One major barrier to a drug's access to target is the endothelium's plasma membrane (PM). In metazoans, the four major classes of phospholipids include phosphatidylcholine (PC) and sphingomyelin (SM), which are enriched in the outer leaflet of the PM, and phosphatidylserine and phosphatidylethanolamine, which are enriched in the inner leaflet. How different phospholipids impact the PM's permeability to drugs is not fully understood. We previously identified 275 synthetic small molecules that kill C. elegans (PMID 26108372). We discovered that a subset of these, including a molecule called wact-190
, accumulate as crystals within the marginal cells of the anterior pharynx and perforate the luminal PM. Dose-response analyses indicate a tight correlation (r=0.99) between crystal formation and death. Towards understanding crystal formation, we screened for mutants that resist wact-190
's lethality and identified 46 resistant mutants. We found that reduction-of-function of a presumptive sphingomyelin (SM) synthesis pathway and a p-glycoprotein pump called PGP-14 are each sufficient to confer resistance to both the lethality and crystal formation that results from wact-190
and other crystal-forming compounds. Mutants in the SM pathway and pgp-14
not only resist the lethality induced by crystal-forming compounds, but are hypersensitive to relatively smaller molecules. Mass-spectrometry revealed that 'resistant' molecules accumulate less in the mutants, while 'hypersensitive' molecules accumulate more in the mutants relative to wild type. We found that the SM synthase, which is the terminal enzyme in the SM synthesis pathway, is expressed exclusively in the pharynx and the spermatheca, while pgp-14
is expressed exclusively in the marginal cells of the anterior pharynx. Both proteins function cell-autonomously in the marginal cells with respect to wact-190
resistance. A human homolog of PGP-14 is ABCB4, which translocates PCs from the inner to the outer leaflet of the PM. SM synthases use the choline head from PCs to make SM. Hence our current model is that PGP-14 provides SM synthase with PCs to generate SM in the PM. Indeed, preliminary analyses indicate that both pgp-14
mutants and an SM synthase mutant are deficient in SM in anterior pharynx. Together, our work suggests that SM abundance in the PM is a key determinant of small molecule accumulation within animal cells. Further implications will be discussed.