Polylysine-coated beads induce functional presynaptic specializations at axon-bead contacts. (A) Widefield fluorescence image of cultured neurons seeded with polylysine-coated beads (gray) after 8 div and fixed 48 h later at 10 div, labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). (B) Zooms corresponding to the areas highlighted in A: top row, natural synapse at axon-dendrite contact (NS); middle row, induced presynapse at an axon-bead contact (S+); bottom row, axon-bead contact with no induced presynapse (S−). The position of the bead is indicated by the dashed gray circle. (C) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), vamp2 (orange), and map2 (gray). (D) Zooms corresponding to the natural synapse, S+ and S− axon-bead contacts highlighted in C. (E) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), map2 (gray), and feeding with anti-synaptotagmin antibody (syt) during constitutive vesicular cycling (orange). (F) Zooms corresponding to the natural synapse (NS), S+, and S− axon-bead contacts highlighted in E. Scale bars for A, C, and E: 20 µm; for B, D, and F: 2 µm. (G) Quantification of the proportion of S+ (blue) and S− (gray) axon-bead contacts. (H) Quantification of the labeling intensity for bassoon (dark purple), synapsin (orange), synaptophysin (purple), and vamp2 (yellow) 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. (I) 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. (J) Quantification of the labeling intensity after syt feeding for constitutive (orange) and stimulated (KCl-induced, yellow) vesicular cycling 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. Significance signs on graphs compare to the value for the same labeling in bead-induced presynapses (S+, normalized to 1.0). See Data S1 file for detailed statistics.
Figure 1.

Polylysine-coated beads induce functional presynaptic specializations at axon-bead contacts. (A) Widefield fluorescence image of cultured neurons seeded with polylysine-coated beads (gray) after 8 div and fixed 48 h later at 10 div, labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). (B) Zooms corresponding to the areas highlighted in A: top row, natural synapse at axon-dendrite contact (NS); middle row, induced presynapse at an axon-bead contact (S+); bottom row, axon-bead contact with no induced presynapse (S−). The position of the bead is indicated by the dashed gray circle. (C) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), vamp2 (orange), and map2 (gray). (D) Zooms corresponding to the natural synapse, S+ and S− axon-bead contacts highlighted in C. (E) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for actin (green), synaptophysin (purple), map2 (gray), and feeding with anti-synaptotagmin antibody (syt) during constitutive vesicular cycling (orange). (F) Zooms corresponding to the natural synapse (NS), S+, and S− axon-bead contacts highlighted in E. Scale bars for A, C, and E: 20 µm; for B, D, and F: 2 µm. (G) Quantification of the proportion of S+ (blue) and S− (gray) axon-bead contacts. (H) Quantification of the labeling intensity for bassoon (dark purple), synapsin (orange), synaptophysin (purple), and vamp2 (yellow) 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. (I) 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. (J) Quantification of the labeling intensity after syt feeding for constitutive (orange) and stimulated (KCl-induced, yellow) vesicular cycling 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. Significance signs on graphs compare to the value for the same labeling in bead-induced presynapses (S+, normalized to 1.0). See Data S1 file for detailed statistics.

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