Evidence of triglyceride-phase incorporation into artificial bilayers for studying lipid droplet biogenesis

CARUSO B; PERILLO MA; MANGIAROTTI A; WILKE N

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

Lipid Droplets (LD) are intracelularstructures consisting on an apolar lipid core -composed mainly of triglycerides(TG) and steryl esters- which is surrounded by a phospholipid and proteinmonolayer. LDs originate in  the ER bilayer, where TG synthesis concludes.The mechanisms underlying TG nucleation, size maduration and budding-off fromthe ER membrane are a matter of current investigations and the role ofdewetting from cytosolic-bilayer interface appears to play a critical role. Inorder to contrast the  nano-sized "blisters" of TG that someauthors predict [1], here we formed free-standing bilayersby transferring films of a monolayer of mixed phosphatidylcholine(EPC)/TG incoexistence with TG microlenses (i.e.an excluded TG phase floating in the surface). These membranes werecharacterized by adding them the solvatochromic fluorescent probe Nile Red(NR) and observing them under spectral confocal microscope. Such bilayersexhibit fluorescence emission spectra comparable of bilayers of vesicles withsimilar composition (POPC and TO). By comparison with literature data  andfluorescence spectra of EPC and TG monolayers, the peaks could be assigned todifferent phases, namely 1) PC membranes (λemmax=630 nm ) bilayer and bilayer) and 2) TG isotropic phase(λemmax=570 nm ). No microscopic structures could be observedat  λemmax=570 nm. Diffusion of NR under this TG phase wascharacterized using FRAP analysis yielding values (D=2 μm2s )typical of model bilayer membranes, suggesting that the probe is diffusing in a2D structure. This system appears appropiate for describing whichis the distribution of the TG phase, that is, homogeneously among theintrabilayer space or in nanoscopic "blisters", by evaluatingdiffusion times obtained by FCS and FRAP.