Project description:The aim of this work is to investigate whether A. thaliana senses low temperature by perceiving changes in membrane fluidity. To this end, we have performed an experiment to test whether mutant or transgenic plants with altered membrane lipid composition, regulate their gene expression in the same manner as wild type plants in response to cold. Previous work has demonstrated that a change in the expression levels of a number of genes is important in acquiring tolerance to low temperatures. Chemicals which rigidify cell membranes in such a way as to mimic the effects of cold have been shown to be able to induce the expression of such genes. However, because of the non-specific nature of such chemical treatments, it has not been possible to demonstrate unequivocally that the changes in gene expression observed were the result of changes in membrane fluidity. All of the mutants used in our experiment, fab1, fad2-2 and the fad3/fad7/fad8 mutant, have increased lipid saturation levels compared to wild type plants and are thought to have reduced membrane fluidity. The fab1 mutant is also known to be sensitive to chilling. In the fab1 mutant the elongation of 16:0 fatty acids to 18:0 is reduced. The fad2-2 mutant has reduced 18:1 desaturase activity and hence reduced amounts of polyunsaturated phospholipids. The fad3/fad7/fad8 triple mutant is deficient in 18:2 desaturase activity and consequently unable to synthesise trienoic fatty acids. The transgenic line used contained a 35S::FAD3 transgene and in contrast to the mutants tested, should have increased lipid desaturation and increased membrane fluidity. A. thaliana ecotype Col-0 was used as the wild type control for the fab1 and fad2-2 mutants, in addition to the 35S::FAD3 line. The fad3/fad7/fad8 mutant had previously been transformed with the 35S::apoaequorin transgene and a Columbia line expressing apoaequorin under the control of the same promoter, was included to control for the presence of aequorin. Nine day old seedlings grown in petri-dishes on MS were transferred from their growth room (20 oC, 16 h photoperiod, 100 E m-2 s-1) to a growth cabinet (20 oC, 16 h photoperiod,160 m-2 s-1) 24 hours before the experiment began. The next day, one petri-dish of seedlings from each line of plants used was transferred to a cabinet running at 5 oC (16 h photoperiod,160 E m-2 s-1). Control plates remained at 20 oC. Seedlings were harvested after three hours and frozen in liquid nitrogen.

Project description:The aim of this work is to investigate whether A. thaliana senses low temperature by perceiving changes in membrane fluidity. To this end, we have performed an experiment to test whether mutant or transgenic plants with altered membrane lipid composition, regulate their gene expression in the same manner as wild type plants in response to cold. Previous work has demonstrated that a change in the expression levels of a number of genes is important in acquiring tolerance to low temperatures. Chemicals which rigidify cell membranes in such a way as to mimic the effects of cold have been shown to be able to induce the expression of such genes. However, because of the non-specific nature of such chemical treatments, it has not been possible to demonstrate unequivocally that the changes in gene expression observed were the result of changes in membrane fluidity. All of the mutants used in our experiment, fab1, fad2-2 and the fad3/fad7/fad8 mutant, have increased lipid saturation levels compared to wild type plants and are thought to have reduced membrane fluidity. The fab1 mutant is also known to be sensitive to chilling. In the fab1 mutant the elongation of 16:0 fatty acids to 18:0 is reduced. The fad2-2 mutant has reduced 18:1 desaturase activity and hence reduced amounts of polyunsaturated phospholipids. The fad3/fad7/fad8 triple mutant is deficient in 18:2 desaturase activity and consequently unable to synthesise trienoic fatty acids. The transgenic line used contained a 35S::FAD3 transgene and in contrast to the mutants tested, should have increased lipid desaturation and increased membrane fluidity. A. thaliana ecotype Col-0 was used as the wild type control for the fab1 and fad2-2 mutants, in addition to the 35S::FAD3 line. The fad3/fad7/fad8 mutant had previously been transformed with the 35S::apoaequorin transgene and a Columbia line expressing apoaequorin under the control of the same promoter, was included to control for the presence of aequorin. Nine day old seedlings grown in petri-dishes on MS were transferred from their growth room (20 oC, 16 h photoperiod, 100 E m-2 s-1) to a growth cabinet (20 oC, 16 h photoperiod,160 m-2 s-1) 24 hours before the experiment began. The next day, one petri-dish of seedlings from each line of plants used was transferred to a cabinet running at 5 oC (16 h photoperiod,160 E m-2 s-1). Control plates remained at 20 oC. Seedlings were harvested after three hours and frozen in liquid nitrogen. 12 samples were used in this experiment

Project description:Dendritic cells (DCs) are professional antigen-presenting cells of the immune system. Upon sensing pathogenic material in their environment, DCs start to mature, which includes cellular processes, such as antigen uptake, processing and presentation, as well as upregulation of costimulatory molecules and cytokine secretion. During maturation, DCs detach from peripheral tissues, migrate to the nearest lymph node, and find their way into the correct position in the net of the lymph node microenvironment to meet and interact with the respective T cells. We hypothesize that the maturation of DCs is well prepared and optimized leading to processes that alter various cellular characteristics from mechanics and metabolism to membrane properties. Here, we investigated the mechanical properties of monocyte-derived dendritic cells (moDCs) using real-time deformability cytometry to measure cytoskeletal changes and found that mature moDCs were stiffer compared to immature moDCs. These cellular changes likely play an important role in the processes of cell migration and T cell activation. As lipids constitute the building blocks of the plasma membrane, which, during maturation, need to adapt to the environment for migration and DC-T cell interaction, we performed an unbiased high-throughput lipidomics screening to identify the lipidome of moDCs. These analyses revealed that the overall lipid composition was significantly changed during moDC maturation, even implying an increase of storage lipids and differences of the relative abundance of membrane lipids upon maturation. Further, metadata analyses demonstrated that lipid changes were associated with the serum low-density lipoprotein (LDL) and cholesterol levels in the blood of the donors. Finally, using lipid packing imaging we found that the membrane of mature moDCs revealed a higher fluidity compared to immature moDCs. This comprehensive and quantitative characterization of maturation associated changes in moDCs sets the stage for improving their use in clinical application.

Dataset Information

The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness.

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The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness.

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