Ilayers, these do not authentically reflect biological membranes, including the diversity of membrane lipid and protein composition, the complexity of lateral and transversal lipid asymmetries and the influence of the cytoskeleton on membrane organization. Bacteria represent an alternative model to study membrane lipid organization. E. coli for example is very well characterized, grows rapidly and allows easy genetic manipulations, popular for functional studies. Nevertheless, bacteria are small (less than few m) and require high-resolution microscopy to be properly analyzed. Moreover, the PM composition of prokaryotes is quite different from the mammalian PM (Table 3), leading to different organization and functions. Yeast represents another powerful model to investigate membrane lipid organization and especially the importance of proteins in this process. Saccharomyces Cerevisiae is one of the most intensively used eukaryotic models due to comprehensive banks of mutants, a size ( 10m) compatible with conventional microscopy and a rapid growth. However, the yeast cell wall limits penetration of molecules larger than 700Da [158], preventing incorporation into intact yeast of fluorescent analogs of polar lipids mixed with BSA as lipid carriers. Other labeling order Pan-RAS-IN-1 approaches, such as expression of lipid specific markers, have to be developed to circumvent this difficulty (see Section 3.1.2). Mammalian nucleated cells offer the possibility of co- and 3D-culture and an easy growth. However, they usually present considerable limitations to study membrane lipid lateral organization due to lipid metabolism, endocytosis and a tortuous surface due to vesicular trafficking and membrane protrusions, which can lead to false interpretations. This is why our group focuses on RBCs [26, 27, 29, 30, 146]. RBC is the simplest and best characterized eukaryotic cell system, both at lipid and protein levels [159, 160]. Moreover, for practical purposes, RBCs (i) are easily available and robust; (ii) are highly homogenous in size and shape due to rapid clearance of damaged RBCs by the spleen; (iii) present a flat surface without membrane projections or protrusions, avoiding confusion between domains and lipid enrichment in membrane ruffles; (iv) do not metabolize lipids; and (v) do not make endocytosis, avoiding any confusion between domains and endosomes. Whereas all membranes described above represent interesting models to visualize lipid organization, it has to be kept in mind that their composition is quite different. Table 3 gives the PM composition of different cell types. For instance, SM and cholesterol contents of the RBC PM are particularly high, as compared to the PM of human alveolar macrophages. Since cholesterol plays a dominant role in the regulation of membrane fluidity, changes in cholesterol levels will differentially modulate membrane organization into domains in theseAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptProg Lipid Res. Author manuscript; available in PMC 2017 April 01.AZD4547 chemical information Carquin et al.PagePMs. It is also important to note that yeast PM exhibits ergosterol instead of cholesterol, while most bacteria PMs do not contain sterols. Other factors such as the PM anchorage to the underlying cytoskeleton, which is about 20-fold stronger in RBC than in fibroblasts, and the presence of a cell wall, as in yeast, should also be considered (see Section 5.2). Therefore, generalized discussions of submicrometric membrane domains.Ilayers, these do not authentically reflect biological membranes, including the diversity of membrane lipid and protein composition, the complexity of lateral and transversal lipid asymmetries and the influence of the cytoskeleton on membrane organization. Bacteria represent an alternative model to study membrane lipid organization. E. coli for example is very well characterized, grows rapidly and allows easy genetic manipulations, popular for functional studies. Nevertheless, bacteria are small (less than few m) and require high-resolution microscopy to be properly analyzed. Moreover, the PM composition of prokaryotes is quite different from the mammalian PM (Table 3), leading to different organization and functions. Yeast represents another powerful model to investigate membrane lipid organization and especially the importance of proteins in this process. Saccharomyces Cerevisiae is one of the most intensively used eukaryotic models due to comprehensive banks of mutants, a size ( 10m) compatible with conventional microscopy and a rapid growth. However, the yeast cell wall limits penetration of molecules larger than 700Da [158], preventing incorporation into intact yeast of fluorescent analogs of polar lipids mixed with BSA as lipid carriers. Other labeling approaches, such as expression of lipid specific markers, have to be developed to circumvent this difficulty (see Section 3.1.2). Mammalian nucleated cells offer the possibility of co- and 3D-culture and an easy growth. However, they usually present considerable limitations to study membrane lipid lateral organization due to lipid metabolism, endocytosis and a tortuous surface due to vesicular trafficking and membrane protrusions, which can lead to false interpretations. This is why our group focuses on RBCs [26, 27, 29, 30, 146]. RBC is the simplest and best characterized eukaryotic cell system, both at lipid and protein levels [159, 160]. Moreover, for practical purposes, RBCs (i) are easily available and robust; (ii) are highly homogenous in size and shape due to rapid clearance of damaged RBCs by the spleen; (iii) present a flat surface without membrane projections or protrusions, avoiding confusion between domains and lipid enrichment in membrane ruffles; (iv) do not metabolize lipids; and (v) do not make endocytosis, avoiding any confusion between domains and endosomes. Whereas all membranes described above represent interesting models to visualize lipid organization, it has to be kept in mind that their composition is quite different. Table 3 gives the PM composition of different cell types. For instance, SM and cholesterol contents of the RBC PM are particularly high, as compared to the PM of human alveolar macrophages. Since cholesterol plays a dominant role in the regulation of membrane fluidity, changes in cholesterol levels will differentially modulate membrane organization into domains in theseAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptProg Lipid Res. Author manuscript; available in PMC 2017 April 01.Carquin et al.PagePMs. It is also important to note that yeast PM exhibits ergosterol instead of cholesterol, while most bacteria PMs do not contain sterols. Other factors such as the PM anchorage to the underlying cytoskeleton, which is about 20-fold stronger in RBC than in fibroblasts, and the presence of a cell wall, as in yeast, should also be considered (see Section 5.2). Therefore, generalized discussions of submicrometric membrane domains.
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