PDF(Pubmed)
Abstract：
Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is crucial for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna-/-) MEFs, and all LaA constructs prevented increased nuclear envelope ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit emerin to the nuclear membrane in Lmna-/- MEFs. Our finding that tags impede some LaA functions but not others might explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.
摘要：
Lamins是中间丝蛋白，有助于许多细胞功能，包括核形态和机械稳定性。Lamin的N端头域对于高阶细丝组装和功能至关重要，然而，常用的N端标签对lamin功能的影响仍未被研究。这里,我们系统地研究了两个不同大小的标签对哺乳动物细胞模型中LaminA（LaA）功能的影响，该模型被设计为允许精确控制标记的lamin蛋白的表达。未标记,带有FLAG标签的,和GFP标记的LaA完全拯救了核形状缺陷，当在层粘连蛋白A/C缺陷（Lmna-/-）MEF中以相似的水平表达时，和所有LaA构建体防止这些细胞中增加的核膜(NE)破裂。N端标签,然而,改变了LaA的核定位，并损害了LaA恢复核变形能力和将Emerin招募到Lmna-/-MEF中的核膜的能力。我们的发现，标签阻碍了一些LaA功能，而不是其他可能解释了当标记的层蛋白在模型生物中表达时功能表型的部分丧失，并且应该提醒研究人员使用标记的层蛋白来研究细胞核。
