Acute actin disassembly and over-stabilization lower the accumulation of presynaptic components at induced presynapses. (A) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, control treated with vehicle (DMSO) for 1 h and labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). Bottom, panels showing A+ presynapses labeled for actin, presynaptic components, and after syt feeding. The first panel is a zoom of the area highlighted in the top image. (B) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 1 µM swinholide A (swin) for 1 h, and labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). Bottom, panels showing A− presynapses labeled for actin, presynaptic components, and after syt feeding. The first panel is a zoom of the area highlighted in the top image. (C) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 5 µM cucurbitacin E (cuc) for 3 h, and labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). Bottom, panels showing A+ presynapses labeled for actin, presynaptic components, and after syt feeding. The first panel is a zoom of the area highlighted in the top image. Scale bars on large images in A–C, 20 µm; on zoomed images, 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 actin-enriched presynapses (A+) and induced presynapses with no actin enrichment (A−) in the control condition, and at A− presynapses after swin treatment. (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 actin-enriched presynapses (A+) and induced presynapses with no actin enrichment (A−) in the control condition, and at A+ presynapses after cuc treatment. Significance signs (bottom: *, ns) on graphs compare to the value for the same labeling in the control condition for A+ presynapses (normalized to 1.0); significance signs (top: °, ns) compare to the value for the same labeling in the control condition for A− presynapses. See Data S1 file for detailed statistics.
Figure 4.

Acute actin disassembly and over-stabilization lower the accumulation of presynaptic components at induced presynapses. (A) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, control treated with vehicle (DMSO) for 1 h and labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). Bottom, panels showing A+ presynapses labeled for actin, presynaptic components, and after syt feeding. The first panel is a zoom of the area highlighted in the top image. (B) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 1 µM swinholide A (swin) for 1 h, and labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). Bottom, panels showing A− presynapses labeled for actin, presynaptic components, and after syt feeding. The first panel is a zoom of the area highlighted in the top image. (C) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 5 µM cucurbitacin E (cuc) for 3 h, and labeled for actin (green), bassoon (purple), synapsin (orange), and map2 (gray). Bottom, panels showing A+ presynapses labeled for actin, presynaptic components, and after syt feeding. The first panel is a zoom of the area highlighted in the top image. Scale bars on large images in A–C, 20 µm; on zoomed images, 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 actin-enriched presynapses (A+) and induced presynapses with no actin enrichment (A−) in the control condition, and at A− presynapses after swin treatment. (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 actin-enriched presynapses (A+) and induced presynapses with no actin enrichment (A−) in the control condition, and at A+ presynapses after cuc treatment. Significance signs (bottom: *, ns) on graphs compare to the value for the same labeling in the control condition for A+ presynapses (normalized to 1.0); significance signs (top: °, ns) compare to the value for the same labeling in the control condition for A− presynapses. See Data S1 file for detailed statistics.

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