Arp2/3 and formins inhibitors have distinct effects on actin and 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), and map2 (gray). Bottom, panels showing A+ and A− presynapses labeled for actin, presynaptic components, and after syt feeding. The top panels are zooms of the areas highlighted in the top image. (B) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 50 μM CK666 (Arp2/3 inhibitor) for 1 h, and labeled for actin (green), bassoon (purple), and map2 (gray). Bottom, panels showing A+ and A− presynapses labeled for actin, presynaptic components, and after syt feeding. The top panels are zooms of the areas highlighted in the top image. (C) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 30 µM SMIFH2 (formins inhibitor) for 3 h, and labeled for actin (green), bassoon (purple), and map2 (gray). Bottom, panels showing A+ and A− presynapses labeled for actin, presynaptic components and after syt feeding. Top panels are zooms of the areas 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+) in the control condition, and after CK666 or SMIFH2 treatment. Significance signs on graphs compare to the value for the same labeling in the control condition for A+ presynapses (normalized to 1.0). See Data S1 file for detailed statistics.
Figure 5.

Arp2/3 and formins inhibitors have distinct effects on actin and 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), and map2 (gray). Bottom, panels showing A+ and A− presynapses labeled for actin, presynaptic components, and after syt feeding. The top panels are zooms of the areas highlighted in the top image. (B) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 50 μM CK666 (Arp2/3 inhibitor) for 1 h, and labeled for actin (green), bassoon (purple), and map2 (gray). Bottom, panels showing A+ and A− presynapses labeled for actin, presynaptic components, and after syt feeding. The top panels are zooms of the areas highlighted in the top image. (C) Top, widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, treated with 30 µM SMIFH2 (formins inhibitor) for 3 h, and labeled for actin (green), bassoon (purple), and map2 (gray). Bottom, panels showing A+ and A− presynapses labeled for actin, presynaptic components and after syt feeding. Top panels are zooms of the areas 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+) in the control condition, and after CK666 or SMIFH2 treatment. Significance signs on graphs compare to the value for the same labeling in the control condition for A+ presynapses (normalized to 1.0). See Data S1 file for detailed statistics.

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