Abstract
Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio parahaemolyticus VopL depends on its three W domains and on its dimerization through a unique VopL C-terminal domain (VCD). The VCD shows a previously unknown all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.
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Acknowledgements
Supported by US National Institutes of Health (NIH) grant R01 GM073791 to R.D. and R01 GM079265 to D.R.K. We thank N. González-Escalona (US Food and Drug Administration) for providing the V. parahaemolyticus genomic DNA and T. Irving and L. Guo for assistance during SAXS data collection at the BioCAT beamline. BioCAT research is supported by NIH grant RR-08630 and work at the Advanced Photon Source at Argonne National Laboratory by US Department of Energy contract W-31-109-Eng-38. Crystal data collection at CHESS was supported by National Science Foundation grant DMR-0936384 and NIH grant RR-01646.
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S.N. expressed proteins, did the sedimentation, crystallization, structure determination and SAXS experiments, and participated in most of the other experiments; M.B. carried out pyrene actin polymerization assays; G.R. did ITC and SEC-MALS experiments; M.J.G., J.D.W. and D.R.K. conducted and analyzed TIRF experiments; R.D. did the crystallographic studies, supervised the research and wrote the paper.
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Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Tables 1 and 2. (PDF 2942 kb)
Supplementary Video 1
Visualization by TIRF microscopy of actin assembly. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin alone (33% Oregon Green-labeled). Video corresponds to Fig. 1e (top panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 522 kb)
Supplementary Video 2
Visualization by TIRF microscopy of actin assembly induced by P-3W-VCD.Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCD. Video corresponds to Fig. 1e (second panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 449 kb)
Supplementary Video 3
Visualization by TIRF microscopy of actin assembly induced by 3W-VCD. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCD. Video corresponds to Fig. 1e (third panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 174 kb)
Supplementary Video 4
Visualization by TIRF microscopy of actin assembly induced by VCD. Timelapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct VCD. Video corresponds to Fig. 1e (bottom panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 539 kb)
Supplementary Video 5
Visualization by TIRF microscopy of actin assembly induced by immobilized P-3W-VCD (corresponding to Fig. 2f top panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) on a coverslip pre-coated with VopL construct P-3W-VCD. Video corresponds to Fig. 2f (top). A yellow dot marks the immobilized pointed end, and a yellow arrow marks the growing barbed end of one of the filaments that was initially anchored to the coverslip by its pointed end, but then dissociated. Images were captured every 10 s for 5 min, and are displayed at 10 frames s−1. Filaments were considered tethered to the coverslip if the position of one of the ends of the filament did not change for at least five frames. (MOV 632 kb)
Supplementary Video 6
Visualization by TIRF microscopy of actin assembly induced by immobilized P-3W-VCD (corresponding to Fig. 2f bottom panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) on a coverslip pre-coated with VopL construct P-3WVCD, corresponding to Fig. 2f (bottom). A yellow dot marks the immobilized pointed end, and a yellow arrow marks the growing barbed end of one of the filaments that remained anchored to the coverslip by its pointed end. Images were captured every 10 s for 5 min, and are displayed at 10 frames s−1. Filaments were considered tethered to the coverslip if the position of one of the ends of the filament did not change for at least five frames. (MOV 259 kb)
Supplementary Video 7
Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g top panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows a Qdot that nucleates the assembly of 15 actin filaments (marked with arrowheads and numbered). (MOV 1299 kb)
Supplementary Video 8
Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g top panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows another example of a Qdot that nucleates the assembly of multiple (8) actin filaments (marked with arrowheads and numbered). (MOV 652 kb)
Supplementary Video 9
Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g middle panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Video corresponds to Fig. 2g. Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows Qdots that nucleate the assembly of actin filaments that remain attached through their pointed ends (marked by circles) after nucleation and whose barbed ends (marked by arrows) elongate freely. (MOV 1277 kb)
Supplementary Video 10
Visualization by TIRF microscopy of actin assembly induced by Qdot-coupled 3W-VCD (corresponding to Fig. 2g bottom panels). Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM 3W-VCD coupled with Qdot-625 (red). Images were captured every 2 s for 5 min, and are displayed at 10 frames s−1. The movie shows a Qdot that nucleates the assembly of an actin filament that remains attached through its barbed and displays accelerated barbed end elongation. The filament buckles similarly to filaments elongated from immobilized formin. The arrow and circle indicate the barbed and pointed ends, respectively. (MOV 1632 kb)
Supplementary Video 11
Animation of the crystal structure of VCD. The video shows ribbon and surface representations colored according to three different criteria: domain organization (core, magenta and green; arm, pink; coiled coil, gold), electrostatic charge distribution (blue, positively charged to red, negatively charged), and temperature factor (B-factor) values (blue, lowest to red, highest). (MOV 5233 kb)
Supplementary Video 12
Visualization by TIRF microscopy of actin assembly in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-Actin (33% Oregon Greenlabeled) with 2.5 μM profilin. Video corresponds to Fig. 4b (top panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 318 kb)
Supplementary Video 13
Visualization by TIRF microscopy of actin assembly induced by P-3W-VCD in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCD and 2.5 μM profilin. Video corresponds to Fig. 4b (second panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 498 kb)
Supplementary Video 14
Visualization by TIRF microscopy of actin assembly induced by 3W-VCD in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCD and 2.5 μM profilin. Video corresponds to Fig. 4b (third panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 309 kb)
Supplementary Video 15
Visualization by TIRF microscopy of actin assembly induced by P-3WVCDP191E without profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCDP191E in the absence (control) of profilin. A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 405 kb)
Supplementary Video 16
Visualization by TIRF microscopy of actin assembly induced by P-3WVCDP191E in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATPactin (33% Oregon Green-labeled) with 0.1 nM VopL construct P-3W-VCDP191E in the presence of 2.5 μM profilin. Video corresponds to Fig. 4b (fourth panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 665 kb)
Supplementary Video 17
Visualization by TIRF microscopy of actin assembly induced by 3W-VCDP191E without profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCDP191E in the absence of profilin. A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 608 kb)
Supplementary Video 18
Visualization by TIRF microscopy of actin assembly induced by 3W-VCDP191E in the presence of profilin. Time-lapse TIRF microscopy of the assembly of 1.5 μM Mg-ATP-actin (33% Oregon Green-labeled) with 0.1 nM VopL construct 3W-VCDP191E in the presence of 2.5 μM profilin. Video corresponds to Fig. 4b (fifth panel). A yellow dot marks the pointed end, and a yellow arrow marks the growing barbed end of one of the filaments. Images were captured every 10 s for 10 min, and are displayed at 10 frames s−1. (MOV 649 kb)
Supplementary Video 19
Model of nucleation by VopL. Proposed assembly of actin subunits with VCD at the pointed end of the polymerization nucleus. The video shows ribbon and electrostatic surface representations (blue, positively charged to red, negatively charged). The two chains of VCD are colored green and yellow and the first W-actin complex at the pointed end of the polymerization nucleus is colored blue and red. Note the existing shape and charge complementarity between VCD and the first W-actin complex in the proposed model of interaction. A second W-actin complex, positioned with respect to the first one according to the structure of the actin filament, is show (actin, gray; W domain, red). For the second W-actin complex, the distance between the C-terminus of the W domain and the N13 terminus of VCD is ~40 Å. Not shown in this model are 12 amino acids of the flexible linker between the third W domain and VCD, which with rearrangement of the termini would be sufficient to bridge thedistance between the W domain and VCD. (MOV 6388 kb)
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Namgoong, S., Boczkowska, M., Glista, M. et al. Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation. Nat Struct Mol Biol 18, 1060–1067 (2011). https://doi.org/10.1038/nsmb.2109
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DOI: https://doi.org/10.1038/nsmb.2109
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