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F. Yang et al.
increased rapidly in wild-type cells, but this increase was significantly blunted in cKO cells (Figure 4C and D). Interestingly, the flippase activity was mainly compro- mised in the R1 to R3 cell populations (Figure 4E and F), indicating correlation between flippase activity and cell development. We also analyzed the percentage of NBD-PS fluorescence positive cells in the S1 to S3 cell population and found that the flippase activity was mainly compro- mised in S1 and S2 cell populations (Online Supplementary Figure S4A and B). These data demonstrate that Tmem30a is crucial for phospholipid flipping in erythroid cells.
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Tmem30a deficiency compromises lipid raft clustering upon erythropoietin treatment
The above data demonstrate that Tmem30a is required for phospholipid flipping. PS is essential for the coupling between actin and lipid anchored proteins, and thereby the formation of functional local raft-like domain at the plasma membrane. One of the most important roles of lipid rafts is to separate and regulate specific membrane components with other components and thereby increas- ing the concentration of signaling molecules. To examine whether depletion of Tmem30 affects lipid raft formation
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Figure 2. Tmem30a-deficient mice (cKO) embryos are defective in definitive erythropoiesis. (A) Representative flow cytometric profiles of control and Tmem30a cKO fetal liver single cells stained with CD71 and Ter119. Gates from R1 to R5 were set as indicated. (B) Absolute number of cells in each R population was calculated in the fetal liver from each embryo. Data are represented as mean±Standard Error of Mean (SEM) of the cell count of six fetal livers for each embryonic data set. (C) Representative CD71/FSC profiles of Ter119hi cells sorted into three populations according to cell size. The percentage of cells in each S population with respect to total Ter119hi cells are indicated for one representative fetal liver. (D) Comparison of the number of S1, S2, and S3 populations. (E) Representative flow cytometry of enucleated cells in the Ter119 hi population of fetal livers, using Syto-16 for nuclei and DAPI for cell viability. (F) Percentages of enucleated cells in the Ter119 hi populations. Data are presented as Mean±SEM of the cell count of six fetal livers for each embryonic data set. *P<0.05; **P<0.01; ***P<0.001.
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haematologica | 2019; 104(10)