We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-ΔNLSΔCLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin.

中文翻译：

---

光，机械应力和pH诱导的盘基网柄菌层状突变体的体内组装。

我们表达了盘基网柄菌Lamin（NE81）缺少功能性核定位信号和C端脂质修饰的CAAX框。该lamin突变体在胞质溶胶中组装成超分子三维簇，在有丝分裂开始时分解，并在末期后期重新组装，从而模仿了内源蛋白的行为。由于拆卸是由CDK1介导的丝氨酸122磷酸化调节的，我们生成了一个模拟磷酸S122E突变体，称为GFP-NE81-S122E-ΔNLSΔCLIM。出乎意料的是，在成像过程中，融合蛋白组装成胞质簇，类似于缺乏磷酸模拟突变的蛋白。团簇在黑暗中再次分解。可以用蓝色而不是绿色或接近紫外光诱导组装，并且它与融合标签无关。组装类似地在细胞扁平化时发生。较早的报道和自己的观察表明，蓝光和细胞扁平化都可能导致细胞内pH降低。实际上，将细胞保持在低pH值也可逆地诱导簇形成。我们的结果表明lamin组装可以被各种压力因素诱导，并且这些是通过细胞内酸化来转导的。尽管这些作用已在拟南芥的磷酸化CDK1突变体中显示Dictyostelium lamin，它们也可能与野生型lamin有关。