Figure 3. Farp1 regulates the motility,... | Rockefeller University Press

Figure 3.

$Figure 3. Farp1 regulates the motility, turnover, and morphology of dendritic filopodia. (A) Profile of Farp1 in rat forebrain at indicated embryonic (E) and postnatal (P) days detected by immunoblotting of equal protein amounts. SynCAM 1 appears at multiple molecular weights due to N-glycan modifications (Fogel et al., 2007). (B) Confocal imaging of live hippocampal neurons at 9 div coexpressing GFP-Farp1 (green) with the F-actin probe UtrCH-Cherry (red) visualizes Farp1 in dendritic filopodia (asterisks). Grayscale, individual channels. Bar, 2.5 µm. (C) Confocal images of live hippocampal neurons at 9 div transfected with a shFarp1 knockdown vector (left), shScramble control (center), or shScramble plus Farp1sh res (right). Images were taken at 3-min intervals for 30 min. Asterisks mark filopodia tracked throughout the imaging period. Bars, 2.5 µm. (D) Filopodial tip motility imaged as in C. Knockdown of Farp1 reduces filopodial dynamics, whereas overexpressing Farp1 increases it (D’Agostino and Pearson test, distributions are significantly different; shScramble, 92 filopodia; shFarp1, 100; shScramble + Farp1sh res, 91; three independent experiments). n.s., not significant. (E) Filopodial turnover imaged as in C. 100 filopodia per condition were tracked from t0. Stability is plotted as a survival curve. Farp1 overexpression doubles the fraction of stable filopodia, defined as present for 30 min (three independent experiments). (F) Farp1 knockdown shortens filopodia (D’Agostino and Pearson test, distributions were significantly different; shScramble, 1.7 ± 0.2 µm length from n = 108 filopodia; shFarp1, 1.4 ± 0.1 µm from n = 135; shScramble + Farp1sh res, 2.5 ± 0.2 µm from n = 119; P = 0.002 and 0.005, respectively; three independent experiments).$

Farp1 regulates the motility, turnover, and morphology of dendritic filopodia. (A) Profile of Farp1 in rat forebrain at indicated embryonic (E) and postnatal (P) days detected by immunoblotting of equal protein amounts. SynCAM 1 appears at multiple molecular weights due to N-glycan modifications (Fogel et al., 2007). (B) Confocal imaging of live hippocampal neurons at 9 div coexpressing GFP-Farp1 (green) with the F-actin probe UtrCH-Cherry (red) visualizes Farp1 in dendritic filopodia (asterisks). Grayscale, individual channels. Bar, 2.5 µm. (C) Confocal images of live hippocampal neurons at 9 div transfected with a shFarp1 knockdown vector (left), shScramble control (center), or shScramble plus Farp1^{sh res} (right). Images were taken at 3-min intervals for 30 min. Asterisks mark filopodia tracked throughout the imaging period. Bars, 2.5 µm. (D) Filopodial tip motility imaged as in C. Knockdown of Farp1 reduces filopodial dynamics, whereas overexpressing Farp1 increases it (D’Agostino and Pearson test, distributions are significantly different; shScramble, 92 filopodia; shFarp1, 100; shScramble + Farp1^{sh res}, 91; three independent experiments). n.s., not significant. (E) Filopodial turnover imaged as in C. 100 filopodia per condition were tracked from t₀. Stability is plotted as a survival curve. Farp1 overexpression doubles the fraction of stable filopodia, defined as present for 30 min (three independent experiments). (F) Farp1 knockdown shortens filopodia (D’Agostino and Pearson test, distributions were significantly different; shScramble, 1.7 ± 0.2 µm length from n = 108 filopodia; shFarp1, 1.4 ± 0.1 µm from n = 135; shScramble + Farp1^{sh res}, 2.5 ± 0.2 µm from n = 119; P = 0.002 and 0.005, respectively; three independent experiments).