Cellular localization and tissue distribution of endogenous DFCP1 protein
Cellular localization and tissue distribution of endogenous DFCP1 protein
复制标题
DOI：
10.2220/biomedres.36.121
复制
发表时间：
2015-04-01
影响因子：
1.2
通讯作者：
Uchiyama, Yasuo
中科院分区：
文献类型：
--
作者：
Nanao, Tomohisa;Koike, Masato;Uchiyama, Yasuo
自噬对于维持细胞新陈代谢是必不可少的。一旦在细胞中诱导了自噬，隔离膜就形成了一个所谓的噬菌体。内质网(ER)是作为吞噬细胞膜来源的几个候选细胞之一。最近，从DFCP1(双FYVE结构域包含蛋白)阳性的ER相关隔室中发现了LC3阳性的分离膜，称为omegasome。尽管GFP标记的DFCP1蛋白已经在培养细胞中被检测到，但对这种内源性蛋白在细胞和组织中的准确分布知之甚少。为了确定内源性DFCP1蛋白的表达，我们制备了针对小鼠DFCP1蛋白的抗体。该抗体可识别人和鼠的DFCP1蛋白，这两种蛋白的分子质量都约为87 kDa。在正常情况下的HeLa细胞中，DFCP1的免疫反应沿Tom20阳性的丝状线粒体呈点状或管状分布，仅部分共存于内质网或高尔基体。此外，在饥饿条件下，不同的DFCP1阳性结构变得更加点状和散布在细胞质中，而Lc3阳性自噬小体的一部分对DFCP1免疫阳性。这些结果表明，针对DFCP1的抗体可能是解释omegasome隔间噬菌体形成机制的有用工具。
Autophagy is essential for the maintenance of cellular metabolism. Once autophagy is induced in cells, the isolation membrane forms a so-called phagophore. The endoplasmic reticulum (ER) is one of several candidates for the membrane source for phagophores. Recently, LC3-positive isolation membranes were found to emerge from a DFCP1 (double FYVE domain-containing protein)positive, ER-associated compartment called the omegasome. Although the GFP-tagged DFCP1 protein has been examined in cultured cells, little is known about the precise cellular and tissue distribution of this endogenous protein. To determine the expression of the endogenous DFCP1 protein, we produced antibodies specific to mouse DFCP1 protein. The antibody recognized both human and mouse DFCP1 proteins, both of which have molecular masses of approximately 87 kDa. In HeLa cells under normal conditions, immunoreactivity for DFCP1 was found dotted or tubular along Tom20-positive filamentous mitochondria and was only partially co-localized in the ER or Golgi apparatus. Moreover, under starved conditions, distinct DFCP1-positive structures became more dotted and scattered in the cytoplasm, while one part of the LC3-positive autophagosomes were immunopositive for DFCP1. These results indicate that an antibody raised against DFCP1 could be a useful tool in explaining the mechanism of phagophore formation from omegasome compartments.