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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

Disruption of poles by antidynein antibodies. The monoclonal antibody (mAb 70.1) that recognizes the intermediate chain of  cytoplasmic dynein was added to extracts containing chromatin bead spindles (A), DMSO asters (B), or sperm DNA spindles (C) with  centrosomes. Pole structures were disrupted within 10 min. Bar, 5 μm.
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Figure 4: Disruption of poles by antidynein antibodies. The monoclonal antibody (mAb 70.1) that recognizes the intermediate chain of cytoplasmic dynein was added to extracts containing chromatin bead spindles (A), DMSO asters (B), or sperm DNA spindles (C) with centrosomes. Pole structures were disrupted within 10 min. Bar, 5 μm.

Mentions: The similar behavior of stable microtubules added to spindles and asters suggested that this movement was due to a common mechanism. We have shown that seed movement on chromatin bead spindles is dependent on cytoplasmic dynein. Inhibition of dynein with vanadate or by addition of a monoclonal antibody to the dynein intermediate chain (mAb 70.1) not only blocked seed translocation but caused dissolution of the poles (Heald et al., 1996; and Fig. 4 A). We wanted to test whether dynein was also involved in the organization of DMSO asters and in spindle poles containing centrosomes. The addition of mAb 70.1 to DMSO asters caused disruption of aster integrity (Fig. 4 B), confirming a requirement for dynein in spontaneous aster assembly shown previously (Verde et al., 1991; Gaglio et al., 1996). Furthermore, addition of mAb 70.1 also disrupted sperm DNA spindle poles containing centrosomes, causing them to splay outwards and become disorganized within 10 min (Fig. 4 C). In all cases, addition of antibody prevented seed movement towards poles (data not shown). When added before initiation of spindle assembly reactions, mAb 70.1 blocked pole formation, yielding similar structures with frayed, unfocused ends (Heald et al., 1996; see Fig. 8 a, D). Therefore, these results indicate that dynein-dependent translocation of microtubules is required for pole formation and maintenance both in the presence and absence of centrosomes.


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)

Disruption of poles by antidynein antibodies. The monoclonal antibody (mAb 70.1) that recognizes the intermediate chain of  cytoplasmic dynein was added to extracts containing chromatin bead spindles (A), DMSO asters (B), or sperm DNA spindles (C) with  centrosomes. Pole structures were disrupted within 10 min. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Disruption of poles by antidynein antibodies. The monoclonal antibody (mAb 70.1) that recognizes the intermediate chain of cytoplasmic dynein was added to extracts containing chromatin bead spindles (A), DMSO asters (B), or sperm DNA spindles (C) with centrosomes. Pole structures were disrupted within 10 min. Bar, 5 μm.
Mentions: The similar behavior of stable microtubules added to spindles and asters suggested that this movement was due to a common mechanism. We have shown that seed movement on chromatin bead spindles is dependent on cytoplasmic dynein. Inhibition of dynein with vanadate or by addition of a monoclonal antibody to the dynein intermediate chain (mAb 70.1) not only blocked seed translocation but caused dissolution of the poles (Heald et al., 1996; and Fig. 4 A). We wanted to test whether dynein was also involved in the organization of DMSO asters and in spindle poles containing centrosomes. The addition of mAb 70.1 to DMSO asters caused disruption of aster integrity (Fig. 4 B), confirming a requirement for dynein in spontaneous aster assembly shown previously (Verde et al., 1991; Gaglio et al., 1996). Furthermore, addition of mAb 70.1 also disrupted sperm DNA spindle poles containing centrosomes, causing them to splay outwards and become disorganized within 10 min (Fig. 4 C). In all cases, addition of antibody prevented seed movement towards poles (data not shown). When added before initiation of spindle assembly reactions, mAb 70.1 blocked pole formation, yielding similar structures with frayed, unfocused ends (Heald et al., 1996; see Fig. 8 a, D). Therefore, these results indicate that dynein-dependent translocation of microtubules is required for pole formation and maintenance both in the presence and absence of centrosomes.

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