Figure 5.
Figure 5. Sema3A-induced repulsion requires Syb2-dependent traffic of its receptor Nrp1/PlexA1. (A) E15 dissociated neurons from Syb2+/+ and Syb2−/− embryos were grown on poly-l-lysine and laminin coverslips. At DIV2, 3 µg/ml of control or Sema3A-Fc was added to the medium. After 3 h of treatment, neurons were fixed and immunostained for MAP2 (green) and Tau (red). (B) The graph displays mean ± SEM values (error bars) of axonal length quantified in each condition (control: Syb2+/+, n = 47; Syb2−/−, n = 79; Sema3A: Syb2+/+, n = 30; Syb2−/−, n = 59 axons). Bar, 10 µm. (C) Mean values ± SEM (error bars) of axonal length quantified in each condition (control, n = 88; control + BoNT/D, n = 101; 3 µg/ml Sema3A, n = 97; 3 µg/ml Sema3A + BoNT/D, n = 99 axons). ***, P < 0.0005 by Student’s t test. (D) Immunofluorescence of PlexA1 and Tau in dissociated cortical neurons cultured from Syb2−/− or Syb2+/+ embryos and treated for 30 min with or without 1 µg/ml Sema3A. Bar, 2 µm. (E) Mean intensity of PlexA1 in soma of cortical neurons (control: Syb2+/+, n = 129; Syb2−/−, n = 109; Sema3A: Syb2+/+, n = 129; Syb2−/−, n = 162 somas in two embryos per genotype). (F) Mean intensity of PlexA1 in soma of cortical neurons (control, n = 41; control + BoNT/D, n = 50; Sema3A, n = 64; Sema3A + BoNT/D, n = 55 somas). (G) Scheme of Syb2-SEP exocytosis protocol. Cos7 cells were cotransfected with PlexA1-FLAG, Syb2-SEP, and Nrp1-mRFP full length or Nrp1ΔCyto-mRFP, and exocytic events of Syb2-SEP were recorded for 3 min. After 30 min of treatment with control (Fc) or Sema3A (Sema3A-Fc), the same cell was recorded for an additional 3 min. Kymographs of recorded cells before and after treatment are shown; exocytic events appear as bright spots, marked with arrowheads. (H) Quantification of Syb2-SEP exocytic events. To evaluate the effect of treatment, exocytic events in the second recording are expressed as a percentage of the first recording for each cell (Nrp1 full length in control, n = 4; and Sema3A condition, n = 8 cells; Nrp1ΔCyto in Sema3A condition, n = 8 cells). Vertical bar, 10 s; horizontal bar, 10 µm. Error bars indicate SEM. (I) Proposed model: Sema3A induces a local disequilibrium (endocytosis → exocytosis) in the growth cone, generating a local surface shrinkage, and prevents progression of the growth cone in the direction of the source, favoring turning to the opposite direction. Syb2 interacts with Nrp1 and PlexA1 and is required for their traffic.

Sema3A-induced repulsion requires Syb2-dependent traffic of its receptor Nrp1/PlexA1. (A) E15 dissociated neurons from Syb2+/+ and Syb2−/− embryos were grown on poly-l-lysine and laminin coverslips. At DIV2, 3 µg/ml of control or Sema3A-Fc was added to the medium. After 3 h of treatment, neurons were fixed and immunostained for MAP2 (green) and Tau (red). (B) The graph displays mean ± SEM values (error bars) of axonal length quantified in each condition (control: Syb2+/+, n = 47; Syb2−/−, n = 79; Sema3A: Syb2+/+, n = 30; Syb2−/−, n = 59 axons). Bar, 10 µm. (C) Mean values ± SEM (error bars) of axonal length quantified in each condition (control, n = 88; control + BoNT/D, n = 101; 3 µg/ml Sema3A, n = 97; 3 µg/ml Sema3A + BoNT/D, n = 99 axons). ***, P < 0.0005 by Student’s t test. (D) Immunofluorescence of PlexA1 and Tau in dissociated cortical neurons cultured from Syb2−/− or Syb2+/+ embryos and treated for 30 min with or without 1 µg/ml Sema3A. Bar, 2 µm. (E) Mean intensity of PlexA1 in soma of cortical neurons (control: Syb2+/+, n = 129; Syb2−/−, n = 109; Sema3A: Syb2+/+, n = 129; Syb2−/−, n = 162 somas in two embryos per genotype). (F) Mean intensity of PlexA1 in soma of cortical neurons (control, n = 41; control + BoNT/D, n = 50; Sema3A, n = 64; Sema3A + BoNT/D, n = 55 somas). (G) Scheme of Syb2-SEP exocytosis protocol. Cos7 cells were cotransfected with PlexA1-FLAG, Syb2-SEP, and Nrp1-mRFP full length or Nrp1ΔCyto-mRFP, and exocytic events of Syb2-SEP were recorded for 3 min. After 30 min of treatment with control (Fc) or Sema3A (Sema3A-Fc), the same cell was recorded for an additional 3 min. Kymographs of recorded cells before and after treatment are shown; exocytic events appear as bright spots, marked with arrowheads. (H) Quantification of Syb2-SEP exocytic events. To evaluate the effect of treatment, exocytic events in the second recording are expressed as a percentage of the first recording for each cell (Nrp1 full length in control, n = 4; and Sema3A condition, n = 8 cells; Nrp1ΔCyto in Sema3A condition, n = 8 cells). Vertical bar, 10 s; horizontal bar, 10 µm. Error bars indicate SEM. (I) Proposed model: Sema3A induces a local disequilibrium (endocytosis → exocytosis) in the growth cone, generating a local surface shrinkage, and prevents progression of the growth cone in the direction of the source, favoring turning to the opposite direction. Syb2 interacts with Nrp1 and PlexA1 and is required for their traffic.

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