Levels of LCBs determine nuclear shape and volume in yeast. (a) De novo synthesis pathway of sphingolipids in yeast. See Fig. S1 b for a more detailed description of the pathway. Genes targeted in our experiments are highlighted in blue. SPT, serine palmitoyltransferase; SphK, sphingosine kinase (LCB4); CerS, ceramide synthase (LAG1); LCBs, long-chain bases; LCB-P, LCB-1-phosphate. (b) Live-cell microscopy of yeast cells expressing Heh1-GFP. DIC, differential interference contrast. Scale bar, 5 µm. (c) Area of nuclei of WT yeast and cells harboring the tsc3∆ (n > 100 cells). *P < 1 e-4, unpaired t test. (d) Representative electron microscopy image of WT yeast and TSC3 deletion (tsc3∆). NE contour labeled with a white dotted line for visualization. Scale bars, 400 nm. (e) Live-cell microscopy of yeast cells expressing Heh1-GFP harboring the lcb4∆ or lag1∆. Scale bar, 5 µm. Changes in the levels of LCBs relative to WT cells in these strains are shown below (data from Hwang et al. [2019]). (f) Area of isolated nuclei of WT yeast and cells harboring the lcb4∆ or lag1∆. Box plots of quantification of nuclear areas (n = 200 cells). *P < 1 e-4, one-way ANOVA test. (g) Coulter counter profiles of purified yeast nuclei (n = 10,000). (h) Coulter counter profiles of yeast cells (n = 10,000). (i) Model of how changing the levels of LCBs affects the nucleus.