Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function - PubMed (original) (raw)

Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function

Adam C Martin et al. J Cell Biol. 2005.

Abstract

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.

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Figures

Figure 1.

Figure 1.

Arp2 and Arp3 residues chosen for mutagenesis. Crystal structure of actin and NBPs of actin, Arp2, and Arp3 (Kabsch et al., 1990; Robinson et al., 2001). Subdomains of actin are indicated with Roman numerals. Because subdomains I and II of Arp2 are not present in the crystal structure, the backbone of actin was substituted. Altered residues, ATP (blue), and Ca2+ (orange) are identified by colors. Residue numbers correspond to the yeast actin peptide sequence.

Figure 2.

Figure 2.

ATP binding of Arp2 and Arp3 mutants. (A) Coomassie staining of purified mutant Arp2/3 complexes. Asterisk indicates Arp3 degradation. (B) ATP cross-linking to purified Arp2/3 complex mutants. Arp2/3 complex subunits were separated by SDS-PAGE and visualized by autoradiography (top) and Coomassie staining (bottom). Asterisks indicate Arp3 degradation. See Table II for quantification. (C and D) Etheno-ATP binding to purified Arp2/3 complex mutants. Data points represent averages ± SEM from two experiments. See Table II for K d values.

Figure 3.

Figure 3.

Actin cytoskeleton and endocytosis defects of Arp2 and Arp3 mutants. (A) Rhodamine phalloidin staining of yeast expressing indicated Arp2 and Arp3 mutants. Cells were grown at 25°C, or shifted to 37°C for 1 h. Arrows indicate depolarized cells. Arrowheads indicate abnormal actin aggregates. (B) Arp2/3 complex localization in Arp2 and Arp3 mutants. Actin and Arp2/3 were visualized by indirect immunofluorescence after growth at 25°C. HA-tagged Arc18 was used to immunolocalize the Arp2/3 complex. Arrowheads indicate examples of colocalization. (C) Analysis of endocytosis by fluorescence microscopy using LY uptake. Cells were incubated with LY for 1 h at 25°C. Quantification is in Table II.

Figure 4.

Figure 4.

Arp2 and Arp3 mutants affect actin patch protein internalization. (A) Tracking of individual cortical Abp1 patches. Abp1-GFP was visualized every 0.25 s and patch movement traces were obtained for the entire life of patches. Green and red dots indicate first and last positions, respectively. Dotted line indicates plasma membrane. (B) Internalization of Sla1 patches. Single frames (left) and kymographs (right) of time-lapse images collected at 0.5-s intervals using a line drawn perpendicular to the plasma membrane (e.g., yellow). Dotted line: plasma membrane. (C) Fluorescence intensity of individual Abp1-GFP patches measured over time. (D) Single frames (left) and time series of single cortical patches (right) of cells expressing Abp1-GFP (red) and Sla1-CFP (green). Time-lapse between images was ∼4.55 s. Average times and SDs between Sla1 appearance and Abp1 recruitment were calculated from at least 20 patches.

Figure 5.

Figure 5.

Arp2 and Arp3 mutants affect the continuity of sla2 Δ actin comet tails. (A) Quantification of actin treadmilling rates using FRAP in _sla2_Δ cells expressing the indicated Arp mutant and Act1-GFP. Average treadmilling rates and SDs were calculated from 10 separate actin tail clusters. (B) Confocal images of whole cells (left) and magnified views of actin comet tail clusters (right). Arrowheads indicate actin clumps. (C) Kymograph of time-lapse images collected at 1-s intervals using a line drawn perpendicular to the plasma membrane (e.g., gray line, B). A section of the actin comet tail cluster (e.g., white box, B) was photo-bleached after 10 s. Arrowheads indicate where the actin network appears to separate from the plasma membrane.

Figure 6.

Figure 6.

Effects of Arp2 and Arp3 mutants on Arp2/3 complex nucleation and branching. (A and F–H) Nucleation activity of Arp2 and Arp3 mutants. 2 μM actin (5% pyrene labeled) was polymerized with the indicated concentration of Arp2/3 complex and, when indicated, 50 nM Las17 or 400 nM WCA. 500 μM ATP was present in nucleation reactions unless otherwise stated. (B and C) Barbed end concentrations at 50% polymerization generated by Arp2/3 (20 nM) and different concentrations of Las17 (B) or with 50 nM Las17 and 7, 57, 107, or 507 μM ATP (C). (D) Branching activity of Arp2 and Arp3 mutants. Actin filaments polymerized with 20 nM mutant Arp2/3 complex (2 nM wild-type Arp2/3) and 200 nM Las17 were visualized with rhodamine phalloidin. Average branching efficiency ± SD was calculated as branch number per micrometer filament length, corrected for differences in nucleation activity. (E) WCA binding to Arp2/3 complex mutants. 40 nM Arp2/3 complex was incubated with various concentrations of Las17 WCA-coated beads and the fraction of bound Arp2/3 was calculated by quantifying Arp3 left in the supernatant by immunoblotting. Data points represent averages ± SEM from three experiments. K d values of 4.5 ± 0.9 μm, 3.9 ± 1.0 μm, and 4.5 ± 1.0 μm were calculated for wild-type, arp2-G302Y, and arp3-G302Y Arp2/3 complexes, respectively.

Figure 7.

Figure 7.

Single-particle reconstructions of Arp2/3 complexes. (A) Docking of the nonactivated bovine Arp2/3 crystal structure (ribbon diagram) into the reconstruction of nonactivated wild-type yeast Arp2/3 complex (chicken-wire representation). Color scheme follows C. The view in the second row is rotated by 90° horizontally from that in the first row; the view in the third row is rotated by 180° horizontally from that in the first row. (B) Three views of the reconstructions (solid blue surface representation) from nonactivated wild-type Arp2/3 complex (WT), nonactivated arp2-Y306A mutant (arp2-Y306A), and activated wild-type Arp2/3 complex (WT-WCA). The views match those in A. Red arrows indicate the region of Arp2 that shows differences between WT and WT-WCA/arp2-Y306A. Gray arrows indicate the differences in the Arc15 region. (C) Solvent exposed surface of the crystal structure; view matches third row in A. (D) Projection views of the three Arp2/3 reconstructions. Red arrows indicate the Arp2 region, highlighting differences seen in B. (E) Fourier shell correlation graphs for the three reconstructions calculated from two randomly selected half sets of each respective data set. The 0.5 criterion indicates ∼2.5 nm resolution for all reconstructions.

Figure 8.

Figure 8.

Model for nucleotide's role in Arp2/3 complex function. (A) Proposed relative contributions of Arp2 and Arp3 nucleotide binding to actin filament nucleation and a yet to be determined structural activity. (B) Proposed steps in an endocytic pathway (Merrifield et al., 2002; Kaksonen et al., 2003) affected by Arp2 and Arp3 nucleotide-binding mutants. Both Arp2 and Arp3 mutants affect the initiation and the rate of actin assembly at endocytic sites (i), but Arp3 mutants more dramatically inhibit internalization of endocytic proteins as a consequence of defects in actin network structure and integrity (ii).

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References

    1. Amann, K.J., and T.D. Pollard. 2001. The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments. Nat. Cell Biol. 3:306–310. - PubMed
    1. Belmont, L.D., and D.G. Drubin. 1998. The yeast V159N actin mutant reveals roles for actin dynamics in vivo. J. Cell Biol. 142:1289–1299. - PMC - PubMed
    1. Belmont, L.D., A. Orlova, D.G. Drubin, and E.H. Egelman. 1999. a. A change in actin conformation associated with filament instability after Pi release. Proc. Natl. Acad. Sci. USA. 96:29–34. - PMC - PubMed
    1. Belmont, L.D., G.M. Patterson, and D.G. Drubin. 1999. b. New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites. J. Cell Sci. 112(Pt 9):1325–1336. - PubMed
    1. Bernheim-Groswasser, A., S. Wiesner, R.M. Golsteyn, M.F. Carlier, and C. Sykes. 2002. The dynamics of actin-based motility depend on surface parameters. Nature. 417:308–311. - PubMed

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