2C and D). during cell migration, development, homeostasis, and ensuring the equal distribution of genetic material during cell division (13). Most nuclear movements are microtubule-mediated; however, growing numbers of actin-dependent nuclear movements have been recognized (1,2,410). Mechanisms for actin-dependent nuclear movement are unclear. In NIH3T3 fibroblasts polarizing for migration into in vitro wounds, an actin-dependent nuclear movement is triggered by serum or the serum factor lysophosphatidic acid (LPA) and this reorients the centrosome toward the leading edge (5). Nuclear movement and actin retrograde flow occur at the same rate, but how actin is coupled to the nucleus is unknown. We explored the possible involvement of the LINC (linker of nucleoskeleton and cytoskeleton) complex, which spans the inner and outer nuclear membranes (INM and ONM, respectively). LINC complexes consist of ONM nesprin and INM SUN proteins and have been implicated in microtubule-dependent, but not actin-dependent, nuclear movements (2,1113). Nevertheless, the largest splice forms of two mammalian nesprins, nesprin1 and nesprin2, contain cytoplasmically oriented paired actin-binding calponin homology (CH) domains (2). To test whether nesprins were involved in nuclear movement, we initially expressed dominant negative constructs [red fluorescent protein-spectrin repeat-Klarsicht/ANC-1/Syne homology (RFP-SR-KASH) and RFP-KASH] of the LINC complex in wound-edge NIH3T3 fibroblasts and then stimulated nuclear movement with LPA. Expression of these constructs, known to disrupt LINC complexes and displace nesprins from the nuclear envelope (2,3,14), inhibited centrosome orientation and rearward nuclear positioning, while a control construct (RFP-KASHL) lacking the lumenal SUN-binding domain had no effect (Fig. 1AC, andfig. S1). Live cell imaging showed that RFP-KASH blocked nuclear movement (Fig. 1Dandmovie S1). Thus, nesprins and the LINC complex are involved in centrosome orientation and nuclear movement. We cannot exclude the possibility that nesprins function in centrosome positioning, as nuclear movement is needed to observe centrosome centration defects (5,15). == Fig. 1. == Nuclear movement requires Terbinafine hydrochloride (Lamisil) 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–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 inB. 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–tubulin NIH3T3 fibroblasts. (Left) Phase contrast image from the start ofmovie S1. Boxes indicate regions used for the GFP–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 forB and C; N>33 forE). Error bars indicate SEM. Scale bars inA,15 m;D,10 m. Expression analysis and immunoblotting showed that NIH3T3 fibroblasts express only one of Terbinafine hydrochloride (Lamisil) the actin-binding, giant nesprin isoforms, nesprin2G RH-II/GuB (fig. S2A to C). Depletion of nesprin2G with siRNA blocked centrosome orientation due to defective rearward nuclear Terbinafine hydrochloride (Lamisil) movement, whereas control siRNAs had no effect (Fig. 1E,figs. S2 to 4andmovies S2andS3). These effects of nesprin2G-depletion were not due to gross alterations in the nuclear envelope because the levels and localization of five other nuclear envelope proteins were not greatly altered (fig. S4). The centrosome and nuclear defects in nesprin2G-depleted cells were rescued by expression of GFP-mini-N2G, which contains the Terbinafine hydrochloride (Lamisil) N-terminal CH domains and a C-terminal region containing spectrin repeats and the KASH domain of nesprin2G (Fig. 1E,fig S5 and S6). GFP-mini-N2G lacking the CH domains (CH) or mutated to reduce F-actin-binding [Ile128Ala128 and Ile131Ala131 (abbreviated as I128, 131A inFig. 1)] failed to rescue the polarity defects in nesprin2G-depleted cells (Fig.1E,figs. S5 and S6). Thus, nesprin2G and its actin-binding CH domains are necessary for nuclear movement. We next asked whether moving nuclei associated with actin filaments. LPA stimulates actin filament formation in serum-starved cells (5,1618), and we found that an irregular actin meshwork formed near the nucleus at early times after LPA-stimulation (Fig..