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. 2013 Nov 15;342(6160):856-60.
doi: 10.1126/science.1243147.

Cytoplasmic Volume Modulates Spindle Size During Embryogenesis

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Free PMC article

Cytoplasmic Volume Modulates Spindle Size During Embryogenesis

Matthew C Good et al. Science. .
Free PMC article

Abstract

Rapid and reductive cell divisions during embryogenesis require that intracellular structures adapt to a wide range of cell sizes. The mitotic spindle presents a central example of this flexibility, scaling with the dimensions of the cell to mediate accurate chromosome segregation. To determine whether spindle size regulation is achieved through a developmental program or is intrinsically specified by cell size or shape, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes. Spindle size was observed to shrink with decreasing compartment size, similar to what occurs during early embryogenesis, and this scaling trend depended on compartment volume rather than shape. Thus, the amount of cytoplasmic material provides a mechanism for regulating the size of intracellular structures.

Figures

Figure 1
Figure 1. Spindle Length Scales with Compartment Size In Vitro and In Vivo
(A) System for creating cell-like compartments in vitro, including a passivated boundary, cell-free cytoplasm capable of assembling metaphase spindles (Xenopus egg or embryo extracts), and tunable compartment size. (B) Spindles in droplets – compressed to improve image quality - corresponding to spheres 80, 55, and 40 μm in diameter. Uneven shading is due to image stitching. (C) Spindle length in encapsulated X. laevis egg extract scaled with droplet size in vitro. Left: Linear scaling regime. Inset: scaling prediction. Raw data (orange circles), and average spindle length (orange squares) +/- SD across 5 μm intervals in droplet diameter are shown. P-value (< 10-60) and R2 (0.34) calculated from linear fit to raw droplet data in 20-80 μm diameter range. Right: full scaling curve in vitro. For comparison, gray bars indicate two standard deviations from average embryo data in D. (D) Spindle length scaling in vitro mirrored length scaling in the X. laevis embryo through Stage 8 with similar linear scaling regimes and a plateau where spindle size was uncoupled from compartment size. Raw data from embryos across 5 μm intervals in cell diameter (gray circles), and average spindle length (black squares) +/- 2 SD (thick error bars) are shown. Scale bar 20 μm.
Figure 2
Figure 2. Cytoplasmic Volume Sets Spindle Size In Vitro
To distinguish between boundary- and volume-sensing models, spindle length scaling was compared in uncompressed (spherical) and compressed (disk-like) droplets (details in fig. S4B). Spindle length scaling in both droplet geometries appeared identical when plotted as a function of droplet volume, supporting a volume-sensing mechanism. Spindle scaling curves did not overlay when plotted as a function of projected (imaged) droplet diameter, ruling out boundary-sensing. Raw data points (circles: gray = uncompressed, red = compressed) and spindle length, averaged across ten droplets (squares: black = uncompressed, red = compressed), are shown. Raw data was fit to a log function in volume plot and linear function in diameter plot (black line, R2 = 0.45 (uncompressed), and red line, R2 = 0.79 (compressed)). P-values indicate statistical difference between y-intercepts of compressed vs. uncompressed regression lines, calculated using an analysis of covariance.
Figure 3
Figure 3. A Limiting Component Model for Spindle Size Regulation
(A) Schematic of limiting component model (for more details, see fig. S5A and supplemental text). (B) Limiting tubulin model accurately predicted X. Laevis spindle length from droplet volume in vitro. Raw data from droplets (blue circles) and binned averages (dark blue squares) was compared to the model. Shaded gray regions represent model predictions across a range of parameter values (fig. S5B); the red line shows the prediction for intermediate values. (C) Cytoplasmic tubulin became significantly depleted as cell size decreased during X. laevis embryogenesis. Comparison of model prediction (red) and experimental data (gray) for the fraction of total cellular tubulin incorporated in the spindle as a function of cell volume. Model used parameter values that gave best agreement in fig. S5C and D.
Figure 4
Figure 4. Cell Volume and Composition Control Spindle Size During Xenopus Early Embryogenesis
(A) Spindle length scaled linearly with cell volume across a broad range of developmental stages during early X. laevis embryogenesis (Stages 5-10). Spindle length had an upper limit and was uncoupled from cell volume in Stages 2-4. Raw data (colored circles) and stage-averaged cell diameter and spindle length (black squares) +/- SD are shown. (B) Despite having distinct maximum spindle lengths, coupled to developmental stage (Stage 4 = green, Stage 8 = red), the length of X. laevis embryo extract mitotic spindles scaled with compartment volume in vitro. This result suggested that changes in cytoplasmic volume and composition work in concert to regulate spindle size. Raw data points (light circles) and bin-averaged spindle length (squares) were calculated for 5 μm intervals in droplet diameter across the 20-80 μm range of droplet diameters (wider interval were used for averaging in largest droplets because data was sparse).

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