School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455.
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455.
Mol Biol Cell. 2018 May 1;29(9):1003-1011. doi: 10.1091/mbc.E17-04-0233. Epub 2018 Mar 22.
Linker-of-nucleoskeleton-and-cytoskeleton (LINC) complexes are conserved molecular bridges within the nuclear envelope that mediate mechanical force transmission into the nucleoplasm. The core of a LINC complex is formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Mammals encode six KASH proteins and five SUN proteins. Recently, KASH proteins were shown to bind to the domain interfaces of trimeric SUN2 proteins in vitro. However, neither the existence of SUN2 trimers in living cells nor the extent to which other SUN proteins conform to this assembly state have been tested experimentally. Here we extend the application of fluorescence fluctuation spectroscopy to quantify SUN protein oligomerization in the nuclear envelopes of living cells. Using this approach, we demonstrate for the first time that SUN2 trimerizes in vivo and we demonstrate that the in vivo oligomerization of SUN1 is not limited to a trimer. In addition, we provide evidence to support the existence of potential regulators of SUN protein oligomerization in the nuclear envelope. The differential SUN protein oligomerization illustrated here suggests that SUN proteins may have evolved to form different assembly states in order to participate in diverse mechanotransduction events.
核骨架-核膜连接蛋白（LINC）复合物是核膜内保守的分子桥，介导机械力传递到核质。LINC 复合物的核心由核膜外膜和内膜的 KASH 和 SUN 蛋白分别在腔隙中的相互作用形成。哺乳动物编码六种 KASH 蛋白和五种 SUN 蛋白。最近，KASH 蛋白被证明在体外与三聚体 SUN2 蛋白的结构域界面结合。然而，SUN2 三聚体在活细胞中的存在以及其他 SUN 蛋白在多大程度上符合这种组装状态都尚未经过实验验证。在这里，我们扩展了荧光波动光谱的应用，以定量测量活细胞核膜中 SUN 蛋白的寡聚化。通过这种方法，我们首次证明 SUN2 在体内三聚化，并且证明 SUN1 的体内寡聚化不仅限于三聚体。此外，我们提供了证据支持核膜中 SUN 蛋白寡聚化的潜在调节剂的存在。这里显示的不同的 SUN 蛋白寡聚化表明，SUN 蛋白可能已经进化为形成不同的组装状态，以便参与不同的机械转导事件。
