Feric M , Broedersz CP , Brangwynne CP .

DP2 GM105437 NIGMS NIH HHS , 1DP2GM105437-01 NCCDPHP CDC HHS

	Figure 1. Non-uniform distribution of nuclear bodies is dependent on a nuclear actin meshwork.(a,b) Top images are maximum intensity XY projections of the entire nucleus containing nucleoli labeled with NPM1::GFP (green) and HLBs labeled with TagRFP::coilin (red); bottom images are maximum intensity XZ projections. Scale bar = 100 μm. (a) An emulsion of nuclear bodies is stabilized by a nuclear actin network. (b) A few massive nuclear bodies are found at the bottom -plane after actin disruption by Lat-A. (c,d) XY views of Lifeact::GFP labeled actin network (green) and NPM1::RFP labeled nucleoli (red) in untreated (c) and Lat-A treated nuclei (d). Scale bar = 20 μm. (e) Schematic showing how a nuclear body (red) is subjected to a downward gravitational force, but is held in place by an actin network (green). (f) Disruption of the actin network results in nuclear body sedimentation. Faded sphere represents initial position. (g) Schematic illustrating the distance, z0, as the difference from the center of the nucleolar distribution, znucleoli, and the centroid of the nucleus, zc. (h) The normalized number density, as a function of vertical z-position, where the lowest -position,  = 0, corresponds to the bottom of the native nucleus (n = 43 nuclei). Inset contains comparison of normalized number density as a function of -position for untreated nuclei (filled symbols) and Lat-A treated nuclei (unfilled symbols) (n = 12 nuclei).
	Figure 2. Nuclear actin forms a soft, viscoelastic network.(a) Schematic diagram of active microrheology experimental set-up with two opposing electromagnets. Inset shows XY image of Lifeact::GFP labeled actin network (green) with an embedded magnetic microsphere (red, R = 1.5 μm). Scale bar = 10 μm. (b) An example of sinusoidal applied force (black) and response of magnetic microsphere (red). (c) Lissajous (stress-strain) plot of applied magnetic force as a function of measured displacement of microspheres in one nucleus. Error bars are s.e.m. Curves are fit to an ellipse and color denotes applied frequency. (d) Viscoelastic moduli, storage modulus (red) and loss modulus (blue), as a function of frequency in the untreated nucleus (n = 9 nuclei). Error bars represent s.e.m. Solid line is from fit determined by viscoelastic model in Fig. 3 inset.
	Figure 3. Nuclear actin undergoes creep under constantly applied stress.(a) Creep response for an individual bead at a force of 1.4 pN. Top shows applied force, and bottom shows displacement. (b) Averaged creep compliance as a function of time for different applied force (blue = 0.6 pN, green = 0.8 pN, and red = 1.4 pN) for the untreated nucleus, where n = 6, 4, and 7 nuclei, respectively. Black line represents fit for combined data based on viscoelastic model. Inset shows schematic of extended Kelvin-Voigt model.
	Figure 4. Centrifugation changes nuclear body spatial and size distributions in a force dependent manner.(a–d) Top images are maximum intensity XY projections of the entire nucleus containing nucleoli labeled with NPM1::GFP (green) and HLBs labeled with TagRFP::coilin (red); middle images are maximum intensity XZ projections of the same nucleus; and bottom images are XY views of the Lifeact::GFP labeled actin network with NPM1::RFP labeled nucleoli. For top and middle rows, scale bar = 100 μm, and for bottom row, scale bar = 20 μm. (a) The untreated nucleus contains suspended nuclear bodies at 1 g. For (b–d), the nucleus was centrifuged at 10 g (n = 21 nuclei), 100 g (n = 22 nuclei), or 1,000 g (n = 15 nuclei), for twenty minutes, respectively and nuclear bodies and nuclear actin were subsequently imaged. (e) Schematic illustrating relative displacement, Δz, between the average center of the nucleolar distribution in the native nucleus, , and the average center of the nucleolar distribution after centrifugation, . (f) The normalized number density, as a function of vertical -position of nucleoli, where the lowest -position,  = 0, corresponds to the bottom of the nucleus for untreated nuclei (black) and nuclei centrifuged at 10 g (blue), 100 g (green), and 1,000 g (red) for twenty minutes. Inset shows the relative sedimentation displacement, Δz, as a function of the applied gravitational force. Error bars represent s.e.m. (g) The normalized probability distribution of nucleolus size (volume) for untreated nuclei (black) and nuclei centrifuged at 10 g (blue), 100 g (green), and 1,000 g (red) for twenty minutes. Solid line shows a slope of −1.5, indicative of power-law observed in native nuclei. Inset shows the average size of nucleoli vs. applied gravitational force. Error bars represent s.e.m.
	Figure 5. Spatial distributions evolve in time, consistent with creep behavior.(a–d) Top images are maximum intensity XY projections of the entire nucleus containing nucleoli labeled with NPM1::GFP (green); bottom images are maximum intensity XZ projections of same nuclei. Scale bar = 100 μm. (a) The untreated nucleus at 1 g. For (b–d), the nucleus was centrifuged at 10 g for 3 hours (n = 14 nuclei), 8 hours (n = 9 nuclei), and 14 hours (n = 9 nuclei), respectively. (e) The relative sedimentation displacement, Δz, as a function of centrifugation time at centrifugation forces of 10 g (blue), 100 g (green), and 1,000 g (red). Error bars represent s.e.m. Black solid line presents prediction at 1 g using Stokes law and average viscosity determined from centrifugation experiments. Dashed line represents a distance of 200 μm, representing the radius of the nucleus. Inset shows the creep velocity as function of effective gravitational force for 10 g, 100 g and 1,000 g. Error bars represent 95% confidence interval from the fit. (f) The relative sedimentation displacement, Δz, as a function of the scaled force for centrifugation forces of 10 g (blue), 100 g (green), and 1,000 g (red). Error bars represent s.e.m. Black solid line is the best-fit line for the data, and the slope is the inverse of the viscosity. Inset shows the viscosity individually determined for effective gravitational forces with error bars representing 95% confidence interval. Black solid line indicates the viscosity determined from the creep compliance fit by the viscoelastic model (Fig. 3b).
	Figure 6. Signatures of aging due to gravitational creep.(a) Brownian dynamics simulation results (XZ plane) of gravitational creep over time. Scale bar = 100 μm. (b) The correlation between the distance, of the nucleoli from the center of the simulation box compared to the mean nucleolar size. Inset shows the distance, of the simulated nuclear bodies from (a) as a function of time. (c–e) Top images are maximum intensity XY projections of the entire (untreated) nucleus containing nucleoli labeled with NPM1::GFP (green); bottom images are maximum intensity XZ projections. Scale bar = 100 μm. From left to right, the spatial asymmetry of nuclear bodies becomes more pronounced and the average size of nuclear bodies increases. (f) The distance, of the center of the nucleolar distribution from the center of the nucleus compared to the median nucleolar size for all the centrifugation data combined; dashed black line is the best-fit line (correlation coefficient, ρ = 0.67). Arrow points in the direction of increasing effective force, , and/or centrifugation time, . Inset shows the correlation between the distance, and median nucleolar size for only the native data; dashed black line is the best-fit line (correlation coefficient, ρ = 0.46). Symbols are: native 1 g (black circles, n = 43 nuclei), 10 g 20 min (blue circles, n = 21 nuclei), 10 g 3 h (blue squares, n = 14 nuclei), 10 g 8 h (blue triangles, n = 9 nuclei), 10 g 14 h (blue diamonds, n = 9 nuclei), 100 g 10 min (green squares, n = 9 nuclei), 100 g 20 min (green circles, n = 22 nuclei), 100 g 30 min (green triangles, n = 9 nuclei), 100 g 50 min (green diamonds, n = 9 nuclei), 100 g 100 min (green pentagons, n = 12 nuclei), 1,000 g 2 min (red squares, n = 11 nuclei), 1,000 g 5 min (red triangles, n = 11 nuclei), 1,000 g 10 min (red diamonds, n = 11 nuclei), and 1,000 g 20 min (red circles, n = 15 nuclei).

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