B-50/GAP-43-induced formation of filopodia depends on Rho-GTPase

Abstract

In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50-induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177-226). Cotransfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50-induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.

Figure 1

Morphologies of Rat-1 fibroblasts during spreading, stained for f-actin. Class I, cells with few protrusions; Class II, irregularly shaped cells; Class III, filopodial cells; and Class IV, lamellar cells.

Figure 2

B-50–induced formation of filopodia and focal adhesions in spreading Rat-1 fibroblasts. Rat-1 fibroblasts were transfected with wild-type B-50-cDNA (in pcDNA1) using lipofectin and grown for 30 h in the absence of serum. Subsequently, cells were replated onto glass coverslips using trypsin/EDTA and fixed after 40 min. Cells were stained for B-50 (left panels) and for f-actin (a) or vinculin (b and c) (right panels) and observed by confocal laser scanning microscopy. Each figure shows a projection of several confocal planes. Inserts show enlargements of the indicated boxes. Scale bar, 10 μm.

Figure 3

Dynamics of B-50/EGFP–induced filopodial extensions in living Rat-1 fibroblasts. Rat-1 fibroblasts were transfected as described in the legend to Figure 2. Upon trypsinization, cells were replated on 24-mm glass coverslips, mounted in a microchamber, and transferred to the microscopic stage of the confocal microscope where they were kept at 37°C and 7% CO₂. Upper frames show GFP-fluorescence in pB-50/GFP-transfected cells; lower frames show the corresponding phase contrast images. (a) Frames at selected timepoints after replating showing the spontaneous extrusion of long, stable, substrate-attached filopodia in a pB-50/EGFP–transfected cell. (b) Time lapse sequence of a pB-50/EGFP–transfected cell showing the transition from a filopodial to a ruffled or lamellar phenotype. Note that untransfected cells do not show a filopodial phenotype preceding lamellar spreading. Scale bar, 10 μm.

Figure 4

Morphologies of cells expressing B-50 mutants. Rat-1 fibroblasts were transfected with [S3G4]B-50 (a), [D41]B-50 (b), [A41]B-50 (c), or [1–176]B-50 (d) (in pcDNA1) and processed as described in the legend to Figure 2. Cells were stained for B-50 (left panels) and for f-actin (right panels) and observed by confocal laser scanning microscopy. Typical examples for cells expressing the various constructs are shown. Scale bar, 10 μm. (e) Quantitation of filopodial cell formation (type III; Figure 1). Values represent means ± SEM from three independent experiments for each construct (**, p < 0.001; *, p < 0.01).

Figure 5

Effects of interference with Cdc42, Rac, or Rho function on the B-50–induced morphological changes. Rat-1 fibroblasts were cotransfected with wild-type B-50/GAP-43 and myc-epitope–tagged N17Cdc42 (a), N17Rac (b), or N19Rho (c) and processed as described in the legend to Figure 2. Cells were triple-stained for B-50 (left panels), myc-epitopes (middle panels), and f-actin (right panels) and visualized by confocal laser scanning microscopy. Scale bar, 10 μm.

Figure 6

Quantitation of filopodial cells after cotransfection with B-50 and GTPase inhibitors. Rat-1 fibroblasts were treated as described in the legend to Figure 5. Percentages of filopodial cells (type III, Figure 1) were scored by examining the f-actin staining. Values represent means ± SEM from three independent experiments for each construct (**, p < 0.001).

Figure 7

Morphological changes induced by expression of activated Rho in Rat-1 fibroblasts during spreading. Rat-1 fibroblasts were transfected with myc-epitope–tagged V14Rho and processed as described in the legend to Figure 2. Cells were stained for myc-epitopes (left panels) and FITC-coupled phalloidin (right panels) and observed by confocal laser scanning microscopy. Scale bar, 10 μm.

Figure 8

B-50–induced shifts in morphological categories of Rat-1 fibroblasts stably expressing mutant Rac- or Rho-GTPase constructs. Rat-1 fibroblasts stably expressing control plasmids (a), N17Rac (b), V12Rac (c), N19Rho (d), or V14Rho (e) were transfected with B-50 and processed as described in the legend to Figure 2. Untransfected (open bars) or wild-type B-50 transfected cells (closed bars) were classified into four morphological categories based on their f-actin staining (see Figure 1). Values represent means ± SEM from three independent experiments for each cell line (*, p < 0.05).