One of the central challenges in cell biology is to understand the precise function and regulation of the huge variety of complex lipids found in animal cells. The complex lipids long-chain polyunsaturated fatty acids (PUFAs) and glycosphingolipids (GSLs) are strictly regulated in a cell-type specific manner suggesting that the precise control of these molecules is important for normal cell function. Indeed, altered levels of PUFAs and GSLs lead to serious and complex human diseases including an X-linked form of mental retardation and inherited fatal metabolic disorders such as Tay-Sachs disease.
My laboratory focuses on understanding the cellular roles played by PUFAs and GSLs in the belief that these molecules play a fundamental and as yet poorly understood role in controlling cell signalling and function. We are using a genetic approach, which benefits from the numerous experimental advantages of the nematode C. elegans. Understanding the function of PUFAs and GSLs will clarify how altered levels of these complex lipids contribute to cellular dysfunction and disease. Since both PUFAs and GSLs are largely conserved between nematodes and mammals, it is likely that these molecules have evolutionarily conserved functions. Therefore the expectation is that our findings in C. elegans will be relevant to humans. Long-chain polyunsaturated fatty acids (PUFAs)
We have developed the nematode C. elegans as a model system to understand how PUFAs modulate neurotransmitter release. There is a wealth of evidence showing that the basic mechanisms of synaptic vesicle exocytosis and endocytosis are extremely well conserved between C. elegans and mammals. These organisms share a highly conserved set of proteins, which are used in similar ways in the two systems. However, prior to our work, little attention was paid to the possibility of using C. elegans as a model to investigate the critical roles of specific lipids at the synapse.
Synaptojanin levels at sites of neurotransmitter release are severely decreased in fat-3 mutants. (A) The green box in this schematic indicates the portion of the dorsal nerve cord imaged. (B) Visual quantification of GFP::SYNAPTOJANIN at sites of neurotransmitter release; yellow indicates high concentration and red low concentration. (C) Line graphs representing the fluorescence intensity of GFP::SYNAPTOJANIN in the dorsal nerve cord. In fat-3 mutants the peaks are weaker and similar in intensity to the fluorescence measured in intersynaptic regions of the cord.
PUFAs are enriched in synaptic membranes, including synaptic vesicles, and have critical synaptic functions in both humans and C. elegans. For example, alteration of PUFA metabolism causes a form of mental retardation, and diets deficient in essential PUFAs are associated with deficits in behaviour and brain function in infants. All these defects are likely caused by abnormal neurotransmitter release. Similarly, we have recently shown in C. elegans that PUFA depletion results in abnormally low levels of neurotransmitter release from synapses. However, almost nothing is known about the mechanistic basis of PUFAs’ synaptic functions. To address this problem, we generated C. elegans fat-3 mutants that cannot synthesize PUFAs. These mutant animals are depleted of synaptic vesicles and, as a result, display striking behavioural phenotypes caused by insufficient release of acetylcholine and serotonin at neuromuscular junctions (determined electrophysiologically, in collaboration with the Jorgensen’s lab at the University of Utah.
We have recently found that the impaired neurotransmission observed in fat-3 mutants is caused, at least in part, by defects in synaptic vesicle endocytosis. The mutants have unusually low levels of the phosphoinositide phosphatase synaptojanin at synaptic release sites and exposure to exogenous PUFAs, such as arachidonic acid, restored normal synaptojanin localization. Since synaptojanin is essential to ensure normal synaptic vesicle endocytosis, these findings suggest that PUFAs are required for efficient synaptic vesicle endocytosis because they regulate synaptojanin localization or stabilization at release sites. Previous work in other labs demonstrated that synaptojanin is localized to synaptic membranes by specific synaptojanin-localizing proteins, so it is possible that PUFAs control synaptojanin localization via one or more of these proteins. However, we have genetic and localization data indicating that PUFAs do not act through the major synaptojanin-localizing proteins, endophilin and amphiphysin, suggesting that PUFAs contribute to synaptojanin localization via novel protein(s).
Identifying these proteins and elucidating the mechanisms by which PUFAs influence synaptic vesicle recycling would be a major advance in understanding synaptic function.
The level and localisation of the endophilin-localising proteins amphiphysin and endophilin at sites of neurotransmitter release are normal in fat-3 mutants, suggesting that synaptojanin mislocalization does not depend upon these two proteins.
GSLs are glycosylated derivatives of ceramide in the lipid bilayer. Their ubiquitous distribution and complexity suggest that they have important functions, but what these are in vivo is still poorly understood. We have characterized the phenotype of C. elegans mutants with essentially no GSLs. The C. elegans genome encodes three ceramide glucosyltransferase (cgt) genes, which are required for GSL biosynthesis. Animals lacking cgt function do not synthesize GSLs, arrest growth at the first larval stage, and display defects in a subset of cells in their intestine while the cells connecting the intestine with the pharynx or the anus appear normal; these defects impair larval feeding, resulting in a starvation-induced growth arrest. Restoring cgt function in this limited number of cells —but not in a variety of other tissues— is sufficient to rescue the phenotypes associated with loss of cgt function. These surprising findings suggest that GSLs are dispensable in most C. elegans cells, including those of the nervous system.
cgt mutants are unable to ingest food normally.
Most control animals can ingest small fluorescent beads in the intestinal lumen (dashed yellow line; A-C and G), whereas most cgt mutant worms cannot (D-F and G).
Very recently we have discovered that cgt mutants display trafficking defects specifically in the cells that require GSLs and that GSL-depleted HeLa cells (a human cell line) show similar defects, suggesting that GSL function is conserved between humans and C. elegans. We are now concentrating on clarifying which trafficking pathway(s) is affected in GSL-deficient cells.
Our long-term goal is to combine the powerful genetics of C. elegans with the many tools available to study intracellular trafficking in human cells to understand the molecular basis of GSL function.
cgt mutants show abnormal intestinal morphology.
In control L1 animals the intestinal lumen (dashed yellow line) appears thin and continuous (A-C). In cgt mutants the anterior intestinal cells appear swollen and vacuolated (red arrow, E), and the intestinal lumen has mostly disappeared (D-F).