Daam1 is required for activity dependent assembly of presynaptic F-actin.

<p>(<b><i>A</i></b>) Western blot analysis of extracts (5 μg total protein) from cultured hippocampal neurons (DIV15) infected with lentiviral vectors expressing EGFP-Actin alone (control) or with the indicated shRNA targeting Daam1 (sh428, sh880, or sh1272). Antibodies against Daam1 confirm that the shRNAs markedly reduce expression of the protein relative to a control protein (tubulin). Analysis of neomycin expression from the plasmid confirms similar levels of transfection efficiencies. <b>(<i>B</i>)</b> Live cell fluorescent images of cultured hippocampal neurons (14DIV), infected with lentiviral vectors expressing EGFP-Actin (green) alone or with shRNA against Piccolo (Pclo28) or Daam1 (sh880 or sh1272). Cells were stimulated with 90mM KCl for 60 sec in the presence of FM4-64 (red) to promote the assembly of presynaptic F-actin and detect presynaptic sites capable of recycling their synaptic vesicles pools. EGFP-Actin expressed alone (top panels) readily accumulates at FM4-64 dye uptake sites, while only modest accumulation is observed in neurons lacking Piccolo (Pclo28 labeled panels) or Daam1 (sh880, sh1272 labeled panels). Arrows indicate examples of co-localization of EGFP-Actin and FM4-64 positive puncta post stimulation. <b>(<i>C</i>)</b> Quantitation of the change in EGFP-Actin fluorescence at the presynaptic boutons measured pre-and post stimulation at the FM dye uptake sites in presence of Piccolo (TS28) or Daam1 (sh1272, sh880) shRNAs demonstrates a marked decrease in Actin clustering with stimulation when either protein is targeted. Data are expressed as mean with error bars representing standard deviation. For statistical analysis, comparison was made between the control and the specific shRNA samples using a two-tailed t-test (*** p<0.001).