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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: We have found that poles are morphologically similar regardless of their origin.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.

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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Related in: MedlinePlus

Video analysis of seed movement on DMSO asters. (a) Successive video frames at 20-s intervals showing that individual seeds  move poleward. (b) Rates of seed movement over 5-s intervals and distances of individual seeds from the pole over time. (c) Polarity-marked microtubule seeds are oriented with their minus ends directed toward the center of the aster and its focus of microtubule minus  ends. Bars: (a) 5 μm; (c) 8 μm.
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Figure 3: Video analysis of seed movement on DMSO asters. (a) Successive video frames at 20-s intervals showing that individual seeds move poleward. (b) Rates of seed movement over 5-s intervals and distances of individual seeds from the pole over time. (c) Polarity-marked microtubule seeds are oriented with their minus ends directed toward the center of the aster and its focus of microtubule minus ends. Bars: (a) 5 μm; (c) 8 μm.

Mentions: While the visual similarity of these microtubule arrays was striking, it did not indicate whether pole assembly occurred by a common mechanism. To characterize how poles form, we took advantage of an assay developed to follow microtubule movements during spindle assembly. We have shown previously that stable polarity-marked microtubule “seeds,” containing a brightly labeled minus end and a dimly labeled plus end, act as markers for the movement of endogenous microtubules during spindle assembly around chromatin beads (Heald et al., 1996). Seeds added to extracts bind to and translocate along microtubules towards poles with their minus ends leading (Fig. 2 a). We compared seed motility on the three types of polarized microtubule arrays (Fig. 2 b). Just as on chromatin bead spindles, microtubule seeds translocated poleward in DMSO asters, forming bright foci at the poles. A complete analysis of seed movement on DMSO asters is shown in Fig. 3. Poleward seed movement was saltatory, with an average speed of 6 μm/min and a peak velocity of 30 μm/min. Polarity-marked seeds moved poleward with their brightly labeled minus ends leading. Since both chromatin bead and DMSO aster pole structures are generated by microtubule self-organization, these results were not surprising. Importantly, however, seeds also translocated poleward on sperm DNA spindles containing poles derived from centrosomes (Fig. 2 b). In all three cases, seed movement was qualitatively and quantitatively similar (data not shown). Therefore, minus end–directed microtubule movement is a general property of poles, whether or not centrosomes are present.


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)

Video analysis of seed movement on DMSO asters. (a) Successive video frames at 20-s intervals showing that individual seeds  move poleward. (b) Rates of seed movement over 5-s intervals and distances of individual seeds from the pole over time. (c) Polarity-marked microtubule seeds are oriented with their minus ends directed toward the center of the aster and its focus of microtubule minus  ends. Bars: (a) 5 μm; (c) 8 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141625&req=5

Figure 3: Video analysis of seed movement on DMSO asters. (a) Successive video frames at 20-s intervals showing that individual seeds move poleward. (b) Rates of seed movement over 5-s intervals and distances of individual seeds from the pole over time. (c) Polarity-marked microtubule seeds are oriented with their minus ends directed toward the center of the aster and its focus of microtubule minus ends. Bars: (a) 5 μm; (c) 8 μm.
Mentions: While the visual similarity of these microtubule arrays was striking, it did not indicate whether pole assembly occurred by a common mechanism. To characterize how poles form, we took advantage of an assay developed to follow microtubule movements during spindle assembly. We have shown previously that stable polarity-marked microtubule “seeds,” containing a brightly labeled minus end and a dimly labeled plus end, act as markers for the movement of endogenous microtubules during spindle assembly around chromatin beads (Heald et al., 1996). Seeds added to extracts bind to and translocate along microtubules towards poles with their minus ends leading (Fig. 2 a). We compared seed motility on the three types of polarized microtubule arrays (Fig. 2 b). Just as on chromatin bead spindles, microtubule seeds translocated poleward in DMSO asters, forming bright foci at the poles. A complete analysis of seed movement on DMSO asters is shown in Fig. 3. Poleward seed movement was saltatory, with an average speed of 6 μm/min and a peak velocity of 30 μm/min. Polarity-marked seeds moved poleward with their brightly labeled minus ends leading. Since both chromatin bead and DMSO aster pole structures are generated by microtubule self-organization, these results were not surprising. Importantly, however, seeds also translocated poleward on sperm DNA spindles containing poles derived from centrosomes (Fig. 2 b). In all three cases, seed movement was qualitatively and quantitatively similar (data not shown). Therefore, minus end–directed microtubule movement is a general property of poles, whether or not centrosomes are present.

Bottom Line: We have found that poles are morphologically similar regardless of their origin.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.

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