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首页> 外文期刊>Nucleus >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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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

机译：层粘连蛋白A和层粘连蛋白C形成同二聚体，并以较高的复杂形式共存于培养的人类细胞的核质级分和层中

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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.
机译：我们已经研究和量化了人类HeLa S3悬浮细胞的核A型核纤层蛋白池在去污剂可溶性和不溶性组分中的分布。我们设计了一种顺序提取方案，发现在可溶性部分中最多可回收10％的A型lamin。值得注意的是，lamin？C富含低洗涤剂含量，仅回收0.5％的Nonidet P-40 lamin？A和C，其回收率几乎与全细胞提取物和层板级分中发现的比率相同。真核质蛋白（如LAP2a，pRB和p53）与这些部分中的A型lamins一起被共提取。通过蔗糖密度离心，我们发现大多数lamins与人IgG共同沉淀，表明它们在二聚体和稍大的复合物范围内形成了相当小的复合物。另外，以更快的沉降速率获得了一些层粘蛋白A-但没有层粘蛋白C-。在轻质和重质蔗糖级分以及层粘蛋白A中都发现了真实的核蛋白，例如PCNA，p53和LAP2a。最后但并非最不重要的是，从可溶性级分和全细胞RIPA裂解物中进行的免疫沉淀实验表明，层粘蛋白A和层粘连蛋白C不形成异二聚体，但实际上完全分离。相应地，甲醛固定细胞的免疫荧光显微术清楚地表明，层粘连蛋白A和C至少部分定位于层板内的不同斑块。因此，核纤层蛋白A和C的结构分离确实在某种程度上仍保留在核膜中。

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