The C-terminal (PH+C) region of EFA6A leads to an increase in spine density. A, Cultured neurons were transfected at DIV 12 with FLAG-EFA6A PH+C and EGFP, or EGFP alone, and labeled at DIV 19 for EGFP and FLAG. Scale bar, 5 μm. B–D, Quantitation of the effect of EFA6A PH+C expression on the linear density of spines, filopodia, and total protrusions. EGFP alone (protrusion density, 44.3 ± 1.19; spine density, 42.05 ± 1.24; filopodia density, 2.25 ± 0.69; length, 1.58 ± 0.05; width, 1.04 ± 0.02); EFA6A PH+C (protrusion density, 54.85 ± 1.40; spine density, 51.93 ± 1.35; filopodia density, 3.22 ± 0.34; length, 1.59 ± 0.03; width, 0.97 ± 0.02). E–H, Quantitation of the effect of EFA6A expression on the lengths and widths of protrusions. I, Cultured neurons expressing FLAG-EFA6A PH+C were double labeled for EGFP or EFA6A and PSD-95. Scale bar, 5 μm. J, Quantitation of the effect of EFA6A PH+C expression on spine localization of PSD-95. EGFP alone (53.31 ± 2.94); EFA6A PH+C (50.55 ± 2.36). Data were collected from 800–1500 protrusions on 20–27 dendrites of 16–20 neurons for each group in the three independent experiments. *p < 0.05; ***p < 0.001 (Student’s t test).