Figure 3.

Influence of TgFRM1-FH1-FH2, TgFRM2-FH1-FH2, and TgPRF on formation of TgACTI filaments revealed by microscopy. (A) In vitro polymerization of TgACTI with formin domains. TgACTI (25 μM) was incubated in F buffer alone, with 500 nM TgFRM1-FH1-FH2, 60 nM TgFRM2-FH1-FH2, or 500 nM TgFRM1-FH1-FH2 combined with 60 nM TgFRM2-FH1-FH2 for 1 h then visualized by fluorescence microscopy using 0.33 μM Alexa 488-phalloidin. Scale bar, 5 μm. (B) In vitro polymerization of TgACTI with formin domains and profilin. TgACTI (25 μM) was incubated in F buffer alone, with 500 nM TgFRM1-FH1-FH2, 60 nM TgFRM2-FH1-FH2, or 500 nM TgFRM1-FH1-FH2 and 60 nM TgFRM2-FH1-FH2 with 25 μM TgPRF for 1 h then visualized by fluorescence microscopy using 0.33 μM Alexa 488-phalloidin. A representative of 2 similar experiments is shown. Same scale as A. (C) Electron micrographs of replicas of TgACTI polymerized alone or in the presence of TgFRM1-FH1-FH2 or TgFRM2-FH1-FH2. TgACTI (25 μM) was polymerized by addition of F buffer and 500 nM TgFRM1-FH1-FH2 (TgACTI + FRM1) or 60 nM TgFRM2-FH1-FH2 (TgACTI + FRM2) for 1 h then fixed and frozen followed by quick-freeze, deep-etch and platinum replica formation. TgACTI alone (top left panel) formed long straight filaments that often bundled into clusters. Enlarged view (bottom left panel) shows singlet filaments (arrowheads) as well as bundles. In the presence of FRM1 (top central panel), filaments formed interconnected honeycomb networks that were decorated by globular proteins. Enlargement (bottom central panel) of the filaments revealed they were short, formed interconnected networks, and were heavily decorated along their sides as well as containing globular clusters at nodes or intervals along the filaments (arrowheads). In the presence of FRM2 (top right panel), filaments were typically straight and collected into bundles, which on higher magnification show extensive decoration (bottom right panel). Scale bars = 100 nm.