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Mechanisms of actin filament severing and elongation by formins

Nature volume 632, pages 437–442 (2024)Cite this article

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Abstract

Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction1,2. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. Whereas formins are known for their ability to nucleate and elongate F-actin3,4,5,6,7, some formins can additionally depolymerize, sever or bundle F-actin. Two mammalian formins, inverted formin 2 (INF2) and diaphanous 1 (DIA1, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly8,9,10,11 whereas DIA1 has potent elongation activity but does not sever4,8. Using cryo-electron microscopy (cryo-EM) we show five structural states of INF2 and two of DIA1 bound to the middle and barbed end of F-actin. INF2 and DIA1 bind differently to these sites, consistent with their distinct activities. The formin-homology 2 and Wiskott–Aldrich syndrome protein-homology 2 (FH2 and WH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.

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Fig. 1: Cryo-EM structures of INF2 and DIA1 bound to F-actin.
Fig. 2: Hand-over-hand severing mechanism.
Fig. 3: Load, exchange, release and advance elongation mechanism.

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Data availability

Molecular models and cryo-EM density maps have been deposited with the following accession codes: INF2 middle up (PDB code 9B03; EMDB codes EMD-44026, EMD-44020, EMD-44022, EMD-44023, EMD-44024, EMD-44025, EMD-44948); INF2 middle down (PDB code 9B0K; EMDB codes EMD-44045, EMD-44027, EMD-44030, EMD-44031, EMD-44032, EMD-44033, EMD-44943); DIA1 middle (PDB code 9B3D; EMDB codes EMD-44135); INF2 barbed end (PDB code 9AZ4; EMDB codes EMD-44012, EMD-44009, EMD-44010, EMD-44011, EMD-44950, EMD-44951); DIA1 barbed end (PDB code 9B27; EMDB code EMD-44099); INF2 with incoming profilin-actin (PDB code 9AZP; EMDB codes EMD-44018, EMD-44956, EMD-44958); INF2 with incoming actin (PDB code 9AZQ; EMDB codes EMD-44019, EMD-44973, EMD-44972); previously published structures used in model building (PDB codes 2PAV and 8F8P).

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Acknowledgements

We thank H. Higgs for providing the INF2 and Diaph1 genes and for valuable discussions. This study was supported by National Institutes of Health grant nos. R01 GM073791 and RM1 GM136511 to R.D. and T32 AR053461 to N.J.P. Data collection was supported by the Electron Microscopy Resource Lab and The Beckman Center for Cryo-Electron Microscopy, University of Pennsylvania (Research Resource Identifier SCR_022375) and the National Center for CryoEM Access and Training through National Institutes of Health grant no. U24 GM129539.

Author information

Authors and Affiliations

  1. Department of Physiology and Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Nicholas J. Palmer, Kyle R. Barrie & Roberto Dominguez

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  1. Nicholas J. Palmer
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  2. Kyle R. Barrie
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Contributions

N.J.P., K.R.B. and R.D. conceived and designed the study. N.J.P. performed sample preparation and cryo-EM data collection. N.J.P. and K.R.B. processed the cryo-EM data and generated three-dimensional reconstructions. N.J.P., K.R.B. and R.D. conducted structural analyses and interpretations. N.J.P., K.R.B. and R.D. prepared figures and videos. R.D. wrote the initial draft, acquired funding and supervised the work. N.J.P., K.R.B. and R.D. edited the final manuscript.

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Correspondence to Roberto Dominguez.

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Nature thanks Edward Egelman and Thomas Pollard for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Definitions and nomenclatures used.

(a) Domain diagrams of INF2 and Dia1, with zooms defining sub-domains within the FH2 domain. (b) The FH2 dimer forms a ring-like structure mediated by head-to-tail lasso-post interactions (left). FH2 subdomains colored as in part a (right). The newly-defined HCBH is highlighted in red. (c) Actin filament (PDB code: 8F8P) indicating short and long-pitch helices, pointed and barbed ends, plug, H-cleft, and D-loop. (d) Two perpendicular views of the actin monomer (PDB code: 1J6Z) with subdomains 1–4 labeled in blue circles. The H-cleft, nucleotide cleft, D-loop, and plug are indicated. (e) G- (blue, twisted) to F-actin (gray, flat) conformational transition, characterized by a 20° rotation of subdomains 1 and 2 relative to 3 and 4.

Extended Data Fig. 2 Cryo-EM maps around FH2 and HCBH.

(a-g) FH2 fit to the cryo-EM maps for the seven structures described here (as indicated). (h) HCBH of the mobile FH2 of INF2 fit to the cryo-EM map in the up and down mid-filament-bound states.

Extended Data Fig. 3 F-actin-FH2 contact surface areas.

(a-g) For each interaction, the contact area in F-actin is colored according to the interacting FH2. On the FH2 side, contact areas are colored cyan, marine blue, blue, and white depending on which actin subunit it contacts. Contact surface areas (given for each interaction in black boxes) were calculated using the server GetArea (https://curie.utmb.edu/getarea.html).

Extended Data Fig. 4 Comparisons between INF2 and Dia1.

(a) Sequence alignment of WASP WH2 (UniProt code: P42768) with the WH2s of INF2 and Dia1. Note the absence of the LKKT motif in Dia1. (b) Superimposition of AlphaFold models of the FH2-WH2 regions of INF2 (632–991) and Dia1 (833–1200), highlighting differences in the length of the antennas and WH2-FH2 linkers. (c-d) Comparisons of the positions of the FH2s of INF2 (magenta) and Dia1 (orange) bound to A5 and A6 at the barbed end. The comparison is based on a superimposition of the corresponding actin subunits in the two structures.

Extended Data Fig. 5 Long-pitch interactions.

(a-c) Long-pitch interactions and corresponding cryo-EM maps of subdomain 2 in F-actin (a), incoming profilin-actin (b) and incoming actin (c). In all the structures, the D-loop inserts into the H-cleft of the subunit above. However, for the incoming monomer (blue), subdomain 4 contacts subdomain 3 of A5 only after a 14° rotation and the G- to F-actin transition (see also Fig. 3e,f in the main text).

Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics for INF2
Full size table
Extended Data Table 2 Cryo-EM data collection, refinement, and validation statistics for Dia1
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–14.

Reporting Summary

Supplementary Video 1

Structures of INF2 and Dia1 in the middle of F-actin. Comparison of the structures of INF2 and Dia1 bound in the middle of F-actin, highlighting differences in position between the up and down states of INF2 with Dia1 in the middle.

Supplementary Video 2

Proposed hand-over-hand mechanism of F-actin severing by INF2. The video shows cryo-EM maps and models of proposed steps of the severing mechanism (see also Fig. 2 in main text).

Supplementary Video 3

Structures of INF2 and Dia1 at the barbed end. Comparison of the structures of INF2 and Dia1 at the barbed end, highlighting differences in position between their FH2s.

Supplementary Video 4

Proposed LERA F-actin elongation mechanism by formins. The video shows cryo-EM maps and models of proposed steps of the elongation mechanism (see also Fig. 3 in main text).

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Palmer, N.J., Barrie, K.R. & Dominguez, R. Mechanisms of actin filament severing and elongation by formins. Nature 632, 437–442 (2024). https://doi.org/10.1038/s41586-024-07637-0

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