ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning

Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.

Figs. S1 to S18 Table S1 Legend for table S2 Legends for movies S1 to S4 Other Supplementary Material for this manuscript includes the following:

Table S2
Movies S1 to S4 Allele 1, C insertion, frame shift starting from residue 12, STOP after residue 19, 18 of 31 reads ATGTCGAAGAACACGGTGTCGTCGGCCCGCTTCCCGGAAGGTGGACGTGGACGAATATGATGAGAACAAGTTCGTGGACGAGGAGGACGG… M S K N T V S S A R F P E G G R G R I STOP Allele 2, C deletion, frame shift starting from residue 12, STOP after residue 50, 13 of 31 reads ATGTCGAAGAACACGGTGTCGTCGGCCCGCTTCGGAAGGTGGACGTGGACGAATATGATGAGAACAAGTTCGTGGACGAGGAGGACGGC…

M S K N T V S S A R F G R W T W T N M M R T S S W T R R T …
RnC5KO #4 Allele 1, C deletion, frame shift starting from residue 12, STOP after residue 50, 30 of 30 reads ATGTCGAAGAACACGGTGTCGTCGGCCCGCTTCGGAAGGTGGACGTGGACGAATATGATGAGAACAAGTTCGTGGACGAGGAGGACGGC… M S K N T V S S A R F G R W T W T N M M R T S S W T R R T … RnC5KO #8 Allele 1, G deletion, frame shift starting from residue 12, STOP after residue 50, 14 of 29 reads ATGTCGAAGAACACGGTGTCGTCGGCCCGCTTCCGAAGGTGGACGTGGACGAATATGATGAGAACAAGTTCGTGGACGAGGAGGACGGC… M S K N T V S S A R F R R W T W T N M M R T S S W T R R T … Allele 2, CT insertion, frame shift starting from residue 12, STOP after residue 50, 15 of 29 reads

Figure S5: Expression levels of ArpC5 and ArpC5L in rescue experiments
Quantification of Western blots detecting ArpC5 and ArpC5L shows that the average abundance of these proteins per cell in the C5/C5LKO #20 background upon overexpression is ~4 times higher than in B16-F1 wildtype cells. GAPDH was used as loading control. Quantification of overexpression is related to data shown in Figure 2. and Rat2 C5KO and C5LKO cells compared to their respective wildtype counterparts. Two versions with different signal amplifications are shown for the ArpC1b and the polyclonal ArpC5/ArpC5L antibody to allow visualization in both cell types on the same membrane. Vinculin was used as loading control. Polyclonal ArpC5/ArpC5L antibody was employed to verify the sample genotypes. Quantification results are given in Table S1.  C5KO cells compared to the respective C5LKO and WT cells. GAPDH was used as loading control. Western blot quantification shown in Table S1. All scale bars, 20µm.

Figure S13: A non-modeled linker region proximal to ArpC1 harbors the largest differences between the primary structures of ArpC5 and ArpC5L
ArpC5 is color-coded according to the sequence homology between ArpC5 and ArpC5L. This reveals that most of the interaction surface with ArpC1 is highly conserved for those proteins. Sequence alignment of a non-modeled linker likely contacting ArpC1 is shown on the right. Residues with positively charged side chains are given in blue, and residues with negatively charged side chains are given in red. The molecular model of the branch junction used for representation is derived from PDB 7TPT (12). Western blot quantification shown in Table S1. and Rat2 C5KO cells is due to reduced recruitment and not due to reductions at the protein level. Two versions with different signal amplifications are shown for the VASP and the polyclonal ArpC5/ArpC5L antibodies to allow visualization in both cell types on the same membrane. Vinculin and GAPDH were used as loading controls. Polyclonal ArpC5/ArpC5L antibody was employed to verify the sample genotypes. Scale bars, 20µm in standard panels and 2µm in enlarged insets, n.s. = not significant. Western blot quantification shown in Table S1

Figure S18: Uncropped Western blots
Chemiluminescence signals and color images of the PVDF membranes are overlaid. Corresponding figures, employed antibodies, and identity of the marker bands are annotated next to and on the blots, respectively. Red rectangles highlight areas shown in the corresponding figures. Yellow rectangles indicate repetitions used for statistical quantification of Western blots.

Table S1: Quantification of Western blots
Average band intensities of 3 independent biological replicates after background subtraction, normalization via loading control and displayed as fraction/multiple of the respective WT control. Standard Errors of the Mean (SEM) are provided for all non-control groups. Reduction of expression below 0.7 times of the respective control is highlighted by ↓. Increase of expression above 1.3 times and 2 times are highlighted by ↑ and ↑↑, respectively.