## 3 Role of Lipids for Membrane Function and Intracellular Transport
### Effect on Endocytosis and/or Retrograde Transport due to Manipulations of the Lipidome
#### 3.7.2 Studies Related to Ether Lipids
In addition, the large increase in LPI obtained as a result of the HG treatment [176] might contribute to this protection (see discussion about addition of lysolipids to cells in Section 3.7.5). We found it remarkable that an inhibition of GSL synthesis by Fumonisin B1, but not with PDMP, resulted in increased levels of ether lipids [164], and that increasing ether lipids by adding the ether lipid precursor HG also resulted in a decrease of GSLs [176]. Addition of HG to PC-3 cells (which do not contain measurable amounts of Gb3) also resulted in an increase of ether lipids and a decrease in GSLs [170]. Moreover, this treatment of PC-3 cells resulted in an increase of cholesterol, indicating that cholesterol is important for interactions with ether lipids, which has been similarly described for interactions between cholesterol and sphingomyelin [131]. Importantly, these three studies all showed an inverse correlation between the levels of ether lipids and glycosphingolipids. In the next Section 3.7.3, changes in the retrograde transport and lipidome of HEp-2 cells with increased cell density are discussed. Interestingly, this study also showed a small increase in GSLs and a simultaneous small decrease in ether lipids [33], further supporting this apparent co-regulation of ether lipids and sphingolipids. The reason for such a co-regulation is unknown, but it should be noted that the changes in the cell density study were observed without any treatment of cells, and the cells were only grown for an extra day or two. In summary, the studies discussed in this paragraph demonstrate that investigation of Shiga toxin transport has revealed a co-regulation between the levels of sphingolipids and ether lipids. Recently, [PERSON] and colleagues reported a similar correlation between ether lipids and sphingolipids in four cell lines in an extensive study. They used a CRISPR library to repress the expression of 16,000 genes, combined with analyses of sphingolipid-depleted cells (they used myriocin, an inhibitor of the first enzyme in the synthesis of the sphingolipids). They reported a slight but significant increase in PC ether lipids, thus providing another line of evidence for a co-regulation between ether lipids and sphingolipids [184]. It should also be noted that there was no obvious correlation between the amplitude of the decrease in sphingolipids and increase in ether lipids in the four cell lines studied. In addition, alkylhydroxyacetonephosphate synthase (AGPS), a key enzyme in synthesis of ether lipids, was found to be important in promoting the survival of sphingolipid-depleted cells [184]. These authors also showed that ELOV5, an enzyme which is mainly involved in elongation of PUFAs, contributed to making their cells more sensitive to sphingolipid depletion, whereas ELOV6, which mainly acts on elongation of C16-C18 fatty acids, rendered cells more resistant to sphingolipid depletion. These results are in agreement with the changes observed in the cell density study discussed in the next section, where less PUFA-containing and more C18-containing ethers were found at increased cell density [33]. In summary, several studies from two groups have demonstrated an inverse relationship of ether lipids and sphingolipids [33; 164; 170; 176; 184]. Furthermore, such changes do not only affect the total amount of ether lipids, but also the species composition. There are, however, major differences between the results reported from the two groups when it comes to the amount of the various ether lipids, and more studies are needed to understand these differences. Regarding discussions about ether-linked PLs, it is interesting that they behave differently from their corresponding ester-linked analogs regarding how the hydrophobic chains enter membranes. It is now 30 years since magnetic resonance spectroscopy analyses comparing PC with two acyl chains and PE alkenly ethers indicated that these hydrophobic chains enter membranes differently.