Journal: Science Advances

Article Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

doi: 10.1126/sciadv.aaz7651

Figure Lengend Snippet: ( A ) SDS-PAGE of recombinantly expressed human Arp2/3 complex. ( B ) Sequence alignment of human and budding yeast Arp2 and Arp3, showing the three amino acids mutated to generate the diCys mutant used in the cross-linking assay by analogy with the yeast complex . ( C ) Illustration of the cross-linking assay; Arp2 L199 and Arp3 L115 (both mutated to cysteine residues) are within BMOE cross-linking distance (~8 Å) only in the short-pitch conformation, in which Arp2 is moved alongside Arp3, analogous to two adjacent subunits of the actin filament. The C408A mutation in Arp3 was introduced to prevent spurious cross-linking. ( D ) Time course of actin polymerization by diCys Arp2/3 complex mutant and tissue-purified bovine Arp2/3 complex over a range of concentrations ( n = 3, per concentration). ( E ) Western blot analysis (immunoblotting: Arp3) and densitometric quantification ( n = 3) of the cross-linked fraction of diCys Arp2/3 complex in the absence of NPFs and with either WCA or actin-GCA and different BMOE treatment times (as indicated). Data were fit to a first-order exponential function to obtain the kinetic parameters.

Article Snippet: Samples were loaded onto 4 to 15% SDS-PAGE gradient gels (Bio-Rad), transferred onto nitrocellulose membranes (Criterion Blotter, Bio-Rad), blocked with Odyssey Blocking Buffer (LI-COR Biosciences), and probed with anti-Arp2 (1:1000 dilution, Cell Signaling Technologies) and anti-Arp3 (1:200 dilution Santa Cruz Biotechnology) antibodies.

Techniques: SDS Page, Sequencing, Mutagenesis, Purification, Concentration Assay, Western Blot

Journal: Science Advances

Article Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

doi: 10.1126/sciadv.aaz7651

Figure Lengend Snippet: ( A ) SDS-PAGE of diCys Arp2/3 complex with actin-GCA bound to a single, high-affinity site after glycerol gradient fractionation (1:1:1 complex, see also fig. S2, A and B). ( B ) Western blot analysis (IB: Arp3) and densitometric quantification ( n = 3) of the cross-linked fraction of diCys Arp2/3 complex as a function of BMOE treatment time in the 1:1:1 complex or in the presence of a high molar excess (25 μM) of actin-GCA (2:2:1 complex). ( C ) Time course of actin polymerization by diCys Arp2/3 complex (middle) as a function of cross-linking time (left) and in the presence or the absence of N-WASP WCA (as indicated). (Right) Relative polymerization rates were calculated by determining the first derivative of the curve and normalizing the maximum derivative value to that of actin alone ( n = 3). The statistical significance of the measurements was determined using an unpaired two-sided Student’s t test (n.s., P ≥ 0.05; *, P < 0.05; **, P ≤ 0.01).

Article Snippet: Samples were loaded onto 4 to 15% SDS-PAGE gradient gels (Bio-Rad), transferred onto nitrocellulose membranes (Criterion Blotter, Bio-Rad), blocked with Odyssey Blocking Buffer (LI-COR Biosciences), and probed with anti-Arp2 (1:1000 dilution, Cell Signaling Technologies) and anti-Arp3 (1:200 dilution Santa Cruz Biotechnology) antibodies.

Techniques: SDS Page, Fractionation, Western Blot

Journal: Science Advances

Article Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

doi: 10.1126/sciadv.aaz7651

Figure Lengend Snippet: ( A ) Cryo-EM map of human Arp2/3 complex with bound N-WASP CA colored by resolution (as indicated in the side bar). The inset illustrates the presence of side-chain densities in the core region of the map. Arp2 is more mobile and therefore less well defined. ( B ) Two perpendicular orientations of the cryo-EM map fit with the Arp2/3 complex structure (subunits are labeled), showing two CA chains (magenta), one bound to Arp2 (cyan) and ArpC1 (yellow), and the other bound to Arp3 (green). See also movie S1. ( C and D ) Close-up views of the difference map (Arp2-ArpC1 pathway, 3.8 σ; Arp3 pathway, 4.0 σ) showing specific interactions of the C helix, D/E loop, and 498 EWE 500 motif along each CA pathway. Amino acids mutated in this study are highlighted red (see also fig. S6). ( E ) Electrostatic surface representation of the 498 EWE 500 motif-binding pockets in ArpC1 and Arp3. In both cases, the conserved tryptophan of this motif is sandwiched in between a proline and an arginine residue, and positively charged side chains surround the pocket, favoring the interaction with negatively charged amino acids of the A region.

Article Snippet: Samples were loaded onto 4 to 15% SDS-PAGE gradient gels (Bio-Rad), transferred onto nitrocellulose membranes (Criterion Blotter, Bio-Rad), blocked with Odyssey Blocking Buffer (LI-COR Biosciences), and probed with anti-Arp2 (1:1000 dilution, Cell Signaling Technologies) and anti-Arp3 (1:200 dilution Santa Cruz Biotechnology) antibodies.

Techniques: Cryo-EM Sample Prep, Labeling, Binding Assay

Journal: Science Advances

Article Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

doi: 10.1126/sciadv.aaz7651

Figure Lengend Snippet: ( A ) Superimposition of Arp2 (cyan) and Arp3 (green), showing how the orientation of the C helix (magenta) of NPFs is different for the two Arps (numbers in italic designate subdomains 1 to 4). The inset shows that the hinge helix is one helical turn longer in Arp3 than in Arp2, which would create a clash with the C helix if it were to bind to Arp3 the way it binds to Arp2. ( B ) The interaction of the C helix with Arp2 (top row) and Arp3 (bottom row) differs from those of the WH2 domain (left column, yellow) and GAB domain (middle column, yellow) with actin (blue) and from the crystal contact made by the protrusion helix of ArpC1 with Arp3 in the structure of inactive Arp2/3 complex (right column, yellow). ( C ) The N-terminal end of the C helix on Arp3 clashes with the C-terminal tail of Arp3, which would have to move for the C helix to bind. The inset (right) shows a close-up view of this clash, with the cryo-EM map contoured at 6.6 σ.

Article Snippet: Samples were loaded onto 4 to 15% SDS-PAGE gradient gels (Bio-Rad), transferred onto nitrocellulose membranes (Criterion Blotter, Bio-Rad), blocked with Odyssey Blocking Buffer (LI-COR Biosciences), and probed with anti-Arp2 (1:1000 dilution, Cell Signaling Technologies) and anti-Arp3 (1:200 dilution Santa Cruz Biotechnology) antibodies.

Techniques: Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

doi: 10.1126/sciadv.aaz7651

Figure Lengend Snippet: ( A ) Description of structure-inspired Arp2/3 complex mutants M1 to M5 targeting the two CA binding pathways (color-coded, see also fig. S6). These mutants were generated in the background of the diCys mutant (called WT in this figure for simplicity) used in cross-linking assays and whose activity is indistinguishable from that of tissue-purified bovine Arp2/3 complex . ( B and C ) Cross-linking of Arp2/3 complex (1 μM) mutants targeting the Arp2-ArpC1 and Arp3 pathways (color-coded as indicated) as a function of BMOE treatment time and in the presence of actin-GCA (2.5 μM), expressed relative to the spontaneous cross-linking of diCys Arp2/3 complex in the absence of actin-GCA (black trace). The data were fit to a first-order exponential function. ( D ) (Left) Time course of actin polymerization of Arp2/3 complex mutants (as indicated) under the conditions listed above the figure. (Right) Relative polymerization rates calculated by determining the first derivative of the curve and normalizing the maximum derivative value to that of actin alone ( n = 3).

Article Snippet: Samples were loaded onto 4 to 15% SDS-PAGE gradient gels (Bio-Rad), transferred onto nitrocellulose membranes (Criterion Blotter, Bio-Rad), blocked with Odyssey Blocking Buffer (LI-COR Biosciences), and probed with anti-Arp2 (1:1000 dilution, Cell Signaling Technologies) and anti-Arp3 (1:200 dilution Santa Cruz Biotechnology) antibodies.

Techniques: Binding Assay, Generated, Mutagenesis, Activity Assay, Purification

Journal: Science Advances

Article Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

doi: 10.1126/sciadv.aaz7651

Figure Lengend Snippet: ( A ) (Left) Design of the dimeric N-WASP WWCA construct (see also fig. S9B). (Right) Schematic illustration of the hypothetical delivery of an actin subunit at the barbed end of Arp3 in mutant M5 (with a disrupted 498 EWE 500 motif-binding pocket in Arp3) through GST-mediated NPF dimerization, which cannot be accomplished with monomeric NPFs. ( B ) (Left) Time course of actin polymerization by Arp2/3 complex (WT and M5) in the presence of N-WASP constructs WCA, WWCA, and GST-WWCA. (Right) Relative polymerization rates calculated by determining the first derivative of the curve and normalizing the maximum derivative value to that of actin alone ( n = 3). The statistical significance of the measurements was determined using an unpaired two-sided Student’s t test (n.s., P ≥ 0.05; *** P < 0.001). ( F ) Model of Arp2/3 complex activation and branch formation steps (left to right) by clusters of NPFs at membranes.

Article Snippet: Samples were loaded onto 4 to 15% SDS-PAGE gradient gels (Bio-Rad), transferred onto nitrocellulose membranes (Criterion Blotter, Bio-Rad), blocked with Odyssey Blocking Buffer (LI-COR Biosciences), and probed with anti-Arp2 (1:1000 dilution, Cell Signaling Technologies) and anti-Arp3 (1:200 dilution Santa Cruz Biotechnology) antibodies.

Techniques: Construct, Mutagenesis, Binding Assay, Activation Assay