## 3 Role of Lipids for Membrane Function and Intracellular Transport
### Effect on Endocytosis and/or Retrograde Transport due to Manipulations of the Lipidome
#### 3.7.5 Substances Affecting the Membrane Fluidity  
Addition of several lysolipids to HEp-2 cells resulted in a decreased cellular binding of Shiga toxin, Stx2, and anti-Gb3 IgM [180]. The effect increased with the increasing size of the head groups, i.e., LPI > LPC > LPS > LPE >> LPA, with no effect observed for LPA (Table 1). The effect was also dependent on the fatty acyl groups. A stronger effect was obtained with LPC 18:0 than with LPC 18:1, further evidencing the importance of the conical structure for this effect. Most studies were performed using LPI 18:0. The experiments showed that prebound Shiga toxin was released following addition of the lysolipid, and that the LPI effect was observed both in fixed cells or after depletion of ATP, demonstrating that the effect was independent of signaling or membrane turnover. LPI addition resulted in the cell surface rounding up, with less philopodia. Furthermore, studies with the fluorescent probe NR12S, which is predicted to stay in the outer leaflet, revealed that LPI induced lipid disorder in the PM. There was no change in lipid disorder after addition of LPE. LPI and methyl-\(\beta\)-cyclodextrinrin both increased the lipid disorder, but led to opposing effects on toxin binding, showing that the reduction in lipid order did not seem to be important for the binding. Addition of methyl-\(\beta\)-cyclodextrinrin did not affect binding of Shiga toxin but reversed the effect of LPI, suggesting that LPI altered Gb3 receptor conformation and/or distribution. The observed decrease in lipid packing might facilitate interactions of Gb3 with cholesterol, binding the carbohydrate and thereby inhibiting the Shiga toxin binding, an effect which was reversed by addition of methyl-\(\beta\)-cyclodextrinrin. Addition of LPI did not have any effect on binding of ricin [180]. In a follow-up study, lysolipids with large head groups were shown to perturb clathrin-mediated endocytosis. The effect depends on the cells used. The largest effects were observed for HEp-2, HeLa, and SUM-159 cells, and smaller effects were observed for SK-BR3, U-2 OS, and PC-3 cells. Less force (50% reduction) was needed to pull tubules outward from HEp-2 cells following the addition of LPI, whereas no reduction in this force was observed following the addition of LPE [181]. Moreover, the addition of LPI resulted in increased lifetimes for AP-2 pits, and the effect on ricin uptake was much smaller than for transferrin, indicating that the lysolipid effect was larger for clathrin-dependent than for clathrin-independent endocytosis. In this study, the effect was also lower when using lysolipids with smaller head groups (LPE and LPA) or unsaturated lipids (LPC 18:1). As expected, there was a stronger effect of Latrunculin B on transferrin endocytosis after LPI addition, which agrees with data showing that actin requirement for endocytosis is increased at high membrane tension [191]. In the studies with lysolipids and toxins described above, these lipids did not only seem to affect binding but also had an additional effect on retrograde transport and toxicity. A study in yeast showed that lysolipids facilitated COPII vesicle formation and anterograde transport from ER to the Golgi [192]. An effect of lysolipids on anterograde and retrograde transport may be related. In both studies, it was speculated that the conical structure of the lysolipids is important for these effects, but further studies are needed to describe the precise mechanism. After preincubation with HeLa cells, the antitumor drug 2-hydroxyoleic acid (OHOA) was shown to potentially increase retrograde transport of ricin from endosomes to Golgi and further to the ER and result in a major increase in sensitivity to this toxin, whereas Shiga toxin transport to the Golgi was slightly reduced as evaluated with sulfation experiments [171]. After incubation of the cells with 12.5 mM OHOA for 3 h, results showed that 6% of the cellular lipids contained this fatty acyl group.