. Second, amoebae increasing on bacteria as a food source strongly downregulate
. Second, amoebae developing on bacteria as a meals source strongly downregulate the transcription of enzymes involved in de novo fatty acid synthesis (47). Understanding about the path and kinetics of fatty acid flow will further help upcoming studies on the effect of therapeutically helpful substances on fatty acid metabolism applying Dictyostelium as a model technique (48).November 2013 Volume 12 Numberec.asm.orgDu et al.FIG 3 Dictyostelium lipid droplets contain steryl esters. (A to D) Confocal images from fixed cells expressing steryl methyltransferase 1 (Smt1) tagged with GFP (green channel) at the N-terminal finish (A and B) or at its C terminus (C and D) and incubated with (B and D) or with no (A and C) fatty acid (FA). The endoplasmic reticulum was revealed by virtue of an antibody directed against PDI that appears red in panels A and C. Alternatively, lipid droplets were stained by LD540 (red in B and D). The overlaid images (OL) appear in the third column (scale bar, 5 m), exactly where for row B the image from transmitted light can also be shown to demonstrate the outline of the otherwise barely visible cell. (E and F) Optical sections by means of living wild-type (WT) cells stained with LD540 (red) to reveal lipid droplets (dots in panel F) in cells fed with cholesterol ( CHL) for three h. In handle cells ( CHL) the dye associates nonspecifically with organelle membranes such as the nuclear envelope and also the closely related Golgi apparatus (E). Scale bar, 5 m. (G) Thin-layer ERRĪ² Formulation chromatography of lipid samples extracted from wild-type cells grown in axenic medium with no additional additives (Ctrl), with 200 M palmitic acid added ( FA), with one hundred M cholesterol ( CHL) added, or with both ( CHL FA). Substances in the marker lane (M) are labeled as in Fig. 1D. Here, only steryl esters (SE) are relevant. An unknown lipid species (UKL) is additional discussed in the text.Composition of lipid droplets. For experimental purposes, we have chosen to induce lipid droplet formation by the addition of palmitic acid and of cholesterol towards the medium. Our quantitative analysis of lipid composition suggests that no fundamental variations exist in comparison to lipid droplets from other organisms. By far, the big Bim Purity & Documentation neutral lipid species in Dictyostelium lipid droplets is TAG, comprising roughly 57 from the constituent molecules. When cholesterol is offered as well as palmitic acid, the TAG level drops to about 48 , whilst steryl ester (SE) molecules enhance from 4 to 16 . A equivalent TAG-to-SE ratio of 15 was seen in lipid droplets in the yeasts Yarrowia lipolytica (49) and Pichia pastoris (50) as well as in mammalian adipocytes (51). The first consequence of cholesterol addition is the look of a band that migrates slightly below the marker cholesteryl palmitate. Additional addition of palmitate to the medium produces a Second band that matches the marker completely (Fig. five). Certainly, closer evaluation (Table 2) reveals that 43 of this lipid is cholesteryl palmitate, apparently lacking any further modifications. Conjugated to palmitate along with other acyl chains, the added cholesterol tends to make up 92 with the steryl esters within lipid droplets (Table 2), whereas it contributes roughly only 35 with the absolutely free sterol molecules (data not shown). The membrane in the lipid droplet appears to become mostly composed of phospholipids, with either ethanolamine or choline as head groups in roughly equal amounts (data not shown). This composition, also as the total amount, falls within the range of 1 to2 as estim.