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Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1.

DeGeer J, Kaplan A, Mattar P, Morabito M, Stochaj U, Kennedy TE, Debant A, Cayouette M, Fournier AE, Lamarche-Vane N - J. Cell Biol. (2015)

Bottom Line: Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio.Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase-deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1-induced Rac1 activation.Hsc70 was required for netrin-1-mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada The Research Institute of McGill University Health Centre, Montreal, Quebec H4A 3J1, Canada.

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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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fig3: 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).

Mentions: Because Hsc70 is a molecular chaperone, we assessed whether it may function to regulate Trio localization within cortical growth cones. To first establish Trio localization, cortical neurons were treated with netrin-1 for 5 min, and then fixed and stained (Fig. 3 A). Trio and F-actin growth cone localizations were assessed and the intensity of each signal was measured along a 10-µm segment of the distal growth cone (Fig. 3 A, right). Upon netrin-1 treatment, the intensity of Trio shifted to the growth cone periphery compared with untreated growth cones, similar to F-actin (Fig. 3, A and B).


Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1.

DeGeer J, Kaplan A, Mattar P, Morabito M, Stochaj U, Kennedy TE, Debant A, Cayouette M, Fournier AE, Lamarche-Vane N - J. Cell Biol. (2015)

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).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: 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).
Mentions: Because Hsc70 is a molecular chaperone, we assessed whether it may function to regulate Trio localization within cortical growth cones. To first establish Trio localization, cortical neurons were treated with netrin-1 for 5 min, and then fixed and stained (Fig. 3 A). Trio and F-actin growth cone localizations were assessed and the intensity of each signal was measured along a 10-µm segment of the distal growth cone (Fig. 3 A, right). Upon netrin-1 treatment, the intensity of Trio shifted to the growth cone periphery compared with untreated growth cones, similar to F-actin (Fig. 3, A and B).

Bottom Line: Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio.Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase-deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1-induced Rac1 activation.Hsc70 was required for netrin-1-mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada The Research Institute of McGill University Health Centre, Montreal, Quebec H4A 3J1, Canada.

Show MeSH
Related in: MedlinePlus