Mimori-Kiyosue Y, Shiina N, Tsukita S
Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Shimogyo-ku, 600-8813, Japan.
Curr Biol. 2000 Jul 13;10(14):865-8. doi: 10.1016/s0960-9822(00)00600-x.
Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under restricted conditions.
腺瘤性结肠息肉病蛋白（APC）是一种特征明确的肿瘤抑制蛋白[1][2][3]。我们之前发现，在非洲爪蟾A6上皮细胞中标记有绿色荧光蛋白（GFP）的APC沿着一部分微管移动，并在细胞突起的微管生长正端积累[4]。通过酵母双杂交分析被鉴定为APC结合蛋白的EB1，也被报道与微管有关[6][7][8]。为了研究细胞内APC与EB1之间的相互作用，我们比较了A6转染细胞中EB1-GFP与APC-GFP的动态行为。对处于间期的活细胞进行延时显微镜观察发现，EB1-GFP集中在整个细胞质中所有生长中的微管末端，当微管开始缩短时，它会突然从末端消失。因此，EB1似乎在一部分微管的生长末端与APC共定位并相互作用。当APC-GFP过表达时，内源性EB1被招募到大量积累在微管上的APC-GFP处。另一方面，当用诺考达唑破坏微管时，EB1与集中在基底质膜上的APC-GFP不共定位。在有丝分裂期间，APC似乎与微管解离，而EB1-GFP继续集中在微管生长末端。这些发现表明，APC-EB1相互作用在细胞内受到调控，并且仅在受限条件下才在微管末端附近发生。
