Figure 4

Perinuclear actin revealed with a range of actin staining methods. (A) Relative measure of actin accumulation at the NE inside living cells. Average values (n = 5 cells in each case) were calculated (see methods) from normalised intensity values mapped in three-dimensional image stacks recorded from living cells labelled using various protocols to reveal actin and NE-membranes. Results show averages of the percentage of NE-membrane label (either POM121-GFP or dsRed-laminA) that was not distinguishable in spatial coordinates (given resolution limits) from actin labelling probes (actin568, CD-BODIPY, citrin-actin). Averaged results (n= 5 cells) were compared (P < 0.01; student t-test) with those calculated from samples labelled by cytoplasmic micro-injection of dextran-rhodamine inside cells expressing POM121-GFP (B) Spinning disk confocal image of single living HeLa cell expressing lamin A dsRed (upper panels) and citrin-actin (lower panels). The XY optical section shows accumulation of cortical actin at the plasma membrane (red cursor). The green cursor indicates the faint perinuclear actin accumulation. The right panels show detail from the regions of interest indicated by the white-line dashed boxes in the left-hand panels. Scale bars: left panels 7μm; right panels 0.5μm. (C) Shows the YZ section view reconstructed from the same 3D through-stack dataset as (B) above. The red and green cursors show cortical and perinuclear actin accumulation as for (B). Note also the presence of an interphase nuclear invagination where dsRed-LaminA and citrin-actin both intrude upon the nuclear volume (white arrowhead). Scale bars: 7μm.