Abstract
The Arp2/3 complex consists of seven evolutionarily conserved subunits (Arp2, Arp3 and ARPC1–5) and plays an essential role in generating branched actin filament networks during many different cellular processes. In mammals, however, the ARPC1 and ARPC5 subunits are each encoded by two isoforms that are 67% identical. This raises the possibility that Arp2/3 complexes with different properties may exist. We found that Arp2/3 complexes containing ARPC1B and ARPC5L are significantly better at promoting actin assembly than those with ARPC1A and ARPC5, both in cells and in vitro. Branched actin networks induced by complexes containing ARPC1B or ARPC5L are also disassembled ∼2-fold slower than those formed by their counterparts. This difference reflects the ability of cortactin to stabilize ARPC1B- and ARPC5L- but not ARPC1A- and ARPC5-containing complexes against coronin-mediated disassembly. Our observations demonstrate that the Arp2/3 complex in higher eukaryotes is actually a family of complexes with different properties.
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Acknowledgements
We would like to thank members of the Way laboratory, R. Treisman (The Francis Crick Institute) and H. Walden (University of Dundee) for comments on the manuscript, M. Howell (The Francis Crick Institute) for his helpful discussions, J. Cockburn (University of Leeds) for his structural insights into Arp2/3, R. George (The Francis Crick Institute) for his help with Arp2/3 purification, and B. Wharam for his contribution to this project during his summer studentship. We would like to thank J. Bear (University of North Carolina, Chapel Hill, USA) for coronin antibodies, I. Berger (University of Bristol, UK) for the pFL vector, and E. Benanti and M. Welch (University of California, Berkeley, USA) for helpful discussions concerning production of recombinant Arp2/3. This work was supported by Cancer Research UK (C.G. and M.W.) and by postdoctoral fellowships from FRQS (Fonds de recherche du Québec - Santé), EMBO and the Canadian Institutes of Health Research (CIHR) to J.V.G.A. M.-F.C. was supported by ERC advanced grant no. 249982 and FP7 241548.
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J.V.G.A. and M.W. designed the study and wrote the manuscript. J.V.G.A. performed and analysed the experiments with help from C.G. S.K. purified the recombinant Arp2/3 complexes. D.J.B. provided quantitative analysis of protein localizations in actin tails. J.P. performed the in vitro actin assays with input from M.-F.C. All authors discussed the results and commented on the manuscript.
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Supplementary Figure 1 HeLa cells express both isoforms of ARPC1 and ARPC5.
(A) Sequence alignment of human ARPC5 (NP_005708.1) and ARPC5L (NP_112240.1) (top) and ARPC1A (NP_ 006400.2) and ARPC1B (NP_005711.1) (bottom). Pink lines denote residues that are not present in the bovine crystal structure (PDB 1K8K), green line denotes residues involved in the interface between ARPC5 and Arp2. (B) Isoform specific antibodies do not cross react with the opposite endogenous (upper panels) or GFP-tagged (lower panels) ARPC1 or ARPC5 isoforms in HeLa cells. (C) The graphs show the relative abundance of the different ARPC1 or ARPC5 isoforms in a panel of cell lines normalized to their own ARPC2 levels relative to HeLa cells. Error bars represent s.e.m. from n = 3 independent experiments. (D) Immunoblot analysis of HeLa cells treated with siRNA against ARPC2 results in concomitant loss of all other complex subunits.
Supplementary Figure 2 ARPC1 and ARPC5 isoforms differentially affect actin tail lengths.
(a) Immunofluorescent images from which insets in Fig. 2b were taken. An image of a HeLa cell treated with siRNA against ARPC2 is also included. The virus is labelled in green and actin (phalloidin) in red. Scale bar is 10 μm. (B) Quantification of actin tail lengths of individual siRNA oligonucleotides from the siGenome pools against ARPC5, ARPC5L, ARPC1A and ARPC1B used in Fig. 2. Data from 3 independent experiments were combined and error bars represent s.e.m. from n = 360 tails. (C) Immunoblot analysis of RNAi treated Lifeact-RFP cells used in Fig. 2d to measure virus speeds. (D) HeLa cells stably expressing GFP-tagged ARPC1 and ARPC5 isoforms were treated with 500 nM Latrunculin A for 20 min before imaging. GFP-tagged isoforms recruited to vaccinia virus (red) were bleached and their recovery was measured (ARPC1B is shown). Scale bar is 5 μm. The graph shows a comparison of the recovery kinetics of photobleached GFP-tagged isoforms. Data from 3 independent experiments were combined and error bars represent s.e.m. for the indicated number of virus (n).
Supplementary Figure 3 Cofilin promotes actin tail disassembly independently of either ARPC5 isoform.
(A) Immunofluorescence images of virus (red) induced actin tails (green) in HeLa cells treated with the indicated siRNA. (B) Quantification of actin tail lengths in cells treated with the indicated siRNA (n = 360 tails). The immunoblot shows the degree of knockdown. Scale bar is 5 μm. Data from 3 independent experiments were combined and error bars represent s.e.m. from n = 360 tails. Tukey’s multiple comparisons test was used to determine Statistical significances, where ∗∗∗P < 0.001. Mean and s.e.m. are indicated below graph.
Supplementary Figure 4 Actin tail intensity profiles.
Average fluorescence intensity of GFP-tagged proteins in virus-induced actins tails in HeLa cells as a function of distance from the virus. Each profile represents the average of individual ‘snapshots’ of tails taken from live cell imaging. The data are fit with either a Gaussian (vaccinia), an exponentially modified Gaussian (lifeact, ArpC2, cortactin, coronin1C), or a sum of the two (N-WASP). The vertical dotted line denotes the position of peak fluorescence intensity of the RFP-tagged viral protein, A3. Error bars represent 95% confidence intervals with ‘snapshots’ of Vaccinia Virus (n = 4,050), N-WASP (n = 4,342), Cortactin (n = 7,558), ARPC2 (n = 2,043), LifeAct (nt = 1,314) and Coronin 1C (nt = 2,302).
Supplementary Figure 5 Localisation of cortactin is not dependent on Arp2/3 complex isoforms.
(A–C) Immunoblot analysis of HeLa cells stably expresssing (A) photoactivatable β-actin, (B) photoactivatable ARPC2 or (C) photoactivable Cortactin and treated with the indicated siRNAs for 72 h and probed with the indicated antibodies. (D) Average fluorescence intensity of the Cortactin-GFP in virus-induced actins tails as a function of distance from the virus in cells treated with the indicated siRNAs. Each profile is generated by analysing 900–2,100 ‘snapshots’ of the indicated number of tails in live imaging of the indicated number of cells. The data are fit with exponentially modified Gaussian functions. Error bars represent 95% confidence intervals with cortactin-GFP ‘snapshots’ for Control (n = 2,147), siC5-C1A (n = 1,100) and siC5L-C1B (n = 935).
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Actin based motility of vaccinia in cells lacking ARPC5 or ARPC5L.
The panel of three movies represent HeLa cells stably expressing Lifeact-RFP, infected with Vaccinia expressing YFP-A3 (a core viral protein) for 8 h, post siRNA transfection with either control or siRNA against ARPC5 or ARPC5L for 72 h. Movies correspond to 1 min at 1 s intervals. Movies are representative of the data presented in Fig. 2d. (MOV 382 kb)
Actin based motility of vaccinia in cells lacking ARPC1A or ARPC1B.
The panel of three movies represent HeLa cells stably expressing Lifeact-RFP, infected with Vaccinia expressing YFP-A3 (a core viral protein) for 8 h, post siRNA transfection with either control or siRNA against ARPC1A or ARPC1B for 72 h. Movies correspond to 1 min at 1 s intervals. Movies are representative of the data presented in Fig. 2d. (MOV 382 kb)
Photoactivation of actin in a vaccinia actin tail.
The movie shows a representative photoactivation experiment, from which the panels in Fig. 4a were extracted. HeLa cells stably expressing Cherry-GFPPAβ-actin (red before photoactivation) were infected with Vaccinia expressing YFP-A3 for 8 h, post transfection with RNAi against ARPC1A for 72 h. (MOV 651 kb)
Actin based motility of vaccinia in cells lacking Coronin 1B or IC.
The panel of three movies represent HeLa cells stably expressing LifeAct-RFP, infected with Vaccinia expressing YFP-A3 (a core viral protein) for 8 h, post siRNA transfection with either control or siRNA against coronin1B or coronin1C for 72 h. Movies correspond to 1 min at 2.4 s intervals. Movies are representative of the data presented in Fig. 5c. (MOV 437 kb)
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Abella, J., Galloni, C., Pernier, J. et al. Isoform diversity in the Arp2/3 complex determines actin filament dynamics. Nat Cell Biol 18, 76–86 (2016). https://doi.org/10.1038/ncb3286
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DOI: https://doi.org/10.1038/ncb3286
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