Fig. 3

Candidate screening for functional role in filopodia maturation. a Schematic depicting the depletion and GFP validation vectors for in vitro guide screening. U6 U6 promoter, sgRNA single guide RNA, hSyn human Synapsin I promoter, Cre cre recombinase, β-actin beta actin promoter, GFP green fluorescent protein. b Representative blot for GFP to validate depletion efficiency of sgRNA sequences. c Graphical representation of GFP/β-actin intensity relative to no depletion control (GFP alone) (1.12 ± 0.06 GFP intensity, n=12 HEK cell samples), ArpC3 #1 (0.22 ± 0.02, n=7 samples), ArpC3 #2 (0.41 ± 0.05, n=4 samples), CARMIL3 #1 (0.13 ± 0.02, n=9 samples), CARMIL3 #2 (0.97 ± 0.02, n=3 samples), CARMIL3 #3 (0.10 ± 0.01, n=3 samples), LPPR4 #3 (0.04 ± 0.00, n=6 samples), LRRC7 #3 (0.06 ± 0.01, n=3 samples), and LAP2 #1 (0.02 ± 0.01, n=3 samples). F_8,41=85.192, p<0.0001. d Schematic depicting CRISPR depletion of candidates in cultured hippocampal neurons. e Representative images of dendritic morphology for each group of virally mediated depletions. Scale bars are 5 μm. f Graphical representation of dendritic protrusion density for each group of virally mediated depletions, Control (66 ± 2 protrusions, n=66 neurons), LAP2 (81 ± 2 protrusions, n=22 neurons), LPPR4 (66 ± 2 protrusions, n=27 neurons), LRRC7 (56 ± 2 protrusions, n=30 neurons), CARMIL3 (49 ± 3 protrusions, n=29 neurons). Error bars are standard error of the mean (SEM). F_4,169=24.55, p<0.0001. *p<0.05, ***p<0.001; ****p<0.0001, one-way ANOVAs