Actin mesh, rails and corrals are present in natural presynapses. (A) Widefield fluorescence image of cultured neurons infected after plating with CRISPR AAVs for the knock-in of a FLAG-ALFA tag on endogenous actin, fixed at 9 div and labeled for FLAG (gray), ß2-spectrin (purple), map2 (blue), and synapsin (orange). (B) Zooms corresponding to the area highlighted in A showing the FLAG-ALFA-actin isolated channel and overlay for the axon arborization of a FLAG-ALFA-actin knocked-in neuron forming presynapses on nearby neurons. (C and D) STORM images of actin nanostructures in the presynapses of ALFA-actin knock-in neurons labeled for ALFA using primary and secondary antibodies. The first column shows the widefield image of map2 (blue), synaptophysin (orange), and ALFA-actin (gray). The second and third columns are zooms of the highlighted areas, showing an overlay of widefield images of map2 (blue) and synaptophysin (orange) with the STORM image of ALFA-actin (gray). Fourth column is 3D-STORM image of ALFA-actin, color-coded for depth (with Z scale in the top right corner). Fifth column is the STORM image of ALFA-actin with highlighted presynaptic actin structures: active zone mesh (red), actin rails (green), perisynaptic corral (blue). (E and F) DNA-PAINT images of actin nanostructures in the presynapses of 3ALFA-actin knock-in neurons labeled for ALFA using an anti-ALFA nanobody. The first column shows the overlay of the widefield image of map2 (blue) and synaptophysin (orange) with the PAINT image of 3ALFA-actin (gray). The second column is 3D-STORM image of 3ALFA-actin, color-coded for depth (with Z scale in the top right corner). The third column is the PAINT image of 3ALFA-actin with highlighted presynaptic actin structures: active zone mesh (red), actin rails (green), perisynaptic corral (blue). Scale bars in A, 50 µm; in B, 10 µm; in C, 5 µm (first panel), 2 µm (second panel), 1 µm (other panels); in D, 5 µm (first panel), 2 µm (second panel), 1 µm (third panel), 0.5 µm (fourth and fifth panels); in E and F, 1 µm (first column), 0.5 µm (second and third columns). (G) Cartoon summarizing the identified presynaptic actin nanostructures and their putative role: the branched actin mesh at the active zone where vesicles are exocytosed; linear actin rails (green) help vesicles move between the exocytosis, endocytosis zones, and intracellular pools; the perisynaptic actin corral (blue) scaffolds the vesicular pool.
Figure 10.

Actin mesh, rails and corrals are present in natural presynapses. (A) Widefield fluorescence image of cultured neurons infected after plating with CRISPR AAVs for the knock-in of a FLAG-ALFA tag on endogenous actin, fixed at 9 div and labeled for FLAG (gray), ß2-spectrin (purple), map2 (blue), and synapsin (orange). (B) Zooms corresponding to the area highlighted in A showing the FLAG-ALFA-actin isolated channel and overlay for the axon arborization of a FLAG-ALFA-actin knocked-in neuron forming presynapses on nearby neurons. (C and D) STORM images of actin nanostructures in the presynapses of ALFA-actin knock-in neurons labeled for ALFA using primary and secondary antibodies. The first column shows the widefield image of map2 (blue), synaptophysin (orange), and ALFA-actin (gray). The second and third columns are zooms of the highlighted areas, showing an overlay of widefield images of map2 (blue) and synaptophysin (orange) with the STORM image of ALFA-actin (gray). Fourth column is 3D-STORM image of ALFA-actin, color-coded for depth (with Z scale in the top right corner). Fifth column is the STORM image of ALFA-actin with highlighted presynaptic actin structures: active zone mesh (red), actin rails (green), perisynaptic corral (blue). (E and F) DNA-PAINT images of actin nanostructures in the presynapses of 3ALFA-actin knock-in neurons labeled for ALFA using an anti-ALFA nanobody. The first column shows the overlay of the widefield image of map2 (blue) and synaptophysin (orange) with the PAINT image of 3ALFA-actin (gray). The second column is 3D-STORM image of 3ALFA-actin, color-coded for depth (with Z scale in the top right corner). The third column is the PAINT image of 3ALFA-actin with highlighted presynaptic actin structures: active zone mesh (red), actin rails (green), perisynaptic corral (blue). Scale bars in A, 50 µm; in B, 10 µm; in C, 5 µm (first panel), 2 µm (second panel), 1 µm (other panels); in D, 5 µm (first panel), 2 µm (second panel), 1 µm (third panel), 0.5 µm (fourth and fifth panels); in E and F, 1 µm (first column), 0.5 µm (second and third columns). (G) Cartoon summarizing the identified presynaptic actin nanostructures and their putative role: the branched actin mesh at the active zone where vesicles are exocytosed; linear actin rails (green) help vesicles move between the exocytosis, endocytosis zones, and intracellular pools; the perisynaptic actin corral (blue) scaffolds the vesicular pool.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal