Inhibition of glycosphingolipid biosynthesis induces cytokinesis failure

Abstract

Although cells undergo dramatic shape changes during cytokinesis, the role of the plasma membrane and lipids is poorly understood. We report that inactivation of glucosyl ceramide synthase (GCS), either by RNAi or with the small molecule PPMP, causes failure of cleavage furrow ingression. Using mass-spectrometry-based global lipid profiling, we identify individual lipids that are enhanced or depleted due to GCS inhibition. We show that GCS inhibition results in the mislocalization of actin and the ERM proteins, key cytoskeletal proteins that connect the plasma membrane to the actin cortex. Our data suggest that ceramides participate in mediating the interactions between the membrane and the cortex.

Figure 1

A Structure of PPMP, an inhibitor of glucosyl ceramide synthase that causes cytokinesis failure. B PPMP treatment over 24 hours and RNAi depletion of the target enzyme, GCS, over 72 hours causes an increase in the number of binucleated cells (n=300). C Representative control and binucleated HeLa cells. Microtubules are shown in green, DNA in red (Scalebar = 10 μm).

Figure 2

Ceramides with altered levels as a result of PPMP treatment (2 and 3) and siGCS treatment (4, 5, 6 and 7). Exogenous addition of C12- and C14-ceramide (8 and 9) causes cytokinesis failure.

Figure 3

PPMP treatment changes actin and ERM localization and abolishes small cellular protrusions. A–B Control and PPMP-treated cells were stained for microtubules (anti-tubulin) and ERM (anti-ERM). C–D Control and PPMP-treated cells were stained for microtubules (anti-tubulin) and actin (phalloidin). To illustrate changes in cortical protrusions more clearly, magnified images are shown next to the grey scale ERM and actin images (Scale bar = 10 μm). Representative micrograph is shown for n= 28/33.

Figure 4

PPMP treatment causes immature spreading at the polar regions during cytokinesis, i.e. cells start to attach before completing division. Control (A) and PPMP-treated cells are (B) stained for ERM (anti-ERM) and actin (phalloidin) (Scale bar = 10 μm). Representative micrograph is shown for n=5/13.