cell after 7 d Ncd/Dhc64c RNAi treatment (Fig. S2 A available at http://www.jcb.org/cgi/content/full/jcb.200505107/DC1). Moreover, a synthetic pole focusing effect of double RNAi treatment was even observed after a 2-d RNAi treatment, although fewer cells in the population exhibited spindle defects (Fig. S2 B). These results indicate that the synthetic phenotype of double Ncd/Dhc64c RNAi treatment is not due to polyploidy but rather derives from dysfunction of the motors for 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
Related in: MedlinePlus