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Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization.

Heald R, Tournebize R, Habermann A, Karsenti E, Hyman A - J. Cell Biol. (1997)

Bottom Line: In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not.We have found that poles are morphologically similar regardless of their origin.Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, European Molecular Biology Laboratory, 69117 Heidelberg, Germany. Heald@EMBL-Heidelberg.de

ABSTRACT
In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end-directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

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Microtubules are sorted into antiparallel arrays around chromatin in the absence of pole formation. (a) Immunofluorescent  localization of NuMA to the frayed ends of chromatin bead spindles that have formed in the absence of dynein activity. (b and c) Hooking analysis. (b) Quantification of hook handedness in control and poleless spindles. Percentage of right- and left-handed hooks in sections through spindle centers containing chromatin beads, and spindle ends containing microtubule poles (control), or bundles (+ mAb  70.1). (c) Low magnification micrographs (5-μm width) are shown to give an overall impression of microtubule organization seen in sections through spindle ends containing microtubule poles or bundles and through spindle centers containing beads. (d) Higher magnification micrographs (2–2.5-μm width) show hooks on cross sectioned individual microtubules. In the presence of control antibodies, a section through a pole contains right-handed (clockwise) hooks, while a section containing beads contains both right- and left-handed  hooks. In the presence of mAb 70.1, a section through a microtubule bundle likely to be at the spindle end is shown that contains almost  exclusively left-handed hooks. Note: Hook handedness does not give any information about the polarity of the microtubules in these  sections but indicates the degree to which microtubule polarity is uniform. 50–100 microtubules were evaluated in each section, and  three sections were evaluated for each condition. Bar, 5 μm.
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Figure 6: Microtubules are sorted into antiparallel arrays around chromatin in the absence of pole formation. (a) Immunofluorescent localization of NuMA to the frayed ends of chromatin bead spindles that have formed in the absence of dynein activity. (b and c) Hooking analysis. (b) Quantification of hook handedness in control and poleless spindles. Percentage of right- and left-handed hooks in sections through spindle centers containing chromatin beads, and spindle ends containing microtubule poles (control), or bundles (+ mAb 70.1). (c) Low magnification micrographs (5-μm width) are shown to give an overall impression of microtubule organization seen in sections through spindle ends containing microtubule poles or bundles and through spindle centers containing beads. (d) Higher magnification micrographs (2–2.5-μm width) show hooks on cross sectioned individual microtubules. In the presence of control antibodies, a section through a pole contains right-handed (clockwise) hooks, while a section containing beads contains both right- and left-handed hooks. In the presence of mAb 70.1, a section through a microtubule bundle likely to be at the spindle end is shown that contains almost exclusively left-handed hooks. Note: Hook handedness does not give any information about the polarity of the microtubules in these sections but indicates the degree to which microtubule polarity is uniform. 50–100 microtubules were evaluated in each section, and three sections were evaluated for each condition. Bar, 5 μm.

Mentions: Our results thus far indicate that cytoplasmic dynein plays a central role in spindle organization. We wondered to what extent dynein function and pole assembly contributed to the polarized organization of microtubules in spindles, with microtubule minus ends at the poles and antiparallel microtubule interactions in the center of the spindle. We have shown previously that in the presence of mAb 70.1, a parallel array of microtubules formed around chromatin beads with frayed, unfocused ends, and the beads located in the center of the array (Heald et al., 1996; Fig. 6 a). We wanted to know whether microtubules in such bundles were randomly oriented or sorted into an antiparallel array around chromatin, with plus ends at the chromatin and minus ends at spindle termini. To address this question, we assessed microtubule polarity in spindles assembled in the presence or absence of mAb 70.1 using two independent approaches. First, we examined the localization of NuMA, a protein normally associated with the minus ends of microtubules in polarized arrays (Fig. 1) (Maekawa et al., 1991). We found that NuMA was still localized to the two frayed unfocused ends of the spindle formed in the presence of mAb 70.1, albeit more diffusely (Fig. 6 a). This result indicated that microtubule minus ends were still sorted away from chromatin in the absence of pole formation. Second, we determined directly whether microtubule polarity was uniform or not by the hooking technique. In this technique, microtubules are incubated with pure tubulin under conditions that promote addition of hooked protofilament appendages to the microtubule walls (Heidemann and McIntosh, 1980). The polarity of individual microtubules can then be determined by examination of hook handedness in serial sections by electron microscopy (Euteneuer and McIntosh, 1981; Euteneuer et al., 1982). Here we analyzed single sections to determine hook handedness in chromatin bead spindles assembled in the presence of control antibodies or mAb 70.1 (Fig. 6). Under both conditions, sections that were cut through beads, likely to correspond to the spindle centers, contained microtubules with approximately equal proportions of right- and left-handed hooks, indicating that microtubules were of mixed polarity (Fig. 6, b–d). In control spindles, sections through focused microtubule bundles, corresponding to poles, contained hooks of which 90% were the same handedness, indicating that microtubules were of almost uniform polarity (Fig. 6, b–d). Although spindles assembled in the presence of mAb 70.1 did not contain poles, microtubule bundles, likely to be close to spindle termini, contained microtubules of >95% uniform hook handedness. Therefore, both NuMA staining and hook analysis show that microtubules are sorted into antiparallel arrays around chromatin beads. This sorting is independent of dynein activity and pole formation, indicating that other microtubule-based motors are contributing to the antiparallel organization of microtubules in spindles.


Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization.

Heald R, Tournebize R, Habermann A, Karsenti E, Hyman A - J. Cell Biol. (1997)

Microtubules are sorted into antiparallel arrays around chromatin in the absence of pole formation. (a) Immunofluorescent  localization of NuMA to the frayed ends of chromatin bead spindles that have formed in the absence of dynein activity. (b and c) Hooking analysis. (b) Quantification of hook handedness in control and poleless spindles. Percentage of right- and left-handed hooks in sections through spindle centers containing chromatin beads, and spindle ends containing microtubule poles (control), or bundles (+ mAb  70.1). (c) Low magnification micrographs (5-μm width) are shown to give an overall impression of microtubule organization seen in sections through spindle ends containing microtubule poles or bundles and through spindle centers containing beads. (d) Higher magnification micrographs (2–2.5-μm width) show hooks on cross sectioned individual microtubules. In the presence of control antibodies, a section through a pole contains right-handed (clockwise) hooks, while a section containing beads contains both right- and left-handed  hooks. In the presence of mAb 70.1, a section through a microtubule bundle likely to be at the spindle end is shown that contains almost  exclusively left-handed hooks. Note: Hook handedness does not give any information about the polarity of the microtubules in these  sections but indicates the degree to which microtubule polarity is uniform. 50–100 microtubules were evaluated in each section, and  three sections were evaluated for each condition. Bar, 5 μm.
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Figure 6: Microtubules are sorted into antiparallel arrays around chromatin in the absence of pole formation. (a) Immunofluorescent localization of NuMA to the frayed ends of chromatin bead spindles that have formed in the absence of dynein activity. (b and c) Hooking analysis. (b) Quantification of hook handedness in control and poleless spindles. Percentage of right- and left-handed hooks in sections through spindle centers containing chromatin beads, and spindle ends containing microtubule poles (control), or bundles (+ mAb 70.1). (c) Low magnification micrographs (5-μm width) are shown to give an overall impression of microtubule organization seen in sections through spindle ends containing microtubule poles or bundles and through spindle centers containing beads. (d) Higher magnification micrographs (2–2.5-μm width) show hooks on cross sectioned individual microtubules. In the presence of control antibodies, a section through a pole contains right-handed (clockwise) hooks, while a section containing beads contains both right- and left-handed hooks. In the presence of mAb 70.1, a section through a microtubule bundle likely to be at the spindle end is shown that contains almost exclusively left-handed hooks. Note: Hook handedness does not give any information about the polarity of the microtubules in these sections but indicates the degree to which microtubule polarity is uniform. 50–100 microtubules were evaluated in each section, and three sections were evaluated for each condition. Bar, 5 μm.
Mentions: Our results thus far indicate that cytoplasmic dynein plays a central role in spindle organization. We wondered to what extent dynein function and pole assembly contributed to the polarized organization of microtubules in spindles, with microtubule minus ends at the poles and antiparallel microtubule interactions in the center of the spindle. We have shown previously that in the presence of mAb 70.1, a parallel array of microtubules formed around chromatin beads with frayed, unfocused ends, and the beads located in the center of the array (Heald et al., 1996; Fig. 6 a). We wanted to know whether microtubules in such bundles were randomly oriented or sorted into an antiparallel array around chromatin, with plus ends at the chromatin and minus ends at spindle termini. To address this question, we assessed microtubule polarity in spindles assembled in the presence or absence of mAb 70.1 using two independent approaches. First, we examined the localization of NuMA, a protein normally associated with the minus ends of microtubules in polarized arrays (Fig. 1) (Maekawa et al., 1991). We found that NuMA was still localized to the two frayed unfocused ends of the spindle formed in the presence of mAb 70.1, albeit more diffusely (Fig. 6 a). This result indicated that microtubule minus ends were still sorted away from chromatin in the absence of pole formation. Second, we determined directly whether microtubule polarity was uniform or not by the hooking technique. In this technique, microtubules are incubated with pure tubulin under conditions that promote addition of hooked protofilament appendages to the microtubule walls (Heidemann and McIntosh, 1980). The polarity of individual microtubules can then be determined by examination of hook handedness in serial sections by electron microscopy (Euteneuer and McIntosh, 1981; Euteneuer et al., 1982). Here we analyzed single sections to determine hook handedness in chromatin bead spindles assembled in the presence of control antibodies or mAb 70.1 (Fig. 6). Under both conditions, sections that were cut through beads, likely to correspond to the spindle centers, contained microtubules with approximately equal proportions of right- and left-handed hooks, indicating that microtubules were of mixed polarity (Fig. 6, b–d). In control spindles, sections through focused microtubule bundles, corresponding to poles, contained hooks of which 90% were the same handedness, indicating that microtubules were of almost uniform polarity (Fig. 6, b–d). Although spindles assembled in the presence of mAb 70.1 did not contain poles, microtubule bundles, likely to be close to spindle termini, contained microtubules of >95% uniform hook handedness. Therefore, both NuMA staining and hook analysis show that microtubules are sorted into antiparallel arrays around chromatin beads. This sorting is independent of dynein activity and pole formation, indicating that other microtubule-based motors are contributing to the antiparallel organization of microtubules in spindles.

Bottom Line: In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not.We have found that poles are morphologically similar regardless of their origin.Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, European Molecular Biology Laboratory, 69117 Heidelberg, Germany. Heald@EMBL-Heidelberg.de

ABSTRACT
In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end-directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

Show MeSH
Related in: MedlinePlus