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. 2000 May;11(5):1765-74.
doi: 10.1091/mbc.11.5.1765.

Mammalian spindle orientation and position respond to changes in cell shape in a dynein-dependent fashion

C B O'Connell  1 , Y L Wang
Affiliations
Free PMC article

Mammalian spindle orientation and position respond to changes in cell shape in a dynein-dependent fashion

C B O'Connell et al. Mol Biol Cell. 2000 May.
Free PMC article

Abstract

In animal cells, positioning of the mitotic spindle is crucial for defining the plane of cytokinesis and the size ratio of daughter cells. We have characterized this phenomenon in a rat epithelial cell line using microscopy, micromanipulation, and microinjection. Unmanipulated cells position the mitotic spindle near their geometric center, with the spindle axis lying roughly parallel to the long axis of the cell. Spindles that were initially misoriented underwent directed rotation and caused a delay in anaphase onset. To gain further insight into this process, we gently deformed cells with a blunted glass needle to change the spatial relationship between the cortex and spindle. This manipulation induced spindle movement or rotation in metaphase and/or anaphase, until the spindle reached a proper position relative to the deformed shape. Spindle positioning was inhibited by either treatment with low doses of nocodazole or microinjection of antibodies against dynein, apparently due to the disruption of the organization of dynein and/or astral microtubules. Our results suggest that mitotic cells continuously monitor and maintain the position of the spindle relative to the cortex. This process is likely driven by interactions among astral microtubules, the motor protein dynein, and the cell cortex and may constitute part of a mitotic checkpoint mechanism.

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Figures

Figure 1
Figure 1
Rotation of mitotic spindles in unmanipulated NRK cells. Time-lapse phase-contrast images (A–F) or microinjected rhodamine-tubulin images (G–L) were recorded in cells where the initial spindle orientation deviates from the long axis of the cell (A and G). Rotation of the spindle in the upper cell took place mostly in anaphase (C–E). Spindle rotation in the lower cell took place during metaphase (H–J). Both reached an orientation roughly along the longest axis of the cell (E and K). Time in minutes relative to metaphase is shown in the upper right corner. Asterisks and dotted lines indicate initial positions of the spindle poles and axis, respectively. Circles and solid lines indicate current positions of the spindle poles and axis, respectively. Bars, 20 μm.
Figure 2
Figure 2
Time-lapse images of control manipulation performed on metaphase cells. The membrane was pushed perpendicular to the cell axis near the metaphase plate (B), causing no change in the orientation of the long axis of the cell. No change in the position or orientation of the spindle took place, and both anaphase and cytokinesis progressed normally (B–E). Time in minutes relative to metaphase is shown in the upper right corner. Asterisks and dotted lines indicate initial positions of the spindle poles and axis, respectively. Circles and solid lines indicate current positions of the spindle poles and axis, respectively. Bar, 20 μm.
Figure 3
Figure 3
Rotation of mitotic spindle in a cell manipulated to alter the orientation of the long axis. A blunted microneedle was gently placed against the membrane and moved forward to push the cortex (B). Soon after, deformation spindles began to rotate (C–D). By mid-anaphase, the spindle was aligned along the longest axis of the cell (E). The same approach was also used to deform the cortex of a cell microinjected with rhodamine-labeled tubulin (G–L), which had a shape similar to that for the cell in A–F. The position of the needle is shown with an arrow (H). The spindle rotated and the cell proceeded through anaphase and telophase, with no apparent damage to the spindle apparatus. Time relative to metaphase is shown in the upper right corner. Asterisks and dotted lines indicate initial positions of the spindle poles and axis, respectively. Circles and solid lines indicate current positions of the spindle poles and axis, respectively. Bars, 20 μm.
Figure 4
Figure 4
Spindle translocation in a cell manipulated to alter the position of the cell center. The cortex of this elongated cell was constricted near one end, such that the cortex became much closer to the upper spindle pole than to the lower spindle pole (B, arrowhead). The spindle gradually moved through the cytoplasm to a central position within the main cell mass (B–E). A cleavage furrow formed between the chromosomes at the center of the cell. The constriction disappeared upon removal of the needle, generating two daughter cells of unequal sizes (H). Time relative to metaphase is shown in the upper right corner. Asterisks indicate the center of the metaphase plate before manipulation. Circles denote the current central position of the spindle. Bar, 20 μm.
Figure 5
Figure 5
Pattern of dynein localization during mitosis. NRK cells were fixed and stained for the intermediate chain of dynein (green) and chromosomes (blue). The cells were optically sectioned, and images were deconvolved and reconstructed to show the 90° view of structures on all focal planes. During early prometaphase (A), dynein is localized primarily at the kinetochores of the chromosomes. By late prometaphase (B), the distribution has changed dramatically. Dynein is now visible primarily on spindle poles and astral microtubules. Throughout metaphase and early anaphase (C and D), dynein is predominantly localized in a discontinuous pattern along astral microtubules. Some localization was also observed along interzonal microtubules. Bar, 10 μm.
Figure 6
Figure 6
Organization of dynein and microtubules in a control cell (A–C) and a cell treated overnight with 100 nM nocodazole (D–F). The cells were microinjected with fluorescent tubulin at prometaphase and fixed and stained at metaphase. Stacks of optical sections were deconvolved and reconstructed into the 90° view. Microtubules in the mitotic spindle of the treated cell (D) had a typical bipolar arrangement, similar to those in control cells (A). However, both the spindle length and the number and length of astral microtubules are reduced. Enhanced views of astral microtubules are shown in the insets of A and D. The distribution of dynein also appeared disorganized after nocodazole treatment, with no accumulation where the astral microtubules would be (E and F), as seen in control cells (B and C). Bars, 10 μm.
Figure 7
Figure 7
Time-lapse images of a dividing NRK cell treated with 100 nM nocodazole for 24 h. The metaphase spindle appeared normal under phase optics but was located away from the geometric center of the cell, with an orientation misaligned relative to the long axis of the cell (A and B). The cell proceeded into anaphase without correcting the position of the spindle (C). In this instance cytokinesis was inhibited, resulting in a binucleated cell (D). Time in minutes relative to metaphase is shown in the upper right corner. Bar, 20 μm.
Figure 8
Figure 8
The organization of dynein (A′) and microtubules (B′) in NRK cells microinjected with the 70.1 monoclonal antibody against dynein intermediate chain. Stacks of optical sections were deconvolved and used for reconstruction of the 90° view. Dynein staining revealed no localization along astral microtubules, although some staining is visible at the spindle poles (A and A′, arrows). The injection caused no apparent disruption of the spindle, as shown by immunofluorescence of tubulin in a separate cell (B and B′). Bars, 10 μm.
Figure 9
Figure 9
Inhibition of spindle rotation and migration by anti-dynein antibodies. Two NRK cells were injected with 70.1 antibodies against the dynein intermediate chain and deformed with a microneedle as in Figures 3 and 4. In the first cell (A–E), the needle created a highly asymmetric cell shape. Throughout metaphase and anaphase the spindle remained close to its original orientation (B–D), such that one set of chromosomes bumped into the needle (D, arrowhead). A cleavage furrow developed along the longest axis of the cell between separating chromosomes. Asterisks and dotted lines indicate the initial positions of the spindle poles and axis, respectively. Circles and solid lines indicate current positions of the spindle poles and axis, respectively. In the second cell (F–I), the needle was used to create a constriction near one end of the cell, such that the cortex became much closer to one of the spindle poles. No repositioning of the spindle was detected. A cleavage furrow developed between separating chromosomes, creating two daughter cells of unequal sizes. Time relative to metaphase is shown in the upper right corner. Bars, 20 μm.
Figure 10
Figure 10
Models for spindle positioning in mitotic mammalian cells based on pulling forces exerted on astral microtubules. (A) When the spindle is misaligned relative to the long axis of the cell, the lengths of astral microtubules become asymmetric with respect to the spindle axis. Longer microtubules, which interact with more cortically associated dynein, exert stronger forces on the centrosomes and generate a torque toward the long axis of the cell. The forces become symmetric once the spindle is aligned with the long cellular axis, eliminating the torque. (B) A similar mechanism may also center the spindle at the geometric center of the cell. Shorter astral microtubules on the side of the spindle proximal to the cortex exert weaker forces than do longer ones on the opposite side. This produces a net force toward the distal cortex until the spindle reaches a central location.
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