Figure 3. Hsc70 is required for the netrin-1–induced enrichment of Trio at the growth cone periphery. (A) Dissociated E17.5 rat cortical neurons were left untreated or stimulated with netrin-1 for 5 min before fixation. Endogenous Trio and F-actin localization were assessed by epifluorescence microscopy. Bar, 10 µm. (right) The intensity of Trio and F-actin fluorescence along a 10-µm linescan oriented in the central-to-peripheral axis of the growth cone was calculated using Metamorph software. Trio (green) and F-actin (red) pixel intensities from the adjacent images were plotted against distance. (B) The mean pixel intensity of Trio (green) and F-actin (red) along a 10-µm linescan were plotted against distance as in A. The data shown are the mean of >130 growth cones, from four independent experiments. Error bars indicate the SEM. (C) Dissociated E17.5 rat cortical neurons were electroporated with a GFP-reporter and Hsc70 siRNA. At DIV1, cultures were fixed and levels of endogenous Hsc70 were assessed by indirect immunofluorescence. Bar, 15 µm. (D) The mean pixel intensity of Hsc70 was calculated. Error bars indicate the SEM (n = 3; GFP-negative neurons = 19; GFP-positive neurons = 20; ***, P < 0.001; unpaired student’s t test). (E) As in C, dissociated E17.5 rat cortical neurons were electroporated with a GFP-reporter and control siRNA or Hsc70 siRNA and left untreated or treated with netrin-1 for 5 min before fixation. Trio localization was assessed by indirect immunofluorescence. Bars: (main) 25 µm; (inset) 5 µm. (F, left) Schematic of Trio localization showing dispersed localization versus peripheral enrichment. (right) The proportion of electroporated neurons with growth cones harboring a distinct peripheral Trio localization was calculated for each treatment in E (>70 neurons assessed per condition, from four independent experiments). Error bars indicate the SEM (***, P < 0.001; unpaired student’s t test). (G) As in B, the mean pixel intensity of Trio was calculated along 10-µm linescans oriented in the central-to-peripheral axis of each growth cone from E (n > 65 growth cones per condition).
Figure 3.

Hsc70 is required for the netrin-1–induced enrichment of Trio at the growth cone periphery. (A) Dissociated E17.5 rat cortical neurons were left untreated or stimulated with netrin-1 for 5 min before fixation. Endogenous Trio and F-actin localization were assessed by epifluorescence microscopy. Bar, 10 µm. (right) The intensity of Trio and F-actin fluorescence along a 10-µm linescan oriented in the central-to-peripheral axis of the growth cone was calculated using Metamorph software. Trio (green) and F-actin (red) pixel intensities from the adjacent images were plotted against distance. (B) The mean pixel intensity of Trio (green) and F-actin (red) along a 10-µm linescan were plotted against distance as in A. The data shown are the mean of >130 growth cones, from four independent experiments. Error bars indicate the SEM. (C) Dissociated E17.5 rat cortical neurons were electroporated with a GFP-reporter and Hsc70 siRNA. At DIV1, cultures were fixed and levels of endogenous Hsc70 were assessed by indirect immunofluorescence. Bar, 15 µm. (D) The mean pixel intensity of Hsc70 was calculated. Error bars indicate the SEM (n = 3; GFP-negative neurons = 19; GFP-positive neurons = 20; ***, P < 0.001; unpaired student’s t test). (E) As in C, dissociated E17.5 rat cortical neurons were electroporated with a GFP-reporter and control siRNA or Hsc70 siRNA and left untreated or treated with netrin-1 for 5 min before fixation. Trio localization was assessed by indirect immunofluorescence. Bars: (main) 25 µm; (inset) 5 µm. (F, left) Schematic of Trio localization showing dispersed localization versus peripheral enrichment. (right) The proportion of electroporated neurons with growth cones harboring a distinct peripheral Trio localization was calculated for each treatment in E (>70 neurons assessed per condition, from four independent experiments). Error bars indicate the SEM (***, P < 0.001; unpaired student’s t test). (G) As in B, the mean pixel intensity of Trio was calculated along 10-µm linescans oriented in the central-to-peripheral axis of each growth cone from E (n > 65 growth cones per condition).

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