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. 2012 Mar 23;287(13):10684-10692.
doi: 10.1074/jbc.M111.317792. Epub 2012 Feb 8.

Multiple forms of Spire-actin complexes and their functional consequences

Christine K Chen  1 , Michael R Sawaya  1 , Martin L Phillips  1 , Emil Reisler  2 , Margot E Quinlan  3
Affiliations

Multiple forms of Spire-actin complexes and their functional consequences

Christine K Chen et al. J Biol Chem. .

Abstract

Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

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Figures

FIGURE 1.
FIGURE 1.
Rapid actin filament depolymerization by Spir. A, a co-sedimentation assay of 6 μm Spir-CD added to 6 μm F-actin. Spir-CD can be seen in the supernatant (S) (but not in the pellet (P)) with actin that has mostly depolymerized after a 15-min incubation. Representative EM image of actin incubated with a stoichiometric amount of Spir-CD for 15 min. Some short filaments remain, but the majority of the fields imaged have no F-actin. B, fluorescence curves of 5 μm F-actin (2.5% pyrene labeled actin) upon the addition of Spir-ABCD (concentrations as indicated) show rapid depolymerization. The actin polymerization was monitored for 1200 s before Spir was added. Depolymerization is dose-dependent and is complete above stoichiometric Spir concentrations. Filaments exhibit faster depolymerization in the presence of Spir than in the presence of sequestration agent, LatA.
FIGURE 2.
FIGURE 2.
Spir severs weakly in bulk assays. A, 2.5% pyrene-labeled actin was polymerized for ∼9 min before the addition of either Spir-CD or cofilin. The polymerization rate accelerates strongly after the addition of 0.2 μm cofilin, but only a small rate increase is apparent upon the addition of 0.1 or 0.2 μm Spir-CD. B, weak nucleation is apparent when Spir-CD is added to 1.2 μm G-actin, the amount remaining at the time of addition in A.
FIGURE 3.
FIGURE 3.
Spir severs and depolymerizes actin filaments from the barbed end. A, Cy3b-labeled actin filaments were diluted to 200 nm in TIRF buffer before immobilization in flow cells. Time lapse images were recorded for 3 min immediately after 100 nm Spir-ABCD was added. Severing events were observed, and filaments depolymerized faster at one end. Images at 20-s intervals are shown for one filament. B, polarity marked filaments are grown off of AlexaFluor488-phalloidin-stabilized seeds. The addition of 100 nm Spir-ABCD induced both severing and depolymerization of the barbed ends. C, typical Cy3b-labeled actin filament. Time course of filament changes after the addition of 150 nm Spir-CD. Phalloidin was added after 180 s to stabilize existing filaments, and then 250 nm labeled G-actin was added. Growth at one side of the cut site and one end of the filament confirms the polarity of the filament. D, to mimic the effect of severing combined with sequestering, we mechanically sheared 5 μm F-actin by pipetting several times and then adding LatA. The combination of these treatments led to depolymerization that was much faster than the addition of LatA alone. The addition of stoichiometric Spir-ABCD is shown for comparison.
FIGURE 4.
FIGURE 4.
Spir-ABCD and Spir-CD seeds enhance actin polymerization. Spir-ABCD and Spir-CD seeds, created by incubating stoichiometric ratios of Spir and actin in 2 mm MgCl2 and 50 mm KCl (blue and magenta traces), exhibit faster nucleation of filaments as indicated by the shorter lag times compared with actin alone (black) and actin mixed with either Spir-ABCD or Spir-CD at time 0 (green and red).
FIGURE 5.
FIGURE 5.
Longitudinal and lateral actin contacts induced by Spir. A, yeast actin mutants Q41C and S265C incubated with Spir-ABCD or Spir-CD were monitored for excimer formation under nonpolymerizing and polymerizing conditions. Excimer fluorescence is plotted relative to that of the corresponding F-actin controls (100%). Stoichiometric concentrations of Spir-ABCD and Q41C showed excimer fluorescence under nonpolymerizing conditions. Minimal excimer formation was visible in all other Ca-G-actin cases. Under polymerizing conditions, Spir-ABCD incubated with either Q41C or S265C produced high excimer fluorescence. B, cross-linking of Q41C in the presence of Spir-ABCD or Spir-CD is shown. 5 μm yeast actin mutant Q41C converted to Mg-G-actin was incubated with stoichiometric concentrations of either Spir-ABCD or Spir-CD and the cross-linker MTS1. In the presence of Spir-CD, actin dimer formation is strong compared with the Mg-G-actin (with a trace level of cross-linking) and F-actin (cross-linked into higher order oligomers) controls. In contrast, little cross-linking is evident when Q41C is incubated with Spir-ABCD. C, cross-linking of S265C in the presence of Spir-ABCD or Spir-CD. Conditions were the same as in B. Cross-linking with S265C and Spir-ABCD or Spir-CD both result in higher order oligomers. A band between actin monomer and dimer is detected only for Spir-CD and S265C. We interpret this as cross-linking between Spir and actin. D, cross-linking with Spir-CD and wild type yeast actin. A band above the actin monomers confirms that Spir-CD cross-links to actin Cys-374. A indicates molecular weight consistent with an actin monomer. AA indicates an actin dimer. SA indicates Spir-CD-actin.
FIGURE 6.
FIGURE 6.
Crystal structure of Spir-D (magenta) with ECP-cleaved actin (cyan) forming an anti-parallel dimer. Spir-D is positioned next to the W-loop of actin between subdomains 1 and 3. Subdomains are indicated on the left actin monomer. Part of linker 3 between Spir-C and -D is shown. Spir-C and part of actin subdomain 2 are disordered in the crystal. Actin Cys-374 and Spir Cys-459 (shown as yellow balls) form a disulfide bond, creating an anti-parallel dimer.
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References

    1. Quinlan M. E., Heuser J. E., Kerkhoff E., Mullins R. D. (2005) Drosophila Spire is an actin nucleation factor. Nature 433, 382–388 - PubMed
    1. Ahuja R., Pinyol R., Reichenbach N., Custer L., Klingensmith J., Kessels M. M., Qualmann B. (2007) Cordon-Bleu is an actin nucleation factor and controls neuronal morphology. Cell 131, 337–350 - PMC - PubMed
    1. Liverman A. D., Cheng H. C., Trosky J. E., Leung D. W., Yarbrough M. L., Burdette D. L., Rosen M. K., Orth K. (2007) Arp2/3-independent assembly of actin by Vibrio type III effector VopL. Proc. Natl. Acad. Sci. U.S.A. 104, 17117–17122 - PMC - PubMed
    1. Tam V. C., Serruto D., Dziejman M., Brieher W., Mekalanos J. J. (2007) A type III secretion system in Vibrio cholerae translocates a formin/spire hybrid-like actin nucleator to promote intestinal colonization. Cell Host Microbe 1, 95–107 - PubMed
    1. Chereau D., Boczkowska M., Skwarek-Maruszewska A., Fujiwara I., Hayes D. B., Rebowski G., Lappalainen P., Pollard T. D., Dominguez R. (2008) Leiomodin is an actin filament nucleator in muscle cells. Science 320, 239–243 - PMC - PubMed

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