Proton-induced endocytosis is dependent on cell membrane fluidity, lipid-phase order and the membrane resting potential

AbstractRecently it has been shown that decreasing the extracellular pH of cells stimulates the formation of inward membrane invaginations and vesicles, accompanied by an enhanced uptake of macromolecules. This type of endocytosis was coined as proton-induced uptake (PIU). Though the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the dependence of the phenomenon on plasma membrane characteristics is still unknown. The present study shows that depolarization of the membrane resting potential elevates PIU by 25%, while hyperpolarization attenuates it by 25%. Comparison of uptake in suspended and adherent cells implicates that the resting-potential affects PIU through remodeling the actin-cytoskeleton. The pH at the external interface of the cell membrane rather than the pH gradient across it determines the extent of PIU. PIU increases linearly upon temperature increase in the range of 4–36°C, in correlation with the membrane fluidity. The plasma membrane fluidity and the lipid phase order are modulated by enriching the cell's membrane with cholesterol, tergitol, dimethylsulfoxide, 6-ketocholestanol and phloretin and by cholesterol depletion. These treatments are shown to alter the extent of PIU and are better correlated with membrane fluidity than with the lipid phase order. We suggest that the lipid phase order and fluidity influence PIU by regulating the lipid order gradient across the perimeter of the lipid-condensed microdomains (rafts) and alter the characteristic tension line that separates the higher ordered lipid-domains from the lesser ordered ones

Enhancement of Cell Membrane Invaginations, Vesiculation and Uptake of Macromolecules by Protonation of the Cell Surface

The different pathways of endocytosis share an initial step involving local inward curvature of the cell’s lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH≤3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell’s surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake

Dependence of PIU on extracellular pH.

<p>Adherent cell cultures (HaCaT, Caco-2/TC7, COS-7 and HT29/mtx) and non-adherent cultures (TK6) were exposed to solutions of different pH in the presence of dextran-FITC for a period of 5 minutes, before being washed, harvested and analyzed. Uptake, based on flow cytometry, is plotted as function of the external pH from 3 independent experiments per cell line (A) Extent of uptake in terms of fold of uptake (mean ± SD) relative to the constitutive uptake at physiological pH 7.4 (n = 9 for each cell line). (B) Fold of uptake (mean ± SD) relative to the constitutive uptake at physiological pH 7.4 (n = 9 for each cell line). At pH 6, the difference in intracellular dextran concentration among the cell lines is of border line significance (P = 0.056 by one way ANOVA). At pH 5, HaCaT and TK6 cells have 2 folds higher dextran concentration then Caco2/TC7 and COS-7 (P<0.001, <i>t</i>-test), which in turn have 2 folds higher dextran concentration than HT29 cells (P<0.001, <i>t</i>-test).</p

Determination of adsorbed VS internalized fraction of Dextran-FITC following uptake induced by low pH.

<p>(A1) HaCaT cells exposed to pH 5.25 in the presence of dextran-FITC were washed with K⁺PBS, and were analyzed three times by FACS, first at pH 7.4, second at pH 6.6 and third in the presence of 10 µM nigericin. The cells’ fluorescence intensities in the second and third analyses are presented as fraction of the first analysis. *P<0.05, n = 9) (A2) HaCaT cells exposed to pH 5.25 in the presence of dextran-FITC were washed with K⁺PBS, and were analyzed twice by FACS; first time at pH 7.4 and second time in the presence of 10 µM nigericin. The cells’ fluorescence intensities in the second analysis (in the presence of nigericin) are presented relative to the first analysis. *P<0.05, n = 9) (B) Microscopic images were acquired in the fluorescence (Em 530 nm) and DIC channels at X100 magnification. COS-7 cells were grown on glass bottom 96 wellplate and exposed to a solution of HBSS at pH 5.25 in the presence of 70 kD dextran-FITC (5 µM) for 15 minutes period, followed by washing with K⁺PBS at pH 7.4. (B1) Cultures incubated in K⁺PBS at pH 7.4. (B2) Cultures incubated in K⁺PBS at pH 6.0 (B3) Cultures incubated in K⁺PBS at pH 6 with 10 µM Nigiricin.</p

PIU as function of exposure to low pH in cells with depleted ATP or in the presence of verapamil.

<p>Suspended HaCaT cells, treated by 10 µM verapamil or depleted of their ATP pool, were exposed to extracellular pH 5.25 in the presence of dextran-FITC (70 kD, 10 µM), for 5–30 min durations. Results from FACS analyses are presented as fold induction relative to the constitutive uptake at pH 7.4 in terms of geometrical mean±SD. Linear regression of the treated cells with either verapamil or ATP depletion is R² = 0.99. n = 9.</p

Images of cells during exposure to low pH with the plasma membrane labeled by tannic acid.

<p>Areas of the plasma membrane comprising high incidence of inward budding (A and B, bar size = 500 nm). Plasma membrane invaginations exist in different morphologies such as rosettes (C) or chains (D) (bar size = 200 nm). Higher magnification of membrane buds in intricate arrangements (E and F, bar size = 100 nm). Ex – Extracellular space.</p

Cellular uptake of macromolecules following exposure to low pH.

<p>(A) Fluorescent images of HaCaT cells that were incubated with 10 µM dextran-FITC at either pH 7.4 (A1) or pH 5.25 (A2) at RT (bar size = 20 µm). The cells were incubated for 15 minutes in DMEM before being imaged. (B) SCLM images of HaCaT cells that were incubated with unspecific IgG-Cy5 (pseudo-colored in green) for 30 minutes at either pH 7.4 (B1) or pH 5.25 (B2). The cells were fixed with 4% paraformaldehyde and stained with DAPI (colored blue) and phalloidin-TRITC (colored red). Bar size for X–Z = 10 µm and for X–Y = 25 µm. (C) TEM images depicting gold particles (Au) in HaCaT cells that were incubated with unspecific IgG-Gold for 10 minutes at pH 5.25 and then fixed with karnovsky solution. C-2 (bar size = 200 nm) is a magnified section of C-1 (bar size = 500 nm).</p

Kinetic profile of PIU.

<p>(A) The relative intensities of dextran-FITC (70 kD, 10 µM) in HaCaT cells are presented as function of external pH and duration of exposure. Suspensions of cells were exposed to solutions of varying pH (7.4, 6.4, 6, 5.5 and 5) in the presence of dextran-FITC for 6 time periods (1, 5, 10, 20, 30 and 60 min). Results from FACS analyses are presented as fold induction relative to the constitutive uptake at physiological pH 7.4, in terms of geometrical mean±SD (n = 15 per each pH level). (B) The rate of dextran PIU is presented as function of the time the cells were exposed to low pH. Rate was determined by introducing Dextran-FITC as a short pulse for the last 10 min of the cells’ exposure to pH 5.25. Results from FACS analyses are presented as fold induction relative to the constitutive uptake at physiological pH 7.4, in terms of geometrical mean±SD (n = 9). (C) The relative attenuation of the intracellular intensity of dextran-FITC, as function of incubation time and temperature. The cells were initially loaded with Dextran-FITC by mean of PIU. The time dependent exponential decrease in cellular fluorescence suggests the involvement of efflux mechanisms which are concentration dependent, while the dependence on temperature suggests the efflux to be a methabolic driven process. Cell suspensions were pre-loaded with dextran-FITC (70 kD, 10 µM) by exposure to extracellular pH 5.25 for 10 min. The cultures were then washed in fresh HBSS and divided into two groups, incubated at either 37°C or 4°C for four time periods (15, 60, 120 and 180 min). Results from FACS analysis are presented as fold induction relative to the fluorescent intensity of cells that were analyzed 15 min after extracellular solution was restored to pH 7.4, in terms of geometrical mean±SD (n = 9 in 3 independent experiments).</p

The rate of PIU as function of exposure time to low pH.

<p>HaCaT cells were harvested, suspended in HBSS and exposed to pH 5.25 in the presence of 1µM 43kDa dextran-FITC. Following treatment cells were analyzed by FACS calibrated with MESF microspheres. (A) The cells were exposed to pH 5.25 for total periods of 5, 10 15 and 20 minutes where dextran-FITC was added only for the last 5 minutes. The exposure was terminated by dilution in a large volume of cold DMEM. Results of the FACS analysis are given in terms of the number of dextran molecules per cell (mean±SD). The difference between the four groups is insignificant (P>0.05 in One-way ANOVA, n = 12).</p

Different morphologies of membranous structures induced by low pH.

<p>TEM images from cells exposed to external low pH (A to E, bar size = 200 nm). (F) Schematic representation of membranous structures of different morphologies: plasma membrane buds (a), tubules (b), vesicles connected to the plasma membrane (c), clusters of membrane vesicles (d) and inward protrusions of the plasma membrane with buds and connected vesicles (e). Ex – Extracellular space.</p