Acute actin nucleators inhibition effect on the accumulation of presynaptic components is similar at natural synapses and induced presynapses; effect of CK666 on additional presynaptic components. (A–C) Representative images of natural presynapses in control condition (A) or after treatment with CK666 (B) or SMIFH2 (C) labeled for actin, bassoon, and synapsin (green, purple, and orange, respectively, top-left image); actin, synaptophysin, and vamp2 (green, purple and orange respectively, top-right image); actin, synaptophysin and constitutive feeding with anti-synaptotagmin antibody (green, purple, and orange, respectively, bottom-left image); actin, synaptophysin and stimulated feeding with anti-synaptotagmin antibody (green, purple, and orange, respectively, bottom-right image). Zooms are taken from the images shown in Fig. 5, A–C. Scale bars, 2 µm. (D) Quantification of the labeling intensity for actin (green), bassoon (dark purple), synaptophysin (purple), synapsin (dark blue), vamp2 (blue), and after syt feeding for constitutive (orange), and stimulated (yellow) vesicular cycling at induced presynapses devoid of actin enrichment (A−) in the control condition and after CK666 or SMIFH2 treatment, normalized to control A+ presynapses. Significance signs on graph compare to the value for the same labeling in the control condition for A− presynapses (normalized to 1.0). (E) Quantification of the labeling intensity for actin (green), bassoon (dark purple), synaptophysin (purple), synapsin (dark blue), vamp2 (blue), and after syt feeding for constitutive (orange), and stimulated (yellow) vesicular cycling at natural synapses in the control condition and after CK666 or SMIFH2 treatment, normalized to control natural synapses. Significance signs on the graph compare to the value for the same labeling in the control condition for natural synapses (normalized to 1.0). (F and G) Representative images of induced and natural presynapses in the control condition (F) or after treatment with CK666 (G) labeled for actin, syntaxin, and synapsin (green, purple, and orange respectively, top row); actin, SV2 and munc13 (green, purple, and orange, respectively, bottom row). Scale bars, 2 µm. (H) Quantification of the labeling intensity for actin (green), syntaxin (dark purple), SV2 (purple), synaptophysin (dark blue), and munc13 (orange), at induced presynapses enriched in actin (A+) or devoid of actin enrichment (A−), in the control condition and after CK666 treatment, normalized to control A+ presynapses. Significance signs (bottom: *, ns) on graphs compare to the value for the same measurement in the control condition for the same type of presynapse (A+ or A−, normalized to 1.0); significance signs (top: °, ns) compare to the value for the same measurement in the control condition for A+ presynapses. (I) Quantification of the labeling intensity for actin (green), syntaxin (dark purple), SV2 (purple), synaptophysin (dark blue), and munc13 (orange), at natural presynapses (NS), in the control condition and after CK666, normalized to control natural synapses. Significance signs on graphs I and J compare to the value for the same labeling in the control condition for natural synapses (normalized to 1.0). See Data S1 file for detailed statistics.
Figure S7.

Acute actin nucleators inhibition effect on the accumulation of presynaptic components is similar at natural synapses and induced presynapses; effect of CK666 on additional presynaptic components. (A–C) Representative images of natural presynapses in control condition (A) or after treatment with CK666 (B) or SMIFH2 (C) labeled for actin, bassoon, and synapsin (green, purple, and orange, respectively, top-left image); actin, synaptophysin, and vamp2 (green, purple and orange respectively, top-right image); actin, synaptophysin and constitutive feeding with anti-synaptotagmin antibody (green, purple, and orange, respectively, bottom-left image); actin, synaptophysin and stimulated feeding with anti-synaptotagmin antibody (green, purple, and orange, respectively, bottom-right image). Zooms are taken from the images shown in Fig. 5, A–C. Scale bars, 2 µm. (D) Quantification of the labeling intensity for actin (green), bassoon (dark purple), synaptophysin (purple), synapsin (dark blue), vamp2 (blue), and after syt feeding for constitutive (orange), and stimulated (yellow) vesicular cycling at induced presynapses devoid of actin enrichment (A−) in the control condition and after CK666 or SMIFH2 treatment, normalized to control A+ presynapses. Significance signs on graph compare to the value for the same labeling in the control condition for A− presynapses (normalized to 1.0). (E) Quantification of the labeling intensity for actin (green), bassoon (dark purple), synaptophysin (purple), synapsin (dark blue), vamp2 (blue), and after syt feeding for constitutive (orange), and stimulated (yellow) vesicular cycling at natural synapses in the control condition and after CK666 or SMIFH2 treatment, normalized to control natural synapses. Significance signs on the graph compare to the value for the same labeling in the control condition for natural synapses (normalized to 1.0). (F and G) Representative images of induced and natural presynapses in the control condition (F) or after treatment with CK666 (G) labeled for actin, syntaxin, and synapsin (green, purple, and orange respectively, top row); actin, SV2 and munc13 (green, purple, and orange, respectively, bottom row). Scale bars, 2 µm. (H) Quantification of the labeling intensity for actin (green), syntaxin (dark purple), SV2 (purple), synaptophysin (dark blue), and munc13 (orange), at induced presynapses enriched in actin (A+) or devoid of actin enrichment (A−), in the control condition and after CK666 treatment, normalized to control A+ presynapses. Significance signs (bottom: *, ns) on graphs compare to the value for the same measurement in the control condition for the same type of presynapse (A+ or A−, normalized to 1.0); significance signs (top: °, ns) compare to the value for the same measurement in the control condition for A+ presynapses. (I) Quantification of the labeling intensity for actin (green), syntaxin (dark purple), SV2 (purple), synaptophysin (dark blue), and munc13 (orange), at natural presynapses (NS), in the control condition and after CK666, normalized to control natural synapses. Significance signs on graphs I and J compare to the value for the same labeling in the control condition for natural synapses (normalized to 1.0). See Data S1 file for detailed statistics.

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