Koivusalo M, Welch C, Hayashi H et al.Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J Cell Biol 188:547-563

Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
The Journal of Cell Biology (Impact Factor: 9.83). 02/2010; 188(4):547-63. DOI: 10.1083/jcb.200908086
Source: PubMed


Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na(+)/H(+) exchange. Yet, the functional relationship between Na(+)/H(+) exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na(+)/H(+) exchange, elevating cytosolic pH and stimulating Na(+) influx. Remarkably, although inhibition of Na(+)/H(+) exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na(+) influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na(+)/H(+) exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na(+)/H(+) exchange.

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    • "Indeed, while both shLuc and shR1 cells had similar amounts of total Rac1 (Input), shR1 cells had twice as much Rac1-GTP than shLuc cells (Fig. 4E). Therefore, we used the Rac1 blocker amiloride (Koivusalo et al., 2010) to assess whether Rac1 inhibition can change the effect of reggie-1 knockdown on FAs. The number of FAs in shR1 cells (but not in shLuc cells) was reduced to near normal, when cells were treated for 1 h with amiloride (Fig. 4F, G). "
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    ABSTRACT: Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and β1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled β1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or β1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and β1-integrins to FAs to regulate FA formation and membrane dynamics. Copyright © 2015 Elsevier GmbH. All rights reserved.
    European Journal of Cell Biology 07/2015; DOI:10.1016/j.ejcb.2015.07.003 · 3.83 Impact Factor
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    • "Transgenic Drosophila lines expressing the pH sensor soluble mCherry-pHluorin (Koivusalo et al., 2010) (UAS-mCherry-pHluorin) were crossed to GMR-GAL4 to express the pH biosensor specifically in the developing eye. pH measurement were determined as described (Grillo-Hill et al., 2014). "
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    ABSTRACT: Intracellular pH (pHi) dynamics is increasingly recognized as an important regulator of a range of normal and pathological cell behaviors. Notably, increased pHi is now acknowledged as a conserved characteristic of cancers and in cell models is confirmed to increases proliferation and migration as well as limits apoptosis. However, the significance of increased pHi for cancer in vivo remains unresolved. Using Drosophila melanogaster, we show that increased pHi is sufficient to induce dysplasia in the absence of other transforming cues and potentiates growth and invasion with oncogenic Ras. Using a genetically encoded biosensor we also confirm increased pHi in situ. Moreover, in Drosophila models and clonal human mammary cells we show that limiting H(+) efflux with oncogenic Raf or Ras induces acidosis and synthetic lethality. Further, we show lethality in invasive, primary tumor cell lines with inhibiting H(+) efflux. Synthetic lethality with reduced H(+) efflux and activated oncogene expression could be exploited therapeutically to restrain cancer progression while limiting off-target effects.
    eLife Sciences 03/2015; 4(4). DOI:10.7554/eLife.03270 · 9.32 Impact Factor
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    • "At lower concentrations (0.2–0.5 μM), ES3H was likely to enter the cells through a direct, energy-free mechanism; but at higher concentrations (0.5–2 μM), its translocation was an energy-dependent process, likely endocytosis induced by a specific agonistreceptor binding. In one previous study, stimulation by EGF in A431 cells was able to induce macropinocytosis (Hamasaki et al. 2004), a special mode of endocytosis that can be inhibited by actin and Na + /H + exchange inhibitors (Koivusalo et al. 2010). The translocation of S3-HBD Fig. 5 Internalization kinetics and pathway of ES3H. a Time dependency of ES3H. "
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    ABSTRACT: Cell-penetrating peptides (CPPs) are well known as intracellular delivery vectors. However, unsatisfactory delivery efficiency and poor specificity are challenging barriers to CPP applications at the clinical trial stage. Here, we showed that S3, an EGFR-binding domain derived from vaccinia virus growth factor, when fused to a CPP such as HBD or TAT can substantially enhance its internalization efficiency and tumor selectivity. The uptake of S3-HBD (S3H) recombinant molecule by tumor cells was nearly 80 folds increased compared to HBD alone. By contrast, the uptake of S3H by non-neoplastic cells still remained at a low level. The specific recognition between S3 and its receptor, EGFR, as well as between HBD and heparan sulfate proteoglycans on the cell surface was essential for these improvements, suggesting a syngeneic effect between the two functional domains in conjugation. This syngeneic effect is likely similar to that of the heparin-binding epidermal growth factor, which is highly abundant particularly in metastatic tumors. The process that S3H entered cells was dependent on time, dosage, and energy, via macropinocytosis pathway. With excellent cell-penetrating efficacy and a novel tumor-targeting ability, S3H appears as a promising candidate vector for targeted anti-cancer drug delivery.
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