Resolution of NucQuant after correction of the aberration along the z axis. (A) Nuclear pore complex (NPC) architecture and nucleoporin localization in the NPC. (B–E) Cumulative frequency of distances to the nuclear center (left panels) and of distances to the fitted ellipsoid approximation of the nuclear envelope (right panels) using GFP or mRFP-tagged nucleoporins. (B) GFP–Nup49 and mRFP–Nup57, strain yRW3-1a. (C) GFP–Nup159 and mRFP–Nup2, strain yRW7-1a. (D) GFP–Nup49 and mRFP–Nup2, strain yRW4-1a. (E) GFP–Nup159 and mRFP–Nup57, strain yRW8-1a.

Extrapolation of nuclear envelope using detected NPCs. (A) 2D models building nuclear envelopes are represented onto electron microscopic micrographs of nuclear sections on which the positions of NPCs are visually detected (black arrows in the left panel). Strain BY4741. In the second image, the nuclear envelope is built by connecting adjacent NPCs. In the third image, the nuclear envelope is built by spline interpolation. In the right panel, the nuclear envelope is fitted by generating anchoring spots in nuclear envelope. Scale bars: 100 nm. (B,C) Based on the 3D confocal microscopic images, we could detect the NPC positions (blue spheres). Strain yCNOD99-1a. Using the spline-NE model (B), we refined the connection to get a smooth nuclear envelope. Red circles represent the spots that were used to refine the connection. 3D-NE model (C) generates additional anchoring spots (blue empty circle) to get an accurate extrapolation of the nuclear envelope. (D) The fitted nuclear envelope based on 3D-NE model for the nuclei characterized by one (left) or two (right) NPCs clusters. Upper graphs: x, y, z coordinates of detected NPCs; black cross, centroid of detected cluster(s); cluster 1 in red, cluster 2 in green. Strain yCNOD99-1a. (E) The fitted nuclear envelope based on 3D-NE model for the anaphase nuclei characterized using three NPCs clusters. Cluster 3 in blue. Strain yCNOD203-1a. Scale bars in D,E: 1 µm.

Living yeast nuclear geometry during the cell cycle. (A) Time course during a complete cell cycle of a single cell immobilized in a microfluidic device. NPCs in green and nucleolus in red (maximum intensity projections of a 3D image stack). Strain yCNOD99-1a. Note that a black rectangle of equivalent size was used as a background for cropped images. (B) The fitted nuclear envelope based on 3D-NE model for the nuclei in the different cell cycle phases. The surface of the nuclear envelope and the volume of nuclei allowed calculation of sphericity. (C) 3D-NE model fitting of different nuclear shapes (stages 1 to 6) throughout the cell cycle. The cell cycle is represented as a circle; the percentage of cells in each cell cycle phase from a large population was converted to duration (min). For each stage (panels 1 to 6), the DIC and the fluorescent (GFP–Nup49 and Bim1–tDimerRFP) pictures are displayed (stages 1 to 6). Strain yCNOD203-1a. The fitting using the 3D nuclear envelope model is also shown for each stage and was used to calculate sphericity. Scale bars: 1 µm.

The nuclear geometry according to the carbon sources. (A) Cumulative distribution of sphericity of the interphase nuclei cultured in different carbon sources. Strain yCNOD99-1a. (B) Cylindrical coordinates system with an oriented axis in which the position of the SPB is described by its distance from the nuclear center (R) and the angle from the central axis (α). Nucleolus is displayed in red. Angle φ represent rotation around central axis. (C) Cumulative frequency of the angle α between SPB and the central axis. Strain yRW11-1a. (D) SPB probability density maps based on analysis of nuclei comparing glucose with different carbon sources containing media. In glucose: dashed yellow circle, nuclear envelope determined according to the 3D-NE method; red curve, median nucleolus; red dot, median nucleolar centroid. Compare nucleolar size in glucose (red) with nucleolar size in other carbons sources (white). N represents the number of nuclei used to generate the cumulative percentage maps. (E) NPCs probability density maps based on analysis of nuclei in exponential phase cells growing in different carbon sources. Strain yCNOD99-1a. Compare median nuclear size in glucose (white dashed circle) with other carbon sources (yellow dashed circles). N represents the number of cNPCs used to generate the cumulative percentage maps. (F) Plotted variation of NPC density along the central axis in response to different carbon sources. (G) Heterogeneity of NPC distribution in interphasic cells. Nuclei were sorted according to their size (1/3 small, 1/3 medium, 1/3 large nuclei. Strain yCNOD99-1a. Scale bars: 1 μm.

The reorganization of the nuclear central axis during quiescence. (A) The nuclear central axis (SPB–nuclear-center–nucleolar-centroid) is broken after the cells enter quiescence. Red ellipse, nucleolus; green circle, SPB; black cross, nucleus centroid; blue cross, nucleolus centroid; α, angle of SPB to the nuclear–nucleolar­centroid axis. (B) SPB probability density maps based on analysis of nuclei after indicated time of starvation (see Materials and Methods). Representative fluorescent pictures (GFP–Nup49, Spc42–GFP and mCherry–Nop1) are displayed. Scale bars: 1 μm. (C) Cumulative frequency of the angle α upon incubation in glucose-depleted medium (from 2 h to 7 days). Strain yRW11-1a.

The nuclear envelope structure and NPC distribution during quiescence. (A) NPC probability density maps using the nucleolus as a secondary landmark upon time-progressive incubation in glucose-depleted medium. Representative fluorescent pictures (GFP–Nup49 and mCherry–Nop1) are displayed. Strain yCNOD99-1a. (B) Plotted variation of NPC density along the central axis during progressive starvation. (C) NPC probability density maps using SPB as a secondary landmark upon time­-progressive incubation in glucose-­depleted medium. Small red dot, SPB median position. Representative fluorescent pictures (GFP–Nup49 and SPC42–mRFP) are displayed. Strain yRW9-1a. (D) Maximum radial distance ratio of cNPC along x and y axis. Strain yRW9-1a. (E) After the cells enter quiescence, the percentage of different nuclear geometries at incubation times in carbon-depleted medium. Strain yCNOD99-1a. (F) Percentage of elongated nuclei versus sphere-like nuclei in the dyn1Δ mutant after 48 h to 7 days of carbon depletion. Strain yRW19-1a. Results in E,F are represented as mean±s.e.m. (G) SPB probability density maps based on analysis of nuclei from dyn1Δ mutant cells after indicating time of starvation. Strain yRW20-1a. Scale bars: 1 μm.