Division of Molecular Genetics, German Cancer Research Center (DKFZ), INF280, D-69120 Heidelberg, Germany.
Nucleus. 2011 Sep-Oct;2(5):425-33. doi: 10.4161/nucl.2.5.17765.
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 LAP2a, 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 LAP2a 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.
我们研究并定量了来自人 HeLa S3 悬浮细胞的核 A 型层粘连蛋白池，以了解其在去污剂可溶性和不溶性部分的分布情况。我们设计了一个连续提取方案，发现最多只有 10%的 A 型层粘连蛋白可以回收至可溶性部分。值得注意的是，层粘连蛋白 C 在低去污剂部分中富集，只有 0.5%的非离子型去垢剂 NP-40 才能回收与全细胞提取物和核层部分中几乎相等比例的 A 型层粘连蛋白 A 和 C。在这些部分中，像 LAP2a、pRB 和 p53 这样的真正核质蛋白与 A 型层粘连蛋白一起被大量共同提取。通过蔗糖密度离心，我们发现大多数层粘连蛋白与人类 IgG 一起共沉降，表明它们在二聚体和稍大的复合物范围内形成相当小的复合物。除了 A 型层粘连蛋白外，还有一些 lamin A （但不是 lamin C ）可以在沉降速度更快的部分中获得。像 PCNA、p53 和 LAP2a 这样的真正核蛋白，与 lamin A 一起，在轻和重蔗糖部分中都有发现。最后但并非最不重要的是，来自可溶性部分和整个细胞 RIPA 裂解物的免疫沉淀实验表明，lamin A 和 lamin C 不形成异二聚体，而是几乎完全分离。相应地，固定甲醛的细胞免疫荧光显微镜清楚地表明，lamin A 和 lamin C 至少部分定位于核层内的不同斑块中。因此，lamin A 和 lamin C 的结构分离在核膜中也在一定程度上得到保留。
