Beyond apoptosis: the mechanism and function of phosphatidylserine asymmetry in the membrane of activating mast cells

Abstract

Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and loss of asymmetry can contribute to numerous cellular functions and responses that are independent of programmed cell death. Exofacial exposure of phosphatidylserine occurs in lymphocytes and mast cells after antigenic stimulation and in the absence of apoptosis, suggesting that there is a functional requirement for phosphatidylserine exposure in immunocytes. In this review we examine current ideas as to the nature of this functional role in mast cell activation. Mechanistically, there is controversy as to the candidate proteins responsible for phosphatidylserine translocation from the internal to external leaflet, and here we review the candidacies of mast cell PLSCR1 and TMEM16F. Finally we examine the potential relationship between functionally important mast cell membrane perturbations and phosphatidylserine exposure during activation.

Keywords: ABCA, ABC binding cassette family A; CRAC, calcium release activated channel; GPMV, giant plasma membrane vesicle; ITIM, immunoreceptor tyrosine based inhibitory motif; PLA2, phospholipase A2; PLSCR, phospholipid scramblase; PMA, phorbol 12,13-myristate acetate; RBL, rat basophilic leukemia; RFU, relative fluorescence units; ROI, region of interest; TMEM, transmembrane protein; TMEM16F; WGA, wheat germ agglutinin; mast cells; membrane lipids; phosphatidylserine.

Figure 1

(See previous page). Characteristics of phosphatidylserine exposure in activating model mast cells. (A) Kinetics of PS exposure and intracellular free calcium mobilization in RBL2H3 mast cells. Live cell images of Fluo-4 loaded (4 μM for 30 min) RBL2H3 cells (stimulated with PMA/ionomycin (500 nM/500 nM) or via FcεRI (IgE anti-DNP/250 ng/ml KLH-DNP in the presence of 0.1 μg/ml Alexa 568-Annexin V and 1 mM external CaCl₂) were captured using laser scanning confocal microscopy (Nikon Ti INSPIRE, 150 nm optical sections gathered every 30s). Whole cell and membrane regions of interest (ROI) were analyzed for average fluorescence intensity in the FITC (Fluo-4) and Texas Red (Alexa 568-Annexin) channels. Scale bar in B is 7 microns. (B) Patchy presentation of AnnV positive regions in activated RBL2H3. Left Panel. Maximum intensity projection (NIS Elements, Nikon, San Diego, CA) of 10 150 nm optical sections for cell stimulated and stained as in A, image captured at 450s. Right Panel. Intensity profile of Alexa 568-Annexin V fluorescence in a selected 3 micron length of membrane at the indicated time points after stimulation as in A. (C) Process of PS exofacial exposure in activating RBL2H3 cell. Intensity surface plots were used to visualize Alexa 568-Annexin V fluorescence (NIS Elements) of a single 150 nm z disc from RBL2H3 cell stimulated via FcεRI (IgE anti-DNP/250 ng/ml KLH-DNP in the presence of 0.1 μg/ml Alexa 568-Annexin V and 1 mM external CaCl₂). Time after initial exposure to stimulus is indicated in seconds.

Figure 2.

Schematic summary of the functional consequences of exofacial PS exposure.

Figure 3.

PLSCR1 expression and localization in RBL2H3 (A and B). PLSCR1 expression and calcium-induced tyrosine phosphorylation in RBL2H3. (A) Total cellular lysates were extracted from RBL2H3 (ATCC CRL-2256) in a buffer containing 50 mM Hepes pH 7.4, 250 mM NaCl, 20 mM NaF, 10 mM iodoacetamide, 0.5%(w/v) Triton X100, 1 mM PMSF (phenylmethylsulfonylfluoride), 500 mg/ml aprotinin, 1.0 mg/ml leupeptin and 2.0 mg/ml chymostatin. Following acetone precipitation, 10 μg of protein were loaded in duplicated lanes of a 10% SDS-PAGE gel run under reducing conditions. Following electrotransfer to PVDF membrane, a Western blot using 0.1 μg/ml anti rat PLSCR1 (mouse monoclonal 13A6, 1 h, RT in 0.05% Tween 20 and 0.1% BSA) was performed. Developing antibody was goat anti-rat IgG conjugated to HRP (Amersham, Piscataway, NJ), and signal was visualized using Enhanced Chemiluminescence (Amersham) and Kodak Biomax film. Duplicate lanes shown. (B) Cells were stimulated for the indicated times (in min) with 500 nM ionomycin, or 200 ng/ml KLH-DNP. For the latter, cells were pre-incubated for 16 h with 0.1 μg/IgE anti-DNP (clone SPE-7, Sigma, St. Louis, MO). Total lysates from 10 million RBL2H3 per lane were produced using a high salt (see above) lysis buffer. NaCl was normalized to 75 mM before pre-clearing (murine IgG2b) and immunoprecipitation with 1 μg per lane of anti-phosphotyrosine (clone 4G10, Cell Signaling Technologies, Danvers, MA). Western blotting for the presence of PLSCR1 was performed as described above. (C) Immunofluorescent localization of PLSCR1 in RBL2H3. Cells were grown on glass coverslips, fixed in 0.4% (w/v) paraformaldehyde (30 min, RT) and permeabilized (0.4% Triton X100 for 4 min). After blocking with 0.75% (w/v) Fish skin gelatin, immunocytochemistry was performed with anti-PLSCR1 (0.1 μg/ml for 45 min) followed by washing and secondary antibody staining with Alexa-conjugated IgG. Lower panel shows matched exposure of staining were primary antibody was omitted but all other conditions were equivalent. Counterstains were ER Tracker and DAPI (4 μM/30 min and 10 nM for 4 min, Molecular Probes Eugene, OR). Three separate cell nuclei are shown at left (scale bar 4 microns) and digitally zoomed 2X is shown at right. Arrows indicate putative nuclear bodies. Images were acquired through a Plan Apo VC 100X 1.40 oil objective (Nikon). Imaging was performed on a Nikon Ti Eclipse C1 epi-fluorescence and confocal microscopy system. Pinhole size was 60 microns. (D) TMEM16F expression in RBL2H3. Western blotting protocol as in Figure 1, with probing antibody anti-TMEM16F (0.1 μg/ml G-14, Santa Cruz Biotechnology, Dallas, TX). Duplicate lanes shown. (E) Immunofluorescent localization of TMEM16F in RBL2H3. Staining and imaging protocols as in Figure 1 with Alexa-488 conjugated wheat germ agglutinin and Alexa 568 anti-rabbit IgG (Molecular Probes). Scale bar is 8 microns.