Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling

Koivusalo, Mirkka; Welch, Christopher; Hayashi, Hideharu; Scott, Cameron C.; Kim, Moshe; Alexander, R. Todd; Touret, Nicolas; Hahn, Klaus M.; Grinstein, Sergio

doi:10.1083/jcb.200908086

Cited by 755 publications

(564 citation statements)

References 82 publications

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“…Ecliptic pHluorin has a pK a of $7.1 and fluorescence also increases with increasing pH at 410 nm excitation; however, its emission spectrum does not have an isosbestic point to normalize intensity to protein abundance. In more recent studies, this limitation has been addressed by fusing ecliptic pHluorin and pH-insensitive mCherry (Koivusalo et al, 2010). This construct is easy to use with most fluorescence microscopes because ratiometric images can be obtained with GFP and mCherry filter sets, while filter sets for ratiometric pHluorin are distinct.…”

Section: Genetically Encoded Ph Sensorsmentioning

confidence: 99%

“…23.2C; Choi, Webb, Chimenti, Jacobson, & Barber, 2013;Miesenböck et al, 1998) reveals an increase of focal adhesion pH during cell migration of mammalian cells. Further, targeting mCherry-pHluorin to the membrane by fusing the probe to the membrane-targeting sequence of Lyn demonstrated that changes in pH during macropinocytic lamellipodia extension are more profound in the immediate vicinity of the membrane (Koivusalo et al, 2010).…”

Section: Subcellular Ph Measurementsmentioning

confidence: 99%

“…• Plasmid encoding a genetically encoded pH biosensor, such as mCherrypHluorin (Denker et al, 2011;Koivusalo et al, 2010) • Polypropylene round-bottom tube, 5 ml (Falcon, catalog number 352063) • Warm media to 37 C in a water bath before use.…”

Section: Materials Requiredmentioning

confidence: 99%

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Ratiometric Imaging of pH Probes

Grillo-Hill

Webb

Barber

2014

Methods in Cell Biology

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Section: Genetically Encoded Ph Sensorsmentioning

confidence: 99%

Section: Subcellular Ph Measurementsmentioning

confidence: 99%

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Ratiometric Imaging of pH Probes

Grillo-Hill

Webb

Barber

2014

Methods in Cell Biology

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“…Next, we assessed the internalization mechanisms of pep- 15) Therefore, in order to confirm the internalization pathway of peptide 8, the cells were incubated with the tested peptides in the presence of several endocytosis inhibitors (amiloride, 16) an inhibitor of macropinocytosis; nystatin, 17) an inhibitor of caveolae-mediated endocytosis; and sucrose, 18) an inhibitor of clathrin-mediated endocytosis), and the intracellular uptake was measured by flow cytometry (Fig. 5a).…”

Section: Resultsmentioning

confidence: 99%

Structural Development of Cell-Penetrating Peptides Containing Cationic Proline Derivatives

Kobayashi

Misawa²,

Oba

et al. 2018

CHEMICAL & PHARMACEUTICAL BULLETIN

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We designed and synthesized a series of cell-penetrating peptides containing cationic proline derivatives (Pro Key words cell-penetrating peptide; cationic proline derivative; cellular environment; drug delivery system (DDS) carrier Cell penetrating peptides (CPPs) are useful tools in delivering hydrophilic cargo molecules such as proteins and nucleic acids into cells. It is well known that human immunodeficiency virus (HIV)-1 Tat and oligoarginine (Rn), which are representative CPPs contain cationic amino acids.1,2) Recently, it has been reported that the CPPs exert their cell membrane permeability based on the interaction between the side-chain guanidino groups of arginine and acidic groups existing on the cell surface.3,4) However, the influences of the secondary structure of CPPs on their cell membrane permeability has not been fully elucidated. To data, several methods to control peptide secondary structures have been developed. The introduction of unnatural amino acids, such as α,α-disubstituted amino acids (dAAs) or cyclized β-amino acids have been shown to enhance peptide helical structures. 5-7)To investigate the effects of the secondary structure of CPPs on their cell membrane permeability, we designed and synthesized novel CPP derivatives containing dAAs residues such as α-amino-isobutyric acid (Aib). 8) For example, the nonapeptides FAM-βAla-(L-Arg-L-Arg-Aib) 3 -NH 2 (1, FAM: 6-fluorescein), FAM-βAla-(L-Arg-D-Arg-Aib) 3 -NH 2 (2) and FAM-βAla-(D-Arg-D-Arg-Aib) 3 -NH 2 (ent-1) were synthesized, and their cell-penetrating ability were evaluated. The studies revealed that the peptides 1 and ent-1, which formed a righthanded (P) and a left-handed (M) helical structure, respectively, showed higher cell-penetrating ability than peptide 2, which formed a random structure. These results demonstrated that formation of a helical structure is favorable to exert the cell permeability of Arg based CPPs. Furthermore, we reported that peptides containing cationic cyclic dAA residue) 3 -NH 2 ] formed a stable helical structure and efficiently delivered plasmid DNA (pDNA) into cells.9) Moreover, we also reported that a series of CPPs containing the cationic proline derivative FAM-βAla-(L-Arg-L-Arg-Pro 4SGu

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“…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.…”

Section: Role Of Rac1 Activationmentioning

confidence: 99%

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

Hülsbusch

Solis

Katanaev

et al. 2015

European Journal of Cell Biology

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a b s t r a c tReggies/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 ␣5-and ␤1-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 ␤1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and ␣5-or ␤1-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 ␣5-and ␤1-integrins to FAs to regulate FA formation and membrane dynamics.

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Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling

Abstract: Inhibitors of Na+/H+ exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases.

Cited by 755 publications

References 82 publications

Ratiometric Imaging of pH Probes

Ratiometric Imaging of pH Probes

Structural Development of Cell-Penetrating Peptides Containing Cationic Proline Derivatives

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

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