KDM3A is required for actin polymerization. (A) 60 min after plating on glass coverslips, 3D reconstruction reveals cytoplasmic distribution of Halo-KDM3A with strong presence at cellular edges and apical cap. (B) Schematic of stages requiring fast actin polymerization occurring from initial attachment (Phase 0 [P0]) to early spreading (P1). P2 represents fully spread cells. (C) Bright-field image and number of RPE1^WT and KDM3A^null cells that remain round (P0) or began spreading (P1) over plastic plates after suspension in the absence of serum for 2 h. Red arrow and green encircling indicate cells at P0, defined by birefringence at edges, and P1, defined by flattened edges, respectively. (D) Proportion of cells that remain in P0 when cultures are plated immediately after trypsinization (N = 3, n > 50 cells scored/genotype). Error bars: mean ± SD. *, P < 0.0001, from χ². (E) Wide-field images illustrate differences in cellular edges between RPE1^crWT and KDM3A^null. (F) Schematic of actin filament capping or branching leading to changes in cell morphology. As KDM3A binds actin, the rounder cellular edges of KDM3A cells suggest an imbalance between capping (yellow) and ARP2/3 mediated branching (green). (G) Median and range of the distance migrated by attached RPE1^WT cells and KDM3A^null (lines 1, 2, and 8; 4 h after plating). Dots represent measurement of distance moved by a single cell. . (H) Confocal images illustrate the distinct cellular edges of RPE1^WT cells transfected with Halo vector or Halo-KDM3A. (I) Hyperfilopodia-like projections are most prominent at the apical pole of Halo-KDM3A transfected cells (plane 2 in diagram). (J) Median and range of membrane lengths at cellular edge of Halo transfected cells measured as described in Fig. S2 D. N = 2 transfections. Statistics from G and J are from unpaired t tests.