Nakayama Y, Hisano T, Okimoto T, Tanaka Y, Ishikawa T, Himeno M, Ono M, Kuwano M
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Cell Struct Funct. 1994 Dec;19(6):397-409. doi: 10.1247/csf.19.397.
The epidermal growth factor (EGF)-induced endocytosis of its receptor is an obligatory pathway for the cellular regulation of the EGF-specific receptor (EGF-R). BNER4 is a mouse Balb/3T3 cell line transfected with human EGF-R complementary DNA (cDNA). B4/src-13 and B4/src-24 are BNER4 cells transfected with a viral oncogene v-src. Indirect immunofluorescence study demonstrated that EGF-R was mostly localized at the perinuclear region in BNER4 cells at 60 min after EGF addition, whereas it was diffusely distributed throughout the cytoplasm in its v-src transfectants. Double indirect immunofluorescence study further confirmed that EGF-R was localized in lysosomes in BNER4 and B4/src-13 cells at 60 min after EGF addition. Intracellular distribution of the Golgi apparatus, clathrin-coated vesicles and early endosomes were similar in all cell lines. However, the lysosomes detected by anti-lysosomal membrane protein (LGP85) antibodies were diffusely distributed throughout the cytoplasm in the v-src transfectacts. By contrast, in the parental BNER4 cells, the lysosomes were mostly localized in the perinuclear region. The organization of microtubules, but not of actin, was markedly different between BNER4 cells and its v-src transfectants. Nocodazole, which depolymerizes microtubules, altered the distribution of the lysosomes and EGF-R in BNER4 cells. Both intracellular lysosome distribution and microtubule organization in nocodazole-treated BNER4 cells were found to be similar to those in its v-src transfectants without nocodazole treatment. These findings support the notion that changes in lysosome distribution may be correlated with microtubule reorganization by v-src in mouse Balb/3T3 cells.
表皮生长因子（EGF）诱导其受体的内吞作用是细胞调节EGF特异性受体（EGF-R）的必经途径。BNER4是转染了人EGF-R互补DNA（cDNA）的小鼠Balb/3T3细胞系。B4/src-13和B4/src-24是转染了病毒癌基因v-src的BNER4细胞。间接免疫荧光研究表明，添加EGF 60分钟后，EGF-R在BNER4细胞中大多定位于核周区域，而在其v-src转染细胞中则弥漫分布于整个细胞质中。双重间接免疫荧光研究进一步证实，添加EGF 60分钟后，EGF-R在BNER4和B4/src-13细胞中定位于溶酶体。所有细胞系中高尔基体、网格蛋白包被小泡和早期内体的细胞内分布相似。然而，用抗溶酶体膜蛋白（LGP85）抗体检测到的溶酶体在v-src转染细胞中弥漫分布于整个细胞质中。相比之下，在亲代BNER4细胞中，溶酶体大多定位于核周区域。BNER4细胞与其v-src转染细胞之间微管（而非肌动蛋白）的组织明显不同。使微管解聚的诺考达唑改变了BNER4细胞中溶酶体和EGF-R的分布。发现在用诺考达唑处理的BNER4细胞中，细胞内溶酶体分布和微管组织与未用诺考达唑处理的v-src转染细胞相似。这些发现支持这样一种观点，即溶酶体分布的变化可能与小鼠Balb/3T3细胞中v-src介导的微管重组相关。
