A General Method for Definition of Microextensions and for Analysis of Dynamic Changes in the Actin Cytoskeleton
The changes in cell surface topography and movement of cell surface microextensions in isolated cultured cells cannot easily be captured by images of fixed cells. To appreciate the many changes in shape and in the actin cytoskeleton that take place simultaneously, even within a relatively small area of the cell surface and within short time spans time lapse recording of live cells is necessary. This method clearly visualizes the dynamic nature of changes virtually over the entire cell surface the entire of the cell surface consisting of protrusion and retraction of microspikes, filopodial and lamellipodial structures. These changes occur continuously and do not appear to be synchronized . At any given time, the cells appear irregularly shaped, with microspikes, broad lamellae, filopodia and retraction fibers extending from the main body towards the substrate, as well as others protruding from their dorsal cell surfaces. These protrusions are difficult to differentiate from one another functionally on the basis of shape alone, but time-lapse video microscopy at a focal plane near the surface of the culture dish allowed us to distinguish between growing and motile, or stable and retracting microextensions. We refer to advancing, motile and elongated microextensions as filopodia, and to fixed and static structures as retraction fibers. In the recent literature, such protrusions are summarily and often interchangeably described as filopodia, microvilli, retraction fibers, or pseudopodia without regard to potential structural or functional differences [19,20]. This deficiency is particularly evident, when comparing immunofluorescence results from different laboratories, because of the complex architecture of the cell surface and the multitude of shapes of cells of different types and from different organisms.
GFP was chosen as the fluorescent tag for imaging, because we found immunofluorescence of fixed cells to inadequately convey cytoskeletal dynamics of living cells and because of limited stability of the actin cytoskeleton during fixation and staining. The tagging of GFP has been successfully applied to a large number of cytoskeletal proteins . Previous studies with ezrin-GFP showed its localization in ruffles and the leading edge of pseudopodia . Relevant to our work were also experiments in D. melanogaster with the C-terminal fragment of a moesin-related protein fused to GFP . The coding region was placed under the control of the hsp 70 promoter for high level induction by heat shock of embryos. By Western blot flies with two copies of the transgene expressed detectable protein by 90 min, but 1-2 hours were required for fluorescence detection. Expression of the C-terminal domain had two early effects: 1) Upon induction, flies became paralyzed, but they soon recovered. This was thought to be caused by a temporary mechanical disruption of neuronal structures. 2) Long membrane processes appeared on specific cell surfaces within 2 hours after induction, but it could not be determined whether C-moesin stimulated growth of new structures, or whether it accumulated in normal structures that were difficult to discern by other means. Most importantly, however, the development of the embryo proceeded normally. This suggested that, at least in Drosophila, the C-moesin-GFP fusion protein did not interfere with cellular functions. In recent fibroblast experiments, however, Shaw et al.  noted that the C-domain was co-localized with stress fibers by immunofluorescence in unstimulated cells, but in response to the constitutively active form of Rho, RhoAV14, abnormally long and apical processes were formed. In insect cells, overexpression of the C-domain of ezrin enhanced cell adhesion and elicited membrane spreading that was accompanied by microspike and lamelliopodial extension and the formation of unusual, microtubule-containing thin processes, up to 200 μm in length . Such unusual microextensions were not observed in our experiments.
The capability for direct analysis of living cells has significant and important advantages over immunofluorescence techniques. Results do not depend on exposed or available epitopes for antibody detection, and imaging of live cells is more reliable, since loss of fragile microextensions does not become an issue. We have observed such loss by direct microscopic observation of cells during "on stage" fixation and staining procedures for retrospective immunofluorescence analysis and have found that we could monitor, but not prevent such loss (our unpublished observations).
Cytoskeletal Dynamics in Transient Microextensions
The intracellular distribution of C-moesin-GFP and imaging of stress fibers and microextensions with this probe depended on filamentous actin as shown by the disruption of the normal pattern of F-actin in subcortex and stress fibers with the drug cytochalasin D. The changes faithfully reproduce what is typically seen by staining cells with phalloidin, namely withdrawal of actin from microextension and clumping within the cytoplasm. In our retrospective double-staining experiments we saw precise correspondence between the C-moesin-GFP fluorescence signal in the living cell and phalloidin in the same cell before, during, and after drug treatment. This strongly suggested that phalloidin and C-moesin do not compete for binding and probably occupy different binding sites on the filament.
Given the central importance of microfilament dynamics for cell movement, many attempts have been made to study actin rearrangements in vivo. Previous observations have mostly dealt with actin dynamics in lamellipodia using fluorescent analogues of actin and caged resorufin actin that were either microinjected or introduced into permeabilized cells [25,26]. These studies have shown that actin monomers are added at the distal edge of lamellipodia, probably contributing to the protrusive force at the cellular edge, and that actin filaments of the cortex are treadmilling or are in constant centripetal flux [27,28]. Filopodia and retraction fibers were rarely imaged, either because of choice of cell type, or because of difficulties in incorporating sufficient amount of labeled actin into sparse filaments of growing microextensions. More recent work with GFP-actin fusions, however, indicates that this probe is incorporated into filopodial tips .
Our images obtained with C-moesin-GFP indicate that cycles of microfilament protrusion and retraction occur in stationary retraction fibers, as well as in protruding and retracting microextensions. This may occur by actin polymerization or forward and backward movement of filaments, as has been observed in photoactivation experiments of fluorescent labeled actin in retraction fibers of spreading postmitotic PtK2 cells [30,31]. The rate of protrusion and retraction of lamellipodia and filopodia has been measured by many authors and it varied in different cell types [18,28,32,33,34]. The classical studies by Abercrombie et al.  reported measurements of fibroblast lamellar growth ranging from stationary to 1.8 μm/min. These authors also first documented that movement of the cell edge is oscillatory and is accomplished by advance and retraction of thin lamellae. The same phenomenon was observed in the movement of growth cones [35,36]. Our measurements of the rates of advance and retraction of a few microextensions determined from the C-moesin-GFP images are in accord with many of these earlier observations. This implies that the C-moesin-GFP distribution fairly accurately reflects the organization of the actin cytoskeleton and that it does not interfere with filament functions in filopodia. It also suggests that C-moesin-GFP may provide a sensitive new tool for studying spatial and temporal control mechanisms that regulate the actin cytoskeleton and its interactions with the plasma membrane in small segments of the cell cortex. The Rho family-Rho-GDI system (37,38), Ca(2+) (39,40) and phosphatidyl 4,5-biphosphate (41) are prime candidates for driving cellular processes by filamentous actin. Although as yet unknown, it is quite likely that filopodia play an important role in signaling and motility of fibroblasts similar to their function in neurite outgrowth.