1. Physics of Living Systems
  2. Structural Biology and Molecular Biophysics
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Nucleolar dynamics and interactions with nucleoplasm in living cells

  1. Christina M Caragine
  2. Shannon C Haley
  3. Alexandra Zidovska  Is a corresponding author
  1. New York University, United States
Research Article
Cite this article as: eLife 2019;8:e47533 doi: 10.7554/eLife.47533
7 figures, 2 tables and 1 additional file

Figures

Nucleolar coalescence.

(A) Time lapse of a nucleus with fluorescently labeled chromatin (H2B-GFP) and two fusing nucleoli (NPM-mApple). Time points depict: pre-fusion (t = 0 min), with two distinct nucleoli, fusion in progress (t = 33 min), with a clearly visible neck connecting the two nucleoli, and post-fusion (t = 273 min), where a resultant nucleolus can be seen still rounding up. (B) Time series showing the growth of the neck connecting two coalescing nucleoli. At t = 0 s, both the fluorescently labeled chromatin (H2B-GFP) and coalescing nucleoli (NPM-mApple) are depicted, the later frames, 20–600 s, show the progress of the nucleolar coalescence (NPM-mApple). Parts of (B) adapted from Figure 3b in Caragine et al. (2018). Scale bar, 2 µm.

Figure 2 with 1 supplement
Nucleolar size distribution as a function of cell cycle.

(A–D) Micrographs of HeLa cell nuclei with fluorescently labeled chromatin (H2B-GFP) and nucleoli (NPM-DsRed) under the following conditions: unsynchronized cells (A), synchronized cells 1.5 hr (B) and 3 hr (C) after mitosis, and cells arrested at G2/M checkpoint (D). (1–4) enlarged view of the boxed nuclei from (A–D). (E) Average nucleolar area AN as a function of number of nucleoli per nucleus NN for all conditions from (A–D). For unsynchronized cells, total number of nucleoli analyzed NN = 1331 in 228 nuclei, for t = 1.5 hr, NN = 275 in 42 nuclei, for t = 3 hr, NN = 257 in 51 nuclei, and for t = G2/M, NN = 497 in 124 nuclei. (F) Distributions of nucleolar volume VN and their fit to f(VN)VNα for all conditions from (A–D). For all fits, the goodness-of-fit, R2 > 0.98. Scale bar, 15 µm.

Figure 2—figure supplement 1
Nucleolar area distributions, p(AN), for all conditions shown in Figure 2E: unsynchronized cells, synchronized cells 1.5 hr after mitosis, synchronized cells 3 hr after mitosis, and cells arrested in G2/M.

For unsynchronized cells, number of analyzed nucleoli is NN = 1331 in 228 nuclei, for t = 1.5 hr, NN = 275 in 42 nuclei, for t = 3 hr, NN = 257 in 51 nuclei, and for t = G2/M, NN = 497 in 124 nuclei.

Figure 3 with 1 supplement
Nucleolar internal structure.

(A) Micrographs of HeLa nuclei with fluorescently labeled chromatin (H2B-GFP, green), nucleolar granular component (NPM-DsRed, red) and nucleolar dense fibrillar component (DFC) (FBL-mCerulean, blue) and overlays of all three signals (green, red, blue) and red and blue signal. The insets in overlay images present an enlarged view of a nucleolus from the image. (B) Distributions measured for semi-major axis aDFC (red) and semi-minor axis bDFC (green) of single DFCs (NDFC = 1035). The solid red and green lines correspond to the Gaussian fits of distributions of aDFC and bDFC, respectively, with aDFC ≈ 210 nm and bDFC ≈ 180 nm. (C) Nucleolar area, AN, as a function of DFC number per nucleolus, NDFC, with a linear fit AN ≈ 0.92NDFC. We evaluated 1279 DFCs over 114 nucleoli in 63 nuclei. Scale bar, 5 µm.

Figure 3—figure supplement 1
Distributions of DFC eccentricity, area and volume.

(A) Distribution of the measured eccentricities, e=a/b, where a and b are the DFC semi-major and semi-minor axes, respectively (NDFC= 1035). (B) Distribution of the DFC area, A=πab, measured for single DFCs (NDFC= 1035). (C) DFC volume distribution, p(VDFC). DFC volume, VDFC, was calculated as VDFC=4πa3/3, where a is the semi-major axis of the DFC, providing the upper boundary on the volume estimate. p(VDFC) exhibits a sharp maximum at VDFC = 0.030 μm3, suggesting largely a monodisperse population of DFCs (NDFC= 1035).

Comparison of size, shape and nuclear positioning between fusing and nonfusing nucleoli.

(A) Micrographs of a nucleus with fluorescently labeled chromatin (H2B-GFP), where two void spaces (labeled by yellow triangle and yellow circle) correspond to two nucleoli that did not fuse between t = 0 and 60 min. (B) Schematics of measured variables. (C) Measured variables for nonfusing nucleoli: nucleolar area, AN, nucleolar eccentricity, e, shortest distance from the nucleolar centroid to the nuclear envelope, De, and the angle between the major nuclear and nucleolar axes, α (NN = 17, Ncell = 6). All characteristics are calculated in the nucleolar focal plane. (D) Micrographs of a nucleus with fluorescently labeled chromatin (H2B-GFP), where two void spaces (labeled by yellow triangle and yellow cross) correspond to two nucleoli before fusion at t = 0 min, while at t = 60 and 120 min they can be seen fusing (yellow circle). (E) Measured variables for fusing nucleoli: AN, e, De and α (NN = 12, Ncell = 7). The dashed red line at t = 0 min indicates fusion. All measurements are carried out in the nucleolar focal plane. Scale bar, 5 µm.

Figure 5 with 1 supplement
Comparison of dynamics between fusing and nonfusing nucleoli.

(A) Trajectories of two nonfusing nucleoli color-coded by their temporal evolution (blue to red). The time step is 5 min. (B) Histogram of the velocity magnitude, v, for the nonfusing nucleoli (NN = 17, Ncell = 6). (C) Velocities for pairs of nonfusing nucleoli, where vmax and vmin is the larger and the smaller nucleolar velocity, respectively. (D) Histogram of radial velocity, vrad, for nonfusing nucleoli, with vrad calculated with respect to the midpoint distance between nucleoli. (E) vrad as a function of time for nonfusing nucleoli. (F) Velocity angle, αv, in polar coordinates as a function of time for nonfusing nucleoli. (G) Trajectories of pair of fusing nucleoli color-coded by their temporal evolution (blue to red). The pre-fusion nucleoli are visible at earlier times (blue to yellow), while the post-fusion nucleolus appears at later times (orange to red). The time step is 15 and 16 min. (H) Histogram of the v for the fusing nucleoli (NN = 12, Ncell = 7). (I) Velocities for pairs of fusing nucleoli, where vmax and vmin is the larger and the smaller nucleolar velocity, respectively, with a linear fit vmax 1.74vmin. (J) Histogram of vrad for fusing nucleoli. (K) vrad as a function of time for fusing nucleoli. (L) αv for fusing nucleoli as a function of time.

Figure 5—figure supplement 1
Additional nucleolar trajectories and enlarged view of nucleolar trajectories from Figure 5A and Figure 5G.

(A) Trajectories of nucleoli which do not fuse, time increases from blue to red (enlarged view of Figure 5A). (B) The trajectory areas determined as the convex hull of the trajectories of nucleoli from A. (C) Trajectories of nucleoli which fuse, time increases from blue to red (enlarged view of Figure 5G). The post fusion nucleolus is in between the other two trajectories. (D) The trajectory areas determined as the convex hull of the trajectories of nucleoli from C.

Nucleoli under physiological conditions and upon ATP-depletion.

(A–B) Micrographs of HeLa nuclei with fluorescently labeled chromatin (H2B-GFP, green) and nucleoli (NPM-DsRed, red), their color overlay and z-projections of nucleolar and nuclear contours: (A) under physiological conditions (control) and (B) after ATP depletion. (C) Distributions of the following nucleolar measurements under physiological conditions (NN= 648, NCell= 208) vs. upon ATP depletion (NN= 345, NCell= 127): nucleolar area normalized by nuclear area, AN/ANuc, nucleolar eccentricity, e, angle between the major nuclear and nucleolar axes, α, the shortest distance from the nucleolar centroid to the nuclear envelope normalized by the nuclear circumference, de, fraction of the nucleolar contour with negative curvature, fneg, and number of continuous regions of negative curvature along the nucleolar contour, Nneg. All measurements are carried out in the nucleolar focal plane. Scale bar, 5 µm.

Figure 7 with 1 supplement
Nucleoli upon biochemical perturbations.

(A) Micrographs of HeLa nuclei with fluorescently labeled chromatin (H2B-GFP, green) and nucleoli (NPM-DsRed, red), and the overlay, under the following conditions: control, upon addition of blebbistatin, latrunculin A, nocodazole, α-amanitin, flavopiridol, trichostatin A, and cycloheximide (at t1= 30 min and t2= 6.5 hr). (B) Histograms of the following measurements for all conditions (width indicates probability): nucleolar area normalized by nuclear area, AN/ANuc, nucleolar eccentricity, e, angle between the major nuclear and nucleolar axes, α, the shortest distance from the nucleolar centroid to the nuclear envelope normalized by the nuclear circumference, de, fraction of the nucleolar contour with negative curvature, fneg, and number of continuous regions of negative curvature along the nucleolar contour, Nneg. All data collected in the nucleolar focal plane. Red dot, solid red line and dotted red lines indicate the mean, median and quartiles, respectively. Table 1 provides p-values for all measured data with respect to the control. The number of nucleoli and cells are as follows: control (NN= 648, NCell= 208), blebbistatin (NN= 399, NCell= 127), latrunculin A (NN= 307, NCell= 104), nocodazole (NN= 310, NCell= 106), α-amanitin (NN= 268, NCell= 95), flavopiridol (NN= 309, NCell= 105), trichostatin A (NN= 278, NCell= 95), and cycloheximide at t1= 30 min (NN= 291, NCell= 91), and cycloheximide at t2= 6.5 hr (NN= 294, NCell= 105). Scale bar, 5 µm.

Figure 7—figure supplement 1
Nucleoli upon biochemical perturbations, including z-projections.

Micrographs of HeLa nuclei with fluorescently labeled chromatin (H2B-GFP, green) and nucleoli (NPM-DsRed, red), the two signal overlay, and z-projections of nuclear and nucleolar contours, under the following conditions: control, upon addition of blebbistatin, latrunculin A, nocodazole, α-amanitin, flavopiridol, trichostatin A, and cycloheximide (at t1= 30 min and t2= 6.5 hr). Scale bar, 5 µm.

Tables

Table 1
Statistical characteristics of distributions for physical quantities evaluated for nucleoli upon biochemical perturbations (see Figures 67).
Mean ± standard deviation
NNucleoliNCellsAN/ANuc e α de fneg Nneg
Control6482080.052 ± 0.0401.30 ± 0.3247 ± 260.076 ± 0.0290.033 ± 0.0660.66 ± 1.37
ATP-depletion3451270.054 ± 0.0421.29 ± 0.2848 ± 260.082 ± 0.0280.038 ± 0.0640.82 ± 1.39
Blebbistatin3991270.052 ± 0.0411.29 ± 0.2846 ± 260.076 ± 0.0280.036 ± 0.0670.72 ± 1.38
Latrunculin A3071040.061 ± 0.0441.31 ± 0.3647 ± 250.071 ± 0.0280.030 ± 0.0630.56 ± 1.21
Nocodazole3101060.052 ± 0.0371.28 ± 0.2550 ± 280.074 ± 0.0290.032 ± 0.0610.67 ± 1.38
α-amanitin268950.060 ± 0.0471.34 ± 0.2944 ± 270.074 ± 0.0290.046 ± 0.0720.99 ± 1.71
Flavopiridol3091050.052 ± 0.0401.34 ± 0.3443 ± 270.074 ± 0.0280.048 ± 0.0800.92 ± 1.67
Trichostatin A278950.044 ± 0.0321.29 ± 0.3643 ± 280.073 ± 0.0260.025 ± 0.0630.53 ± 1.35
Cyclohex I291910.053 ± 0.0371.32 ± 0.2946 ± 260.076 ± 0.0250.038 ± 0.0690.76 ± 1.49
Cyclohex II2941050.052 ± 0.0391.35 ± 0.3847 ± 250.074 ± 0.0290.040 ± 0.0670.74 ± 1.33
p-values (with respect to control)Relative difference of mean [%]
AN/ANuc e α de fneg NnegAN/ANuc e α de fnegNneg
ATP-depletion0.3590.6740.5130.0090.2580.0934%−1%2%8%15%24%
Blebbistatin0.8930.5150.6940.9110.5820.4950%−1%−2%0%9%9%
Latrunculin A0.0020.7140.9400.0110.4640.24617%1%0%−7%−9%−15%
Nocodazole0.9850.3760.1690.3350.7340.9250%−2%6%−3%−3%2%
α-amanitin0.0110.0890.0900.2800.0170.00615%3%−6%−3%39%50%
Flavopiridol0.7890.0890.0590.2720.0060 .0180%3%−9%−3%45%39%
Trichostatin A0.0020.9060.0510.0990.0870.160−15%−1%−9%−4%−24%−20%
Cyclohex I0.6540.2570.6330.9320.3550.3592%2%−2%0%15%15%
Cyclohex II0.9840.0350.9820.2840.1630.3810%4%0%−3%21%12%
Kullback-Leibler divergence (with respect to control)Skew
AN/ANuc e α de fneg NnegAN/ANuc e α de fnegNneg
Control1.103.07−0.09−0.042.172.68
ATP-depletion0.0260.0110.0210.0320.0510.0181.012.23−0.19−0.161.801.87
Blebbistatin0.0230.0250.0310.0360.0220.0071.312.26−0.110.032.002.14
Latrunculin A0.0590.0290.0280.0400.0500.0150.793.01−0.15−0.072.062.36
Nocodazole0.0220.0140.0420.0250.0330.0100.871.84−0.280.071.902.55
α-amanitin0.0600.0430.0570.0320.0620.0360.981.950.080.081.612.26
Flavopiridol0.0380.0270.0440.0350.0530.0211.282.360.03−0.021.622.09
Trichostatin A0.0560.0460.0620.0590.0470.0340.853.150.17−0.252.953.15
Cyclohex I0.0410.0320.0430.0510.0430.0151.132.13−0.08−0.121.982.70
Cyclohex II0.0130.0260.0490.0210.0400.0140.984.19−0.080.011.662.14
Table 1—source data 1

Statistical characteristics of distributions measured in Figures 6 and 7.

https://cdn.elifesciences.org/articles/47533/elife-47533-table1-data1-v1.xlsx
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line
(H. sapiens)
HeLaATCCATCC:CCL2
RRID:CVCL_0030
Stable H2B-GFP cell line
Transfected construct
(H. sapiens)
NPM-DsRedAddgene34553
RRID:Addgene_34553
Transfected construct
(H. sapiens)
mCerulean-Fibrillarin-7Addgene55368
RRID:Addgene_55368
Transfected construct
(H. sapiens)
NPM-mAppleCaragine et al., 2018n/aGenerated by Zidovska Lab – published in Caragine et al., 2018
Chemical compound, drugRO-3306Enzo Life SciencesALX-270–463
Chemical compound, drug2-Deoxy-D-Glucose (DOG)Millipore SigmaD8375
Chemical compound, drugTrifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP)Millipore SigmaC2920
Chemical compound, drugLatrunculin AMillipore SigmaL5163
Chemical compound, drugBlebbistatinMillipore SigmaB0560
Chemical compound, drugNocodazoleMillipore SigmaM1404
Chemical compound, drugα-AmanitinSanta Cruz Biotechnologysc-202440
Chemical compound, drugCycloheximideSanta CruzBiotechnologysc-3508
Chemical compound, drugFlavopiridolSanta Cruz Biotechnologysc-202157
Chemical compound, drugTrichostatin AMillipore SigmaT8552
Software,
algorithm
MatlabMathWorks2017a, 2019a
Software, algorithmAdobe Illustrator, PhotoshopAdobe Inc.CC2018

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

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