Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C

Abstract

Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85 alpha constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and phosphatidylinositol-specific phospholipase C (PI-PLC), as assayed by 3-phosphoinositide synthesis in situ and in vitro and inositol 1, 4,5 trisphosphate steady-state levels, respectively; they were abolished by stable transfection of v-Src-transformed cells for dominant-negative truncated p85 alpha expression and by pharmacological inhibitors of PI3K and PI-PLC, indicating a requirement for both enzymes. Whereas PI3K activation resisted PI-PLC inhibition, PI-PLC activation was abolished by a PI3K inhibitor and dominant-negative transfection, thus placing PI-PLC downstream of PI3K. Together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of the actin cytoskeleton in oncogene-transformed fibroblasts, resulting in constitutive ruffling and macropinocytosis.

Figure 1

Constitutive stimulation of fluid-phase, but not receptor-mediated, endocytosis by v-Src and K-Ras. Control (○), v-Src–transformed (▪), or K-Ras–transformed (●) Rat-1 fibroblasts were transferred for 30 min in DMEM without serum and then incubated in DMEM supplemented with 4 mg/ml HRP for the indicated intervals of pulse (A) or 30 min (C) or with 50 nM ¹²⁵I-transferrin and 1% BSA for the indicated intervals of pulse (B) or 7 min (D). Cells in C and D were briefly washed at 4°C and reincubated in DMEM at 37°C for the indicated intervals of chase. Finally, cells were washed extensively at 4°C and surface-digested with pronase. Values of intracellular accumulation of HRP were normalized as indicated in MATERIALS AND METHODS. Intracellular ¹²⁵I-transferrin accumulation was divided by pronase-releasable counts (considered as surface-bound) to yield receptor-mediated endocytosis efficacy. In C and D, residual intracellular contents nor-malized as described above were expressed as percentages of corresponding values at the end of the chase, and curves were fitted to monoexponential decays. Values shown are means ± SD of three dishes from one experiment. Kinetics of uptake and chase was reproduced at least three times and twice, respectively.

Figure 2

Scanning electron microscopy. (A) Control Rat-1 fibroblasts; (B and F) v-Src fibroblasts; (C and G) K-Ras fibroblasts; (D and H) Wp85α fibroblasts; (E) v-Src/Δp85α fibroblasts.

Figure 3

Formation of a macropinosome by membrane ruffling in a v-Src–transformed fibroblast. (A) Bright-field contrast image of overlapping cellular extensions from two adjacent v-Src fibroblasts. The extension in the focal plane contains four round electron-lucent vacuoles of irregular size, and its tip shows active ruffling. The 10 smaller images below are taken from the same field at 10-s intervals. At the ruffling membrane, a large lamellipodium (arrows in B–E) appears to fold back and to generate an ovoid macropinosome (arrow in G), which rounds up within ∼10 s (H), then docks to (I) and fuses with (J) a preexisting macropinosome. The fused organelle similarly rounds up within 10 s (K). Bars, 10 μm.

Figure 4

Filling of macropinosomes by a fluorescent fluid-phase tracer. The indicated cells were incubated for 7 min in DMEM supplemented with 1 mg/ml Texas Red–dextran, washed, and examined immediately in the confocal microscope by fluorescence and bright-field imaging. Fused images are presented. Extensions with ruffled ends that contain several aligned macropinosomes are evident in v-Src (B) and especially Wp85α (C) fibroblasts. They are not seen in control (A) and v-Src/Δp85α (D) fibroblasts. The two nonfluorescent distal macropinosomes in B were presumably formed after washing (see also Veithen et al., 1998).

Figure 5

Actin cytoskeleton reorganization. F-actin was decorated with rhodamine–phalloidin in untreated control (A), v-Src (B), K-Ras (E), Wp85α (C), v-Src/Δp85α (D), or wortmannin-treated (100 nM for 30 min) Wp85α (F) fibroblasts. Stress fibers are almost absent in v-Src (B), K-Ras (E), and Wp85α (C) fibroblasts but reappear in these cells within 30 min after PI3K inhibition by wortmannin (F) and are prominent in stable v-Src/Δp85α transfectants (D). Bars, 10 μm.

Figure 6

Constitutive macropinocytosis is abrogated by cytochalasin E in transformed fibroblasts. Control (white bars), v-Src (black bars), and K-Ras (hatched bars) fibroblasts were preincubated for 30 min in DMEM without serum supplemented with the indicated concentrations of cytochalasin E and then incubated for another 30 min in the same medium supplemented with 4 mg/ml HRP. Peroxidase accumulation is calculated as in Figure 1. Values are means ± SD of three dishes.

Figure 7

Constitutive macropinocytosis, but not receptor-mediated endocytosis, is abrogated by pharmacological inhibition of PI3K in transformed fibroblasts. Control (○), v-Src (▪), and K-Ras (●) fibroblasts were preincubated for 30 min in DMEM without serum supplemented with the indicated concentrations of wortmannin or LY294002 and then incubated for another 30 min in the same medium with 4 mg/ml added HRP (A and B) or for 10 min with 50 nM added ¹²⁵I-transferrin and 1% BSA (C). Intracellular tracer accumulation is calculated as in Figure 1. Values are means ± SD of three dishes. The concentration-dependence of the effects of PI3K inhibitors on control and v-Src–transformed fibroblasts was reproduced at least three times.

Figure 8

p85 expression. Equal loads of the indicated cell lysates were analyzed by Western blotting with a polyclonal antiserum against rat p85.

Figure 9

Stable Wp85α transfection of control Rat-1 fibroblasts accelerates fluid-phase endocytosis. Control (○) or Wp85α fibroblasts (▴) were preincubated for 30 min in DMEM without serum, further incubated for the indicated times of pulse in the same medium containing HRP (A) or for 30 min with HRP, washed, and then chased for the indicated times in DMEM (B). Intracellular tracer accumulation is calculated as in Figure 1. Values are means ± SD of four dishes.

Figure 10

PI-PLC inhibitors abrogate constitutive macropinocytosis in v-Src–transformed and Wp85α stably transfected fibroblasts. (A and B) Control or Src fibroblasts were preincubated for 30 min in DMEM without serum supplemented with the indicated concentrations of inhibitors and then incubated for another 30 min in the same medium with 4 mg/ml added HRP. (C) Rat-1/v-Src and Rat-1/Wp85α fibroblasts were preincubated for the indicated times with 100 μM NCDC, and HRP was added for the last 30 min of incubation. Values are means ± SD of four dishes. The concentration-dependence of the effects of PI-PLC inhibitors on control and v-Src–transformed fibroblasts was reproduced at least three times.

Figure 11

PI3K activity correlates with constitutive macropinocytosis. (A) PI3K was assayed in situ and compared with peroxidase accumulation. The indicated cells were incubated for 6 h in phosphate-free DMEM containing 0.5% dialyzed FCS and 250 μCi/ml [³²P]orthophosphate. Inhibitors were added 30 min before this incubation and maintained throughout. After a brief wash, cell lipids were extracted and phosphoinositides were analyzed by HPLC and expressed in absolute counts. ND, not detectable. Data for PtdIns(3,4)P2 levels are from one experiment representative of two to five experiments. Counts of PtdIns(3,4,5)P3 are from the same experiment, which provided the best resolution of this minor compound. These values are compared with macropinocytosis, measured after cells were incubated for 30 min in DMEM without serum in the presence or absence of the inhibitors and then for another 30 min with HRP, as in Figure 1. Values are means ± SEM of 16 to 89 dishes compiled from all experiments performed for this paper. (B) PI3K was assayed in vitro in the indicated cell lines after IRS-1 immunoprecipitation. Values are means ± SEM of five to nine dishes per condition.

Figure 12

PI-PLC is downstream of PI3K in the same signaling pathway to constitutive macropinocytosis. In situ phospholipase activity was determined by intracellular IP3 levels with the use of a competitive radioligand-binding assay. The indicated cells were preincubated at 37°C for 1 h with DMEM without serum supplemented with the indicated concentrations of inhibitors. All values are means ± SEM of four dishes pooled from two separate experiments.