Our studies reveal that Daam1 physically interacts with Piccolo and is present at synapses. Moreover, similar to loss of Piccolo andProfilin2, Daam1 loss of function impairs Alizarin presynaptic F-actin assembly, suggesting that within AZs Piccolo/Profilin/Daam1could serve as a regulated nucleation site for F-actin assembly. To identify more precisely the component of Phalloidin labeling that is dependent upon Piccolo and Daam1, we established an imaging assay based on the acute addition of beads to live cells. To monitor F-actin in these experiments we expressed the F-actin binding domain from Utrophin tagged with RFP. Here, we observed low levels of RFP-Utrophin CHD around CD4-EGFP or CD4-EGFP-Pclo1980-2553 positive beads when expressed with Myc-C-Daam1.While the reappeared to be higher levels of RFP-Utrophin CHD around beads in cells expressing CD4-EGFP-Pclo1980-2553, we sought to increase the specificity of the assay. To achieve this we acutely treated cells with a low concentration of Latrunculin A to attenuate the assembly of F-actin. This dramatically abolished the accumulation of RFP-Utrophin CHD near beads dropped onto CD4-EGFP/ Myc-C-Daam1 transfected cells, while prominent labeling remained at CD4-EGFP-Pclo19802553/Myc-C-Daam1beadclusters. This is consistent with a higher kinetic assembly rate of F-actin surrounding CD4-EGFP-Pclo1980-2553/Myc-C-Daam1 beads over coming the partial inhibition by the low concentration of Latrunculin-A. Dynamic assembly of F-actin has an established role in Peramivir Trihydrate release of neuro transmitter, synaptic vesicle poolsize, and the activity dependent recycling of SVs, but the molecular mechanisms guiding temporal and spatial patterns of dynamic assembly of F-actin at the presynaptic bouton have been unclear. In this study, we provide evidence that Piccolo binds Daam1,a processive Formin, and that this interaction can lead to localized dynamic assembly of F-actin. Coupled with previous findings demonstrating interactions between Piccolo and other proteins involved in Actin regulation including Profilin2,GIT1,Epac2, and Abp1,thisworksupports a model in which a molecular complex centered around Piccolo directs the formation of Actin filaments from the active zone and provides a mechanisms for regulating synaptic transmission through Actin dynamics.