Presynapses induced for 48 and 96 h show a similar proportion of actin enrichment with similar presynaptic content. (A) Widefield fluorescence image of cultured neurons 96 h after bead seeding at 6 div, labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). (B) Zooms corresponding to the areas highlighted in A: top row, actin-enriched induced presynapse at an axon-bead contact (A+); second row, induced presynapse at an axon-bead contact with no actin enrichment (A−); third row, axon-bead contact with no induced presynapse (S−); bottom row, natural synapse at axon-dendrite contact (NS). (C) Widefield fluorescence image of cultured neurons 96 h after bead seeding at 6 div, labeled for actin (green), synaptophysin (purple), vamp2 (orange), and map2 (gray). (D) Zooms corresponding to the A+, A−, S− axon-bead contacts and natural synapses (NS) highlighted in C. Scale bars in A and C: 20 µm; B and D: 2 µm. (E) Quantification of the proportion of A+ (dark blue) and A− (blue) at axon-bead contacts that resulted in an induced presynapse for neurons after 48 and 96 h incubation with beads. (F) Quantification of the labeling intensity for actin (green), bassoon (dark purple), synaptophysin (purple), synapsin (orange), and vamp2 (yellow) at actin-enriched presynapses (A+), induced presynapses with no actin enrichment (A−), and axon-bead contacts devoid of presynapse (S−), 48 h (left), and 96 h (right) after bead seeding, normalized to the intensity of A+ presynapses after 48 h. Significance signs on graphs (bottom: *, ns) compare to the value for the labeling on bead-induced presynapses (S+, normalized to 1.0 at 48 h); significance signs (top: °, ns) compare to the value for the same labeling and type of induced presynapse (A+ or A−) between 48 and 96 h. See Data S1 file for detailed statistics.
Figure S4.

Presynapses induced for 48 and 96 h show a similar proportion of actin enrichment with similar presynaptic content. (A) Widefield fluorescence image of cultured neurons 96 h after bead seeding at 6 div, labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). (B) Zooms corresponding to the areas highlighted in A: top row, actin-enriched induced presynapse at an axon-bead contact (A+); second row, induced presynapse at an axon-bead contact with no actin enrichment (A−); third row, axon-bead contact with no induced presynapse (S−); bottom row, natural synapse at axon-dendrite contact (NS). (C) Widefield fluorescence image of cultured neurons 96 h after bead seeding at 6 div, labeled for actin (green), synaptophysin (purple), vamp2 (orange), and map2 (gray). (D) Zooms corresponding to the A+, A−, S− axon-bead contacts and natural synapses (NS) highlighted in C. Scale bars in A and C: 20 µm; B and D: 2 µm. (E) Quantification of the proportion of A+ (dark blue) and A− (blue) at axon-bead contacts that resulted in an induced presynapse for neurons after 48 and 96 h incubation with beads. (F) Quantification of the labeling intensity for actin (green), bassoon (dark purple), synaptophysin (purple), synapsin (orange), and vamp2 (yellow) at actin-enriched presynapses (A+), induced presynapses with no actin enrichment (A−), and axon-bead contacts devoid of presynapse (S−), 48 h (left), and 96 h (right) after bead seeding, normalized to the intensity of A+ presynapses after 48 h. Significance signs on graphs (bottom: *, ns) compare to the value for the labeling on bead-induced presynapses (S+, normalized to 1.0 at 48 h); significance signs (top: °, ns) compare to the value for the same labeling and type of induced presynapse (A+ or A−) between 48 and 96 h. See Data S1 file for detailed statistics.

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