TAGLN2 polymerizes G-actin in a low ionic state but blocks Arp2/3-nucleated actin branching in physiological conditions

Figures

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  Figure 1. TAGLN2 polymerizes G-actin in low-salt buffer. (a) TAGLN2 (TG2) binding to pre-polymerized F-actin or G-actin was assessed after incubation for 30 min. S, supernatant; P, pellet. Full-length blots/gels are presented in Supplementary Fig. S5. (b) Actin beads were incubated with gelsolin (2.5 μg), actin (2.5 μg), and increasing amounts of TAGLN2 (1, 2.5, 5, 10 μg) in G-buffer for 60 min. The samples were assessed for G-actin binding activity as described in the Methods. Full-length blots/gels are presented in Supplementary Fig. S5. (c) Affinity of TAGLN2 to G-actin was measured as described in the Methods. Blue line, binding to G-actin; orange line, binding to BSA. (d–f) Time-based fluorometric analysis of pyrene-labelled actin polymerization. Reactions were as follows: (d) presence or absence of TAGLN2 in low-salt G-buffer or high-salt F-buffer (arrow, time of F-buffer addition); (e) increasing concentrations of His-TG2 in G-buffer (T/A = TG2/actin); (f) increasing concentrations of TAGLN1, 2, and 3. Time to half-maximum fluorescence (t1/2) at each TAGLN concentration was plotted. Results are representative of at least three independent experiments; (g) analysis of pre-polymerized…

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  Figure 2. TAGLN2-polymerized F-T/actin complex is resistant to depolymerization and has no preference between ATP-actin and ADP-actin. (a) Actin polymerization reactions containing actin and TAGLN2 (actin + TAGLN2) or actin alone (actin) in G-buffer and actin and TAGLN2 (actin + TAGLN2) or actin and phalloidin (actin + phalloidin) in F-buffer were diluted fivefold with G-buffer to initiate depolymerization in the absence (left) or presence (right) of cofilin. Results are representative of at least three independent experiments. (b) TAGLN2 uses both ATP-actin and ADP-actin for polymerization in G-buffer as monitored by fluorescence (left) or co-sedimentation (right). Results are representative of at least three independent experiments. Full-length blots/gels are presented in Supplementary Fig. S6.

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  Figure 3. Visualization of TAGLN2-mediated G-actin polymerization in low-salt buffer. (a) Electron micrographs of negatively stained actin polymers in G- and F-buffer. Left column represents the control images showing G-actin and the distinctive appearances of F-T/actin complex in G-buffer. Right column shows filamentous (F) actin and TAGLN2 and F-actin complex in F-buffer. Scale bars, 2 μm. (b) Real-time actin assembly in the absence or presence of TAGLN2 in G-buffer was monitored by TIRF microscopy using Atto488-actin. Scale bars, 2 μm. (c) Quantification of average filament length, average filament numbers per field, and branching density in each condition. *p < 0.05 versus without TG2. (d) TAGLN2 (0.4 μM)-Atto594 actin (0.2 μM) complex was formed in the G-buffer condition, diluted 1/10 in G-buffer, and loaded onto an NEM-coated coverglass. Mg2+-exchanged Ca2+-Atto488 actin (0.2 μM) mixture in F-buffer was then loaded onto the coverglass for 10 min and viewed by confocal microscopy. Results are representative of at least three independent experiments. (e) Time-lapse imaging of actin growth from TAGLN2-Atto488 actin seed. The number of actin seeds was increased in the presence of…

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  Figure 4. Three-dimensional reconstruction of F-T/actin reveals that TAGLN2 serves as a molecular staple. (a) Identification of essential actin-binding regions of TAGLN2. Schematic diagram of the TAGLN2 constructs (left) and fluorometric analysis of pyrene-labelled actin polymerization in the presence of the indicated proteins [2 μM actin, 8 μM TAGLN2 (TG2) or TG2 mutants, 20 nM Arp2/3, and 200 nM GST_VCA] (right) are shown. Purified proteins were stained with Coomassie blue (right). Full-length blots/gels are presented in Supplementary Fig. S6. (b) Three-dimensional (3D) reconstruction of TAGLN2/actin filaments (F-T/actin). i, Surface view of 3D F-actin reconstruction was generated with IHRSR using actin as a reference. Actin subdomains are labelled as 1–4. ii, 3D reconstruction of F-T/actin complex. iii, Superimposition of F-T/actin reconstruction (cyan) and the TAGLN2 atomic model (PDB ID: 1WYM, red) was fitted into extra densities existing in the two superimposed models. Arrows point to additional density of decorated filaments. Filaments are oriented with pointed end at top. Numbers indicate actin SDs. Transverse sections of actin and F-T/actin reconstruction showing actin subdomains…

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  Figure 5. TAGLN2 inhibits Arp2/3 complex-mediated nucleation of branched filaments. (a) Time-based fluorometric analysis of pyrene-labelled actin polymerization. Polymerization assay in presence of His-TG2 (0, 2, 4, 8, 16, 32, and 64 μM) in F-buffer. Results are representative of at least three independent experiments. (b) Fitting of TG2 (red) and Arp2/3 (green) to F-actin reconstruction, showing the clash between TG2 and ARP2/3. (c) Dose-dependent inhibition of Arp2/3 and VCA-stimulated actin nucleation by TAGLN2. (0–32 μM TAGLN2 [TG2] or TAGLN2ΔAB, 20 nM Arp2/3, 200 nM GST_VCA). Results are representative of at least three independent experiments. (d) Competition with the Arp2/3 complex for actin binding in the presence of TAGLN2 or TG2ΔAB. Full-length blots/gels are presented in Supplementary Fig. S7. (e) Real-time imaging of actin branching formed by Arp2/3 and VCA with/without TAGLN2 (left). Scale bars, 2 μm. Quantification of branching density for each condition (right). *p < 0.05 versus no addition. (f) Fluorometric analysis of pyrene-labelled actin polymerization in the presence of the indicated proteins (Note, 2 μM actin, 8 μM TAGLN2 [TG2] or TAGLN2 mutants; 20 nM Arp2/3, and 200…

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  Figure 6. TAGLN2 mediates filopodium formation in COS-7 cells. (a) Membrane protrusions in COS-7 cells expressing GFP or TG2_GFP stimulated with PMA. Scale bar, 5 μm. White arrows indicate spiky-like membrane protrusions at lamellipodia. (b) Expression and localization of WT TG2 in COS-7 cells. COS-7 cells were transfected with non-GFP-tagged WT TG2 and analysed as described in (a). Full-length blots/gels are presented in Supplementary Fig. S7. (c and d) COS-7 cells expressing GFP or TG2_GFP were stimulated with PMA and imaged by scanning EM (c) and platinum replica EM (d). Blue and yellow arrows indicate the edges of the cell membrane. Scale bar, 10 μm. *p < 0.05 versus GFP-transfected cells.

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  Figure 7. Knockdown of TAGLN2 reduces filopodia in HeLa cells and mouse T cells. (a) TAGLN1, 2, and 3 expression in HeLa cells with A7R5 cells, Jurkat T cells, and brain lysates as positive controls, respectively. Full-length blots/gels are presented in Supplementary Fig. S9. (b) TAGLN2 expression after siRNA knockdown in HeLa cells. Full-length blots/gels are presented in Supplementary Fig. S8. (c) Effect of TAGLN2 knockdown on filopodium formation in HeLa cells. (d) Quantification of filopodia and their average length by FiloQuant analysis. *p < 0.05 versus scrambled siRNA-transfected cells. (e) SEM micrographs recorded after 10-min co-incubation of Jurkat T cells and SEE-loaded Raji B cells. I, Elongated actin-rich filopodia projections (green arrow) were observed during antigen recognition in the early stage of the immunological synapse. ii, The purple arrow indicates the ruffled leading edge of a T cell forming a mature immunological synapse. (f) TG2_GFP-expressing Jurkat T cells were incubated for 20 min with SEE-loaded Raji B cells (red: ICAM1-Cy5), and then the cells were imaged by confocal microscopy. 3D rotating views (90 °C) of the top images are shown. Accumulation of TAGLN2…