Revealing presynaptic actin enrichment in natural synapses and its role using CRISPR-tagging of endogenous actin. (A) Widefield fluorescence image of cultured neurons infected after plating with CRISPR AAVs for the knock-in of an HA tag on endogenous actin, fed with a synaptotagmin antibody for 1 h (orange) and fixed at 9 div, then labeled for HA (green), synaptophysin (purple), and map2 (gray). (B) Zooms corresponding to the area highlighted in A showing isolated channels and overlay of the axon of a HA-actin knocked-in neuron forming actin-enriched A+ (1 and 3) and non-enriched A− (2) presynapses. (C) Zooms on individual presynapses highlighted in B. (D) Quantification of the proportion of A+ (dark blue) and A− (blue) presynapses along the axons of HA-actin neurons. (E) Quantification of the labeling intensity for HA-actin (green), synaptophysin (purple), and constitutive synaptotagmin feeding (orange) at HA-actin-enriched presynapses (A+), induced presynapses with no actin enrichment (A−), and axon shafts contacts devoid of presynapse (S−), normalized to the intensity at A+ presynapses. Significance signs on graphs compare to the value for the same labeling in A+ presynapses (normalized to 1.0).