Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement

Abstract

Nuclei move to specific locations to polarize migrating and differentiating cells. Many nuclear movements are microtubule-dependent. However, nuclear movement to reorient the centrosome in migrating fibroblasts occurs through an unknown actin-dependent mechanism. We found that linear arrays of outer (nesprin2G) and inner (SUN2) nuclear membrane proteins assembled on and moved with retrogradely moving dorsal actin cables during nuclear movement in polarizing fibroblasts. Inhibition of nesprin2G, SUN2, or actin prevented nuclear movement and centrosome reorientation. The coupling of actin cables to the nuclear membrane for nuclear movement via specific membrane proteins indicates that, like plasma membrane integrins, nuclear membrane proteins assemble into actin-dependent arrays for force transduction.

Fig. 1

Nuclear movement requires nesprin2G. Wound edge is toward the top of all images. A, Representative wide-field epifluorescence image of centrosome orientation in RFP-KASH-expressing cells (cell expressing RFP-KASH is shown in insert and by arrow). Cells were stained as follows: DNA (blue), centrosomes (anti-pericentrin, yellow) and cell-cell contacts (anti-β-catenin, red). B, Centrosome orientation in cells expressing the indicated constructs. Random reorientation is ~33% (26). C, Average centrosome and nucleus positions along the axis perpendicular to the wound from cells described in B. The cell center is defined as “0”. Positive values are toward the leading edge; negative values away. D, Nuclear movement in RFP-KASH-expressing (insert), GFP-α-tubulin NIH3T3 fibroblasts. (Left) Phase contrast image from the start of movie S1. Boxes indicate regions used for the GFP-α-tubulin fluorescence kymographs on the right. Arrowheads indicate centrosomes. Time is in hour:min. E, Average centrosome and nucleus positions from siRNA-treated cells expressing the indicated GFP-tagged constructs (N2G is nesprin2G). LBR is lamin B receptor; WT wild type; N2G nesprin2G. Experiments were repeated ≥ 3 times (N>30 for B and C; N>33 for E). Error bars indicate SEM. Scale bars in A, 15 µm; D, 10 µm.

Fig. 2

Coupled movement of dorsal actin cables and the nucleus. The wound edge is toward the top of all images. A, Representative wide-field epifluorescence images of nuclei in cells fixed after LPA-treatment. Time is in min. Cells were stained as follows: DNA [4´,6´-diamidino-2-phenylindole (DAPI)] and F-actin (rhodamine-phalloidin). B, Number of dorsal cables spanning the entire nucleus from cells treated as in A. Experiments were repeated 3 times with N > 151. C, Fluorescence kymograph of a Lifeact-mCherry-expressing cell from movie S4 and S5. Fluorescence images were acquired from dorsal and ventral planes of the cell, pseudocolored and merged. Nuclear position, dashed white circle. Arrows indicate dorsal (red) and ventral (green) cables. Images taken every 5 min. D, Fluorescence kymographs of Lifeact-mCherry in GAPDH- or nesprin2G-depleted cells. Nuclear position, red dashed circle. Images are every 10 min. Arrows indicate moving dorsal actin cables. E, LPA-stimulated dorsal cable and nucleus velocities in cells treated as in D. Data are from 5 independent experiments (N=36 (GAPDH) and 27 (nesprin2G)). Error bars indicate SEM. Scale bars for A and C, 5 µm; D, 10 µm.

Fig. 3

Nesprin2G and SUN2 form TAN lines. All images are of the dorsal nuclear surface. A, Top) Fluorescence images of a nucleus from a nesprin2G-depleted cell expressing GFP-mini-N2G and stained with rhodamine-phalloidin (F-actin). (Bottom) Fluorescent images of a nucleus in a cell stained with nesprin2G antibody (N2G) and rhodamine-phalloidin (F-actin). Arrows, colocalized N2G and dorsal actin cables. Wound edges are toward the upper right (top) and right (bottom). B, Fluorescence images of nuclei in nesprin2G-depleted cells expressing GFP-mini-N2G. Cells were stained with GFP antibody (GFP-mini-N2G) and rhodamine-phalloidin (F-actin). Cells were treated with dimethyl sulfoxide (DMSO), 50 µM blebbistatin (BB) or 0.5 µM cytochalasin D (CD) for 1 hr before and during LPA treatment. The wound-edge is toward the top left. C, Fluorescence images of nuclei in nesprin2G-depeleted cells. Cells were stained with SUN1, SUN2, LBR and GFP (GFP-mini-N2G) antibodies. Arrows show N2G colocalizing with SUN2, but not SUN1 or LBR. The wound-edge is toward the top. D, Average centrosome and nucleus positions from SUN2-depleted cells expressing the indicated GFP-tagged constructs. Dashed line represents average nuclear position in non-depleted cells. Experiments were repeated ≥ 3 times with N>201. Error bars, SEM. Bars in A to C, 5 µm.

Fig. 4

TAN lines are coupled with dorsal actin cables during nuclear movement. A, Fluorescence kymograph of GFP-mini-N2G TAN lines in a nucleus from a nesprin2-depleted cell from movie 6. The leading and lagging edges of the nucleus and two TAN lines are indicated. Each image is 5 min. B, Representative distance versus time plot of TAN lines, nuclear centroid, and leading and lagging nuclear edges from a movie of a cell treated as in A. C, Comparison of GFP-mini-N2G TAN line and leading nuclear edge velocities in cells treated as in A. Data are from 30 movies of 30 cells. R² is the coefficient of determination. D, Fluorescence kymograph of GFP-miniN2G and Lifeact-mCherry on the dorsal nuclear surface of a nucleus in a cell treated as in A from movie 7. Arrows indicate examples of TAN lines forming on actin cables. Panels are every 10 min and oriented with the wound-edge at the top. Time is hour:min for A and D. Scale bars in A and D, 5 µm.