**8. Putative mechanism of ELOA-induced cellular toxicity**

670 Lipoproteins – Role in Health and Diseases

already dead cell (Figure 2 C) [21].

single molecule sensitivity, Fluorescence Correlation Spectroscopy (FCS) and Confocal Laser Scanning Microscopy (CLSM) imaging by avalanche photodiodes (APD), so called APD imaging [37], which enable quantitative and nondestructive studies of molecular interactions and mobility in living cells, revealed that ELOA primarily acts on the plasma membrane of PC12 cells, inflicting damage and eventually causing plasma membrane rupture (Figure 2 A and B) followed by a rapid influx and distribution of ELOA inside the

**Figure 2. ELOA interaction with live PC12 cells studied by Fluorescence Correlation Spectroscopy (FCS) and APD imaging. (A)** Schematic presentation of different locations – the cell culturing medium, PC12 cell plasma membrane and nucleus, at which FCS measurements were performed. **(B)** FCS

*medium c* = 2.5 μM). **Insert:** Autocorrelation curves normalized to the same amplitude show that lateral mobility of ELOA in the plasma membrane (red) is significantly slower than in the medium (blue and green), as evident from the shift of the autocorrelation curve recorded at the plasma membrane (red) towards longer characteristic times. ELOA was neither detected in the cell nucleus nor in the cytoplasm. FCS measurements were taken 40-45 min after exposing PC12 cells to fluorescently labeled ELOA. **(C)** APD imaging shows that fluorescently labeled ELOA complexes are not gradually taken up by PC12 cells. Instead, the concentration of ELOA complexes in the immediate cellular surroundings

progressively increases, reaching a local concentration that is several times higher than the

concentration in the bulk medium. At a critical time-point (61.5 min), the plasma membrane ruptures. Only then the ELOA complexes "stream in" and swiftly distribute in the cellular interior, preferring

*medium c* = 670 nM) and the plasma membrane

*medium c* = 240 nM) is lower

measurements show that the concentration of ELOA in the bulk medium ( *bulk*

than in the immediate vicinity of the cell ( *cell surr*.

particularly the cell nucleus. The scale bar is 10 μm.

( *plasma membrane*.

Local rearrangements of lipid organization in the plasma membrane of PC12 cells (Figure 3) observed using a general lipophilic marker that differently partitions between the ordered and disordered phase of the lipid bilayer (1,1'-dioctadecyl-3,3,3',3'-tetramethylind ocarbocyanine perchlorate dye, DiIC18(5)) [21,38], are consistent with the hypothesis that ELOA may form transient pores in the plasma membrane.

**Figure 3. Lipid marker distribution in the plasma membrane of live PC12 treated with ELOA. (A)** Representative image showing uniform distribution of the fluorescent lipid marker DiIC18(5) in PC12 cells not exposed to ELOA (left). In cells exposed to ELOA the distribution of DiIC18(5) becomes patchy, and regions of local accumulation could be observed (right). **(B)** DiIC18(5) partitioning between different regions in the plasma membrane is also affected, as evident from the shifting of the autocorrelation curve to longer characteristic times in cells treated with ELOA. (C) Transmitted-light and APD images of PC12 cells taken 40 min after exposure to ELOA show that the plasma membrane marker DiIC18(5) (red) colocalizes with the fluorescently labeled ELOA complexes (green). The scale bar is 10 μm.
