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Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants

¹ Department of Cell Biology, Duke University Medical Center, NC27710, USA
² Department of Biology, Duke University, NC27708, USA

View larger version (12K): [in a new window]   Fig. 1. Full-length FN and moesin are shown, along with the yfp and cfp chimeras we have constructed. The yfp was fused between FN-III domains 3 and 4, as previously described (Ohashi et al., 1999). The C-terminal actin-binding domain of moesin was fused to the C-terminus of cfp to produce a molecule that binds to actin filaments.

View larger version (99K): [in a new window]   Fig. 2. Dual labeling of the FN matrix with FN-yfp and the actin cytoskeleton with cfp-Moe. Cells were cultured for 48 hours to allow matrix assembly and stained with polyclonal antibody for FN (c) or with phalloidin for actin (e). (a, b and d) show the fluorescence of yfp and cfp as indicated, and in (f), the yfp and cfp images are superimposed. Bar, 50 µm.

View larger version (107K): [in a new window]   Fig. 5. An area of cell culture (after 72 hours of culture) showing some mature FN matrix fibrils and several areas where matrix fibril assembly is being initiated. The cfp-Moe staining of the actin cytoskeleton is shown in red, and the FN-yfp in green. The cfp-Moe expression is variable: it is high for the cell in the center, medium for the one at the top and low for the cell at the bottom right. There is a cell at the middle right that is not expressing cfp-Moe at all (its nucleus is indicated by a dotted line at 0:00 and is faintly visible at 1:00). Five sites of matrix assembly are identified by numbers at 0:00. A movie is available in supplementary results, showing these and intervening frames, carefully aligned to fixed points on the substrate. The most informative view of matrix assembly is obtained by stepping back and forth between frames of the movie. Bar, 50 µm.

View larger version (26K): [in a new window]   Fig. 3. Western blot of FN-yfp. Conditioned media from a 3-day confluent culture was analyzed by SDS-PAGE (5%) under reducing condition followed by western blotting with polyclonal antibodies for FN (a) and gfp (b). (a) shows that non-transfected 3T3 cells express native FN and FN-yfp/cfp-Moe transfected cells express both FN-yfp and native FN. (b) shows that the transfected 3T3 cells secrete about half as much FN-yfp as the FN-gfp secreted by the transfected CHO cells.

View larger version (77K): [in a new window]   Fig. 4. Localization of FN matrix, actin cytoskeleton and ß1 integrin. Cells were cultured for 72 hours to allow matrix assembly and stained using a monoclonal antibody specific for activated ß1 integrin. FN-yfp, cfp-Moe and ß1 images were superimposed. The integrin is prominently localized in spots or small streaks at the ends of actin filament bundles (arrows). Colocalization is sometimes indicated by a yellow color, but because of the variable intensity of fluorescence some FN colocalized with actin appears green (e.g., the FN fibrils indicated by the arrows in a and d). Bar, 20 µm.

View larger version (76K): [in a new window]   Fig. 6. Contraction of FN fibrils following cytochalasin B treatment. Images were taken before and 10 and 20 minutes after adding 4 µM cytochalasin B. Actin filament bundles persisted at 10 minutes but collapsed into irregular islands at 20 minutes. The FN fibril indicated by the arrow was extended from an attachment point on the pseudopod up to the cell body at both 0 and 10 minutes. When the actin filament bundles disappeared at 20 minutes, this fibril contracted towards the bottom attachment point. That bottom attachment point also changed from a curved FN fibril to a straight one at 20 minutes, as other attachment points were lost. The bottom two panels show a superimposition of the FN image only at 0-10 and 10-20 minutes. Bar, 20 µm.

View larger version (139K): [in a new window]   Fig. 7. Some FN fibrils remain fixed and others contract following cytochalasin B treatment. (a,b) show the cell two hours before and immediately before adding 4 µM cytochalasin B. (c,d) panels are 30 minutes after adding cytochalasin. The upper panels (a-c) show actin in red and FN in green; (d) shows staining for ß1 integrin. The elongated fibril (upper arrowhead) contracted before cytochalasin treatment. Another fibril (lower arrowhead) extended during the two hour period, then contracted after the addition of cytochalasin. The fibril indicated by the upper arrow elongated considerably in the two hour period and then remained unchanged, presumably bound to the substrate. This fibril maintained a streak of colocalized ß1 integrin. The lower panels show superimposition of the FN label at two time points: (a,b) shows minus 120 minutes in red and 0 minutes in green; (b,c) shows 0 minutes in green and plus 30 minutes in red. The arrows indicate the elongated fibrils in (a,b) and the contracted fibrils in (b,c). Most FN fibrils appeared to be attached to the substrate, as they did not move before or after the addition of cytochalasin. However, some fibrils showed substantial contraction: the most conspicuous examples are shown at higher magnification (x2) in b' and c' (showing only the FN fluorescence). Bar, 20 µm.