Imaging of Dynamic Changes of the Actin Cytoskeleton in Microextensions of Live NIH3T3 Cells with a GFP Fusion of the F-Actin Binding Domain of Moesin

Abstract

The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin. C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent. Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.

Background

The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin.

Results

C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent.

Conclusions

Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.

Background

], but how moesin interacts with the actin cytoskeleton during the dynamic restructuring of the cell cortex has not been entirely resolved.

].

C-moesin-GFP Binds to Actin Filaments Without Disrupting Microfilament Organization or Cell Behavior

].

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 expressing C-moesin-GFP (b, d). The transfected cell in (b) is attached to several other untransfected cells. Its fluorescence pattern matches filopodia and stress fibers, and is identical in distribution to microfilaments stained with TRITC-phalloidin (a). Cells expressing different levels of C-moesin-GFP are seen within the same field (d, 1-4). The distribution of C-moesin-GFP is the same as that of TRITC-phalloidin (c), regardless of level of expression.

).

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 (a,b) Similar to C-moesin and consistent with identical amino acid sequences of the F-actin-binding region, both C-ezrin and C-radixin bind to actin filaments in stress fibers (sf) (b,c,e,f) and numerous microextensions. This localization is sensitive to cytochalasin D.

C-moesin-GFP as a Probe to Study Filopodial Microfilaments

).

, 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.

, 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.

). 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.

, 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.

Cytochalasin D Changes the Distribution of C-moesin-GFP

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.

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 large arrow points at changes within one pseudopodium. The small arrows point to filopodia and retraction fibers. Note withdrawal and clumping of C-moesin-GFP fluorescence. In (B), cells were treated for 30 minutes with 20 μM cytochalasin D (a, b) and then fixed, or treated for 20 minutes and then allowed to recover for 1 hour after drug washout (c, d) before fixation. They were then stained with TRITC-phalloidin (a, c) and imaged for C-moesin-GFP fluorescence (b, d). Arrows point to identical spots in parallel images.

(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.

, panels c, d). The rate of recovery may have varied, however, since transfected cells tended to have thinner retraction fibers at that time.

A General Method for Definition of Microextensions and for Analysis of Dynamic Changes in the Actin Cytoskeleton

]. 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.

]. 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.

].

) 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.

Conclusions

Imaging of live NIH3T3 cells expressing the C-terminal F-actin binding domain of moesin fused to GFP before, during and after treatment with cytochalasin D, and retrospective analysis with fluorescent phalloidin are consistent with a pattern of actin microfilaments in different regions of the cells. The high sensitivity of this method allowed us to analyze dynamic and diverse changes that occur spontaneously in small areas of the cell surface and to distinguish microextensions according to their F-actin content, motility and life history. C-moesin-GFP may provide a sensitive new tool to study critical regulatory steps required to support the highly dynamic interactions between different cytoskeleton and membrane components, and to unravel spatial and temporal relationships.

Recombinant DNA Constructs

DYE Terminator Cycle Sequencing ready reaction Kit (Perkin Elmer, Norwalk, CT). Plasmid DNA was prepared from Qiagen columns according to the manufacturer's instructions (Qiagen Inc., Chatsworth, CA).

Cell Culture and Electroporation

) for microscopic analysis, or onto 3.5 cm Falcon plastic culture dishes (Beckton Dickinson, Lincoln, NJ) for biochemical analysis.

Antibodies and Reagents

]. For double immunofluorescence experiments, chicken antibodies specific for moesin (ChG1, kindly provided by W. Lankes, Berlin, Germany) and FITC-conjugated goat anti-chicken antibodies were used. The GFP polyclonal antibody reagent was from Clontech Laboratories, Inc. (Palo Alto, CA). Horseradish peroxidase (HRPO)-conjugated goat anti rabbit antibodies were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). TRITC (tetramethylrhodamine isothiocyanate)-labeled phalloidin and rhodamine-conjugated goat anti rabbit antibodies were from Jackson Immunochemicals (West Grove, PA).

Digitally Enhanced Video Differential Interference Contrast (DEV-DIC) microscopy of Live Cells

incubator. The medium was replaced every 5 minutes through the perfusion chamber, and the stage temperature was kept at 37°C with an automatic thermostat. Images were collected using a B&W C2400 CCD camera with on-chip integration and an Argus 20 digital image processor (Hamamatsu Photonics, Japan).

Image Processing

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Additional material

These movie files can be viewed with Quick Time, downloadable for free at

Movie 1 (789KB)

Movie 2 (833KB)

Movie 3 (405KB)

Acknowledgements

This work was supported by funds from the US Public Health Service 1RO1-41045, the Tobacco-Related Disease Research Program Grant 4TR-0316, Training Grants CA09302 and 2T32GM07365 (MRA), and partial fellowship support from the Janet M. Shamberger Fund to PL. We thank Laiqiang Huang for discussions and for providing some of the cDNA clones.

Pninit Litman and Manuel Ricardo Amieva contributed equally to this work.

Abbreviations

BSA, bovine serum albumin; C-terminal, carboxyterminal; DMEM, Dulbecco's minimal essential medium; DEV-DIC, digitally enhanced video-differential interference contrast; GFP, green fluorescent protein; N-terminal, aminoterminal; PBS, phosphate buffered saline; PCR, polymerase chain reaction; SDS PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; TRITC, tetramethyl rhodamine isothiocyanate.