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. Author manuscript; available in PMC: 2011 Jul 14.
Published in final edited form as: Nat Cell Biol. 2010 Oct 24;12(11):1057–1063. doi: 10.1038/ncb2109

Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility

Cat M Haglund 1, Julie E Choe 1, Colleen T Skau 2, David R Kovar 2, Matthew D Welch 1,*
PMCID: PMC3136050  NIHMSID: NIHMS302949  PMID: 20972427

Abstract

Diverse intracellular pathogens subvert the host actin polymerization machinery to drive movement within and between cells during infection. Rickettsia in the spotted fever group (SFG) are Gram-negative, obligate intracellular bacterial pathogens that undergo actin-based motility and assemble distinctive ‘comet tails’ that consist of long, unbranched actin filaments1,2. Despite this distinct organization, it was proposed that actin in Rickettsia comet tails is nucleated by the host Arp2/3 complex and the bacterial protein RickA, which assemble branched actin networks3,4. However, a second bacterial gene, sca2, was recently implicated in actin tail formation by R. rickettsii5. Here, we demonstrate that Sca2 is a bacterial actin-assembly factor that functionally mimics eukaryotic formin proteins. Sca2 nucleates unbranched actin filaments, processively associates with growing barbed ends, requires profilin for efficient elongation, and inhibits the activity of capping protein, all properties shared with formins. Sca2 localizes to the Rickettsia surface and is sufficient to promote the assembly of actin filaments in cytoplasmic extract. These results suggest that Sca2 mimics formins to determine the unique organization of actin filaments in Rickettsia tails and drive bacterial motility, independently of host nucleators.

The geometry of actin filaments in eukaryotic cells is specified by nucleation and elongation factors. These include the Arp2/3 complex and its nucleation-promoting factors (NPFs) that assemble branched networks, and formins and tandem-monomer-binding proteins that assemble unbranched filaments6. The Arp2/3 complex is essential for the motility of diverse microbial pathogens, including Listeria monocytogenes, Shigella flexneri, and vaccinia virus, which each express a factor that mimics or recruits host NPFs7. These pathogens have been useful tools for investigating the assembly of branched actin arrays, such as those in cellular lamellipodia. Although many Rickettsia species express the NPF RickA3,4, the failure to observe Arp2/3 complex subunits in Rickettsia comet tails1,8–10, the ability of Rickettsia to undergo motility in cells in which Arp2/3 is inhibited8–10, and the unbranched organization of Rickettsia tails suggest an Arp2/3-independent polymerization mechanism. Other bacterial pathogens express tandem-monomer-binding nucleators, including VopF from Vibrio cholerae11, VopL from Vibrio parahaemolyticus12, and TARP from Chlamydia trachomatis13. However, these secreted effectors are not implicated in actin-based motility. Because SFG Rickettsia assemble distinctive parallel actin filament arrays to drive motility, they could be used as a model for investigating the forces that such arrays impart on intracellular cargo, or for studying actin assembly pathways in cellular structures such as filopodia and microvilli.

To identify bacterial factors that contribute to actin nucleation, we searched translated Rickettsia genome databases for proteins with WASP (Wiskott-Aldrich syndrome protein) homology 2 (WH2) motifs, which are actin-binding peptides in NPFs, some formins, and tandem-monomer-binding nucleators6. We identified WH2 motifs in Rickettsia Sca2 (surface cell antigen 2), a protein that was recently implicated in R. conorii invasion of mammalian cells14 and, importantly, in R. rickettsii actin tail formation and virulence5. Sca2 has a conserved autotransporter domain, predicted to anchor it in the outer membrane, and a large passenger domain, predicted to be exposed on the bacterial surface15 (Fig. 1a). In R. parkeri, a representative SFG species, Sca2 contains a central cluster of three putative WH2 motifs, similar to tandem-monomer-binding nucleators (Fig. 1a, b; these putative WH2 motifs differ from those proposed in an earlier study.5). This WH2 cluster is flanked by two proline-rich domains (PRDs) that are predicted to interact with the actin monomer-binding protein profilin16, similarly to formin homology 1 (FH1) domains of formins (Fig. 1c). Thus, Sca2 shares sequence motifs with both formins and tandem-monomer-binding nucleators.

Figure 1. Sca2 shares sequence motifs with actin assembly factors.

Figure 1

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(a) Schematic representation of the domain organization of R. parkeri Sca2 and truncation derivatives. SS, signal sequence; PRD, proline-rich domain; WH2, WASP homology 2; AT, autotransporter. (b) Alignment of putative WH2 motifs in R. parkeri Sca2 with WH2 domains in actin nucleating proteins from various species (Rr, Rickettsia rickettsii; Hs, Homo sapiens; Vp, Vibrio parahaemolyticus; Dm, Drosophila melanogaster). Similarity shading is based on the BLOSUM45 matrix. (c) Alignment of the proline-rich domains (PRDs) of R. parkeri Sca2 with FH1 domains in representative formins (Sp, Schizosaccharomyces pombe). Proline residues are shaded in purple, hydrophobic residues in green, and alanine and glycine residues in yellow.

We therefore investigated whether purified recombinant R. parkeri Sca2 passenger domain (tagged with glutathione S-transferase; GST-Sca2) affected the assembly kinetics of pyrene-labelled actin in vitro. In this assay, Sca2 passenger domain exhibited dose-dependent nucleation activity (Fig. 2a, b), eliminating the lag phase of polymerization (Fig. 2a, left panel). Similar kinetics were observed using Sca2 lacking the GST tag (data not shown). The intrinsic nucleating activity of Sca2 distinguishes it from RickA, which requires host Arp2/3 complex to nucleate actin3,4. The combination of purified Sca2 and RickA assembled actin with the same kinetics as Sca2 alone (Fig. 2c), indicating that RickA does not affect nucleation by Sca2.

Figure 2. Sca2 nucleates actin filaments.

Figure 2

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(a) Polymerization of pyrene-actin over time with GST alone (160 nM, black line) or increasing concentrations of GST-Sca2 (indicated on left; green lines). Left panel: magnified view of the first 100 s of the time course shown on the right. Actin polymerization was detected by the increase in pyrene fluorescence that occurs on polymerization (AU, arbitrary units). (b) Effect of Sca2 concentration on the initial polymerization rate of pyrene-actin. Initial polymerization rate was assessed by measuring the gradient of the lines in the left graph of a. (c) Polymerization of pyrene-actin over time with GST-Sca2 alone (40 nM, green) or Sca2 and GST-RickA (40 nM and 200 nM respectively, black). (d) Polymerization of pyrene-actin over time with increasing concentrations of GST-Sca2-1106 (blue). Left panel: magnified view of the first 100 s of the time course shown on the right. (e) Polymerization of pyrene-actin over time with increasing concentrations of GST-Sca2-670 (blue). Left panel: magnified view of the first 100 s of the time course shown on the right. (f) Polymerization of pyrene-actin over time with increasing concentrations of GST-Sca2-646-1106 (red).

To map the domains in Sca2 responsible for nucleation, we tested the activity of truncated Sca2 derivatives (Fig. 1a). A fragment containing the N-terminus, WH2 cluster, and PRDs (GST-Sca2-1106) nucleated actin assembly (Fig. 2d). However, it was less potent than GST-Sca2 and lost activity at salt concentrations above 50 mM, suggesting that the missing repetitive sequences are necessary for optimal activity. The Sca2 N-terminal domain (GST-Sca2-670) also displayed weak nucleation activity, with slightly different assembly kinetics compared to GST-Sca2-N1106 (Fig. 2e). In contrast, a derivative containing only the putative WH2 cluster and PRDs (GST-Sca2-646-1106) caused dose-dependent inhibition of polymerization (Fig. 2f). Potent inhibition required concentrations approaching that of actin, suggesting that it bound and sequestered actin monomers. The failure of the WH2 cluster to nucleate actin implies that Sca2 does not belong to the tandem-monomer-binding class of nucleators, distinguishing it from VopF, VopL, and TARP. Collectively, these results indicate that efficient actin nucleation by Sca2 requires the N-terminal domain, WH2 cluster, and PRDs.

Two features of the bulk polymerization kinetics in the presence of GST-Sca2 and GST-Sca2-1106 suggested that Sca2 might affect actin assembly at fast-growing barbed ends, a property of formins. First, reactions containing low concentrations of Sca2 reached steady state more slowly than reactions lacking Sca2. Second, all concentrations of Sca2 lowered the steady-state level of actin polymer, suggesting that Sca2 raises the critical concentration for actin assembly. We measured the effect of Sca2 on the critical concentration by polymerizing actin overnight in the presence of a range of GST-Sca2 concentrations (Fig 3a). Saturating concentrations of GST-Sca2 raised the critical concentration of actin to 0.7 µM, approximately that of pointed ends, suggesting that Sca2 significantly inhibits barbed-end dynamics. To test this, we monitored pyrene-actin assembly from filament seeds at a monomer concentration below the critical concentration for pointed ends. Under these conditions, low nanomolar amounts of Sca2 inhibited barbed-end elongation in a dose-dependent manner (Fig. 3b, c). Sca2 also slowed the depolymerization of preformed actin filaments (Fig. 3d). The Kd of Sca2 for barbed ends, derived from its inhibition of barbed-end assembly, was 1.5 nM. Thus, Sca2 binds barbed ends with high affinity, a conserved property of formins17.

Figure 3. Sca2 is a profilin-dependent actin filament elongation factor that protects barbed ends from capping protein.

Figure 3

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(a) Actin polymer formed after overnight polymerization of pyrene-actin (1 µM, 10% pyrene labelled) in the presence of a range of GST-Sca2 concentrations. (b) Elongation of preformed actin filaments (unlabelled) in the presence of 0.4 µM pyrene-actin monomers and the indicated amounts of GST-Sca2 (nM). (c) Dependence of the initial elongation rate on the concentration of GST-Sca2. Initial polymerization rate was assessed by measuring the gradient of the lines in the first 100 s from graphs of the pyrene-actin elongation assay that contained the indicated concentrations of Sca2. Dissociation constant calculated from these data is shown at the top. (d) Disassembly of preformed actin filaments following dilution into polymerization buffer containing the indicated concentrations of GST-Sca2 (nM). (e) Actin was polymerized for 10 min in the presence of buffer, GST-Sca2 (10 nM), profilin (4 µM), or profilin and GST-Sca2 together, as indicated. Rhodamine-phalloidin (1 µM) was added, and diluted samples were observed by epifluorescence microscopy (representative images shown). Scale bar, 10 µm. (f) Distribution of filament lengths from actin-polymerization reactions performed as in e. The boxes cover percentiles 25–75 with lines marking the medians. The whiskers mark percentiles 10 and 90. The P values were determined using the Kruskal-Wallis test: Asterisk indicates P < 0.05, triple asterisks indicate P < 0.001. Buffer control, n = 415; profilin, n = 209; Sca2, n = 356; Sca2 plus profilin, n = 351. (g) Elongation of filament seeds after the addition of pyrene-actin monomers in the presence of profilin (0.5 µM), GST-Sca2 (6 nM), or GST-Sca2 and profilin together. (h) Dependence of the initial elongation rate on the concentration of profilin, in the presence of 20 nM GST-Sca2. (i) Elongation of filament seeds in the presence of CapZ (10 nM) or CapZ and GST-Sca2 (12 nM). All reactions included profilin (1 µM).

Sca2 dynamics are similar to the Schizosaccharomyces pombe formin Cdc12p18,19, the Drosophila melanogaster formin DAAM20, and the mammalian formin mDia2 (ref. 21), which markedly slow barbed-end elongation in the absence of profilin. Because profilin enables elongation by these formins21,22 and Sca2 contains predicted profilin binding sites in its PRDs, we tested whether human platelet profilin could accelerate barbed-end elongation in the presence of Sca2. We first measured the lengths of filaments nucleated by GST-Sca2 prior to the reaction reaching steady state (Fig. 3e, f). Control reactions containing actin alone produced filaments with a median length of 8.0 µm. In contrast, filaments nucleated by GST-Sca2 had a median length of only 0.6 µm (p < 0.001, Kruskal-Wallis test). When profilin and Sca2 were both included, the length of filaments was increased to a median of 5.7 µm. The filaments polymerized by Sca2 and profilin were unbranched, like filaments in Rickettsia comet tails. To confirm that profilin stimulated elongation at barbed ends, we monitored pyrene-actin assembly from filament seeds at a monomer concentration below the critical concentration for pointed ends (Fig. 3g). Inclusion of profilin with GST-Sca2 significantly accelerated elongation, although the rate was slower than with actin and profilin alone, suggesting that Sca2 remains bound to polymerizing barbed ends in the presence of profilin. The effect of profilin was dose-dependent and saturated at a 1:1 profilin:actin ratio (Fig. 3h), consistent with the hypothesis that Sca2 assembles profilin–actin complexes more efficiently than actin alone. Thus, Sca2 functions similarly to formins as a profilin-dependent barbed-end elongation factor.

By associating with barbed ends, formins can compete with capping proteins and prevent termination of elongation23. We tested whether Sca2 could compete with the capping protein CapZ using the pyrene-actin elongation assay (Fig. 3i). In control reactions containing CapZ and profilin, CapZ fully capped barbed ends. However, Sca2 and profilin competed with CapZ, permitting elongation at a rate equivalent to that of Sca2 and profilin alone. This further supports the hypothesis that Sca2 remains bound to barbed ends as they polymerize.

To directly test whether Sca2 processively associates with barbed ends, we used TIRF (total internal reflection fluorescence) microscopy to observe the effect of Sca2 on the polymerization of fluorescently labelled actin filaments anchored to coverslips by inactivated myosin. In a control reaction containing actin alone, filaments grew at 11.9 ± 0.3 subunits s−1 (mean ± s.d., n = 8; Fig. 4a–c, and Supplementary Information, Video S1). In the presence of subnanomolar concentrations of GST-Sca2, we observed two filament populations: free filaments that grew at a rate similar to controls (14.4 ± 0.9 subunits s−1, n = 8), and Sca2-bound filaments that grew extremely slowly (0.6 ± 0.2 subunits s−1, n = 8; Fig. 4d–f and Supplementary Information, Video S2). The proportion of slow-growing filaments depended on the dose of Sca2. Elongation was slowed more by Sca2 than by the mammalian formin mDia2 (Supplementary Information, Fig. S1, and Videos S3 and S4), to a rate comparable to that observed with Cdc12p18,19 or DAAM20. In the presence of profilin, filaments in a control reaction behaved similarly to actin alone (Fig 4g–i and Supplementary Information, Video S5). However, inclusion of profilin with subnanomolar amounts of Sca2 caused the Sca2-associated filaments to elongate significantly faster than in the absence of profilin (5.4 ± 0.6 subunits s−1, n = 8; Fig. 4j–l and Supplementary Information, Video S6), confirming that Sca2-mediated elongation is activated by profilin. Additionally, Sca2-associated filaments were less intensely labelled than control filaments, a phenomenon that is observed with formins but not other actin nucleators21. Importantly, Sca2-bound filaments were rarely observed to switch from the dim slow-growing population to the bright fast-growing one, as was observed for mDia2 (Supplementary Information, Fig. S1j–l) and other formins21,24, demonstrating that Sca2 associates with polymerizing barbed ends for extended periods of time (>10 min, >5000 subunits added).

Figure 4. Sca2 processively associates with growing filament barbed ends, and elongation is accelerated by profilin.

Figure 4

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(a) Assembly of individual actin filaments imaged by timelapse TIRF microscopy. A black dot (C) marks the pointed end of a control filament, and an arrow marks the growing barbed end. Elapsed time in seconds is indicated in upper right corner of each image. (b) Kymograph showing growth of the filament depicted in a. (c) Plots of growth over time for eight individual filaments from the reaction pictured in a. Average growth rate is indicated at the top (subunits s−1). (d) Filaments imaged by TIRF microscopy as in a, but with GST-Sca2 included in the reaction. A control filament is labelled in black as in a. Green arrowheads (S1, S2) mark two Sca2-associated filaments. (e) Kymographs of the filaments marked in d. Left, control filament; right, Sca2-associated filaments. (f) Plots of growth over time for eight individual filaments per population from the reaction pictured in d. (g–i) Timelapse images, kymograph, and growth plots of control filaments assembled in the presence of profilin. (j–l) Timelapse images, kymographs, and growth plots of filaments assembled in the presence of GST-Sca2 and profilin. Control filaments are labelled in black and Sca2 filaments in green; dots mark the pointed ends and arrows mark the growing barbed ends. (m) Timelapse images showing two examples of filaments buckling in the presence of immobilized GST-Sca2. Green dots mark the pointed ends and open circles (S) mark the barbed ends. (n) Growth plots of filaments assembled in the presence of immobilized GST-Sca2. Scale bars, 5 µm.

To further confirm that Sca2 processively associates with barbed ends, we immobilized Sca2 on a coverslip and looked for buckling of actin filaments between their growing barbed ends and myosin anchor points along filament sides, which is observed with mDia2 (Supplementary Information, Fig. S1m and Video S7) and other formins25. Notably, in the presence of profilin and immobilized GST-Sca2, we observed actin filaments elongating and buckling (Fig. 4m, n, and Supplementary Information, Video S8). Thus, Sca2 acts as a true functional mimic of host formins.

The activity of purified Sca2 in vitro suggests that it participates in the actin-based motility of Rickettsia, which would require it to localize to the bacterial surface. To test this, we raised antibodies that recognized Sca2 on western blots of R. parkeri-infected cell lysates (Supplementary Information, Fig. S2a). Abundant Sca2 was detected by immunofluorescence microscopy on the surface of R. parkeri in infected Drosophila S2R+ cells (Fig. 5a, b) or Vero cells (Supplementary Information, Fig. S2b), independent of an actin tail. When a tail was present, Sca2 was generally enriched at actin-associated bacterial surfaces. Sca2 was not detected along the length of the tail, suggesting that it is not released from Rickettsia during movement.

Figure 5. Sca2 localizes to actin-associated bacterial surfaces and is sufficient to promote actin polymerization in cell extracts.

Figure 5

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(a) Sca2 (white or green) was immunostained with anti-Sca2 antibodies, and actin (white or purple) was stained with Alexa 488-phalloidin in R. parkeriinfected Drosophila melanogaster S2R+ cells. Imaging was by deconvolution microscopy. Scale bar, 5 µm. (b) Magnified image of the bacterium in the lower left corner of a. Scale bar, 1 µm. (c) Timelapse micrographs of a cluster of polystyrene beads coated with GST-Sca2 and added to Xenopus laevis egg extract supplemented with rhodamine-actin. The elapsed time from adding beads to the extract is indicated (min:s). Scale bar, 20 µm.

To determine whether surface-bound Sca2 is sufficient to assemble actin in cell cytoplasm in the absence of other bacterial factors, we coated polystyrene beads (0.5–2.0 µm diameter) with GST-Sca2 and observed whether polymerization of rhodamine-actin occurred in Xenopus laevis egg extract. Sca2 beads tended to form clumps, from which dense arrays of actin filaments assembled and lengthened over time (Fig. 5c and Supplementary Information, Video S9). Once formed, the actin arrays persisted for extended periods (> 30 min). This demonstrates that Sca2 is sufficient to nucleate actin filaments and promote their elongation in the context of cell cytoplasm.

Our results show that Rickettsia have evolved a mechanism of polymerizing host actin through functional mimicry of eukaryotic formins by the bacterial surface protein Sca2. The ability of Sca2 to nucleate unbranched filaments and protect them from capping corresponds with the long, parallel filament organization in Rickettsia tails1,2. Moreover, the profilin-dependent elongation mechanism is consistent with the requirement for profilin for Rickettsia motility in cells10. Although it has been proposed that both Sca2 and RickA might contribute to Rickettsia motility5, our work, together with other evidence, suggests that Sca2 has a primary role. In particular, the observation that actin tails assemble independently of the host Arp2/3 complex8–10 argues against a RickA-dependent motility mechanism. Furthermore, the recent discovery that a transposon insertion in sca2 abolishes R. rickettsii actin tail formation indicates that Sca2 is required for actin assembly5. Further evidence comes from correlations between the sca2 and rickA gene sequences and the properties of actin-based motility of various Rickettsia species (Supplementary Information, Fig. S3). Both sca2 and rickA are intact and their sequences are conserved in SFG species that undergo motility and polymerize long unbranched comet tails. However, in the typhus group species R. typhi, which undergoes actin-based motility but forms shorter actin tails of unknown filament organization2,9, sca2 encodes a divergent protein, whereas rickA is absent. Conversely, in R. canadensis, a species of uncertain phylogeny26 that does not undergo actin-based motility27, the rickA gene is apparently intact, but sca2 contains deletions compared with R. parkeri sca2, the largest of which occurs in the N-terminal domain. The simplest interpretation of these observations is that Sca2 drives motility and RickA participates in host-cell invasion, a process that is thought to depend on the Arp2/3 complex28.

The striking similarity between the activities of Sca2 and formins raises the question of whether Sca2 structurally mimics formins or functions through a divergent mechanism. The core structural and functional unit of formins is the formin homology 2 (FH2) domain17. Although Sca2 does not share significant primary sequence similarity with this domain, the predicted secondary structure of the Sca2 N-terminus is surprisingly similar to that of FH2 (Supplementary Information, Fig. S4). Thus, the Sca2 N-terminus might be a structural mimic of the FH2 domain, which was previously thought to be exclusively eukaryotic29. Nevertheless, the domain organization of Sca2 is inverted, with its putative FH2-like domain located N-terminal to its PRDs, compared to the arrangement in formins. We propose that the WH2s and/or PRDs deliver actin monomers to the N-terminal domain to enable efficient nucleation and elongation. The molecular details of Sca2-mediated actin assembly remain to be determined and will probably exhibit general similarities and interesting differences with eukaryotic formins.

Adaptation of a formin-dependent mechanism for actin assembly distinguishes Rickettsia from well-studied motile pathogens, which require the host Arp2/3 complex (and in some cases NPFs) for actin-based motility7,30. This raises an intriguing question: what evolutionary advantage is conferred by each strategy? Mimicry of formins enables Rickettsia to bypass a requirement for host protein intermediates, which could be critical for a life cycle that involves infection of diverse species (arthropods and mammals) and cell types. A recent report suggests that cell-to-cell spread of Shigella flexneri is affected by inactivation of the mammalian formins mDia1 and mDia2, although these proteins do not participate in actin-based motility31. Therefore, exploitation of formin-mediated actin assembly pathways might be a more widespread pathogenic strategy. In the future, Rickettsia motility can be exploited to further elucidate the cellular functions of formins and other factors that influence the assembly of long, parallel actin filaments.

Methods

WH2 homology search and sequence alignments

The PHI-BLAST algorithm was used to search NCBI databases (restricted to Rickettsia taxid:780) using the query RxxLLxxIxxxxxxLKKV and the pattern L-[LM]-X(1,3)-I-X(3,8)-L-[KRH]-[KRHQSPN]-[VILATSG]. This query matched a motif in R. felis that was similar to motifs in R. parkeri. Putative WH2 motifs were aligned manually based on characteristics that include a short amphiphilic helix, a variable linker sequence, and an LKkv motif defined by invariant leucine, a basic residue, a basic or variable residue, and a small residue32. These motifs differ from the WH2 sequences proposed in an earlier study.5 Similarity scoring was done in Geneious software v4.5.4 (Biomatters) using the Blosum45 matrix and default settings. Alignment of Sca2 and RickA protein or translated nucleotide sequences was performed in Geneious using the Geneious Align algorithm and corrected manually.

Molecular biology

The sca2 gene was amplified by PCR from boiled R. parkeri and subcloned into either pGEX4T-2, using BamHI and XhoI sites (GST constructs), or pET22b, using NcoI and XhoI sites (for Sca2-500-His used for antibody production). The forward primer for GST-Sca2, GST-Sca2-1106, and GST-Sca2-670 was 5'-GCGTGTGGATCCGCAAGCTTTAAAGATTTAGTTAGTAAAACC-3', and the reverse primers were 5'-GAATAGCTCGAGTTCATCACCGGCCCC-3', 5'-GAATAGCTCGAGAGATGTGGATTTAGTAATCCCTAAAC-3', and 5'-GAATAGCTCGAGGTCGCTTTGTGTTTGTTCTAAATT-3', respectively. Primers for GST-Sca2-646-1106 were 5'-GCGTGTGGATCCAGCAATGAAGCCAATAAAATTTTAG-3' and 5'-GAATAGCTCGAGAGATGTGGATTTAGTAATCCCTAAAC-3'. For Sca2-500-His, primers were 5'-GCGTGTCCATGGTCCATATGGCAAGCTTTAAAGATTTAGTTAGTAAAACC-3' and 5'-GAATAGCTCGAGTAATTGTCCCGTATCATTGGTAAA-3'. For full-length Sca2 (for submission to GenBank) primers were 5'-GCGTGTCCATGGCTCATATGAATTTACAAAATTCCCACTCA-3' and 5'-GAATAGCTCGAGTAGCCCTTGATGGCTTTGATAC-3'.

Protein purification

Sca2 derivatives were expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells (Stratagene) induced with 1 mM IPTG overnight at 16°C. Cell suspensions were sonicated, and proteins were isolated using Glutathione Sepharose 4B (GE Healthcare) or Ni-NTA agarose (Qiagen) affinity chromatography. For some experiments the GST tag was cleaved by incubation with thrombin for 2.5 h at room temperature. Eluted proteins were further purified by gel-filtration chromatography on a Superdex 200 10/300 GL column (GE Healthcare) into protein storage buffer (20 mM MOPS pH 7.0 or HEPES pH 7.4, 100 mM KCl, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 10% (v/v) glycerol). GST-mDia2(FH1FH2), encompassing amino acids 521–1171, was also expressed in E. coli and purified by glutathione affinity chromatography, but eluted protein was further purified by chromatography on a Source Q column (GE Healthcare) and dialyzed against mDia2 storage buffer (5 mM NaH2PO4, pH 7.0, 150 mM NaCl, 0.5 mM EGTA, 0.5 mM DTT). GST-RickA purification was described previously4. Human platelet profilin was purified by passing platelet extracts over a poly-L-proline affinity column as described previously33, washing in wash buffer (20 mM PIPES pH 6.8, 150 mM KCl, 0.2 mM ATP, 0.2 mM DTT, 3 M urea), and then eluting with wash buffer plus 7 M urea. Profilin was dialyzed into storage buffer (20 mM HEPES pH 7.7, 20 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM ATP, 0.2 mM DTT, 100 mM sucrose). Proteins were flash frozen in liquid N2 and stored at −80°C, except GST-mDia2(FH1FH2) was kept at 4° C (however, contrary to a previous report34, the activity of flash-frozen GST-mDia2(FH1FH2) was identical to aliquots stored at 4°).

Pyrene-actin polymerization assays

Rabbit muscle actin and pyrene-labelled actin (Cytoskeleton, Inc) were resuspended in G buffer (5 mM Tris pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT), dialyzed into G buffer for >16 h, and stored at 4°C. Polymerization assays included 1 µM actin monomers, 10% pyrene-labelled. Polymerization was initiated by adding 10x initiation buffer (10 mM MgCl2, 10 mM EGTA, 5 mM ATP, 500 mM KCl), bringing final buffer components to 1 mM MgCl2, 2.2 mM EGTA, 0.7 mM ATP, 0.5 mM DTT, 75 mM KCl. In experiments with GST-Sca2-1106 and GST-Sca2-670, KCl was omitted from initiation buffer, and the final KCl concentration was 25 mM. For kinetic experiments, fluorescence was detected at 20 s intervals on a Fluorolog-3 model FL3–11 spectrofluorometer (Horiba Jobin Yvon Inc) at 365 nm excitation and 407 nm emission, using SpectrAcq v5.20 and DataMax v2.2.12B software. Data were normalized (minimum, first value in each data set; maximum, highest value from complete experiment) and graphed using Prism v5.0b (GraphPad Software). Initial polymerization rates were calculated from the first 80 s or 100 s of normalized data. Elongation assays performed essentially as previously described.35 Briefly, unlabelled actin filaments were added to proteins of interest in F buffer (5 mM Tris pH 8.0, 50 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.7 mM ATP, 0.2 mM DTT) and sheared 5 times through a 27G syringe. Half of this mixture was added to actin monomers (final concentration 0.4 or 0.5 µM, 10% pyrene-labelled), then transferred to a cuvette containing 10× initiation buffer. For competition experiments, proteins (Sca2, CapZ, and profilin) were mixed with F buffer before adding actin seeds. Affinity of Sca2 for barbed ends was estimated from initial elongation rates using Prism software with default ‘One-site binding’ parameters. Depolymerization was induced by diluting preformed actin filaments (1 µM, 50% pyrene-labelled) 1:10 into polymerization buffer (a 9:1 mixture of G buffer and 10x initiation buffer) containing Sca2. To determine the effect of Sca2 on actin critical concentration, actin monomers (10% pyrene-labelled, 1 µM final concentration) were mixed with a range of Sca2 concentrations and initiation buffer, then incubated overnight. Pyrene fluorescence readings were collected on a PerkinElmer Victor X3 plate reader with 355 nm excitation and 405/410 nm emission filters. Control reactions established the baseline fluorescence of pyrene-actin monomers and confirmed that the critical concentration was 0.1 µM as expected. Baseline-corrected fluorescence data were converted to units of actin polymer.

Epifluorescence and TIRF microscopy to visualize actin filaments

For epifluorescence microscopy, pyrene-actin polymerization reactions were stabilized by mixing with equimolar rhodamine-phalloidin (Invitrogen), diluted 1:50 into fluorescence buffer (50 mM KCl, 1 mM MgCl2, 3 mg ml−1 dextrose, 10 mM imidazole pH 7.0, 10 mM DTT, 0.5% (w/v) methylcellulose, 20 µg ml−1 catalase and 25 U ml−1 glucose oxidase) and observed using an Olympus IX71 microscope with a 100× (1.35 NA) PlanApo objective and a Photometrics CoolSNAP HQ camera controlled through MetaMorph v5.0r7 (Molecular Devices). Brightness and contrast were adjusted, greyscale inverted, and dimensions set to 300 dpi in Photoshop CS4 (Adobe). Filament lengths were measured for at least 200 filaments from 6–10 random fields per condition using the ImageJ NeuronJ plugin36. Pixels were converted to microns in Excel (Microsoft). Statistical significance was assessed in Prism using the Kruskal-Wallis test.

TIRF microscopy was performed essentially as described previously21,24. Reactions were carried out with 1 µM actin, 33% of which was labelled with Oregon Green. The growth of eight filaments was measured over the course of 300–600 s for each condition and population using custom ImageJ plugins and Excel macros37. The minimal growth of Sca2-associated filaments in the absence of profilin made it impossible to distinguish barbed from pointed end, so total filament length was measured. Immobilization experiments were performed as described previously25. Images and videos were prepared as described previously21. Profilin was used at 5 µM and GST-Sca2 at 0.5 nM (Fig. 4d) or 0.1 nM (Fig. 4j).

Tissue culture and bacterial growth

R. parkeri str. Portsmouth was a gift from C. Paddock (Centers for Disease Control and Prevention)38. Tissue culture conditions were described previously10. For infections, R. parkeri was purified by Renografin density gradient centrifugation39 and added to the media of cells growing on glass coverslips, and infection continued at 33° C for 2 days.

Detection of Sca2 by Western blot and immunofluorescence

The purified N-terminal 500 amino acids of Sca2 with a 6x His tag (Sca2-500-His) was used to raise antisera in rabbits (Covance Inc). Affinity purification was performed by standard methods using GST-Sca2-670 conjugated to Affigel-10 (Bio-Rad). Eluted antibodies were dialyzed into antibody storage buffer (10 mM MOPS pH 7.0, 150 mM NaCl, 35% (v/v) glycerol). For western blots, proteins were separated by SDS-PAGE, transferred to nitrocellulose, incubated with anti-Sca2 antibodies (0.2 µg ml−1) for 2 h, probed with anti-rabbit HRP secondary antibodies (diluted 1:20,000) for 1 h, then detected with Lumigen TMA-6 (Lumigen). For immunofluorescence, Vero or Drosophila S2R+ cells were fixed 2 days post-infection with 4% (v/v) formaldehyde in PBS and blocked with PBS containing 2% (w/v) BSA (bovine serum albumin) and 0.1% (v/v) Triton X-100. Anti-Sca2 antibodies were used at 2 µg ml−1. Rickettsia were detected with anti-rOmpA 14–13 monoclonal antibodies40 diluted 1:300, and filamentous actin was detected with Alexa 488-phalloidin at 0.5 U ml−1. Secondary antibodies were Alexa 568 goat anti-rabbit (Invitrogen) at 5 µg ml−1 and AMCA donkey anti-mouse (Jackson) at 5 µg ml−1. Coverslips were mounted with ProLong Gold (Invitrogen). S2R+ cells were imaged on an Applied Precision DeltaVision 4 Spectris deconvolution microscope with a 100× (1.4 NA) PlanApo objective and a Photometrics CH350 CCD camera. Images were captured using SoftWoRx v3.3.6 (Applied Precision) and deconvolved with Huygens Professional v3.1.0p0 (Scientific Volume Imaging). Slices covering 4 µm of depth were merged using ImageJ’s ZProjection at maximum intensity. Brightness was adjusted and dimensions set to 300 dpi in Photoshop CS4. Vero cells were imaged by epifluorescence microscopy (described above).

Actin polymerization on beads in Xenopus extract

Polystyrene microspheres (Polysciences, 0.5 µm and 2 µm, non-functionalized) were incubated on ice with 1–10 µM Sca2 for 1 h before adding BSA to 5 mg ml−1 and incubating for 15 min. Beads were washed in CSF-XB (10 mM HEPES pH 7.7, 2 mM MgCl2, 0.1 mM CaCl2, 100 mM KCl, 5 mM EGTA, 50 mM sucrose) and kept at 4°C. Xenopus laevis egg extract was provided by the laboratory of R. Heald, University of California, Berkeley, USA. To 8 µl of extract, 1 µl of actin (3 µM, 20% rhodamine-labelled) and 1 µl of beads were added. 2–3 µl of this dispersion was placed between a slide and coverslip and observed immediately by epifluorescence microscopy, or sealed with nail polish and observed after 2–30 min incubation. For Supplementary Information, Video S9, 1 µl of 1% Triton X-100 was also included. Images were recorded every 20 s (Fig. 5c) or 15 s (Supplementary Information, Video S9). ImageJ was used to adjust brightness and contrast and export QuickTime movies.

Supplementary Material

Supplementary Information
Video S1
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Video S2
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Video S3
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Video S4
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Video S5
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Video S6
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Video S7
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Video S8
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Video S9
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ACKNOWLEDGEMENTS

We thank T. Hackstadt and T. Clark for reagents, C. Paddock for the R. parkeri strain, the R. Heald lab for Xenopus egg extracts, B. Scott for help with alignments, Y. Li for technical assistance, and E. Benanti, K. Campellone, A. Serio, S. Reed and T. Ohkawa for comments on the manuscript. This work was funded by NIH-NIAID grant R01 AI074760 (to M.D.W.) and NIH grant R01 GM079265 (to D.R.K.).

Footnotes

AUTHOR CONTRIBUTIONS C.M.H., D.R.K. and M.D.W. designed the experiments; C.M.H., D.R.K, C.T.S. and J.E.C. performed the experiments; C.M.H. and M.D.W. wrote the manuscript with input from D.R.K.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

Accession numbers. Our sequence of R. parkeri Sca2 is deposited in GenBank under accession number HM055592.

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Supplementary Materials

Supplementary Information
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