Nanoscale packing differences in sphingomyelin and phosphatidylcholine revealed by BODIPY fluorescence in monolayers: physiological implications

Abstract

Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes. However, sphingoid-based lipids, such as SM, rather than saturated-chain PCs have been implicated as key components of lipid rafts in biomembranes. These observations raise questions about the physical packing properties of the phase states that can be formed by these two major plasma membrane lipids with identical phosphocholine headgroups. To investigate, we developed a monolayer platform capable of monitoring changes in surface fluorescence by acquiring multiple spectra during measurement of a lipid force-area isotherm. We relied on the concentration-dependent emission changes of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled PC to detect nanoscale alterations in lipid packing and phase state induced by monolayer lateral compression. The BODIPY-PC probe contained an indacene ring with four symmetrically located methyl (Me) substituents to enhance localization to the lipid hydrocarbon region. Surface fluorescence spectra indicated changes in miscibility even when force-area isotherms showed no deviation from ideal mixing behavior in the surface pressure versus cross-sectional molecular area response. We detected slightly better mixing of Me4-BODIPY-8-PC with the fluid-like, liquid expanded phase of 1-palmitoyl-2-oleoyl-PC compared to N-oleoyl-SM. Remarkably, in the gel-like, liquid condensed phase, Me4-BODIPY-8-PC mixed better with N-palmitoyl-SM than dipalmitoyl-PC, suggesting naturally abundant SMs with saturated acyl chains form gel-like lipid phase(s) with enhanced ability to accommodate deeply embedded components compared to dipalmitoyl-PC gel phase. The findings reveal a fundamental difference in the lateral packing properties of SM and PC that occurs even when their acyl chains match.

Figure 1

Langmuir surface balance modified to acquire surface fluorescence spectra of lipids forming a monolayer at the air/water interface. (A) Structure of Me₄-BODIPY-8-PC. (B) Schematic of modified Langmuir film balance equipped to acquire surface fluorescence.

Figure 2

Compression-induced changes in fluorescence emission properties and force–area isotherms for 1 mol % Me₄-BODIPY-8-PC mixed with either 18:1-SM or POPC. (A, B) Representative emission spectra obtained for mixed monolayers of 1 mol % BODIPY-PC and 18:1-SM (A) or POPC (B). (C) Monolayer isotherms for 18:1-SM and POPC showing surface pressure (π) versus average molecular area in absence and presence of lipid fluorophore. (D) Surface pressure versus BODIPY-PC surface concentration (pmol/cm²) response. (E) Monomer emission intensity (solid line) and emission wavelength maximum (λ_max) (symbol) versus BODIPY-PC surface concentration (pmol/cm²) in 18:1-SM or POPC. The monomer emission intensity represents the integrated area over the 505–535 nm range. (F) Emission peak broadening versus BODIPY-PC surface concentration (pmol/cm²) in 18:1-SM or POPC. The broadening represents the peak width (nm) determined at 50% maximum intensity.

Figure 3

Compression-induced changes in fluorescence emission properties and force–area isotherms for 10 mol % Me₄-BODIPY-8-PC mixed with either 18:1-SM or POPC. (A, B) Representative emission spectra obtained for mixed monolayers of 10 mol % BODIPY-PC and 18:1-SM (A) or POPC (B). (C) Monolayer isotherms for 18:1-SM and POPC showing surface pressure versus average molecular area in absence and presence of lipid fluorophore. (D) Surface pressure (π) versus BODIPY-PC surface concentration (pmol/cm²) response. (E) Monomer emission intensity (solid line) and emission wavelength maximum (λ_max) (symbol) versus BODIPY-PC surface concentration (pmol/cm²) in 18:1-SM or POPC. The monomer emission intensity represents the integrated area over the 505–535 nm range. (F) Emission peak broadening versus BODIPY-PC surface concentration (pmol/cm²) in 18:1-SM or POPC. The broadening represents the peak width (nm) determined at 50% maximum intensity.

Figure 4

Compression-induced changes in fluorescence emission properties and force–area isotherms for 1 mol % Me₄-BODIPY-8-PC mixed with either 16:0-SM or DPPC. (A, B) Representative emission spectra obtained for mixed monolayers of 1 mol % BODIPY-PC and 16:0-SM (A) or DPPC (B). (C) Monolayer isotherms for 16:0-SM and DPPC showing surface pressure versus average molecular area in absence and presence of lipid fluorophore. (D) Surface pressure (π) versus BODIPY-PC surface concentration (pmol/cm²) response. (E) Monomer emission intensity (solid line) and emission wavelength maximum (λ_max) (symbol) versus BODIPY-PC surface concentration (pmol/cm²) in 16:0-SM or DPPC. The monomer emission intensity represents the integrated area over the 505–535 nm range. (F) Emission peak broadening versus BODIPY-PC surface concentration (pmol/cm²) in 16:0-SM or DPPC. The broadening represents the peak width (nm) determined at 50% maximum intensity. Unfilled symbols in panels D, E, and F mark the phase transition region where LE and LC domains coexist.

Figure 5

Compression-induced changes in fluorescence emission properties and force–area isotherms for 10 mol % Me₄-BODIPY-8-PC mixed with either 16:0-SM or DPPC. (A, B) Representative emission spectra obtained for mixed monolayers of 1 mol % BODIPY-PC and 16:0-SM (A) or DPPC (B). (C) Monolayer isotherms for 16:0-SM and DPPC showing surface pressure versus average molecular area in absence and presence of lipid fluorophore. (D) Surface pressure (π) versus BODIPY-PC surface concentration (pmol/cm²) response. (E) Monomer emission intensity (solid line) and emission wavelength maximum (λ_max) (symbol) versus BODIPY-PC surface concentration (pmol/cm²) in 16:0-SM or DPPC. The monomer emission intensity represents the integrated area over the 505–535 nm range. (F) Emission peak broadening versus BODIPY-PC surface concentration (pmol/cm²) in 16:0-SM or DPPC. The broadening represents the peak width (nm) determined at 50% maximum intensity. Unfilled symbols in panels D, E, and F mark the phase transition region where LE and LC domains coexist.

Figure 6

Evaluation of surface concentration-dependent dimer emission by 1 mol % Me₄-BODIPY-8-PC in different monolayer matrices using normalized difference spectra. The normalized emission peak obtained at 5 mN/m (= monomer) was subtracted from spectra obtained at 15, 25, and 40 mN/m for each matrix lipid. (A) POPC; (B) DPPC; (C) 18:1-SM; (D) 16:0-SM. The inflection depth at ∼510 nm reflects the magnitude of the λ_max apparent red-shift. The normalized spectra used to produce the difference spectra are shown in Figure S1. Similar comparisons at 10 mol % Me₄-BODIPY-8-PC are presented in Figures S2 and S3.

Figure 7

Spectra for 1 mol % Me₄-BODIPY-8-PC in different monolayer matrices compared at identical interfacial molecular packing areas. (A) POPC and 18:1-SM at 78 Ų/molecule (Me₄-BODIPY-8-PC = 2.12 pmol/cm²); (B) DPPC and 16:0-SM at 78 Ų/molecule (Me₄-BODIPY-8-PC = 2.12 pmol/cm²); (C) POPC and 18:1- SM at 58 Ų/molecule (Me₄-BODIPY-8-PC = 2.85 pmol/cm²); (D) DPPC and 16:0-SM at 45 Ų/molecule (Me₄-BODIPY-8-PC = 3.75 pmol/cm²).