Xenbase Image ID: 119663

Figure 4. Tracking of NPC and lamina movement in interphase. (A) Time-lapse of a NRK cell expressing POM121-EGFP3 transiently. The lower nuclear surface was followed in a single confocal section every 2 s for a total of 30 min on a real-time confocal microscope. Sequence was averaged with a running window of five frames. Representative frames show numbered NPCs in a time window of ∼2 min used for tracking in B. Time, mm:ss. NPC movement is difficult to appreciate in still images; see Video 3 for the complete sequence. (B) Tracking of NPC movement. NPCs labeled #1–5 in A in a region of local NE movement are tracked together with two NPCs labeled C1 and C2 in A, which reside in an area of little movement and serve to illustrate global nuclear drift. Note the parallel and synchronous tracks of the NPCs. (C) Pattern FRAP of an NRK cell nucleus transiently expressing EGFP–Lamin B1. 33 1 × 0.5-μm regions were photobleached in the lower nuclear surface. Movement of landmarks was followed in single confocal sections every minute for 30 min (top row). Marks in the boxed area were used to track elastic deformations of the lamin lattice. Global cellular movement was corrected with two reference points. Relative position changes are shown in the bottom row as a network connecting the center of the bleach marks. Time, h:mm:ss; horizontal box length, 6 μm. (D) Pattern FRAP of a NRK nucleus transiently coexpressing POM121-YFP3 and ECFP–lamin B1. The outlined 21 0.9 × 0.6-μm regions were photobleached selectively in the lamina using a 413-nm Kr laser line. Movement of the lamina landmarks and the unbleached NPCs was then followed in single double-labeled confocal sections every 31 s for 30 min. Representative frames show distortion of nuclear shape by cell migration (note lamina folding at 07:56 and 12:40). Marks and NPCs used for tracking in E are red. Time, mm:ss. See Video 4 to better appreciate lamina elasticity. (E) Tracking of NPCs and lamina bleachmarks. Exemplary time-space tracks are shown for NPC #1 (green) and lamin grid marks A1–B2 (black) over 22 min in x, t and y, t plots. Global nuclear drift was normalized using to B5 and B7 marks. Note correlation between the x and y lamina and NPC movement. NPCs #2 and 3 and the surrounding marks behaved identically (not shown). Bars, 5 μm. Online supplemental material (Videos 3 and 4) is available at http://www.jcb.org/cgi/content/full/200101089/DC1. Image published in: Daigle N et al. (2001) Copyright © 2001, The Rockefeller University Press. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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