Etics of lipid droplet (LD) formation, palmitic acid was added to a cell culture, and also the well-established lipid droplet dye Nile red was applied to image living cells at various instances. Figure 1A shows that lipid droplet formation in Dictyostelium has some qualities also observed in mammalian cells (34). New lipid droplets kind rapidly, rising initial more than 10-fold in number (Fig. 1B) and after that up to 2-fold in size (Fig. 1C), having a higher cellto-cell variation at 6 or 8 h following feeding. At late time points, the lipid droplets also often kind aggregates (Fig. 1A, eight h). To characterize the lipid goods which are formed upon fatty acid addition, extracts have been analyzed by thin-layer chromatography (TLC) (Fig. 1D). As expected, CYP2 Inhibitor drug essentially the most prominent adjust is noticed in the band identified as triacylglycerol (TAG) by comigration using a typical. More than the first three h, TAG concentration enhanced 23-fold (as determined by ImageJ analysis) and progressed to a plateau. This stage lasted for 6 h and was followed by a steady decline of fat levels more than the subsequent 9 h, reaching the initial value just after 24 h. During this time, 3 cell divisions took place, explaining the gradual consumption of storage fat and its conversion into membrane lipids or metabolic power. As an alternative to TLC densitometry, we employed an assay developed for TAG quantification within serum samples (Fig. 1E). The TAG concentration of total cell extracts collected more than 24 h paralleled the observations made by TLC (Fig. 1E, filled circles). A fatty acid washout experiment led to an immediate decline of TAG levels (Fig. 1E, open circles). One particular feature of this kind of measurement, however, is that the values show only an apparent 2-fold enhance of TAG at peak time (three h over 0 h), that is clearly underestimated. This house is caused by the truth that the enzymatic assay indirectly determines the concentration of glycerol released from fat by lipase action. Simply because the endogenous content material of glycerol and other feasible relevant metabolites in cells from normal medium is unknown, it was arbitrarily set to 1. One particular other function we noticed during the feeding experiments is that the cells grown in medium plus fatty acid appeared smaller sized than those grown in axenic medium lacking fatty acids (Fig. 1A, 0-h versus 8-h panel). To measure the cell volume, we spun cells soon after overnight feeding (14 h) into a glass capillary produced for hematocrit measurements by means of a funnel constructed from a plastic pipette tip. Indeed, the pellet volume occupied by the cells from the fatty acid-containing diet program was decreased to 74 three (n 3). Mainly because these modifications are paralleled in protein content material, we corrected all quantitative data accordingly (see Supplies and Procedures). Purification of lipid droplets and proteomic evaluation. About ten years ago, Miura and coworkers had fused the sequence in the Dictyostelium homologue of perilipin (Plin) to GFP and expressed the construct in mammalian CHO cells, where it localized to lipid droplets (35). To be able to acquire a marker for biochemical Dopamine Receptor Antagonist review preparation of lipid droplets, we constructed a GFP-Plin fusion for the expression in Dictyostelium cells. This strain was fed with fatty acid, and also the cells were mechanically cracked, leaving organelles intact. The preparation was loaded inside the middle of a sucrose step gradient so that only fat-containing organelles had been able to float for the leading throughout ultracentrifugation. In contrast to untreated cells, the cells that were allowed to f.