Glycosphingolipids are the most abundant glycolipids in neuronal membranes, and their metabolic pathways are well characterized.1–4
Briefly, ceramide is synthesized in the endoplasmic reticulum, transported to the Golgi where glycosyltransferases are responsible for the stepwise addition of monosaccharides, generating structures of remarkable complexity. The newly formed glycolipids are transported to the plasma membrane to fulfill their functional roles. Likewise, through endocytosis, the lipids are recycled back into the cell interior, and undergo degradation by stepwise removal of the sugars by glycosidases present in the lysosome and plasma membrane.5
Defects in sphingolipid metabolism are associated with a number of devastating lysosomal storage diseases including Niemann Pick type A,6
and Krabbe diseases.9
We are interested in probing the metabolism of glycolipids within single cells. Immunostaining experiments have revealed that the distribution of lipids in a neuronal population is highly heterogeneous.10, 11
Characterizing glycolipid metabolism in single cells has the potential of unveiling functional differences in these neurons, which may provide insight into the nature of the lysosomal storage diseases and therefore leading to innovative therapies.
We coined the term metabolic cytometry to refer to the use of modern chemical instrumentation to monitor metabolism in single cells.12–14
In our experiments, we incubate cells with a fluorescence substrate, which is taken up and metabolized. A single cell is aspirated within a capillary and lysed. The fluorescence products are separated by capillary electrophoresis and detected with laser-induced fluorescence. As long as those metabolites retain their fluorescent tag, they many be detected with exquisite sensitivity using high sensitivity laser-induced fluorescence.
To date, our efforts have primarily employed a tetramethylrhodamine (TMR)-labeled glycolipid as substrate, which is taken up by cells and is catabolized.13–16
We have recently described the use of a BODIPY-labeled glycolipid as substrate;17
this substrate undergoes both catabolism and anabolism.18–19
In metabolic cytometry, a cell is aspirated within a capillary and lysed by contact with a surfactant. The contents are separated by capillary electrophoresis equipped with an ultrasensitive laser-induced fluorescence detector.
Our instruments have been designed to monitor fluorescence in one spectral channel, which allows use of a single labeled substrate. We have observed a large cell-to-cell variation in uptake of TMR-labeled substrates for both cultured PC12 cells and primary neurons, and significant cell-to-cell variation in metabolism for primary neurons.12–14
To address this very wide variation in uptake and metabolic activity, we have created a set of fluorescence detectors with six to nine orders of magnitude dynamic range.15–16
These instruments have revealed that cultured PC12 cells tend to generate very similar expression profiles of fluorescent glycolipids, which provides confidence in the reproducibility of the analytical method. In constrast, neurons provide a quite diverse population of metabolic products, undoubtedly reflecting differences in their function.
In this paper, we expand our technology to simultaneously monitor glycolipid metabolism in two different pathways. catabolism and anabolism. Our new system employed GM1-TMR and BODIPY-FL-labeled LacCer, which also undergoes anabolism, as substrates. Primary rat cerebella neurons were incubated with these substrates. Single cells were aspirated into a capillary and lysed. Their contents were separated by capillary electrophoresis coupled with a two-spectral channel laser-induced fluorescence detector. One spectral channel was tuned to detect tetramethylrhodamine-labeled metabolites, while the other detected BODIPY-FL labeled metabolites.