Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice

Abstract

This work comprises a structural and dynamical study of monolayers and bilayers composed of native pulmonary surfactant from mice. Spatially resolved information was obtained using fluorescence (confocal, wide field and two photon excitation) and atomic force microscopy methods. Lipid mass spectrometry experiments were also performed in order to obtain relevant information on the lipid composition of this material. Bilayers composed of mice pulmonary surfactant showed coexistence of distinct domains at room temperature, with morphologies and lateral packing resembling the coexistence of liquid ordered (lo)/liquid disordered (ld)-like phases reported previously in porcine lung surfactant. Interestingly, the molar ratio of saturated (mostly DPPC)/non-saturated phospholipid species and cholesterol measured in the innate material corresponds with that of a DOPC/DPPC/cholesterol mixture showing lo/ld phase coexistence at a similar temperature. This suggests that at quasi-equilibrium conditions, key lipid classes in this complex biological material are still able to produce the same scaffold observed in relevant but simpler model lipid mixtures. Also, robust structural and dynamical similarities between mono- and bi-layers composed of mice pulmonary surfactant were observed when the monolayers reach a surface pressure of 30mN/m. This value is in line with theoretically predicted and recently measured surface pressures, where the monolayer-bilayer equivalence occurs in samples composed of single phospholipids. Finally, squeezed out material attached to pulmonary surfactant monolayers was observed at surface pressures near the beginning of the monolayer reversible exclusion plateau (~40mN/m). Under these conditions this material adopts elongated tubular shapes and displays ordered lateral packing as indicated by spatially resolved LAURDAN GP measurements.

Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures

“…Finally, the GP values we measured in intracellular LB-like structures (Figure 4, Table 1) are significantly higher than those for l o -like phases reported from giant vesicles composed of pulmonary surfactant (the very final material after LB secretion) obtained from murine lung lavages [55], suggesting important differences with this material. These last results are in agreement with recent observations from Cerrada et al , where they perform a comparative study using different experimental approaches between secreted LBs and pulmonary surfactant obtained from broncho-alveolar lavages [25].…”

Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order

“…Finally, the distribution of GP values is fitted to either a one or two-peak Gaussian chosen by the lower root-mean squared error. The resultant GP histogram can be used to calculate changes in mean lipid order or, for a well-defined two-peak Gaussian, to indicate the presence of two phases [6]. To facilitate additional spectral analysis, spectra are generated from the mean intensities of images at each wavelength of the stack.…”

“…To facilitate the characterization of such drug-delivery devices and to deepen our understanding of the fundamental biology of the cell membrane, a non-destructive method for evaluating intrinsic membrane physicochemical properties is required. As an example, packing or molecular order of membrane lipids can be sensed by fluorescent polarity-sensitive probes such as Laurdan or Di-4-ANEPPDHQ, whose emission spectrum shifts in response to changes in the molecular order of the membrane environment, usually quantified by a parameter denoted Generalized Polarization (GP) [6–11]. With the advent of commercial microscopes equipped with spectral detectors, shifts in the fluorescence emission spectra, and thus the GP parameter, can now be determined with much higher spatial accuracy using spectral imaging [10].…”

Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches

“…To achieve that function the pulmonary surfactant has a complex mixture of hundreds of lipids and four proteins (pulmonary surfactant proteins, called SP-A, -B, -C, and -D). The thermotropic behavior is controlled by the phospholipid composition and cholesterol content [17]. Figure 4 shows the GP analysis for the LAURDAN emission in a porcine pulmonary surfactant in response to a temperature increase.…”