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![[Figure 2]](tz5079fig2mag.jpg)
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Figure 2
Actin-binding and actin-bundling activities of ABP34. (a) Binding of ABP34 to F-actin in solution. F-actin co-sedimentation assays were analyzed by Coomassie Brilliant Blue-stained 12% SDS–PAGE. Lane M, molecular-weight markers (labelled in kDa). In the other lanes, S and P indicate supernatant and pellet fractions of F-actin co-sedimentation assays, respectively. In the actin-binding co-sedimentation assays with ABP34, 5 µM actin and 0.25 µM ABP34 were used. ABP34, which bound strongly to F-actin in solution, was found with F-actin in the pellet. (b) Distribution of F-actin and ABP34 in vivo. The cells expressing GFP-ABP34 were placed on a glass cover slip in nutrient medium, allowed to adhere, fixed with cold methanol and stained for F-actin with TRITC-conjugated phalloidin (middle). Images of Dictyostelium cells expressing GFP-ABP34 were recorded with a multi-photon confocal laser scanning microscope. GFP-ABP34-expressing cells (left) exhibited a high concentration of GFP-ABP34 in the cortical region along with F-actin according to the merged image (right). The scale bar represents 5 µm. (c) Transmission electron micrographs of the F-actin and ABP34 mixture in buffer consisting of 20 mM PIPES pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM ATP, 5 mM EGTA, 0.25 mM CaCl2. Left, 5 µM F-actin; middle, 5 µM F-actin with 1 µM ABP34 (low magnitude); right, 5 µM F-actin with 1 µM ABP34 (high magnitude). F-actin bundles are formed in the presence of ABP34 (middle, right). Scale bars represent 200 nm (left and middle) and 50 nm (right). |
![[Figure 2]](tz5079fig2.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
