Figure 4.

Quantitative analysis of the behavior of GFP-Lamin C, Alexa488-WGA, and ketel^GFP compared with mRFP-Nup107 during the NE/NPC disassembly process. For each time point, the average intensity of small regions within the NE (4 rectangles) and nuclear interior (N; 2–3 circles) of n = 3–7 nuclei (as indicated in each graph) and within the cytoplasm (C; 6 circles) were measured. Typical regions are depicted in C′ for ketel^GFP. Regions were identified based on the marker with the longest lasting signal at the NE (namely, GFP-Lamin C in A, Alexa488-WGA in B, and mRFP-Nup107 in C) and subsequently transferred to the other fluorescent channel. To allow comparison among markers, these values were normalized at each time point to the mean intensity within the entire field (so that a normalized intensity of one reflects a homogeneous distribution of the marker between the nucleus and the cytoplasm). Graphs represent the mean normalized intensities at the NE (dark blue circles and curves), within the nucleus (cyan dots and curves) and cytoplasm (purple triangles and curves) for mRFP-Nup107 (red, left) and either GFP-Lamin C (A), Alexa488-WGA (B), or ketel^GFP (C) (green, right). Error bars are SD. Time points at which the NE can no longer be discriminated for the intranuclear signal (N = NE) are indicated for both markers. The stars on the mRFP-Nup107 disassembly curves indicate the time point at which the intranuclear signal begins to increase. Quantifications were performed on the movies shown in Figure 3, A–C (Supplemental Movies 2–4), and qualitatively similar results were obtained upon quantification of a distinct movie (data not shown).