Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
J Cell Sci. 2024 Aug 15;137(16). doi: 10.1242/jcs.262207. Epub 2024 Aug 23.
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.
核纤层蛋白是中间丝蛋白，参与多种细胞功能，包括核形态和机械稳定性。核纤层蛋白的 N 端头部结构域对于高级纤维组装和功能至关重要，但常用的 N 端标签对核纤层蛋白功能的影响在很大程度上仍未得到探索。在这里，我们在一个哺乳动物细胞模型中系统地研究了两种不同大小的标签对核纤层蛋白 A（LaA）功能的影响，该模型设计用于精确控制标记核纤层蛋白的表达。在 lamin A/C 缺陷（Lmna-/-）MEF 中以相似水平表达未标记、FLAG 标记和 GFP 标记的 LaA 时，均可完全挽救核形状缺陷，并且所有 LaA 构建体均防止了这些细胞中核包膜破裂的增加。然而，N 端标签改变了 LaA 的核定位，并损害了 LaA 恢复核变形性和将 emerin 募集到核膜的能力在 Lmna-/- MEF 中。我们发现标签会阻碍一些 LaA 的功能，但不会阻碍其他功能，这可能解释了当标记的 lamins 在模型生物中表达时，部分功能丧失表型的原因，并且应该提醒使用标记 lamins 研究核的研究人员注意。
