FAT is a component of glomerular slit diaphragms
FAT is a component of glomerular slit diaphragms
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DOI：
10.1046/j.1523-1755.2001.0590031003.x
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发表时间：
2001-03-01
影响因子：
19.6
通讯作者：
Yamamoto, T
中科院分区：
文献类型：
--
作者：
Inoue, T;Yaoita, E;Yamamoto, T
裂隙横膈膜是肾小球足细胞的细胞间连接处。狭缝隔膜的分子组成仍然是难以捉摸的。狭缝横膈膜的特征是细胞间有较宽的间隙。桥粒和粘附体连接具有相同的形态特征，它们包含钙粘蛋白超家族的成员。因此，我们假设狭缝隔膜的某些成分属于钙粘蛋白超家族。因此，我们利用基于钙粘蛋白分子保守序列的引物，通过同源聚合酶链反应（PCR）从逆转录（RT）肾小球cDNA中分离出编码FAT的cDNA。FAT是钙粘蛋白超家族的新成员，具有34个串联钙粘蛋白样细胞外重复序列，与果蝇的肿瘤抑制脂肪非常相似。采用核糖核酸酶保护法和原位杂交法检测成年大鼠肾小球中脂肪的表达。为了在足细胞中精确定位FAT蛋白，我们通过免疫兔c -末端20个氨基酸对应的寡肽制备了抗FAT的亲和纯化抗体。在肾小球总RNA中检测FAT mRNA的表达。原位杂交显示足细胞中有明显的信号。用溶化肾小球进行Western blot分析显示，其分子量大于500 kD，呈单带。培养的大鼠肾上皮细胞（NRK52E）的免疫染色显示脂肪在细胞-细胞接触部位积累。在肾小球，沿肾小球毛细血管壁可见明显的脂肪染色。单克隆抗狭缝横膈膜双标记免疫染色（mAb 5-1-6）显示抗fat抗体与mAb 5-1-6定位相同。此外，超薄冷冻双标记免疫金技术表明，FAT细胞质结构域的金颗粒位于mAb 5-1-6标记的狭缝膜的底部，并且FAT细胞质结构域与与狭缝膜相关的细胞质成分ZO-1共定位。FAT的分子结构及其与5-1-6抗原和ZO-1的共定位表明FAT是狭缝隔膜的组成部分。
Background Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, we have hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, we have isolated cDNA encoding FAT from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat.Methods. Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, we prepared affinity-purified antibody against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids.Results. Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining was observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrated that gold particles for FAT cytoplasmic domain were located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalized with ZO-1, a cytoplasmic component associated with slit diaphragms.Conclusion. The molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms.