Figure 4. FMN2 localizes in the perinuclear region in fibroblasts migrating through 3D collagen ECMs.

(A–D) MEFs were cultured in 3D collagen gels (3 mg/ml) for 16–18 hr. (A) MEF were fixed and immuno-labeled with antibodies against FMN2 (Endogenous FMN2, green), actin stained with fluorescent phalloidin (actin, red), and the nucleus with DAPI (blue). Cells were imaged by SDC. Left 3 panels: maximal intensity projections of z-stacks, white arrowhead: cortical FMN2. Boxed regions in upper panels (Bar = 20um): zoomed and magnified in lower panels (Bar= 5 um). Right panel: X/Z section (at the white line in left panels) from 3D reconstruction. (B) Time-lapse imaging of a single confocal slice of a MEF co-expressing FMN2-GFP (green), mCardinal histone H2B (blue) and mApple actin (red). Bar= 20um time in minutes. (C,D) Maximal intensity projections of SDC Z-stacks of MEF mock-transfected (Control) or transfected with an siRNAs targeting the 3′untranslated region of FMN2 alone (FMN2 KD) or together with FMN2-GFP (Rescue) fixed and stained with fluorescent phalloidin (actin, green), and the nucleus stained with DAPI (C, red, D, blue) or immuno-labeled with antibodies against vinculin (D, red). In Rescue panels, FMN2-GFP is in blue in the color overlay. Boxed regions in upper panels (Bar= 10 um) shown zoomed below (Bar= 5um). In (C), green arrowheads: perinuclear actin bundles, red arrowhead: lack thereof. Bottom panels: X/Z section (at the white line in upper panels) from a 3D reconstruction. In (D), regions of the yellow (leading edge adhesions) and blue (perinuclear adhesions) boxes zoomed and shown below, green arrowheads: FAs. See also Figure S5 and movie S5