Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation - is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.     

Factor Analysis of the Impact of the Environment on Microbial Communities in the Tvärminne Area, Southern Coast of Finland

Data already examined by regression analysis were subjected to factor analysis to scrutinize the effects of environmental factors on microbial populations in the brackish waters of the Tvärminne archipelago on the southern coast of Finland. Water samples were collected from 1.0-m depth at one point in Tvärminne Storfjärd, 71 times over about 2 years. Twenty-six parameters were determined on each sample, 10 of environmental and 16 of microbiological type. The correlations between the parameters were factorized using the principal axis solution, and eight factors chosen for further consideration were rotated by the varimax method. The major part of the variance (about 90% of the total communality) of the microbiological parameters was covered by five factors, interpreted as phytoplankton blooms, the periods before and after the blooms, freshwater outflows, and water temperature. Wind variables were components in the factors interpreted as freshwater outflows. Rainfall played a minor part in the total variance of the microbial community, but it washed yeasts and proteolytic bacteria from the land into the study area. The eight factors selected covered about 60 to 98% of the variance of the microbiological parameters. The highest values (above 90%) were obtained for direct counts of bacteria, for plate counts of mesophilic and polymyxin-resistant bacteria, and for the two community respiration parameters; the lowest values (60 to 75%) were obtained for H₂S-producing and proteolytic bacteria.