METHODS: We have purified such a construct without the PrLD (PrLD-less TDP-43) and have induced its phase separation using a solution-jump method and an array of biophysical techniques to study the morphology, state of matter and structure of the TDP-43 assemblies.
RESULTS: The fluorescent TMR-labelled protein construct, imaged using confocal fluorescence, formed rapidly (< 1 min) round, homogeneous and 0.5-1.0 µm wide assemblies which then coalesced into larger, yet round, species. When labelled with AlexaFluor488, they initially exhibited fluorescence recovery after photobleaching (FRAP), showing a liquid behaviour distinct from full-length TDP-43 and similar to PrLD. The protein molecules did not undergo major structural changes, as determined with circular dichroism and intrinsic fluorescence spectroscopies. This process had a pH and salt dependence distinct from those of full-length TDP-43 and its PrLD, which can be rationalized on the grounds of electrostatic forces.
CONCLUSIONS: Similarly to PrLD, PrLD-less TDP-43 forms liquid droplets in vitro through liquid-liquid phase separation (LLPS), unlike the full-length protein that rather undergoes liquid-solid phase separation (LSPS). These results offer a rationale of the complex electrostatic forces governing phase separation of full-length TDP-43 and its fragments. On the one hand, PrLD-less TDP-43 has a low pI and oppositively charged domains, and LLPS is inhibited by salts, which attenuate inter-domain electrostatic attractions. On the other hand, PrLD is positively charged due to a high isoionic point (pI) and LLPS is therefore promoted by salts and pH increases as they both reduce electrostatic repulsions. By contrast, full-length TDP-43 undergoes LSPS most favourably at its pI, with positive and negative salt dependences at lower and higher pH, respectively, depending on whether repulsive or attractive forces dominate, respectively.
方法:我们在没有PrLD的情况下纯化了这种构建体(无PrLD的TDP-43),并使用溶液跳跃方法和一系列生物物理技术诱导了其相分离,以研究形态学,TDP-43组件的物质状态和结构。
结果:荧光TMR标记的蛋白质构建体,使用共聚焦荧光成像,快速形成(<1分钟)圆形,均质和0.5-1.0µm宽的组件,然后合并成更大的组件,然而,圆形,种。当用AlexaFluor488标记时,它们最初在光漂白(FRAP)后表现出荧光恢复,显示与全长TDP-43不同的液体行为,与PrLD相似。蛋白质分子没有发生重大的结构变化,用圆二色性和固有荧光光谱法测定。该过程具有与全长TDP-43及其PrLD不同的pH和盐依赖性,这可以在静电力的基础上合理化。
结论:类似于PrLD,无PrLD的TDP-43通过液-液相分离(LLPS)在体外形成液滴,与全长蛋白质不同,后者经历液-固相分离(LSPS)。这些结果提供了控制全长TDP-43及其片段相分离的复杂静电力的基本原理。一方面,无PrLD的TDP-43具有低pI和带正电荷的结构域,LLPS被盐抑制,削弱域间静电吸引力。另一方面,由于高的等离子点(pi),PrLD带正电,因此LLPS被盐促进,并且pH增加,因为它们都减少静电排斥。相比之下,全长TDP-43在其pI下最有利地经受LSPS,在较低和较高的pH值下具有正盐和负盐依赖性,分别,取决于排斥力还是吸引力占主导地位,分别。