The Mitotic Spindle in the One-Cell C. elegans Embryo Is Positioned with High Precision and Stability

Abstract

Precise positioning of the mitotic spindle is important for specifying the plane of cell division, which in turn determines how the cytoplasmic contents of the mother cell are partitioned into the daughter cells, and how the daughters are positioned within the tissue. During metaphase in the early Caenorhabditis elegans embryo, the spindle is aligned and centered on the anterior-posterior axis by a microtubule-dependent machinery that exerts restoring forces when the spindle is displaced from the center. To investigate the accuracy and stability of centering, we tracked the position and orientation of the mitotic spindle during the first cell division with high temporal and spatial resolution. We found that the precision is remarkably high: the cell-to-cell variation in the transverse position of the center of the spindle during metaphase, as measured by the standard deviation, was only 1.5% of the length of the short axis of the cell. Spindle position is also very stable: the standard deviation of the fluctuations in transverse spindle position during metaphase was only 0.5% of the short axis of the cell. Assuming that stability is limited by fluctuations in the number of independent motor elements such as microtubules or dyneins underlying the centering machinery, we infer that the number is ∼1000, consistent with the several thousand of astral microtubules in these cells. Astral microtubules grow out from the two spindle poles, make contact with the cell cortex, and then shrink back shortly thereafter. The high stability of centering can be accounted for quantitatively if, while making contact with the cortex, the astral microtubules buckle as they exert compressive, pushing forces. We thus propose that the large number of microtubules in the asters provides a highly precise mechanism for positioning the spindle during metaphase while assembly is completed before the onset of anaphase.

Figure 1

Definition of spindle coordinates and typical trajectories in control embryos. (A) Trajectories of the anterior (red) and posterior (blue) poles superimposed on a fluorescence micrograph of a one-cell C. elegans embryo labeled with GFP-γ-tubulin. The horizontal magenta line is the A-P axis, and the vertical green line is the transverse axis. The intersection of the axes defines the cell center. Vertical green dashed lines mark the position of centrosomes on the A-P axis when anaphase ends. (B) Definition of the spindle coordinates and the color scheme used in the subsequent panels. (C) The x- (orange) and y coordinates (red) of the anterior centrosome of a typical cell. The dashed khaki line marks NEBD and the dashed cyan line marks anaphase onset. The gray zone marks the maintenance phase, during which the transverse position and the orientation are stationary, and which is analyzed in detail. (D) The x- (light blue) and y coordinates (dark blue) of the posterior centrosome. (E) The x- (magenta) and y coordinates (green) of the spindle center. (F) Spindle length (khaki) and orientation (violet).

Figure 2

Power spectrum of the transverse spindle position in control embryos. (A) Time traces of live and fixed cells. (Green, upper curve) Transverse position of the spindle center of the embryo in Fig. 1 during the maintenance phase. (Blue, lower curve) Spindle transverse position in a methanol-fixed embryo. Note that there is more high-frequency noise in the fixed cell due to the reduction in intensity of the GFP. However, the lower frequency, biological noise is clearly less in the fixed cell. (B) Experimental and theoretical power spectra. (Green circles) One-sided power spectral density of the y component of the spindle center computed during the maintenance phase. (Black line) Average of power spectra from eight embryos. (Blue circles) Power spectrum of the fixed embryo. (Solid red line) Least-squares fit to the Lorentzian model with σ² = 24.0 ± 6.0 × 10³ nm², τ = 14.5 ± 3.8 s, and high-frequency asymptote σ₀² = 36 nm²/Hz. (Dashed red line) Least-squares fit to the second-order model with σ² = 27.1 ± 8.4 × 10³ nm², τ = 18.1 ± 5.7 s, τ₀ = 0.37 ± 0.02 s, and σ₀² = 36 nm²/Hz.

Figure 3

Spindle positioning in gpr-1/2(RNAi) and zyg-9(RNAi) embryos. (A) Time traces of spindle position in a gpr-1/2(RNAi) embryo along the A-P axis (magenta) and the transverse (green) axis showing the loss of transverse oscillations. (B) Time traces of spindle position in a zyg-9(RNAi) embryo along the A-P axis (magenta) and the transverse axis (green). (C) Average power spectra of eight gpr-1/2(RNAi) and eight zyg-9(RNAi) embryos (black solid and purple open circles, respectively). For comparison, the second-order model fit to the control embryos (from Fig. 3) is shown in red. See also Fig. S1 for positioning in lin-5(RNAi) and nmy-2(RNAi) embryos and Fig. S2 for effects of zyg-9(RNAi) on cortical microtubules.

Figure 4

Models of centering. Diagram of a one-cell embryo showing microtubules growing from (green) and shrinking to (red) the centrosomes (circles). If a microtubule continues to grow when it contacts the cortex (the inside of the ellipse), then it will push. If the centrosome is closer to one side, the microtubules on that side will spend less time growing and shrinking (because they do not have to go as far) and so will spend a larger fraction of time pushing: this leads to a centering force. If a microtubule shrinks while still in contact with the cortex, then it will pull. Pulling is often destabilizing, though under some circumstances it can lead to centering. If vesicles are carried by motors toward the centrosome, then the drag force on the vesicle will lead to a reactive force on the centrosome and spindle: if the spindle is displaced, there will be a net force pulling the centrosome toward the center. Buckling microtubules are shown at the ends: the left one cannot slide on the cortex; the right one can slide.