TARP associates with actin but not the Arp2/3 complex. (A) Extracts from HeLa cells were incubated with GST or GST fusions to TARP and specifically bound proteins were resolved by SDS/PAGE and visualized by Coomassie blue staining. In this experiment, Tarp and Tarp-C-domain GST fusion constructs (GST-TARPΔ and GST-C-domainΔ, respectively) were truncated at the C terminus by 150 amino acids to prevent proteolytic breakdown (see Fig. 4A). GST fusions are indicated by open circles. Actin (arrow) from HeLa lysates was specifically bound by GST-TARPΔ or GST-C-domainΔ. Full-length Tarp and Tarp–C domain showed identical results (data not shown). The HeLa lysate shown in the first lane represents 1% of the material used in the +lysate pull-down lanes. Samples identical to those shown in the Coomassie-stained gel were subjected to immunoblotting with actin (α actin), actin-related protein 3 (α Arp3), or formin (α Dia1)-specific antibodies. Molecular mass of protein standards is in kDa. (B) GST-TARP associates with monomeric actin (G actin) directly. Immobilized GST-fusion proteins to full-length TARP, the C-terminal domain of TARP, and the N-terminal domain of TARP were incubated with monomeric actin in G buffer. Bound proteins eluted from glutathione beads were resolved by SDS/PAGE and subjected to immunoblotting with an actin (α actin)-specific antibody.

TARP nucleates new actin filaments. (A) Nucleation of new actin filaments is TARP concentration-dependent in the presence of 1 μM CD (+CD). An increase in pyrene actin fluorescence was observed as actin polymerization was assessed in the presence of 150, 300, 600, and 1,200 nM GST fusions to the C-terminal domain of TARP (C-dom TARP). (B) Pyrene-conjugated actin filaments generated in A were visualized by transmission electron microscopy after initiation of polymerization. Actin filaments generated in the presence of 150 nM C-terminal domain of TARP (150 nM C-dom TARP + CD) and (150 nM C-dom TARP − CD) or actin alone control (actin alone + CD) were collected 30 min postpolymerization initiation and stained with 1% uranyl-acetate. (Scale bar, 0.1 μm.) (C) Actin filament length was measured from micrographs collected in B. Data are represented as the average length of 40 individual actin filaments per condition. Error bars depict one standard deviation from the mean.

The actin-binding domain of TARP oligomerizes to form an actin nucleator. TARP fragments 8d* (* indicates GST is removed) capable of polymerizing actin and 8e* missing a proline-rich domain were analyzed by gel filtration. (A) An additional peak representing oligomerized Tarp (arrow) was detected in the A280 trace of eluted fragment 8d*. Elution times are indicated above the major peaks. The protease used to remove the GST moiety eluted at the 31.5-min peak. (B) Protein fractions were collected in 2-min intervals from the gel filtration column. Protein fractions were resolved by SDS/PAGE and were subjected to immunoblotting with a TARP-specific antibody. Dextran Blue 2000, polyacrylic sizing beads, and protein standards are indicated above the immunoblot with respective molecular weight and peak elution times. (C) Oligomerized TARP peptide polymerizes pyrene actin. GST-TARP peptide 8d* fraction 17 increased actin polymerization compared with the smaller 8d* fraction 27, which polymerized actin to similar levels as the actin alone control. 8e* fraction 29 reduced the rate of actin polymerization.

TARP promotes actin polymerization. (A) Pyrene actin polymerization in the presence of GST-TARP fusions. GST-TARP fusions representing the entire protein (TARP), C-terminal domain of TARP (C-dom TARP), or N-terminal domain of TARP (N-dom TARP) were incubated with 1 μM monomeric pyrene-labeled actin. A TARP-mediated increase in actin polymerization after the addition of polymerization buffer at 600 sec was measured as arbitrary fluorescence intensity (Intensity a.u) over time (seconds) with excitation and emission wavelengths of 365 and 407 nm, respectively. Purified GST (GST) and pyrene actin alone (actin alone) served as negative controls. (B) TARP-mediated actin polymerization occurs in the presence of HeLa extracts. Pyrene actin polymerization as described in A with the addition of 10 μg of HeLa extracts. (C) CD inhibits actin filament growth initiated by TARP. Pyrene assay as in A with the addition of 1 μM CD added to the C-terminal domain of TARP (C-dom TARP + CD) and actin control (actin alone + CD). (D) Pyrene-conjugated actin was visualized by transmission electron microscopy after initiation of polymerization. Actin filaments generated in the presence of the C-terminal domain of TARP (C-dom TARP) or GST control (GST) were collected at 5, 10, and 30 min postpolymerization initiation and stained with 1% uranyl-acetate. (Scale bar, 0.1 μm.)

Schematic of C. trachomatis TARP and GST-TARP fusions used in this study. (A) L2 TARP harbors an N-terminal tyrosine-rich repeat region (green boxes), which are contained within the GST fusions: GST-TARP, GST-TARPΔ, and the GST-N domain. Proline-dense (blue box) and actin-binding domains (red box) are contained within the C-terminal half of TARP located in the GST fusions: GST-C-domain, GST-C-domainΔ, and 8d. The GST fusion 8e contains the actin-binding domain only. (B) TARP harbors a conserved actin-binding domain. Sequence alignment of the actin-binding helix of WAVE2 and the putative actin-binding helix of TARP. Black bars indicate the residues that fall within the actin-binding helix for TARP (above) and WAVE2 (below). Yellow shading indicates identical residues, and green shading indicates similarity. A consensus sequence is shown under the alignment.