Two pathways through Cdc42 couple the N-formyl receptor to actin nucleation in permeabilized human neutrophils

Abstract

We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends.

Figure 1

A, FMLP leads to free barbed ends on actin filaments in neutrophils permeabilized with OG. The increase in free barbed ends was determined. The values represent cytochalasin B-sensitive actin assembly initiated in neutrophils treated with FMLP (30 nM) for 3 min and then OG permeabilized (FMLP-OG), or OG permeabilized and then treated with FMLP for 3 min (OG-FMLP). The results are means ± SEM of ten experiments normalized by setting the control free barbed ends in permeabilized cells at 100%. B, Effect of 2 μM cytochalasin B on rates of pyrene-actin assembly of resting and FMLP-treated OG-permeabilized neutrophils. Cytochalasin B inhibits the FMLP-mediated increase in actin polymerization. There is a small but significant increase in free actin filament pointed ends in FMLP-stimulated permeabilized neutrophils (P < 0.05). The results are means ± SEM of five experiments. C, Relationship of the Octyl glucoside concentration and the retention of FMLP-mediated barb end exposure. Neutrophils were exposed to the indicated amount of OG for 10 s and then treated with 30 nM FMLP for 3 min. The data is from triplicate samples from a single experiment, representative of two experiments; mean ± SD. D, Relationship of the Octyl glucoside permeabilization time and the retention of FMLP-mediated barb end exposure. Neutrophils were exposed to 0.4% OG for the indicated time periods and then treated with 30 nM FMLP for 3 min. The data is from triplicate samples from a single experiment, representative of two experiments; mean ± SD. E, Relationship of FMLP incubation time and detectable free barbed ends. Neutrophils were permeabilized for 10 s with 0.4% OG, were then incubated with 30 nM FMLP for the indicated stimulation time, and then assayed for free barbed ends. The data is from triplicate samples from a single experiment, representative of two experiments; mean ± SD.

Figure 2

A, Effect of OG on the integrity of the resting neutrophil plasma membrane. Electron micrograph showing perforation distribution on plasma membrane of neutrophils permeabilized with 0.4% OG. Neutrophils permeabilized with 0.4% OG for 10 s were placed onto polylysine-coated coverslips. Bar, 200 nM. Inset of whole neutrophil; bar, 2 μm. B, Substrate-attached OG permeabilized neutrophils are able to undergo shape changes upon FMLP exposure. DIC and fluorescent images of attached neutrophils, which were OG permeabilized as described. Bar, 5 μm. C, Incorporation of rhodamine actin in FMLP-stimulated permeabilized neutrophils. Images represent confocal micrographs from the middle third of permeabilized neutrophils. The top shows resting controls; the bottom shows FMLP-treated (30 nM) neutrophils. The phalloidin stain demonstrates both preexisting actin filaments and those filaments formed during the assay after permeabilization. The rhodamine actin represents exogenous actin associated with the cells after permeabilization. In the overlay, yellow represents the rhodamine actin in filamentous form that polymerized after permeabilization from free barbed nuclei. Bar, 5 μm.

Figure 3

Effect of FMLP concentration on barbed-end exposure in OG-permeabilized neutrophils. Cells were exposed to 0.4% OG for 10 s and then exposed to the indicated FMLP concentration for 3 min. The results are means ± SEM of five experiments.

Figure 4

Effect of PIP₂ and phosphoinositide-binding peptide on barbed-end exposure in OG-permeabilized neutrophils. A, Effect of PI(4,5)P₂ concentration on barbed-end exposure in neutrophils. The appearance of nucleation of actin assembly in permeabilized neutrophils is detectable in the presence of 12 μM PI(4,5)P₂, and a maximal increase in nucleation activity follows the addition of 60 μM PI(4,5)P₂. Neutrophils are permeabilized as described and the lipids are added as micelles. The results are mean ± SEM of three experiments. B, Effect of PS and PI, PIP, PI(4,5)P₂, and PI(3,4,5)P₃ on barbed-end exposure in neutrophils. Both PI(4,5)P₂ and PI(3,4,5)P₃ induced a large increase in actin nucleation, whereas PI and PIP induced a small increase in free barbed ends. Phosphatidyl serine induced no change in actin nucleation. The lipids were added at 30 μM. The results are mean ± SEM of three experiments. C, Effect of a phosphoinositide-binding peptide on FMLP-induced barbed-end exposure. A PIP₂-binding 10-mer peptide derived from the gelsolin phosphoinositide-binding site added to permeabilized neutrophils for 30 s before FMLP addition inhibits the nucleation response of the permeabilized neutrophils to FMLP. Peptide concentrations ≥45 μM produce complete suppression of FMLP's effects. A random 10-mer peptide (CP; 45 μM) containing the same residues as the gelsolin 10-mer had no inhibitory activity on the FMLP-mediated increase in free barbed ends. The results are mean ± SEM of three experiments.

Figure 4

Effect of PIP₂ and phosphoinositide-binding peptide on barbed-end exposure in OG-permeabilized neutrophils. A, Effect of PI(4,5)P₂ concentration on barbed-end exposure in neutrophils. The appearance of nucleation of actin assembly in permeabilized neutrophils is detectable in the presence of 12 μM PI(4,5)P₂, and a maximal increase in nucleation activity follows the addition of 60 μM PI(4,5)P₂. Neutrophils are permeabilized as described and the lipids are added as micelles. The results are mean ± SEM of three experiments. B, Effect of PS and PI, PIP, PI(4,5)P₂, and PI(3,4,5)P₃ on barbed-end exposure in neutrophils. Both PI(4,5)P₂ and PI(3,4,5)P₃ induced a large increase in actin nucleation, whereas PI and PIP induced a small increase in free barbed ends. Phosphatidyl serine induced no change in actin nucleation. The lipids were added at 30 μM. The results are mean ± SEM of three experiments. C, Effect of a phosphoinositide-binding peptide on FMLP-induced barbed-end exposure. A PIP₂-binding 10-mer peptide derived from the gelsolin phosphoinositide-binding site added to permeabilized neutrophils for 30 s before FMLP addition inhibits the nucleation response of the permeabilized neutrophils to FMLP. Peptide concentrations ≥45 μM produce complete suppression of FMLP's effects. A random 10-mer peptide (CP; 45 μM) containing the same residues as the gelsolin 10-mer had no inhibitory activity on the FMLP-mediated increase in free barbed ends. The results are mean ± SEM of three experiments.

Figure 4

Effect of PIP₂ and phosphoinositide-binding peptide on barbed-end exposure in OG-permeabilized neutrophils. A, Effect of PI(4,5)P₂ concentration on barbed-end exposure in neutrophils. The appearance of nucleation of actin assembly in permeabilized neutrophils is detectable in the presence of 12 μM PI(4,5)P₂, and a maximal increase in nucleation activity follows the addition of 60 μM PI(4,5)P₂. Neutrophils are permeabilized as described and the lipids are added as micelles. The results are mean ± SEM of three experiments. B, Effect of PS and PI, PIP, PI(4,5)P₂, and PI(3,4,5)P₃ on barbed-end exposure in neutrophils. Both PI(4,5)P₂ and PI(3,4,5)P₃ induced a large increase in actin nucleation, whereas PI and PIP induced a small increase in free barbed ends. Phosphatidyl serine induced no change in actin nucleation. The lipids were added at 30 μM. The results are mean ± SEM of three experiments. C, Effect of a phosphoinositide-binding peptide on FMLP-induced barbed-end exposure. A PIP₂-binding 10-mer peptide derived from the gelsolin phosphoinositide-binding site added to permeabilized neutrophils for 30 s before FMLP addition inhibits the nucleation response of the permeabilized neutrophils to FMLP. Peptide concentrations ≥45 μM produce complete suppression of FMLP's effects. A random 10-mer peptide (CP; 45 μM) containing the same residues as the gelsolin 10-mer had no inhibitory activity on the FMLP-mediated increase in free barbed ends. The results are mean ± SEM of three experiments.

Figure 5

A, Effect of V12Rac1 and V12Cdc42 concentration on barbed-end exposure in OG-permeabilized neutrophils. Cells were exposed to 0.4% OG for 10 s and then exposed to the indicated small GTPase concentration for 3 min. The results are mean ± SEM of at least three experiments. B, Comparison of the effect of dominant negative Rho GTPase mutants of Cdc42 (N17Cdc42) and Rac1 (N17Rac1) on FMLP-induced free barbed-end creation in permeabilized neutrophils. The dominant negative Rac1 inhibited almost two-thirds of the FMLP-mediated increase in free barbed ends, whereas the CDC42 inhibited all the FMLP-mediated increase. The results are mean ± SEM of two experiments. C, The hierarchy of CDC42 and Rac upstream of actin assembly in the permeabilized neutrophil. The dominant negative CDC42 and Rac2 proteins (N17) were tested downstream of the constitutively active version of the opposite partner. N17Rac2 (3 μM) inhibited >50% of the V12CDC42- (150 nM) mediated barbed-end increase, whereas N17CDC42 (3 μM) had no significant inhibitory effect on the Rac2Q61L- (150 nM) mediated increase in free barbed ends. Greater concentrations of either dominant negative did not lead to further inhibition. The results are mean ± SEM of at least three experiments.

Figure 5

A, Effect of V12Rac1 and V12Cdc42 concentration on barbed-end exposure in OG-permeabilized neutrophils. Cells were exposed to 0.4% OG for 10 s and then exposed to the indicated small GTPase concentration for 3 min. The results are mean ± SEM of at least three experiments. B, Comparison of the effect of dominant negative Rho GTPase mutants of Cdc42 (N17Cdc42) and Rac1 (N17Rac1) on FMLP-induced free barbed-end creation in permeabilized neutrophils. The dominant negative Rac1 inhibited almost two-thirds of the FMLP-mediated increase in free barbed ends, whereas the CDC42 inhibited all the FMLP-mediated increase. The results are mean ± SEM of two experiments. C, The hierarchy of CDC42 and Rac upstream of actin assembly in the permeabilized neutrophil. The dominant negative CDC42 and Rac2 proteins (N17) were tested downstream of the constitutively active version of the opposite partner. N17Rac2 (3 μM) inhibited >50% of the V12CDC42- (150 nM) mediated barbed-end increase, whereas N17CDC42 (3 μM) had no significant inhibitory effect on the Rac2Q61L- (150 nM) mediated increase in free barbed ends. Greater concentrations of either dominant negative did not lead to further inhibition. The results are mean ± SEM of at least three experiments.

Figure 5

A, Effect of V12Rac1 and V12Cdc42 concentration on barbed-end exposure in OG-permeabilized neutrophils. Cells were exposed to 0.4% OG for 10 s and then exposed to the indicated small GTPase concentration for 3 min. The results are mean ± SEM of at least three experiments. B, Comparison of the effect of dominant negative Rho GTPase mutants of Cdc42 (N17Cdc42) and Rac1 (N17Rac1) on FMLP-induced free barbed-end creation in permeabilized neutrophils. The dominant negative Rac1 inhibited almost two-thirds of the FMLP-mediated increase in free barbed ends, whereas the CDC42 inhibited all the FMLP-mediated increase. The results are mean ± SEM of two experiments. C, The hierarchy of CDC42 and Rac upstream of actin assembly in the permeabilized neutrophil. The dominant negative CDC42 and Rac2 proteins (N17) were tested downstream of the constitutively active version of the opposite partner. N17Rac2 (3 μM) inhibited >50% of the V12CDC42- (150 nM) mediated barbed-end increase, whereas N17CDC42 (3 μM) had no significant inhibitory effect on the Rac2Q61L- (150 nM) mediated increase in free barbed ends. Greater concentrations of either dominant negative did not lead to further inhibition. The results are mean ± SEM of at least three experiments.

Figure 6

A, Effect of inhibitory peptides on FMLP-induced actin nucleation in permeabilized neutrophils. VCA (400 nM), a peptide derived from WASP, and 30 nM FMLP (F) increase nucleation activity in OG permeabilized when compared with control untreated cells. Addition of the WASP-derived peptide, CA, reduces by ∼50% the ability of FMLP to nucleate actin in permeabilized neutrophils at saturating levels. N17Rac1 (2.7 μM) and CA peptide (3 μM) completely inhibit the FMLP-mediated increase in nucleating activity. The peptide GST-V (3 μM) demonstrated no inhibitory effect on the FMLP-mediated increase in free barbed ends. Data are the mean ± SEM from three separate experiments. B, Dose effect of the WASP-derived CA peptide on FMLP- or V12Rac1-induced actin nucleation. Each data point is from triplicate samples from a single experiment, representative of two experiments; mean ± SD. C, Effect of the WASP-derived CA peptide on actin nucleation induced by V12Rac1, V12CDC42, PIP₂, and GTPγS. CA (3 μM), a peptide derived from WASP, inhibits the actin nucleation activity of V12Rac1 (300 nM), V12CDC42 (300 nM), PIP2 (33 μM), and GTPγS (16 μM) in OG permeabilized by as much as two-thirds compared with control untreated levels.

Figure 6

A, Effect of inhibitory peptides on FMLP-induced actin nucleation in permeabilized neutrophils. VCA (400 nM), a peptide derived from WASP, and 30 nM FMLP (F) increase nucleation activity in OG permeabilized when compared with control untreated cells. Addition of the WASP-derived peptide, CA, reduces by ∼50% the ability of FMLP to nucleate actin in permeabilized neutrophils at saturating levels. N17Rac1 (2.7 μM) and CA peptide (3 μM) completely inhibit the FMLP-mediated increase in nucleating activity. The peptide GST-V (3 μM) demonstrated no inhibitory effect on the FMLP-mediated increase in free barbed ends. Data are the mean ± SEM from three separate experiments. B, Dose effect of the WASP-derived CA peptide on FMLP- or V12Rac1-induced actin nucleation. Each data point is from triplicate samples from a single experiment, representative of two experiments; mean ± SD. C, Effect of the WASP-derived CA peptide on actin nucleation induced by V12Rac1, V12CDC42, PIP₂, and GTPγS. CA (3 μM), a peptide derived from WASP, inhibits the actin nucleation activity of V12Rac1 (300 nM), V12CDC42 (300 nM), PIP2 (33 μM), and GTPγS (16 μM) in OG permeabilized by as much as two-thirds compared with control untreated levels.

Figure 6

A, Effect of inhibitory peptides on FMLP-induced actin nucleation in permeabilized neutrophils. VCA (400 nM), a peptide derived from WASP, and 30 nM FMLP (F) increase nucleation activity in OG permeabilized when compared with control untreated cells. Addition of the WASP-derived peptide, CA, reduces by ∼50% the ability of FMLP to nucleate actin in permeabilized neutrophils at saturating levels. N17Rac1 (2.7 μM) and CA peptide (3 μM) completely inhibit the FMLP-mediated increase in nucleating activity. The peptide GST-V (3 μM) demonstrated no inhibitory effect on the FMLP-mediated increase in free barbed ends. Data are the mean ± SEM from three separate experiments. B, Dose effect of the WASP-derived CA peptide on FMLP- or V12Rac1-induced actin nucleation. Each data point is from triplicate samples from a single experiment, representative of two experiments; mean ± SD. C, Effect of the WASP-derived CA peptide on actin nucleation induced by V12Rac1, V12CDC42, PIP₂, and GTPγS. CA (3 μM), a peptide derived from WASP, inhibits the actin nucleation activity of V12Rac1 (300 nM), V12CDC42 (300 nM), PIP2 (33 μM), and GTPγS (16 μM) in OG permeabilized by as much as two-thirds compared with control untreated levels.

Figure 7

Summary scheme of the pathway from the FMLP receptor to actin filament ends in OG-permeabilized neutrophils. The FMLP receptor leads to the small G protein Cdc42. The pathway then branches into a Rac-dependent path and a Rac-independent path that includes the ARP2/3 complex.