Mapping the electrostatic profiles of cellular membranes

Abstract

Anionic phospholipids can confer a net negative charge on biological membranes. This surface charge generates an electric field that serves to recruit extrinsic cationic proteins, can alter the disposition of transmembrane proteins and causes the local accumulation of soluble counterions, altering the local pH and the concentration of physiologically important ions such as calcium. Because the phospholipid compositions of the different organellar membranes vary, their surface charges are similarly expected to diverge. Yet, despite the important functional implications, remarkably little is known about the electrostatic properties of the individual organellar membranes. We therefore designed and implemented approaches to estimate the surface charges of the cytosolic membranes of various organelles in situ in intact cells. Our data indicate that the inner leaflet of the plasma membrane is most negative, with a surface potential of approximately -35 mV, followed by the Golgi complex > lysosomes > mitochondria ≈ peroxisomes > endoplasmic reticulum, in decreasing order.

FIGURE 1:

Distribution of prenylated charge probes in RAW264.7 cells. Confocal images of RAW264.7 macrophages transiently cotransfected with one of the prenylated surface charge probes (+2-Pre-GFP, leftmost column; +4-Pre-GFP, second column from left; +6-Pre-GFP, third column; +8-Pre-GFP, rightmost column) and an organelle-specific marker (mCherry-Sec61, an ER marker, top row; PM-RFP, a plasmalemmal marker, middle row; sialyltransferase-RFP, a Golgi marker, bottom row). Here and elsewhere, confocal images are representative of at least 20 cells from three different experiments for each condition. The smaller images at the top of each condition show the individual channels (charge probes: green; organellar markers: red), while the main (larger) panels show merged images.

FIGURE 2:

Distribution of prenylated charge probes in HeLa cells. Confocal images of HeLa cells transiently cotransfected with one of the prenylated surface charge probes (+2-Pre-GFP, leftmost column; +4-Pre-GFP, second column from left; +6-Pre-GFP, third column; +8-Pre-GFP, rightmost column) and an organelle-specific marker (mCherry-Sec61, an ER marker, top row; PM-RFP, a plasmalemmal marker, middle row; sialyltransferase-RFP, a Golgi marker, bottom row). The smaller images at the top of each condition show the individual channels (charge probes: green; organellar markers: red), while the main (larger) panels show merged images.

FIGURE 3:

Distribution of amphiphilic helical charge probes in RAW264.7 cells. Confocal images of RAW264.7 macrophages transiently cotransfected with one of the surface charge probes (+2-helix-GFP, leftmost column; +4-helix-GFP, second column from left; +6-helix-GFP, third column; +8-helix-GFP, rightmost column) and an organelle-specific marker (mCherry-Sec61, an ER marker, top row; PM-RFP, a plasmalemmal marker, middle row; sialyltransferase-RFP, a Golgi, bottom row). The smaller images at the top of each condition show the individual channels (charge probes: green; organellar markers: red), while the main (larger) panels show merged images.

FIGURE 4:

Distribution of amphiphilic helical charge probes in HeLa cells. Confocal images of HeLa cells transiently cotransfected with one of the surface charge probes (+2-helix-GFP, leftmost column; +4-helix-GFP, second column from left; +6-helix-GFP, third column; +8-helix-GFP, rightmost column) and an organelle-specific marker (mCherry-Sec61, an ER marker, top row; PM-RFP, a plasmalemmal marker, middle row; sialyltransferase-RFP, a Golgi marker, bottom row). The smaller images at the top of each condition show the individual channels (charge probes: green; organellar markers: red), while the main (larger) panels show merged images.

FIGURE 5:

Quantification of charge probe distribution in different membrane organelles. (A) Diagrammatic representation of the procedure used to quantify the area of the cytosolic aspect of cellular membranes. Membranes are transiently permeabilized by activation of P₂X₇ receptors with ATP, exposed to the impermeant solvochromic dye FM4-64, and then resealed, followed by removal of extracellular FM4-64. (B, C) Representative confocal images of a RAW264.7 cell incubated with FM 4-64 and acquired in the presence of the dye, B, or after washing, C. (D) Representative confocal image of a RAW264.7 cell that was permeabilized by activating P₂X₇ receptors and labeled with FM 4-64 while permeabilized. (E) The cell shown in D was induced to reseal by removal of ATP and reintroduction of divalent cations while in the presence of FM4-64, followed by washing of the extracellular FM4-64. Images in B–E are representative of at least 20 cells from three different experiments for each condition. (F) Quantification of the ratio of the fluorescence of the charge probes to that of FM4-64. Cells were cotransfected with the indicated charge probe and organellar marker, permeabilized to allow partition of FM4-64 to the cytosolic leaflet of all endomembranes, sealed, and washed to remove extracellular FM4-64, before the fluorescence of all three fluorophores was measured. Results are presented as the fraction of the fluorescence intensity of the probe in each organelle divided by the fraction of the surface area occupied by the organelle (FM4-64 labeling).

FIGURE 6:

Measurement of probe dissociation rate using a rapamycin heterodimerization system. (A) Diagrammatic representation of the system used to assess the rate of dissociation of charge probes from the membrane. Surface charge probes tagged with GFP and an FRB domain were transfected into cells, along with RFP and FKBP-tagged Tom70 (FKBP-Tom70-RFP), which localizes to mitochondria. Because the charge probe interacts reversibly with the membrane, it can be recruited to mitochondria irreversibly upon addition of rapamycin. The rate of detachment from the membrane (K_off) is dictated by the membrane surface charge. (B, C) Confocal images of HeLa cells cotransfected with an FRB- and GFP-tagged +8 helical charge probe and FKBP-Tom70-RFP, before, B, and 80 s after addition of 10 µM rapamycin, C. (D, E) Confocal images of HeLa cells cotransfected with a soluble FRB- and RFP-tagged probe (FLEX-RFP) and a nonfluorescent plasma membrane–targeted FKBP-tagged construct (FKBP-Lyn), before, D, and 40 s after addition of 10 µM rapamycin, E. Images in B–E are representative of at least 15 cells from at least three different experiments for each condition. (F) Comparison of the rate of rapamycin-induced dissociation of FRB- and GFP-tagged +8 and +4 helical charge probes from the plasma membrane with the rate of disappearance of FLEX-RFP from the cytosol due to rapamycin-induced recruitment to the plasma membrane. Exponential decay curves were fitted using Prism software. (G) Quantification of the rate of dissociation of the FRB- and GFP-tagged +8 helical charge probe from the membrane as a function of the concentration of rapamycin added to induce its recruitment to mitochondria. The inverse of the t_1/2 for recruitment (in s^–1) is plotted vs. the concentration of rapamycin. Data are means ± SE of three independent experiments and the curve was fitted to a one-site saturable binding model using Prism

FIGURE 7:

Measurement of the dissociation rate of the +8 charge probe from artificial liposomes and its relationship to the zeta potential. (A) Diagrammatic representation of the procedure used to measure the rate of dissociation of the +8 helical charge probe from GUVs. GUVs were generated using varying ratios of PtdCho and PtdSer, containing in addition Cy3-labeled PtdEth to enable visualization by fluorescence microscopy and biotinylated PtdEth to facilitate their attachment to avidin-coated coverslips. The GUVs were allowed to equilibrate with the green fluorescent charge probe and dissociation was initiated by washing the probe off with a large volume of medium. Repeated image acquisition was used to assess the rate of dissociation, ensuring that photobleaching was negligible. (B–D) Dissociation of FITC-labeled +8-helical synthetic peptide from a GUV containing 20% PtdSer. (B) Image of the GUV equilibrated with 50 ng/ml in the labeled probe. The outline of the GUV, visualized as the emission of Cy3-PtdEth, is shown in the inset. (C, D) Images acquired 60 s, C, and 120 s, D, after the unbound probe was washed. Images are representative of 15 similar determinations. (E) Zeta potential measurements of liposomes containing 0, 10, 20, and 30% PtdSer. (F) t_1/2 of the dissociation rate of +8-helical (black bars) and +4-helical synthetic (gray bars) probes from GUVs containing 10, 20 and 30% PtdSer. Data are means ± SE of at least 15 similar determinations for each condition.