Moesin is a major component of delicate microextensions of the cell surface of NIH3T3 cells where it is frequently co-localized by immunofluorescence with ezrin and radixin [12
]. Filopodia-like microextensions can be defined by video microscopy as actively growing and freely motile filopodia, as attached structures that do not move and that remain stable in length, or as structures that are retracting and become shorter. Immunofluorescence analysis of fixed cells does not differentiate between these different forms of microextensions. Furthermore, this technique shows that moesin and F-actin are not uniformly distributed and not even detectable in some instances in these structures (our unpublished results). To substantiate these findings in live cells and to determine the reason for the variation in F-actin content, the present study was undertaken.
C-moesin-GFP Binds to Actin Filaments Without Disrupting Microfilament Organization or Cell Behavior
To study changes in the actin cytoskeleton in microextensions of living cells required a new approach. We utilized the C-terminal domain of moesin fused to GFP for imaging, since the structure contains a high affinity binding site for F-actin and since previous results had shown co-distribution of this domain with filamentous actin [11
]. When expressed in NIH3T3 cells, C-moesin-GFP most prominently decorated stress fibers regardless of level of expression (Figure , ) and regardless of whether GFP was positioned at the N- or C-terminus of the C-moesin fragment. As expected, double-staining of fixed, C-moesin-GFP transfected cells with TRITC-phalloidin yielded precisely overlapping images. The intracellular structures visualized by C-moesin-GFP also included bundles and networks of F-actin in lamellipodia. This pattern could be reproduced by staining with rhodamine-labeled phalloidin. Without any doubt, this distribution was quite distinct from the membrane distribution of full-length moesin-GFP or N-moesin-GFP fusion proteins and from the immunofluorescence localization of the endogenous protein in untransfected control cells [13
Figure 1 C-moesin-GFP is co-localized with the microfilament cytoskeleton. Fields containing transfected and untransfected cells were imaged after staining with TRITC-phalloidin (a, c) and compared with images obtained by fluorescence of the same group of cells (more ...)
Side-by-side comparison of cells either expressing or not expressing C-moesin-GFP did not reveal obvious differences in overall shape, behaviour and surface activity (our unpublished data). Expression of the fusion protein also did not affect quality or intensity of TRITC-phalloidin staining of the cells when compared to adjacent untransfected cells. This implied that C-moesin-GFP does not compete with phalloidin for binding sites and that, at least during the observation period, microfilaments and stress fibers apparently assembled normally in the presence of C-moesin-GFP. A similar distribution to that of C-moesin was observed with corresponding fusion proteins of ezrin or radixin consistent with the structurally conserved F-actin binding sites of these fragments (Figure ).
Figure 2 Comparison of cells expressing the C-terminal domains of moesin, ezrin and radixin. NIH3T3 cells were transfected with C-terminal domain-GFP fusion proteins of ezrin and radixin As with moesin, no effect on cell behavior is seen 6 hours after transfection (more ...)
C-moesin-GFP as a Probe to Study Filopodial Microfilaments
By viewing cells for periods of up to several hours, and by comparing time-lapse video images obtained by DIC with fluorescence images we concluded that the intracellular distribution of C-moesin-GFP reflected the distribution of actin filaments that closely abut the plasma membrane. It is for this reason that the changes in fluorescence intensity and shape parallel the many discrete changes in cell surface architecture that were observed by DIC microscopy. F-actin-containing cellular structures could be monitored with a spatial resolution in the order of 200 nm, and a temporal resolution of 3-5 seconds even without the most sensitive camera (Figure ).
Figure 3 The distribution of C-moesin-GFP parallels dynamic changes in the actin cytoskeleton. In (A), a live, fluorescent cell was imaged for 16 minutes. Note retraction of a lamellipodium (top arrow) and advance of another (lower arrow), as the cell changes (more ...)
An example of a moving pseudopodium, a large cell extension with advancing and retracting lamellae, is presented in Figure , and an example of a smaller cortical region during retraction of a lamella is shown in Figure . Multiple discrete changes occur in this relatively small area of the cell even during the short observation period. In the sequence shown in Figure , the pseudopodium begins to alter its direction of migration. The first frame identifies a small lamella encompassing several ribbed filaments and a thinner pseudopodium terminating in a retraction fiber on the left, and a number of retraction fibers at the bottom edge. Multiple thick filament bundles can be seen within the body of the main pseudopodium. In the 16 minute sequence, the left hand lamella retracted, converting its ribs into retraction fibers. The lamella could be observed withdrawing into the body of the pseudopodium, while maintaining the same fluorescence intensity. The rate of lamellar withdrawal measured over different areas was 1.7 ± 0.5 μm/min.
By two minutes of observation, the cytoskeleton of the structure near the upper arrow in Figure has collapsed and multiple kinks became apparent. A sequence of this process with higher temporal resolution is shown in Figure . The sequence, showing bending and collapse of the microfilament bundle, began 30 seconds after the first panel of Figure , and ended 50 seconds later. Seven minutes after this filopodium began to collapse, fluorescence accumulated in a new filopodium near the upper arrow and 9 minutes later this has disappeared again.
The main portion of the pseudopodium showed complex changes in its cytoskeletal fluorescence as some areas decreased in intensity and changed shape, while the tip maintained the same level of fluorescence. The thick bundles in the center of the main pseudopodium remained relatively constant during this time period. In contrast, the area at the lower edge of the cell began to protrude. Forward moving lamellar veils enveloped short filopodia by advancing at a rate of 0.85-1.5 μm/minute and the latter became ribs within the lamella. By 16 minutes, some of these were observed to bend upwards and to detach from the substrate, collapsing backwards into the cell.
A close-up look at another cell edge is shown in Figure to illustrate that, even within a small area of the cell surface, occupying only a few microns, multiple, independent and simultaneous changes in cytoskeletal architecture could be visualized. The arrowheads in Figure point to the same spot in all frames. The left-most arrowhead illustrates rapid lamellar advance followed by partial retraction and ruffling. The second and third arrowheads point to a filopodial microfilament core that advanced 4.6 μm within 10 minutes. Growth of this filopodial cytoskeleton (at 2nd arrowhead) was plotted over time and the graphic representation showed that elongation was not continuous but oscillatory (Figure ). The maximum rate of growth was 1.74 μm/min, but the overall rate of growth over 21 minutes was only 0.4 μm/min. Although there was considerable variation, similar growth rates and similar oscillatory behavior were observed for numerous other filopodia regardless of whether cells expressed C-moesin-GFP or not. The right most arrow head points to a second filopodial bundle that elongated and then retracted, illustrating that within this 8 μm stretch of the cellular edge at least three independent types of cytoskeletal rearrangements were taking place simultaneously.
Figure 4 Multiple, simultaneous, and independent rearrangements of the microextension cytoskeleton are visualized by C-moesin-GFP. A 10 minute time-lapse sequence of the edge of a live, fluorescent cell is shown at high magnification in (A). The arrowheads point (more ...)
To investigate whether the shape of microextensions depended on microfilament content, we performed parallel analysis by comparing DIC images with fluorescence images of their cytoskeletal cores. As shown in Figure , when a microextension was attached to the substrate, its cytoskeletal core could be withdrawn and microfilaments could reenter the membranous sheath without necessarily inducing retraction or growth of the microextension. This obviously was not the case for all microextensions, since in growing and motile filopodia the fluorescence signal matched their shape very closely indicating that actin filaments filled a large amount of their cytoplasmic space.
Figure 5 DIC and fluorescence time-lapse imaging of microextensions in live C-moesin-GFP transfected cells. C-moesin-GFP fluorescence is seen to withdraw from and to re-enter an attached microextension (arrows). Note that the fluorescence intensities of C-moesin-GFP (more ...)
Cytochalasin D Changes the Distribution of C-moesin-GFP
The distribution of C-moesin-GFP appears to reflect rapid changes in microfilament organization in the moving cell edge. We, therefore, expected this distribution to be influenced by cytochalasin D. To investigate this, cells were treated with varying concentrations of cytochalasin D and imaged for different time periods before, during, and after exposure to the drug. Within seconds after addition of cytochalasin D, protrusive activity at cellular edges stopped as seen both by DIC and by fluorescence imaging. As time progressed, C-moesin-GFP fluorescence began to fade in membrane extensions that were attached to the substrate in a distal-to-proximal direction (Figure ). This fading was incomplete and fluorescence remained associated with short segments and intracellular dots that varied in size and signal intensity that could be stained with phalloidin. Stress fibers also began to disappear, but this required more time than the disruption of microfilaments in microextensions. Bundles of the original stress fibers could still be recognized in a few cells 60 minutes after initiation of cytochalasin D treatment. The example in Figure shows the cellular morphology by DIC and the distribution of C-moesin-GFP fluorescence in a cell treated with 20 μM cytochalasin D for 60 minutes. Other transfected cells showed qualitatively similar responses, but the rate at which dissolution of the cytoskeleton occurred varied.
Figure 6 C-moesin-GFP does not interfere with microfilament rearrangements during and after treatment with cytochalasin D. In (A), a transfected cell was imaged live by DIC (left) and C-moesin-GFP fluorescence (right) during treatment with cytochalasin D. The (more ...)
To test the effects of C-moesin-GFP on microfilament stability and assembly in vivo, we treated cells expressing C-moesin-GFP with cytochalasin D for different periods of time, stained the cells with TRITC-phalloidin, and scored transfected and untransfected cells for absence or presence of stress fibers. The example in Figure (panels a & b) shows marked disruption of microfilament organization, but the distribution of TRITC-phalloidin and C-moesin-GFP was identical in both transfected and untransfected cells. The percentage of cells lacking stress fibers was the same for transfected (97%) and untransfected cells (95.5%) suggesting that C-moesin-GFP did not significantly protect microfilaments from the effects of cytochalasin D.
We also examined whether the presence of C-moesin-GFP interfered with recovery of microfilament structures after removal of cytochalasin D. One hour after drug washout, the cells expressing C-moesin-GFP showed a pattern of cytoskeletal organization that could not be distinguished from that of untransfected control cells (Figure , panels c, d). The rate of recovery may have varied, however, since transfected cells tended to have thinner retraction fibers at that time.