Induced presynapses are similar to natural presynapses between 48 and 96 h after bead seeding and between inhibitory and excitatory presynapses; acute actin perturbation effect on the accumulation of presynaptic components is similar at natural synapses and induced presynapses. (A) Quantification of the labeling intensity for bassoon (dark purple), synaptophysin (purple), synapsin (orange), and vamp2 (yellow) at natural synapses (NS), induced presynapses at axon-bead contacts (S+) and axon-bead contacts devoid of presynapse (S−) 48 h (left) or 96 h (right) after bead seeding, normalized to the intensity at S+ contacts after 48 h. (B) Quantification of the labeling intensity for actin at natural synapses (NS), induced presynapses at axon-bead contacts (S+), and axon-bead contacts devoid of presynapse (S−) 48 h (left) or 96 h (right) after bead seeding, normalized to the intensity at S+ contacts after 48 h. (C) Quantification of the labeling intensity for synapsin (dark purple) and VGLUT (orange) at excitatory natural synapses (NS), induced presynapses at axon-bead contacts (S+), and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at S+ contacts. (D) Quantification of the labeling intensity for synaptophysin (purple) and VGAT (yellow) at inhibitory natural synapses (NS), induced presynapses at axon-bead contacts (S+) and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at S+ contacts. (E) Quantification of the labeling intensity for actin at natural synapses (NS), induced presynapses at axon-bead contacts (S+), and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at S+ contacts for excitatory (left) or inhibitory (right) presynapses. Significance signs on graphs A–E compare to the value for the same labeling in bead-induced presynapses (S+, normalized to 1.0). (F–H) Representative images of natural presynapses in control condition (F) or after treatment with swinholide A (G) or cucurbitacine E (H) 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. 4, A–C. Scale bars, 2 µm. (I) 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 swinholide A treatment, normalized to control natural synapses. (J) 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 cucurbitacin E treatment, 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 S6.

Induced presynapses are similar to natural presynapses between 48 and 96 h after bead seeding and between inhibitory and excitatory presynapses; acute actin perturbation effect on the accumulation of presynaptic components is similar at natural synapses and induced presynapses. (A) Quantification of the labeling intensity for bassoon (dark purple), synaptophysin (purple), synapsin (orange), and vamp2 (yellow) at natural synapses (NS), induced presynapses at axon-bead contacts (S+) and axon-bead contacts devoid of presynapse (S−) 48 h (left) or 96 h (right) after bead seeding, normalized to the intensity at S+ contacts after 48 h. (B) Quantification of the labeling intensity for actin at natural synapses (NS), induced presynapses at axon-bead contacts (S+), and axon-bead contacts devoid of presynapse (S−) 48 h (left) or 96 h (right) after bead seeding, normalized to the intensity at S+ contacts after 48 h. (C) Quantification of the labeling intensity for synapsin (dark purple) and VGLUT (orange) at excitatory natural synapses (NS), induced presynapses at axon-bead contacts (S+), and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at S+ contacts. (D) Quantification of the labeling intensity for synaptophysin (purple) and VGAT (yellow) at inhibitory natural synapses (NS), induced presynapses at axon-bead contacts (S+) and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at S+ contacts. (E) Quantification of the labeling intensity for actin at natural synapses (NS), induced presynapses at axon-bead contacts (S+), and axon-bead contacts devoid of presynapse (S−), normalized to the intensity at S+ contacts for excitatory (left) or inhibitory (right) presynapses. Significance signs on graphs A–E compare to the value for the same labeling in bead-induced presynapses (S+, normalized to 1.0). (F–H) Representative images of natural presynapses in control condition (F) or after treatment with swinholide A (G) or cucurbitacine E (H) 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. 4, A–C. Scale bars, 2 µm. (I) 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 swinholide A treatment, normalized to control natural synapses. (J) 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 cucurbitacin E treatment, 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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