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Graphical Abstract
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Figure 1. Xenopus spindles have the same mass density as the surrounding cytoplasm
(A) Optical setup used to obtain fluorescence and refractive index images of Xenopus spindles. SMFC, single mode fiber coupler; TL, tube lens; CL, condenser lens; OL, objective lens; M, mirror; DM, dichroic mirror; BS, beam splitter.
(B) Phase maps of a representative spindle illuminated at various angles. Scale bar, 10 μm.
(C) Fluorescence images showing microtubules (labeled with TAMRA-tubulin) and chromatin (labeled with Hoechst 33342) of the same spindle. The colored boxes represent regions of interest (5 μm × 5 μm) used for measuring the RI of cytoplasm, spindle, or chromatin (yellow, red, and blue boxes, respectively). Reconstructed ODT image of the spindle’s central plane used for RI and mass density measurements.
(D) Average RI and mass density (mg/mL) of cytoplasm, spindle, and chromatin. Cytoplasm (yellow) 105 ± 0.4 mg/mL, spindle (red) 105 ± 0.4 mg/mL, chromatin (blue) 102 ± 0.6 mg/mL, pcytoplasm, chromatin < 0.0001, Cohen’s d cytoplasm, chromatin = 0.4. n = 320 ROIs from 64 spindles. Data are represented as mean ± SEM. Mann-Whitney test. Black bar and lines indicate mean and standard error of mean. Box and violin plot for each condition indicated, ns p > 0.05, ∗∗∗∗p < 0.0001. See also Figures S1–S3.
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Figure 2. Mass density is stiffness invariant in mechanically distinct spindle regions
(A) Schematic representing three architecturally and mechanically distinct regions (Takagi et al., 2019) within spindles, spindle poles (green), middle of the spindles (blue), and spindle equator (red).
(B) Mass density of the three different regions. Spindle pole: 94 ± 1.0 mg/mL, spindle middle: 94 ± 1.1 mg/mL, and spindle equator: 89 ± 1.2 mg/mL, ppole, equator < 0.001, Cohen’s dpole, equator = 0.6.
(C) Ashby plot of the median dynamic modulus of different spindle regions versus the corresponding mass density. Each circle represents the average value of an ROI (5 μm × 5 μm). n = 80 ROIs from 20 spindles. Data are represented as mean ± SEM. Mann-Whitney test. Black bar and lines indicate mean and SEM. Box and violin plot for each condition indicated, ns p > 0.05, ∗∗∗p < 0.001.
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Figure 3. Spindle mass density correlates with microtubule density
(A) Fluorescence image of a representative Xenopus spindle. Microtubule and mass density were measured along the pole-pole axis (green: spindle poles, blue: spindle middle, red: spindle equator). Scale bar, 10 μm.
(B) Microtubule and mass density vary along the pole-pole axis. Circles and squares represent the average microtubule (red) and mass density (black), respectively. n = 36 spindles.
(C) Representative fluorescence images (top) and corresponding ODT images (bottom) of spindles of different sizes (red, microtubules; blue, DNA). Scale bar, 10 μm.
(D) Average microtubule density of 76 spindles showing natural length variation (20–70 μm).
(E) Average mass density of spindles in (D).
(F) Microtubule mass and spindle dry mass scale with spindle length. Each circle represents the microtubule mass (from fluorescence images) and the dry mass of one spindle. Dotted black line indicates the average dry mass of a wild-type Xenopus spindle (290 pg). Bold lines (D–F) indicate a linear fit of the data, and thin lines indicate the 95% confidence interval of the fitted function. R2 value for each fit indicated.
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Figure 4. Increasing tubulin concentration increases spindle mass density and spindle circularity
(A) Representative fluorescence (top: red, microtubules; blue, DNA) and corresponding ODT images (bottom) of spindles with no (wild type), 2 and 4 μM additional Xenopus tubulin. Scale bar, 10 μm.
(B) Spindle length increases with increasing tubulin concentration (n = 29, 35, and 37 spindles from 3 independent experiments). Wild type: 31 ± 1.3 μm, +2 μM: 40 ± 1.3 μm, pWT, 2 μM < 0.0001, Cohen’s dWT, 2 μM > 1, +4 μM: 47 ± 1.7 μm, pWT, 4 μM < 0.0001, Cohen’s dWT, 4 μM > 1.
(C and D) (C) Microtubule density measured by tubulin fluorescence (wild type: 5 ± 0.4 AU/μm2, +2 μM: 6 ± 0.3 AU/μm2; pWT, 2 μM < 0.05, Cohen’s dWT, 2 μM = 0.7 and +4 μM: 7 ± 0.3 AU/μm2; pWT, 4 μM < 0.0001, Cohen’s dWT, 4 μM > 1) and (D) mass density increases with increasing tubulin concentrations (wild type: 92 ± 1.1 mg/mL, +2 μM: 95 ± 0.5 mg/mL; pWT, 2 μM > 0.05, Cohen’s dWT, 2 μM = 0.6 and +4 μM: 96 ± 0.4 mg/mL; pWT, 4 μM < 0.001, Cohen’s dWT, 4 μM = 0.9).
(E) Supplementing tubulin alters spindle shape (as measured by the circularity shape factor, n = 33, 48, and 47 spindles from 4 independent experiments). Data are represented as mean ± SEM. Mann-Whitney test. Black bar and lines indicate mean and SEM. Box and violin plot for each condition indicated, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. See also Figure S4.
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Figure 4. Increasing tubulin concentration increases spindle mass density and spindle circularity
(A) Representative fluorescence (top: red, microtubules; blue, DNA) and corresponding ODT images (bottom) of spindles with no (wild type), 2 and 4 μM additional Xenopus tubulin. Scale bar, 10 μm.
(B) Spindle length increases with increasing tubulin concentration (n = 29, 35, and 37 spindles from 3 independent experiments). Wild type: 31 ± 1.3 μm, +2 μM: 40 ± 1.3 μm, pWT, 2 μM < 0.0001, Cohen’s dWT, 2 μM > 1, +4 μM: 47 ± 1.7 μm, pWT, 4 μM < 0.0001, Cohen’s dWT, 4 μM > 1.
(C and D) (C) Microtubule density measured by tubulin fluorescence (wild type: 5 ± 0.4 AU/μm2, +2 μM: 6 ± 0.3 AU/μm2; pWT, 2 μM < 0.05, Cohen’s dWT, 2 μM = 0.7 and +4 μM: 7 ± 0.3 AU/μm2; pWT, 4 μM < 0.0001, Cohen’s dWT, 4 μM > 1) and (D) mass density increases with increasing tubulin concentrations (wild type: 92 ± 1.1 mg/mL, +2 μM: 95 ± 0.5 mg/mL; pWT, 2 μM > 0.05, Cohen’s dWT, 2 μM = 0.6 and +4 μM: 96 ± 0.4 mg/mL; pWT, 4 μM < 0.001, Cohen’s dWT, 4 μM = 0.9).
(E) Supplementing tubulin alters spindle shape (as measured by the circularity shape factor, n = 33, 48, and 47 spindles from 4 independent experiments). Data are represented as mean ± SEM. Mann-Whitney test. Black bar and lines indicate mean and SEM. Box and violin plot for each condition indicated, ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. See also Figure S4.
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Figure S2: A custom optical stretcher setup is unable to deform spindles. (A) Schematic showing the components of the custom optical stretcher (OS) including a close-up of an extract droplet showing the two optical fibers and a spindle. (B) A 25 μm vesicle was successfully trapped (0 sec) and stretched (14 sec) using the OS. Upon reducing the laser power to trapping values (from 0.65 W to 0.3 W), the vesicle returned to its original size (28 sec). Black dotted lines show the original vesicle diameter (Loriginal). Scale bar = 15 μm. (C) A spindle placed in between the two fibers is not affected by the increase in laser power (∆ power = 0.35 W), even after 40 seconds. Scale bar = 30 μm. For further details see STAR Methods.
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Figure S3: A high-density region of membranes surrounds Xenopus spindles. Representative fluorescence image of a spindle showing (A) microtubules (labelled with TAMRA-tubulin) and (B) membranes (labelled with DiOC6). Yellow, red, and green boxes represent ROIs (5 μm x 5 μm) for the cytoplasm, spindle, and membranes, respectively used for measuring RI from the ODT image. (C) ODT image of the same spindle. Scale bar = 10 μm. (D) Quantification of RI and mass density of the cytoplasm, the spindle, and membranes from 320 different ROIs from n=64 spindles. Data are represented as mean ± SEM. Mann Whitney test. Black bar and lines indicate mean and standard error of mean. Box and violin plot for each condition also indicated, ns p>0.05, **** p<0.0001, Cohen’s dspindle, membranes > 1. For further details see STAR Methods.
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Figure S4: Addition of tubulin increases spindle mass and alters spindle shape. (A) RI distribution of cytoplasmic ROIs in the control (wildtype) and tubulin-supplemented (+2 μM and +4 μM) extract. Each circle represents a single 5 μm x 5 μm ROI. 150 ROIs evaluated from 3 independent experiments. Mann Whitney test. Black bar and lines indicate mean and standard error of mean. Box and violin plot for each condition also indicated, ns p>0.05. Microtubule mass and spindle dry mass scale with spindle length in the (B) wildtype as well as upon adding 2 μM (C) and 4 μM (D) purified Xenopus tubulin. Red circles: microtubule mass. Black circles: spindle dry mass. n=29, 35 and 37 spindles were measured from 3 independent experiments. Bold lines indicate a linear fit of the data and thin lines indicate the 95% confidence interval of the fitted function. R2 value for each fit indicated. Dotted black line indicates the average dry mass in each case. (E) Schematic representing the circularity (C) of an ellipse-like object and a perfect circle (C = 1). (F) Representative fluorescence images of wildtype and tubulin supplemented spindles (+2 μM and +4 μM), and their respective circularities. For averaged C values also see Figure 4. Red line shows the boundary of an ellipse with the same dimensions as each spindle. Scale bar = 10 μm. For further details see STAR Methods.
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