A unique platform for H-Ras signaling involving clathrin-independent endocytosis

Abstract

Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.

Figure 1.

H-Ras traffics with the CIE pathway. (A) Internalization of MHCI antibody and 633-labeled Tfn for 30 min was performed in HeLa cells expressing GFP-H-Ras. Internalized MHCI was visualized with 594-labeled secondary antibodies. H-Ras was both associated with incoming MHCI-positive endosomes (arrowhead) and the characteristic CI recycling tubular endosomes that lack Tfn (see inset). (B) Coexpression of RFP-H-Ras and Arf6Q67L-GFP was evaluated in live HeLa cells. Inset shows RFP-H-Ras localization to vacuoles. (C) HA-PLD2 was coexpressed with GFP-H-Ras in HeLa cells and processed for immunofluorescence as described in Materials and Methods. Inset shows HA-PLD2 and GFP-H-Ras colocalized at the PM and on the CI recycling tubular endosomes. (D) Activation of RFP-H-Ras in live COS-7 cells was observed using the Ras-GTP detector, RBD (Ras binding domain) fused to GFP. As soon as 30 s after EGF (100 ng/ml) application, the RBD was recruited to the PM. At 310 s, membrane ruffles were seen; at 390 s, a macropinosome was formed and H-Ras-GTP was on it as indicated by the RBD-GFP. Selected frames from a time-lapse movie (Supplemental Video 1) are shown. Note that in the figure, time between frames varies. Bars, 10 μm. (E) H-Ras-GTP was assessed by a pull-down assay with GST-RBD. H-Ras or H-Ras- and Arf6Q67L-expressing cells were serum starved for 1 h, treated with EGF for 10 min as indicated, and subjected to the GST-RBD pull-down assay. The fraction of GTP-bound H-Ras is plotted as the average ± SD of three independent experiments.

Figure 2.

H-RasG12V traffics with the CIE pathway and stimulates macropinocytosis. (A) Internalization of MHCI antibody and 633-Tfn was performed in HeLa cells expressing GFP-H-RasG12V. Internalized MHCI was visualized with 594-labeled secondary antibodies. Macropinosomes were observed after 30 min of internalization and contained MHCI, but not Tfn (see inset). (B) GFP-H-RasG12V was expressed in HeLa cells, and selected frames from a time-lapse movie (Supplemental Video 2) are shown. Note that Video 2 is cropped. (C) HeLa cells expressing GFP-H-RasG12V were treated with 200 nM CD for 20 min, fixed, and examined. (D) GFP-H-RasG12V was expressed with Arf6T27N-HA in HeLa cells and processed for immunofluorescence. (E) RFP-H-RasG12V was coexpressed with Arf6Q67L-GFP in HeLa cells and imaged live. Bars, 10 μm. (F) Quantification of proportion of tubular endosomes observed in cells expressing GFP-tH, GFP-H-Ras, GFP-H-RasG12V, and GFP-H-RasG12V after 20-min treatment with CD. Plotted are the average ± SD from two independent experiments.

Figure 3.

Phosphoinositide dynamics on incoming macropinosomes. Untagged H-RasG12V was expressed in COS-7 cells to induce macropinocytosis together with PH-PLCδ-GFP and PH-Akt-RFP to observe the lipid content of macropinosomes. Selected frames from a time-lapse movie at 20-s intervals (Supplemental Video 3) are shown. Newly formed macropinosomes were labeled both with the PH-PLCδ-GFP and PH-Akt-RFP, indicating the presence of PIP₂ and PIP₃, respectively. Bars, 10 μm.

Figure 4.

Rab5 recruitment onto macropinosomes by H-RasG12V is dependent on loss of PIP₂, but overlaps with PIP₃. (A) GFP-Rab5 was coexpressed in HeLa cells with either RFP-H-Ras (top row) or RFP-H-RasG12V (bottom row). RFP-H-Ras was localized to the PM and CI recycling tubular endosomes (see inset), whereas Rab5 was on discrete endosomes primarily in the juxtanuclear region. When coexpressed with RFP-H-RasG12V, Rab5 distribution was altered; it was recruited onto newly formed macropinosomes (see inset). (B and C) The recruitment of GFP-Rab5 onto macropinosomes formed in COS-7 cells expressing untagged H-RasG12V and either PH-PLCδ-CFP (PIP₂) (B) or PH-Akt-RFP (PIP₃) (C) was examined by live cell imaging (Supplemental Videos 4 and 5, respectively). Selected frames, at 20-s intervals, from a time-lapse movie are shown; arrowheads point to macropinosomes. CFP is pseudocolored red here. Bars, 10 μm.

Figure 5.

Model for macropinosome maturation and distinct signaling platforms for H-Ras. (A) The model depicts three distinct stages of H-RasG12V–induced macropinosome maturation, with specific lipid composition and unique signaling molecules they contain. Actin is required for the formation of a macropinosome (labeled in orange). PIP₂, PIP₃, and H-Ras* (indicating active H-Ras or H-RasG12V) are on the plasma membrane and on nascent macropinosomes. Expression of Arf6Q67L, which blocks CIE right after internalization, captures this newly formed macropinosome. In the second stage of maturation, the macropinosome loses the PIP₂ and acquires Rab5 that overlaps with PIP₃. At the last stage, PIP₃ is lost from the membranes, and Rab5 might contribute to the sorting of the cargo. H-Ras* was observed at all stages of macropinosomes maturation. (B and C) Arf6Q67L vacuoles capture the first stage in macropinosome maturation and help identify unique signaling molecules present on this platform. The distribution of different Ras effectors in cells expressing untagged Arf6Q67L and either H-RasG12V (B) or H-Ras (C). The RBD-GFP was recruited onto Arf6Q67L vacuoles by GFP-H-RasG12V, but not GFP-H-Ras (see insets, top rows, B and C). In B, arrow points to a nontransfected cell labeled with phospho-Erk antibody. Endogenous (active) pErk and pAkt were detected with antibodies on the vacuoles in the presence of GFP-H-RasG12V (insets, middle rows, B), but not GFP-H-Ras (insets, middle rows, C). Neither GFP-H-RasG12V nor GFP-H-Ras could recruit Rab5 onto the Arf6Q67L vacuoles (insets, bottom row, B and C). All images with individual effectors were taken with identical acquisition parameters. Bars, 10 μm.

Figure 6.

Macropinosomes generated by EFA6 contain PIP₂ and PIP₃, and they acquire Rab5 after loss of PIP₂. (A) EFA6 was expressed in COS-7 cells to induce macropinocytosis together with PH-PLCδ-GFP and PH-Akt-RFP to observe the lipid content of macropinosomes. Selected frames from a time-lapse movie at 20-s intervals (Supplemental Video 6) are shown. Newly formed macropinosomes were labeled both with the PH-PLCδ-GFP and PH-Akt-RFP, indicating the presence of PIP₂ and PIP₃, respectively. (B) The recruitment of GFP-Rab5 onto macropinosomes formed in COS-7 cells expressing EFA6 in the background and PH-PLCδ-CFP (PIP₂) was examined by live cell imaging (Supplemental Video 7). Selected frames, at 20-s intervals, from a time-lapse movie are shown; arrowheads point to macropinosomes. CFP is pseudocolored red here. Bars, 10 μm.

Figure 7.

H-RasG12V- but not EFA6-expressing cells have Akt associated with the macropinosomes. (A) RFP-H-RasG12V was expressed in COS-7 cells to induce macropinocytosis together with Akt1-GFP. Selected frames from a time-lapse movie at 50-s intervals (Supplemental Video 8) are shown. Newly formed macropinosomes were labeled both with the RFP-H-RasG12V and Akt1-GFP after the loss of Akt1-GFP (arrowhead). (B) EFA6 was expressed in COS-7 cells to induce macropinocytosis together with RFP-tH that labels macropinosomes and Akt1-GFP. Selected frames from a time-lapse movie at 20-s intervals (Supplemental Video 9) are shown. RFP-tH and Akt1-GFP colocalized in membrane ruffles, whereas newly formed macropinosomes were not labeled with Akt1-GFP (arrowhead). Bars, 10 μm.