Analysis of supramolecular assemblies of NE81, the first lamin-like protein in a unicellular organism

层粘连蛋白构建核层，是细胞机械稳定、染色质组织、基因表达、细胞周期进展和细胞迁移所必需的。这一点在层状蛋白病的发病机制中变得明显，层状蛋白病是由编码层状蛋白A/C的基因突变引起的。尽管这些功能具有普遍的重要性，但迄今为止只在后生动物中发现了层蛋白。在Dictyostelium NE81中，我们现在已经鉴定出一种与核膜相关的蛋白质，其特性证明了在低等真核生物中作为层状蛋白的命名是合理的。这是基于它的主要序列和最初的功能表征。在这里，我们提出了一个工作计划，旨在验证NE81是一个原始的纤层蛋白，并分析其推测的纤层蛋白样功能。首先，我们将生成并分析NE81耗尽突变体、CDK1激酶靶序列突变体和异戊二烯化突变体。其次，我们将在生化和微观水平上检查NE81的预期组装特性。第三，我们将描述前纤层蛋白加工的酶机制的作用，这在Dictyostelium中是完全保守的，并且预计是NE81功能所必需的。第四，我们打算验证假定的NE81在核包膜上的结合伙伴及其与染色质的结合。在单细胞生物中发现层状蛋白不仅从进化的角度来看非常有趣，而且还可以为研究层状蛋白病的分子基础提供一个简单的实验平台。

Lamins build the nuclear lamina and are required for mechanical stabilization of cells, chromatin organization, gene expression, cell cycle progression and cell migration. This became evident by the pathogenesis of laminopathies, which are caused by mutations in genes encoding lamin A/C. Despite these functions of universal importance, lamins have so far only been found in metazoans. With Dictyostelium NE81 we have now identified a protein associated with the inner nuclear envelope, whose properties justify denomination as a lamin-like protein in a lower eukaryote. This is based both on its primary sequence and an initial functional characterization. Here we propose a work schedule that aims at the verification of NE81 as a primitive lamin and the analysis of its putative lamin-like functions. First, we will generate and analyze NE81 depletion mutants, mutants in CDK1 kinase target sequences and isoprenylation mutants. Second, we will examine the expected assembly properties of NE81 on the biochemical and microscopic level. Third, we will characterize the role of the enzymatic machinery for prelamin processing, which is completely conserved in Dictyostelium and is expected to be required for NE81 function. Fourth we intend to verify putative binding partners of NE81 at the nuclear envelope and its binding to chromatin. The discovery of a lamin-like protein in a unicellular organism is not only very interesting in the light of evolution, it could also provide a simple experimental platform for studies of the molecular basis of laminopathies.