Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells
Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells
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DOI：
10.4161/nucl.2.5.17765
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发表时间：
2011-09-01
期刊：
影响因子：
3.7
通讯作者：
Herrmann, Harald
中科院分区：
文献类型：
--
作者：
Kolb, Thorsten;Maass, Kendra;Herrmann, Harald
我们已经调查和量化的核A型核纤层蛋白池从人HeLa S3悬浮细胞相对于它们的分布洗涤剂可溶性和不溶性馏分。我们设计了一个连续的提取方案，并发现，最大10%的A-型核纤层蛋白回收的可溶性馏分。值得注意的是，核纤层蛋白C富集在低去污剂级分中，并且仅用0.5%Nonidet P-40，核纤层蛋白A和C以几乎等同于在全细胞提取物和核纤层级分中发现的比率回收。真正的核质蛋白如LAP 2 α、pRB和p53与这些级分中的A型核纤层蛋白一起被共提取到大部分。通过蔗糖密度离心，我们揭示了大多数核纤层蛋白与人IgG共沉淀，表明它们形成在二聚体范围内的相当小的复合物和稍大的复合物。此外，在更快的沉降部分中获得了一些核纤层蛋白A，而不是核纤层蛋白C。在轻和重蔗糖组分中均发现了真正的核蛋白，如PCNA、p53和LAP 2 α以及核纤层蛋白A。最后但并非最不重要的是，免疫沉淀实验从可溶性级分和RIPA裂解物的全细胞显示，核纤层蛋白A和核纤层蛋白C不形成异二聚体，但实际上完全分离。相应地，甲醛固定的细胞的免疫荧光显微镜清楚地表明，核纤层蛋白A和C至少部分定位于不同的补丁内的层。因此，核纤层蛋白A和C的结构分离确实在一定程度上也保留在核膜中。
We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2 alpha, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A-but not lamin C-is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2 alpha were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.