Bifunctional probes reveal the rules of intracellular ether lipid transport
Böhlig K, Iglesias-Artola JM, Asaro A, Lennartz HM, Link AC, Drobot B, and Nadler A. (2025) Bifunctional probes reveal the rules of intracellular ether lipid transport. Angewandte Chemie
Ether lipids contain a long-chain alcohol that is attached to the glycerol backbone by either an alkyl or a vinyl-ether bond at the sn1 position. In human cells, 10-20% of glycerophospholipids are ether lipids and these levels are higher in the brain, kidneys, heart, and skeletal muscle. Although research has found many cellular functions related to ether lipids levels including membrane trafficking, structure of the endomembrane system, lipid sorting, neurotransmitter release, and ferroptosis, they have been very difficult to directly measure. Due to this, few functions of ether lipids have been studied in mechanistic detail and instead indirect evidence has been used to support these findings. The importance of ether lipids in human cells is demonstrated by the finding that when ether lipid biosynthesis is disrupted this results in hereditary diseases characterized by skeletal, renal, and cerebral abnormalities. Bifunctional probes have been a very versatile tool to examine lipid biology, therefore Böhlig et al were interested in using bifunctional ether lipid probes to investigate the transport of ether lipids. To accomplish this, they used mammalian cancer cell lines (U2OS and HCT-116) and induced pluripotent stem cell (iPSC)-derived neurons and also utilized fluorescence imaging, machine learning-assisted image analysis, and mathematical modelling.
After the bifunctional probes had been synthesized (this is discussed in detail in the article but will not be described in this literature blog), the authors wanted to determine how the type of linkage at the sn1 position affects the transport of phosphatidylcholines (PCs). Two cancer cell lines were chosen due to their previous use in lipid research. The probes were incorporated into the outer layer of the plasma membrane of cells through incubation with liposomes containing the probes, then following the loading pulse, the solution was replaced by cell growth medium and were maintained at 37°C to allow for retrograde lipid transport to occur. The cells were fixed and covalent lipid-protein conjugates were labeled with Cu-mediated click chemistry using Alexa Fluor 594 picolyl-azide dye, thus allowing lipid localization to be observed using fluorescence microscopy. The authors were able to confirm that the lipids were incorporated into the plasma membrane. In addition, they found that the probes used to compare alkyl ether, vinyl-ether, and ester linkage at sn2 demonstrated more fluorescence intensity than the probes used to compare alkyl ether and ester linkages at sn1. The authors suggest that this effect could be due to the chain length influencing the probability of different crosslinking occurring and that there may be a higher probability of proteins interacting with sn2 compared to sn1.
When four-colour fluorescence microscopy experiments were performed with co-staining for the plasma membrane, the endoplasmic reticulum, the Golgi, and endosomes, the lipid signal was measured for each organelle. Pulse-chase experiments were used to evaluate changes in lipid distribution over time. Interestingly, the lipid signal of the plasma membrane decreased for all lipids, suggesting the probe was being internalized by the cells through retrograde trafficking. The plasmalogen probe remained at the plasma membrane to a greater extent than both alkyl ether and ester lipid probes. When internalized, most of the signal for all probes was found in the endoplasmic reticulum, while very little was found in endosomes. Probes with the bifunctional fatty acid at sn2 demonstrated more localization at the Golgi after 30 mins. These findings propose that the destination of lipids during transport is dependent on the presence or absence of a vinyl-ether bond at sn1.
To determine if the lipid localization trends they saw were also present in physiologically relevant systems, iPSC-derived neurons were used in addition to the two cancer cell lines that had different origins. The authors found that two of the probes, the alkyl ether and the ester linkage at sn2, remained at the plasma membrane longer, which is consistent with the observations seen in the two cancer cell lines. This provided further support to the theory that there is a mechanism used to recognize the phospholipid sn1 linkage type that is independent of cell type.
Böhlig et al were interested in evaluating lipid transport in human cell lines and in determining whether this is influenced by the type of linkage present on the glycerol backbone. They developed new bifunctional photoaffinity probes to study the location of phospholipids with alkyl ether, vinyl-ether, and ester bonds at sn1 or sn2 positions. Utilizing fluorescent microscopy also allowed for the lipid localization to be determined when looking at the plasma membrane, endoplasmic reticulum, Golgi, and endosomal structures. They demonstrated similar trends between two cancer cell lines and iPSC-derived neurons, suggesting that there could be a specific mechanism used when transporting lipids that can identify the types of bonds present. Previously, indirect data was collected to support the roles and functions of ether lipids, however discovering techniques that will allow for direct evaluation will increase our knowledge on ether lipids and help determine how disruptions to their levels result in the vast characteristics and symptoms that can be seen.