PC(<i>O</i>-16:0/2:0) disrupts sphingolipid metabolism leading to changes in Mss4-GFP localization.

<p>(<b>A</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) treatment disrupts sphingolipid metabolism.</b> Wild type (BY4741) cells were treated with vehicle or PC(<i>O</i>-16:0/2:0) (20 µM) for the indicated times (min). Lipids were extracted and sphingolipid levels were quantified and expressed as a log₂ fold change of PC(<i>O</i>-16:0/2:0) treated from vehicle treated control. LCB, long chain base; IPC, inositol phosphorylceramide; MIPC, mannosyl phosphorylceramide; DHS(-P), dihydrosphingosine (1-phosphate); PHS(-P), phytohydrosphingosine; LC, long chain (acyl chain is equal to or less than 22 carbons); VLC, very long chain (more than 22 carbons). (<b>B</b>) <b>Treatment with ceramide promotes PES formation and inhibits actin cytoskeleton polarization.</b> Wild type (BY4741) cells expressing GFP-2×PH^PLCδ were grown in YPD in the presence of vehicle (EtOH), PC(<i>O</i>-16:0/2:0), Cer(d18:1/2:0) or Cer(d18:0/2:0) (20 µM, 15 min) prior to imaging live or fixing and staining for acting cytoskeleton polarization as described in methods. The percentage of cells displaying a redistribution of the fluorescent reporter or proper actin polarization is reported in the inset of the respective figure. (<b>C</b>) <b>Inhibition of sphingolipid metabolism prevents the relocalization of Mss4.</b> Mss4-GFP (YKB2955) expressing cells were pretreated with vehicle or myriocin (5 µM) for 30 min and subsequently treated with vehicle or PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) as previously done. Pretreatment with myriocin inhibited PC(<i>O</i>-16:0/2:0)-dependent changes in PES formation.