The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules
The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules
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DOI：
10.1016/s0960-9822(00)00600-x
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发表时间：
2000-07-13
期刊：
影响因子：
9.2
通讯作者：
Tsukita, S
中科院分区：
文献类型：
--
作者：
Mimori-Kiyosue, Y;Shiina, N;Tsukita, S
腺瘤性息肉病线圈蛋白 (APC) 是一种充分表征的肿瘤抑制蛋白 [1-3]。我们之前表明，爪蟾 A6 上皮细胞中带有绿色荧光蛋白 (GFP) 标记的 APC 沿着微管子集移动，并在细胞延伸的生长正端处积累 [4]。 EB1 被酵母双杂交分析鉴定为 APC 结合蛋白 [5]，据报道也与微管相关 [6-8]，为了检查细胞内 APC 和 EB1 之间的相互作用，我们将 EB1-GFP 的动态行为与 A6 转染子中 APC-GFP 的动态行为进行比较，间期活细胞的延时显微镜显示 EB1-GFP 集中在所有生长的细胞中。 微管末端遍及细胞质，当微管开始缩短时，微管末端突然消失。因此，EB1 似乎与 APC 共定位并在微管子集的生长端相互作用。当APC-GFP过表达时，内源EB1被招募到APC-GFP中，并在微管上大量积累。另一方面，当微管被诺考达唑分解时，EB1不与APC-GFP共定位，APC-GFP沿着基底质膜浓缩。在有丝分裂期间，APC 似乎与微管分离，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节，并且仅在受限条件下才允许在微管末端附近发生。
Adenomatous polyposis coil protein (APC) is a well-characterized tumor suppressor protein [1-3]. 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 [6-8], 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.