1. Cell-Size-Dependent Spindle Elongation in the Caenorhabditis elegans Early Embryo 
Yuki Hara, Akatsuki Kimura 
Current Biology 
Volume 19, Issue 18, Pages (September 2009)
DOI: /j.cub
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2. Figure 1
Quantification of Relationship between Spindle Length and Cell Length
(A) Nucleus length (pink, n = 68), metaphase spindle length (green, n = 129), and elongated spindle length (blue, n = 139) in various blastomeres from wild-type embryos were plotted as functions of cell length.
(B) Elongated spindle length (blue), metaphase spindle length (green), and the extent of spindle elongation (yellow) in cells more than 20 μm long were plotted against cell length. Plots in wild-type (solid circles, n = 49), C27D9.1 (RNAi) (open squares, n = 22), F21H12.2 (RNAi) (open diamonds, n = 4), and ima-3 (RNAi) (crosses, n = 5) embryos are shown. Linear regression lines including wild-type and RNAi embryos are shown (±standard errors of the mean, SEM).
(C) The extent of spindle elongation was plotted against cell length in wild-type embryos. Types and embryonic stages of the cells are distinguished by the different shapes and colors of the symbols. Different colors represent the following cell types: blue, P-lineage cells (n = 13); pink, AB-lineage cells (n = 7); and green, EMS-lineage cells (n = 10). Different shapes represent the following embryonic stages: triangles, 2-cell stage; diamonds, 4-cell stage; crosses, 8-cell stage; and circle, 16-cell stage. Black linear regression line is same as the yellow line in (B).
(D) The extents of spindle elongation in wild-type (blue diamonds, n = 30), par-2 (green triangles, n = 14), and par-3 (RNAi) embryos (yellow circles, n = 10) were plotted against cell length, except for 1-cell-stage embryos. In 1-cell-stage embryos, PAR-2 and PAR-3 affect spindle elongation through regulation of the cortical localization of microtubule-pulling force generators [28]. Linear regression line is same as in (C). The residual values of par-2 (RNAi) and par-3 (RNAi) from the regression line were not significantly different from those of the wild-type, as determined by Student's t test (par-2: p = 0.75; par-3: p = 0.076).
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3. Figure 2 Speed of Spindle Elongation also Depends on Cell Size
Speed of spindle elongation was calculated from the centrosome tracking data (Figure S3B) by dividing half the value of the maximum increase by the transit time from the onset of anaphase. Speed was plotted as a function of cell length for various cells (open squares, cells from C27D9.1 (RNAi) embryos, n = 21; solid circles, cells from wild-type embryos, n = 46; crosses, cells from ima-3 (RNAi) embryos, n = 5; open diamonds, cells from F21H12.2 (RNAi) embryos, n = 4). Different colors represent the following cell stages: blue, 1-cell stage (P0 cell); green, 2-cell stage; pink, 4-cell stage; yellow, 8-cell stage; and purple, 16- or more cell stages. Linear regression lines for all samples are shown (±SEM).
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4. Figure 3
Gα-Dependent and -Independent Mechanisms for Cell-Size-Dependent Spindle Elongation
(A) Extents of spindle elongation in gpr-1/2 (RNAi) (solid pink squares, n = 63), goa-1/gpa-16 (RNAi) (open pink squares, n = 19), and control (blue diamonds, “wild-type∗,” n = 82) embryos were plotted as in Figure 1B. The linear regression lines are shown (blue, from the data of the control; pink, from those of goa-1/gpa-16 (RNAi) and gpr-1/2 (RNAi) embryos, ±SEM). The regression coefficients were significantly different (p < 0.001). The correlation between extent and cell size was significant for both control (p < 0.001) and goa-1/gpa-16 (RNAi) and gpr-1/2 (RNAi) embryos (p < 0.001).
(B) Speed of spindle elongation in gpr-1/2 (RNAi) (solid pink squares, n = 63), goa-1/gpa-16 (RNAi) (open pink squares, n = 19), and control (blue diamonds, “wild-type∗,” n = 80) embryos were plotted as in Figure 2. Linear regression lines for each sample are shown (±SEM). The regression coefficients were significantly different (p < 0.001). The correlation between speed and cell size was significant for control embryos (p < 0.001), but not for goa-1/gpa-16 (RNAi) and gpr-1/2 (RNAi) embryos (p = 0.14).
In both (A) and (B), the data obtained with C27D9.1 (RNAi), F21H12.2 (RNAi), and ima-3 (RNAi) background is included for gpr-1/2 (RNAi), goa-1/gpa-16 (RNAi), and control (“wild-type∗”) as in Figures 1B or 2.
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5. Figure 4
Simulation of Spindle Elongation with the Constant-Pulling Model and the Force-Generator-Limited Model
(A–C) The constant-pulling model.
(A) Schematic representation of the constant-pulling model in a large cell (left; P0 cell as example) and a small cell (right; P2 cell as example). Cortical force-generator sites (orange dots) are fixed at the cell cortex. Each force generator pulls the astral microtubules (gray lines) elongating from the spindle (blue object) with a constant magnitude of pulling force (vector of force; orange arrow).
(B and C) Simulation of spindle elongation in embryos with loss of Gα (simulation videos are shown in Movies S3 and S4). The magnitude of pulling force was adjusted to fit the dynamics of elongation in the P0 cell of goa-1/gpa-16 (RNAi) and gpr-1/2 (RNAi) embryos (see Supplemental Experimental Procedures). Comparison of in vivo data (pink dots, goa-1/gpa-16 (RNAi) and gpr-1/2 (RNAi) embryos, same as Figure 3) and simulation data (green dots) determined for the extent (B) and speed (C) of elongation. Linear regression lines are shown (±SEM). The equations are for the simulation data, whereas those for the in vivo data are shown in Figure 3. The slopes of the regression lines for the extent and speed of spindle elongation were not significantly different from those for the in vivo data of goa-1/gpa-16 (RNAi) and gpr-1/2 (RNAi) embryos (extent: p = 0.92; speed: p = 0.46).
(D–F) The force-generator-limited model.
(D) Schematic representation of the force-generator-limited model in a large cell (left) and a small cell (right). The cortical force-generator sites (red dots) are limited and the density of force generators is constant, regardless of the cell size. The number of astral microtubules is constant among cells of different sizes. In the situation depicted, the number of force-generator sites varies according to surface area, so that the magnitude of the pulling force depends on the number of force-generator sites, but not on the number of astral microtubules. Orange arrows represent vectors of force.
(E and F) Simulation of spindle elongation in wild-type cells with the force-generator-limited model as the Gα-dependent mechanism and the constant-pulling model as the Gα-independent mechanism (simulation videos are shown in Movies S5 and S6). The extent (E) and speed (F) of in vivo spindle elongation (blue dots, “wild-type∗,” same as Figure 3) and simulation (green dots) were plotted as a function of cell length, and the linear regression lines for these data are shown (±SEM). The slope of both regression lines for the extent and speed of spindle elongation were not significantly different from those for the in vivo data (extent: p = 0.95; speed: p = 0.89).
(G) Proposed models for regulation of cell-size-dependent spindle elongation. Two mechanisms regulate cell-size-dependent spindle elongation in wild-type embryos: the Gα-dependent mechanism (top) and the Gα-independent mechanism (bottom). The number of Gα-dependent force generators on the cortex is limited, and the total number is proportional to the surface area of the cell. The number of astral microtubules pulled by the Gα-dependent mechanisms will thus be limited. In this figure, we assumed that dynein complexes recruited to the cortex through G proteins and their regulators pull microtubules [13, 14]. Force generation can be mediated by shrinking of the microtubules [15, 16]. By comparison, Gα-independent force generators are abundant enough on the cell cortex so that all astral microtubules that reach the cortex will be pulled with a constant force by the Gα-independent mechanism. Our measurements suggest that the number of astral microtubules does not vary dramatically among cells of different sizes (Figure S9). Simulation studies show that the combination of these mechanisms is sufficient to reproduce the in vivo behavior of cell-size-dependent spindle elongation.
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