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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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Dynein tethers centrosomes to  spindle poles. (a) Cytoplasmic dynein is  eluted from spindles by addition of mAb  70.1. Immunofluorescent localization of  dynein heavy chain to spindle poles disappears within 5 min after mAb 70.1 addition. Microtubules are green, dynein  heavy chain is red, and overlap is yellow.  (b) Centrosomes are released from sperm  DNA spindle poles 3 min after addition of  mAb 70.1. Immunofluorescent localization of γ tubulin on sperm centriolar structures that are dissociating from spindle  poles. (c) Model of how dynein tethers  spindle microtubules to centrosomal microtubules and how this is disrupted by  mAb 70.1. Bars, 5 μm.
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Figure 5: Dynein tethers centrosomes to spindle poles. (a) Cytoplasmic dynein is eluted from spindles by addition of mAb 70.1. Immunofluorescent localization of dynein heavy chain to spindle poles disappears within 5 min after mAb 70.1 addition. Microtubules are green, dynein heavy chain is red, and overlap is yellow. (b) Centrosomes are released from sperm DNA spindle poles 3 min after addition of mAb 70.1. Immunofluorescent localization of γ tubulin on sperm centriolar structures that are dissociating from spindle poles. (c) Model of how dynein tethers spindle microtubules to centrosomal microtubules and how this is disrupted by mAb 70.1. Bars, 5 μm.

Mentions: We wanted to understand how mAb 70.1 was disrupting dynein activity. Microtubule gliding assays performed with purified dynein revealed that mAb 70.1 did not block motility of the motor (data not shown). We therefore examined whether dynein localization was disrupted by addition of mAb 70.1 to extracts. A polyclonal antibody to the dynein heavy chain was used for immunofluorescent analysis of spindles before and after mAb 70.1 addition (Fig. 5). In agreement with published reports, the dynein heavy chain was localized to the poles of spindles, and faint punctate staining was also visible on chromosomes, probably corresponding to kinetochores (Pfarr et al., 1990; Steuer et al., 1990). Upon addition of mAb 70.1, all spindle staining with the heavy chain antibody disappeared within 5 min. Therefore, mAb 70.1 seems to inhibit dynein by preventing localization and/or accumulation of the motor on spindle microtubules, perhaps by disruption of the interaction of dynein with other proteins, such as the dynactin complex or NuMA.


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)

Dynein tethers centrosomes to  spindle poles. (a) Cytoplasmic dynein is  eluted from spindles by addition of mAb  70.1. Immunofluorescent localization of  dynein heavy chain to spindle poles disappears within 5 min after mAb 70.1 addition. Microtubules are green, dynein  heavy chain is red, and overlap is yellow.  (b) Centrosomes are released from sperm  DNA spindle poles 3 min after addition of  mAb 70.1. Immunofluorescent localization of γ tubulin on sperm centriolar structures that are dissociating from spindle  poles. (c) Model of how dynein tethers  spindle microtubules to centrosomal microtubules and how this is disrupted by  mAb 70.1. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Dynein tethers centrosomes to spindle poles. (a) Cytoplasmic dynein is eluted from spindles by addition of mAb 70.1. Immunofluorescent localization of dynein heavy chain to spindle poles disappears within 5 min after mAb 70.1 addition. Microtubules are green, dynein heavy chain is red, and overlap is yellow. (b) Centrosomes are released from sperm DNA spindle poles 3 min after addition of mAb 70.1. Immunofluorescent localization of γ tubulin on sperm centriolar structures that are dissociating from spindle poles. (c) Model of how dynein tethers spindle microtubules to centrosomal microtubules and how this is disrupted by mAb 70.1. Bars, 5 μm.
Mentions: We wanted to understand how mAb 70.1 was disrupting dynein activity. Microtubule gliding assays performed with purified dynein revealed that mAb 70.1 did not block motility of the motor (data not shown). We therefore examined whether dynein localization was disrupted by addition of mAb 70.1 to extracts. A polyclonal antibody to the dynein heavy chain was used for immunofluorescent analysis of spindles before and after mAb 70.1 addition (Fig. 5). In agreement with published reports, the dynein heavy chain was localized to the poles of spindles, and faint punctate staining was also visible on chromosomes, probably corresponding to kinetochores (Pfarr et al., 1990; Steuer et al., 1990). Upon addition of mAb 70.1, all spindle staining with the heavy chain antibody disappeared within 5 min. Therefore, mAb 70.1 seems to inhibit dynein by preventing localization and/or accumulation of the motor on spindle microtubules, perhaps by disruption of the interaction of dynein with other proteins, such as the dynactin complex or NuMA.

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