Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)
Figure 3
The regulation of filopodia density depends on Eps8 actin barbed-end capping activity.
(A–D) Primary eps8−/− hippocampal neurons (3DIV) were infected with either WT Eps8-EGFP (A and C) or control EGFP (B) lentiviral vectors, then fixed and processed for epifluorescence. Neurons expressing Eps8 ([D] left top panel), but not those expressing EGFP ([D] left bottom panel), show a reduction in the density of axonal filopodia ([D] right panel; number of examined neurons: 120 Eps8wt-EGFP, 55 EGFP; Kruskal-Wallis one-way analysis of variance, p<0.001) Data are expressed as the mean±SEM. Of note, occasionally high levels of ectopically expressed Eps8 induced the presence of flattened structures, sometimes with club-like endings, ([C] and inset in [E]). (E–G) Primary eps8−/− hippocampal neurons (3DIV) infected with either Eps8wt-EGFP (E) or Eps8MUT1-EGFP (number of examined neurons: 31 [F]), an Eps8 mutant devoid of actin-capping activity (Figure S3), or control EGFP (G) lentiviral vectors were fixed and processed for epifluorescence or stained with Texas Red phalloidin, as indicated. Merged images are also shown. Eps8MUT1 neurons do not display flattened membrane protrusions and are morphologically undistinguishable from EGFP-infected neurons (see also quantitation [D], right panel). Bar indicates 10 µm for (A, B, and C); 3 µm for the inset in (C), 5 µm for (D), and 8 µm for (E and G).