Abstract
Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development1. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase2,3. The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear. Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo. To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin4. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos. We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior–posterior embryonic axis. Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour. Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.
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Acknowledgements
We thank K. Kemphues for mutants it71 and it5; K. Oegema for experimental assistance; A. Desai, E.-L. Florin and E. Hannak for discussions; R. Pepperkok, K. Schütze and R. Schütze for supplying the laser microdissection setup; and T. Bouwmeester, A. Desai, E. Hannak, E. Karsenti, F. Nédélec and K. Oegema for help with improving the manuscript.
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Fig 2a WT and 2b WTirr
Wild-type (left) and wild-type irradiated (right) embryos, 2 frames per second. Spindle poles are indicated by circles, the region where the spindle is severed is indicated by a flashing bar.
Note the increase in pole-to-pole distance following the cut as well as the asymmetry in movement. On average, the posterior spindle pole travels further and faster than the anterior one (Fig. 3). Furthermore, the posterior spindle pole does not come to a rest, it starts to oscillate transversely as it gets close to the cortex.
Fig2c CeMCAK (RNAi)
CeMCAK (RNAi), 2 frames per second, spindle poles are indicated by circles. Note aberrant polar body at the anterior end.
An increase in pole-to-pole distance following spindle breakage as well as asymmetry in movement can be observed. On average, the posterior spindle pole travels further and faster than the anterior one (Fig. 3). Again the posterior spindle pole oscillates transversely as it gets close to the cortex.
Fig 4a
Model a: Aster geometry is similar for both asters, but all posterior microtubules generate greater pulling forces than the anterior ones. As a result, the posterior spindle pole travels faster than the anterior one, but it does not travel further and it does not start to undergo oscillations.
There is no asymmetry in final position, as both spindle poles move to the point of zero net force, which does not change if the force is increased for all posterior microtubules. Aster geometry defines the position of zero net force.
Fig 4b
Model b: All microtubules generate equal pulling forces. Aster geometry is now changed such that the density of microtubules is increased at the posterior end. As a result, the posterior spindle pole now travels faster and further than the anterior one, but still stably positions itself at the point of zero net force, which is now closer to the cortex.
Fig 4c
Model c: All microtubules generate equal pulling forces and aster geometry is similar for both asters. However, as the posterior spindle pole moves, microtubules of the posterior aster detach from the cortex instead of getting longer (dashed microtubules are inactive for 12 seconds). In this scenario all three features are reproduced, as the posterior spindle pole travels further, faster and undergoes transverse oscillations. The anterior spindle pole again stably positions itself at the point of zero net fore.
Furthermore, net forces are balanced in the static situation before spindle severing. Differences are only apparent in the dynamic situation following the cut.
CeMCAK PAR2 double RNAi
2 frames per second, anterior is on the left. Note aberrant polar body at the anterior end.
No breakage visible.
CeMCAK PAR3 double RNAi
2 frames per second, anterior is on the left. Note aberrant polar body coming into focus at the anterior end.
Spindle breakage occurs.
Zen4 phenotype
2 frames per second, anterior is on the left. Note slightly later and less pronounced breakage in comparison to the CeMCAK phenotype (Fig 2c (3.3Mb)).
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Grill, S., Gönczy, P., Stelzer, E. et al. Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409, 630–633 (2001). https://doi.org/10.1038/35054572
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DOI: https://doi.org/10.1038/35054572
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