Langford Kelly J, Askham Jon M, Lee Tracy, Adams Matthew, Morrison Ewan E
CRUK Clinical Centre, Division of Cancer Medicine Research, St James's University Hospital, Leeds, LS9 7TF, UK.
BMC Cell Biol. 2006 Jan 19;7:3. doi: 10.1186/1471-2121-7-3.
The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented.
Using live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC.
Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.
腺瘤性息肉病大肠杆菌（APC）肿瘤抑制蛋白在哺乳动物细胞中的运输一直是个备受争议的话题。此前已有免疫染色证据表明，肌动蛋白相关的APC定位于细胞间连接，不过哺乳动物连接部位APC的实时成像尚未见报道。
通过对转染的COS-7细胞进行实时成像，我们观察到与细胞间连接相关的绿色荧光蛋白（GFP）-APC池，此外还有先前记录的与微管相关的GFP-APC以及各种次要定位。虽然微管相关和连接相关的群体可以在单个细胞中共存，但它们在亚细胞位置、动态行为和对细胞骨架毒物的敏感性方面存在差异。GFP-APC缺失突变分析表明，在APC犰狳重复结构域后立即截断的蛋白质仍保留在转染细胞中定位于黏附膜的能力。支持这一观点的是，我们在表达截短形式APC的人结肠癌细胞系培养物中也观察到连接部位的APC免疫染色。
我们的数据表明，APC可在细胞周边的两个空间分离的群体中发现，且这些群体可在同一细胞中共存。第一种定位高度动态，与自由边缘附近和细胞顶点的微管相关，而第二种相对静态，与细胞-细胞接触部位的肌动蛋白紧密相关。我们的成像证实，人GFP-APC具有许多先前在非洲爪蟾GFP-APC实时成像中观察到的定位和行为。然而，我们报告了一个新发现，即GFP-APC斑点可与收缩微管的末端保持关联。缺失分析表明，APC蛋白的N端区域介导其连接部位的定位，这与我们观察到结肠癌细胞系中的截短APC蛋白仍能定位于细胞皮层一致。这可能对结直肠癌的发展有影响。
