Removal of Stomatin, a Membrane-Associated Cell Division Protein, Results in Specific Cellular Lipid Changes

Abstract

Lipids are key constituents of all cells, which express thousands of different lipid species. In most cases, it is not known why cells synthesize such diverse lipidomes, nor what regulates their metabolism. Although it is known that dividing cells specifically regulate their lipid content and that the correct lipid complement is required for successful division, it is unclear how lipids connect with the cell division machinery. Here, we report that the membrane protein stomatin is involved in the cytokinesis step of cell division. Although it is not a lipid biosynthetic enzyme, depletion of stomatin causes cells to change their lipidomes. These changes include specific lipid species, like ether lipids, and lipid families like phosphatidylcholines. Addition of exogenous phosphatidylcholines rescues stomatin-induced defects. These data suggest that stomatin interfaces with lipid metabolism. Stomatin has multiple contacts with the plasma membrane and we identify which sites are required for its role in cell division, as well as associated lipid shifts. We also show that stomatin's mobility on the plasma membrane changes during division, further supporting the requirement for a highly regulated physical interaction between membrane lipids and this newly identified cell division protein.

Figure 1

Stomatin is required for cytokinesis in human cells. (A) Representative immunofluorescence images of HeLa cells fixed and stained with anti-α-tubulin (green) and DAPI (magenta) to visualize α-tubulin and DNA, respectively, 72 h after transfection with nontargeting (siNT) or siRNA targeting stomatin (siSTOM1). * indicates bi- or multinucleated cells, scale bar = 10 μm. (B) Cytokinesis failure phenotypes for HeLa cells transfected with siSTOM1 (n = 227 cells) or NT siRNA (n = 204 cells) from three independent experiments. (C) Time taken to progress from anaphase to abscission or cytokinesis failure for siNT (blue) or siSTOM1 treated cells (green) from cells described in (B). **** indicates significance p < 0.0001. Black bars represent median and upper and lower quartiles.

Figure 2

Stomatin depletion induces lipidome changes. (A) Top 25 lipid species that change in siSTOM1- vs siNT-treated cells (fold change > 1.7), sorted by lipid family. The labels on the x axis represent lipid names, with the letters identifying the lipid family and the numbers the length and saturation of the side chains (see structures in Figure S5). *Assignment of this species predicted by MS-FINDER but unconfirmed. Data are presented as mean ± S.D. (N = 9). (B) Selected lipid family changes in siSTOM vs siNT treated cells. An average of the fold changes for each lipid species within a family from all species in our in-house library are shown (lipids with a p-values <0.05 are shown in blue). PC is phosphatidylcholine, PC-O ether PC, PE phosphatidylethanolamine, PE-O ether PE. Triangles show lipids that appear in Figure 2A. Data from Table S2. (C) Quantification of the percentage of binucleated HeLa cells treated with siNT or siSTOM with (green) or without (light blue) addition of 10 μM PC mix. Data represent mean ± S.D. (N = 3), >300 cells scored per experiment, *** indicates p < 0.001.

Figure 3

Stomatin mutants show that specific membrane association is required for cytokinesis. (A) Quantification of binucleation in HeLa stably expressing control membrane marker (MyrPalm-GFP) or GFP-tagged stomatin wt or mutant constructs, 72 h after transfection with nontargeting (NT) or siRNA targeting stomatin (siSTOM1). Data are presented as mean ± S.D. (N = 3), >300 cells scored per experiment, **** indicates p < 0.0001. (B) Lipid analysis from pulldown experiments with GFP-stomatin WT (orange), C30S (blue) or dC (red). Data are presented as mean ± S.D. (N = 3) *, p ≤ 0.05.

Figure 4

Stomatin mutants reveal links between lipid expression and protein functions. (A) and (B) Sum of the average total ion counts of each lipid class in HeLa WT or expressing different stomatin constructs treated with siNT (light blue) or siSTOM (green). HeLa and C30S fail cytokinesis after siSTOM, while GFP-Stom wt and dC do not. High abundance lipids are shown in (A), low abundance lipids are shown in (B). PC, phosphatidylcholine; PC-O, ether PC; PE, phosphatidylethanolamine; PE-O, ether PE; CER, ceramide; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; SM, sphingomyelin; TG, triacylglycerol; TG-O, ether triacylglycerol; DG, diacylglycerol; FA, fatty acid; HexCer, hexosylceramide; and DMPE, dimethyl PE. Data are presented as sum ± S.D. (N = 6). (C) Distribution of mobile (gray columns) and immobile fractions (blue columns) after FRAP treatment of wt or mutant stomatin-GFP, in dividing or nondividing cells. Data are presented as mean (N = 2, cells = 15) and the error indicates S.E.M.