Specific lipids supply critical negative spontaneous curvature--an essential component of native Ca2+-triggered membrane fusion

Abstract

The Ca(2+)-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, alphaT, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary "spontaneous" lipid rearrangements that must occur during native membrane fusion-Ca(2+)-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore.

FIGURE 1

Chemical structures of specific negative curvature lipids, as in Table 1. (A) DOPC. (B) DOPA. (C) Me₂-DOPE. (D) Me-DOPE. (E) DOPE. (F) DOG. (G) αT. (H) CHOL.

FIGURE 2

Confocal analysis of the relative CHOL distribution between the PM and CV of S. purpuratus egg cortices. (A) DiI labeling was carried out on intact oocytes before preparation of cortices, to specifically label the plasma membrane. (B) CV-PM cortex subsequent to labeling with CHOL-B to indicate regions enriched in CHOL. (C) Bright field image of the cortex preparation shown in A and B. (D) Overlay of A and B indicates only partial correlation of DiI label in the PM with the heterogeneous CHOL-B label. (E) Overlay of A, B, and C indicates that CHOL-B labeling correlates primarily with CV-rich regions; areas of the PM denuded of CV have no detectable CHOL-B label. (F) Line traces as indicated in E, reconstructed from a Z stack of A and B, indicate that CHOL-B-enriched regions are localized within and above the plane of the PM, consistent with the position of docked CV. Scale bar in A is 20 μm. Representative of 10 cortices from four separate preparations.

FIGURE 3

Treatment of CHOL-depleted CV with negative curvature analogs rescues the extent but not the Ca²⁺ sensitivity or kinetics of CV-CV fusion. (A) Ca²⁺ activity curve of CV treated with 2 mM mßcd or sequentially with mßcd and 100 μM DOPE, 2 mM CHOL-loaded hpßcd, or 200 μM DOG or αT (n = 3–7). (B) Kinetics of CV-CV fusion in response to 157 ± 17 μM (n = 3); labels as in A. (C) Relationship between total exogenous lipid incorporated into CV membranes, as determined by quantitative analysis of isolated CV membranes, and the extent of fusion after standard mßcd treatment (red triangle). Labels as in A, except open diamond (Me-DOPE), open square (Me₂-DOPE), and open circle (DOPC). (D) The amount of a given reagent incorporated into CV membranes required to fully recover fusion extent correlates linearly with the amount of negative curvature each species contributes. Labels as in A except crosses, which indicate half the minimal quantities of CHOL, DOPE, and DOG required for fusion recovery, to represent the quantity localized to the vesicle outer leaflet (blue fit y = 99.2x + 12.2, R² = 0.92, red fit y = 195x + 25.5, R² = 0.90). (E) Summary of CV fusion recovery after mßcd treatment and subsequent delivery of CHOL or curvature analogs in the quantities shown in D; DOPE, αT, and DOG fully support the ability to fuse, whereas DOPC, Me-DOPE, and Me₂-DOPE do not, and DOPA further inhibits the extent of fusion. *(P < 0.01), **(P < 0.001) indicates difference from control and other conditions (n = 3–7).

FIGURE 4

Inhibition of CV-CV fusion extent by filipin treatment was reversed by the addition of molecules of high negative curvature. (A) Ca²⁺ activity curve of CV treated with 25.3 μM filipin, or sequentially with filipin and 50, 100, or 200 μM αT or C9NBD-T (n = 4). Fluorescence measurements (inset) determined incorporation of 13.4 ± 0.5 amol/CV of C9NBD-T. (B) Kinetics of CV-CV fusion in response to 144 ± 16 μM (n = 4). C. Delivery of αT to filipin-treated CV restores fusion capacity in a dose-dependent manner, correlating with the amount of αT incorporated into the membrane (P < 0.01, n = 4).

FIGURE 5

Phosphatidic acid inhibits fusion. Treatment of CHOL-depleted CV with the curvature analog DOPA did not recover but rather further inhibited the extent of CV-CV fusion (purple). Similarly, supplementing native CV with DOPA (green) inhibited fusion to approximately the same extent as did depletion of CHOL. (A) Ca²⁺ activity curve of CV treated with 200 μM DOPA, 2 mM mßcd or sequentially with mßcd and DOPA (n = 4). (Inset) Kinetics of CV-CV fusion in response to 236 ± 34 μM (n = 4). In a comparable fashion, the generation of endogenous PA in native CV also inhibits the extent, Ca²⁺ sensitivity, and kinetics of CV-CV fusion. (B) Ca²⁺ activity curve of CV treated with increasing concentrations of exogenous PLD as indicated. (Inset) Kinetics of CV-CV fusion in response to 91 ± 15 μM (n = 5).