Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.
Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.
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通过单克隆抗体检测核肌动蛋白和细胞质肌动蛋白之间的构象差异。
作者：
S. Gonsior;S. Platz;Sabine Buchmeier;U. Scheer;B. Jockusch;H. Hinssen
使用 Profilin 和骨骼肌肌动蛋白的重组复合物作为抗原，我们产生了抗肌动蛋白的单克隆小鼠抗体，称为 2G2。蛋白水解肌动蛋白片段的免疫印迹和胃蛋白酶扫描分析表明，该抗体可识别肌动蛋白上的非序列表位，该表位位于序列的三个不同区域内，包括 aa131-139、aa155-169 和 aa176-187。在源自 X 射线衍射的肌动蛋白模型中，这些序列在核苷酸结合裂口区域中在空间上靠近在一起，但不形成连贯的斑块。在免疫印迹中，2G2 与所有 SDS 变性的肌动蛋白亚型以及许多脊椎动物的肌动蛋白发生反应。相反，其免疫荧光反应具有高度选择性和固定依赖性。在用甲醛/洗涤剂固定和提取的成纤维细胞和肌原细胞中，应力纤维或肌原纤维分别保持未染色。同样，在显微注射到活细胞中后，2G2 不与此类微丝束结合。肌球蛋白和原肌球蛋白的提取没有改变这种模式，表明反应性的缺乏可能不是由于肌动蛋白结合蛋白的表位掩蔽所致。更可能的是，与丝状肌动蛋白缺乏反应性的原因是 F-肌动蛋白中无法接近其表位。然而，该抗体在分化的生肌细胞中显示出独特的核点模式，但在成肌细胞中却没有，并且在爪蟾卵母细胞的细胞核中显示出纤维结构。相反，甲醇处理后，观察到应力纤维和肌原纤维的 2G2 特异性染色，但没有核点染色。我们得出的结论是，2G2 除了与 SDS 和甲醇变性的肌动蛋白结合外，还能识别细胞核中存在的天然肌动蛋白的特定构象，并通过将抗体反应区域压缩成连贯的斑块来指定。这种构象显然存在于分化的肌原细胞和卵母细胞中，但不存在于细胞质肌动蛋白丝束中。
Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles.
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肌动蛋白亚型在发育中的大鼠肠上皮中的表达。
DOI：
10.1177/37.8.2754253
发表时间：
1989
期刊：
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
影响因子：
--
作者：
Hartman,AL;Sawtell,NM;Lessard,JL
通讯作者：
Lessard,JL
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Muhlrad,A;Cheung,P;Phan,BC;Miller,C;Reisler,E
通讯作者：
Reisler,E
影响因子：
--
作者：
ONODA, K;YU, FX;YIN, HL
通讯作者：
YIN, HL
影响因子：
56.9
作者：
GILOH, H;SEDAT, JW
通讯作者：
SEDAT, JW
骨骼肌肌动蛋白的蛋白水解和结构。
DOI：
10.1073/pnas.81.12.3680
发表时间：
1984
影响因子：
11.1
作者：
Mornet,D;Ue,K
通讯作者：
Ue,K