Hydration and Supramolecular Organization Studies of Lamellar Bodies in A549 Lung Cells using Laurdan Fluorescence

The pulmonary surfactant is organized at the intracellular level in structures known as lamellar bodies (LBs). These subcellular acidic organelles have a variable size (aprox. 0.5-3 um) and based on the electron microscopy data it is proposed that they possess a concentrically membrane lamellar structure. Although the process of LB secretion by physiological inductors like ATP is well characterized, there is a lack of information related to their physical properties.Using the well known sensitivity of LAURDAN to membrane lateral organization we study LBs organization and dynamic properties of in vivo. We use an immortalized cell line from lung carcinoma, A549, which synthesize pulmonary surfactant and stores it as LBs. The fluorescence signal from LAURDAN was analyzed using the classical generalized polarization function and a newly method based on the Fourier transformation of the emission spectrum (called spectral phasor, Fereidouni-2012). The basic improvement of the spectral phasor is related to the Fourier transformation properties, which allow us the opportunity to decompose complex emission as a linear combination of single decay.Our results indicate that LBs have a highly packedmembrane structure with low extent of hydration in their core. This particular state is shifting to higher levels of fluidity with increased hydration levels at their periphery. The measured values of GP function indicates that the membrane in the core is in a gel like state (or liquid order state), though when the spectral data is analyzed a more complex system is identified. These results show higher lateral packing compared with DPPC vesicles but with a more heterogenic emission pattern.The interpretation opens the possibility to discuss the supramolecular organization and the role of water in these organelles.Fereidouni et al, Optics Express-2012