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. 2020 Dec 22;11(1):6437.
doi: 10.1038/s41467-020-20286-x.

Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction

Florian Fäßler  1 , Georgi Dimchev  1 , Victor-Valentin Hodirnau  1 , William Wan  2 , Florian K M Schur  3
Affiliations

Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction

Florian Fäßler et al. Nat Commun. .

Abstract

The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Subnanometer structure of the actin filament Arp2/3 complex branch junction in cells.
a Isosurface representation of the actin filament Arp2/3 complex branch junction in cells at 9 Å resolution. The structure is shown from three orientations. A guide for orientation is given in b. b The electron microscopy density map (shown transparent) with the flexibly fitted models of the Arp2/3 complex subunits and actin filaments. Schematic guides indicate the positions of the individual subunits of the complex, and the actin subunits of both the mother (M#) and daughter filaments (D#). Subunit colors are annotated in the figure with Arp2 being red, Arp3 orange, ArpC1 green, ArpC2 light blue, ArpC3 violet, ArpC4 dark blue, and ArpC5 yellow. Actin is shown in gray. The color code and legend are used throughout the manuscript to aid the reader.
Fig. 2
Fig. 2. Comparison of Arp2/3 complex in its inactive conformation and in the branch junction in cells.
a Molecular models of the Arp2/3 complex in the inactive (derived from pdb 1TYQ) and the active conformation shown as density maps filtered to 9.5 Å resolution. The models are shown from three orientations, corresponding to the views in Fig. 1. Subunit colors are annotated in the figure with Arp2 being red, Arp3 orange, ArpC1 green, ArpC2 light blue, ArpC3 violet, ArpC4 dark blue, and ArpC5 yellow. Actin is shown in gray. b RMSD values (in Å) calculated between the inactive and active conformations of the individual Arp2/3 subunits (based on the models used in a). Rows indicate which subunit was used for aligning the full models against each other, prior to measurements between individual subunits of the inactive and active Arp2/3 complexes (indicated in the columns). The RMSD analysis reveals that the structural transition upon complex activation is accommodated by two subcomplexes consisting of Arp2, ArpC1, ArpC4 and ArpC5 and Arp3, ArpC2 and ArpC3, respectively, that rotate against each other along an axis formed by the large helices of ArpC2 and ArpC4 (Supplementary Movie 4). RMSD variations between the same subunits in the inactive and active conformations derive from changes upon MD-based modeling of the x-ray crystal structure-derived model into the electron microscopy density map of the branch junction.
Fig. 3
Fig. 3. Actin–Arp2/3 complex interaction surfaces within the branch junction.
a Interaction surfaces between the Arp2/3 complex, and the actin mother and daughter filament. The surfaces for the Arp2/3 complex, mother and daughter filament are shown as density maps at 9.5 Å resolution generated from their respective models. Gray coloring on the surface of Arp2/3 subunits indicates contact sites with actin and coloring of actin subunits in a specific color indicates a contact site with the associated Arp2/3 subunit. Subunit colors are annotated in the figure with Arp2 being red, Arp3 orange, ArpC1 green, ArpC2 light blue, ArpC3 violet, ArpC4 dark blue, and ArpC5 yellow. Actin is shown in gray. The numbering of the mother (M#) and daughter (D#) filament subunits is also indicated. Coloring was applied via the color zone command in ChimeraX in a 5.5 Å radius around each C-alpha atom of the underlying model, which was positioned in a 10 Å radius to a C-alpha atom of its putative interactor. b Surface representation of the interaction of ArpC2 and ArpC4 with the mother filament subunits M6 and M7 (the interaction of ArpC2 with M5 is omitted for clarity). c The protrusion helix of ArpC1 fitted into its density close to subdomains 1 and 3 of M4. d Surface representation of the interactions of Arp3 and ArpC3 with the mother filament. Note the cavity below Arp2, where no contacts between the Arp2 subunit and the mother filament are observed. ArpC3 acts as a linker between Arp2 and the mother filament. e Surface representation of the interaction between Arp2, Arp3, and the first two subunits of the daughter filament.
Fig. 4
Fig. 4. Structural changes in Arp3 and ArpC5 upon branch junction formation.
a Comparison of the conformation of the Arp3 C-terminal tail in the inactive and active conformation. No density is observed for the C-terminal tail of Arp3 in its inactive conformation (top). Instead, the C-terminal tail can flip toward Arp2 and ArpC4, where it is accommodated by an empty density present in the branch junction structure (bottom). b No density is observed for the ArpC5 N-terminus at its binding side in the inactive complex (top). Instead, the ArpC5 N-terminus can be fitted into a density between ArpC1 and Arp2 (bottom). c Positively charged residues in ArpC1 and Arp2 could coordinate the negatively charged N-terminus of ArpC5. d Electrostatic potential map of the area shown in c, with blue color indicating positive and red color indicating negative potentials. Subunit colors are annotated in the figure with Arp2 being red, Arp3 orange, ArpC1 green, ArpC2 light blue, ArpC3 violet, ArpC4 dark blue, and ArpC5 yellow. Actin is shown in gray.
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