Supramolecular Structures of the Dictyostelium Lamin NE81
Supramolecular Structures of the Dictyostelium Lamin NE81
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DOI：
10.3390/cells8020162
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发表时间：
2019-02-01
期刊：
影响因子：
6
通讯作者：
Graef, Ralph
中科院分区：
文献类型：
--
作者：
Grafe, Marianne;Batsios, Petros;Graef, Ralph
核纤层蛋白（Nuclear lamin，核纤层蛋白）是位于核被膜（Nuclear Envelope，NE）内核膜（Internal Nuclear Membrane，INM）上的核特异性中间丝（intermediate filaments，IF）。它们与核膜跨膜蛋白一起形成核纤层，对核和整个细胞的基因调控和机械鲁棒性至关重要。最近，我们的特点是Dictyosteoprotein NE81作为一个进化上保守的层粘连蛋白样蛋白，无论是在序列和功能水平。在这里，我们在结构水平上表明，网骨藻NE81也能够组装成细丝，就像后生动物层蛋白细丝组装一样。使用场发射扫描电子显微镜，我们表明，NE81在非洲爪蟾卵母细胞中表达的形式与整体外观高度联想到非洲爪蟾核纤层蛋白B2的丝状结构。从网骨藻提取物中纯化的重组His标记的NE81的体外组装特性与后生动物核纤层蛋白的那些非常相似。超分辨率受激发射损耗（STED）和扩展显微镜（ExM），以及负染色纯化NE81的透射电子显微镜，证明了其在低离子强度条件下形成丝状结构的能力。这些结果推荐网骨藻作为非哺乳动物模式生物，其具有良好表征的核膜，涉及动物细胞中已知的所有相关蛋白质组分。
Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.