ERM proteins and Odf2 as organizers for apical membranes

1.ERM蛋白敲除小鼠的制备和分析：敲除小鼠的分析表明，radixin通过将胆红素转运蛋白MRP2锚定在肝脏毛细血管胆管的管腔表面，从而维持胆红素转运蛋白的功能。在基因敲除小鼠中，MRP2不能发挥作用，并诱导Dubin-Johnson综合征型高胆红素血症。在这项研究中，我们发现这种综合征的个体组织学和血液学差异可能反映了基底外侧表面MRP3表达水平的遗传背景相关差异。细胞粘附装置组成分析：我们建立了1989年从肝脏制备的毛细血管胆管部分制备浓缩细胞粘附装置的方法，并鉴定了组成该装置的主要蛋白质。在本研究中，我们利用质谱技术的显著进展，鉴定了许多细胞粘附装置的新成分，这是以前的方法所无法实现的。正如预期的那样，许多这些标记的蛋白质定位在细胞粘附装置中。我们制备了针对这些蛋白质的抗体，在培养细胞中的RNAi实验正在进行中。我们正计划研究这些蛋白在细胞间黏附中的协调作用。敲除小鼠中心体成分蛋白Odf2分析：在Odf2条件敲除小鼠中，细胞分裂可能不受影响，只有初级纤毛可能器官特异性消失。利用这些小鼠，可以在体内分析初级纤毛的作用，这一点长期以来一直不清楚。Odf2是精子外致密纤维（ODF）的一种成分，它的消失会导致男性不育。由于只有轻微的表达降低才会导致不育，因此条件敲除小鼠的制备很困难，但我们最终建立了一个杂合子代。由于在细胞培养水平上提出了初级纤毛的一种意想不到的功能，因此对单个动物的分析可能提供重要的信息。少

1.Preparation and analysis of ERM protein knockout mice : Analysis of knockout mice clarified that radixin maintains the function of the bilirubin transporter, MRP2, by anchoring it to the luminal surface of capillary bile ducts in the liver. In knockout mice, MRP2 cannot function, and induces Dubin-Johnson syndrome-type hyperbilirubinemia. In this study, we showed the possibility of individual histological and hematological differences in this syndrome reflecting genetic background-associated differences in the basolateral surface MRP3 expression level.2.Analysis of the composition of cell adhesion apparatus : We established a preparation method of a concentrated cell adhesion apparatus fraction from a capillary bile duct fraction prepared from the liver in 1989, and identified major proteins composing the apparatus. In this study, we identified many novel components of the cell adhesion apparatus, which was not possible using previous methods, by taking advantage of the marked progre … More ss in mass spectrometry. Many of these tagged proteins were localized in the cell adhesion apparatus, as expected. We prepared antibodies against these proteins, and RNAi experiments in cultured cells are now underway. We are planning to investigate how these proteins coordinate in their roles in intercellular adhesion.3.Analysis of centrosome component protein, Odf2, knockout mice : In Odf2 conditional knockout mice, cell division may not be affected, and only the primary cilium may organ-specifically disappear. Using these mice, the role of primary cilia may be analyzed in vivo, which has remained unclear for a long time. Odf2 is a component of outer dense fibers (ODF) of sperms, and its disappearance causes male infertility. Since only a slight decrease in the expression caused infertility, preparation of conditional knockout mice was difficult, but we finally established a heterozygote generation. Since an unexpected function of the primary cilium was suggested at the cell culture level, analysis of individual animals may provide significant information. Less