PDF(Pubmed)
Abstract：
BACKGROUND: Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP), amyotrophic lateral sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE) are associated with deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons. One complexity of this process lies in the ability of TDP-43 to form liquid-phase membraneless organelles in cells. Previous work has shown that the recombinant, purified, prion-like domain (PrLD) forms liquid droplets in vitro, but the behaviour of the complementary fragment is uncertain.
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.
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.
摘要：
背景：泛素阳性包涵体的额颞叶变性（FTLD-TDP），肌萎缩侧索硬化(ALS)和边缘型年龄相关性TDP-43脑病(LATE)与神经元中TARDNA结合蛋白43(TDP-43)的细胞质内含物沉积有关。该过程的一个复杂性在于TDP-43在细胞中形成液相无膜细胞器的能力。以前的工作表明，重组体，纯化,朊病毒样结构域（PrLD）在体外形成液滴，但是互补片段的行为是不确定的。
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。
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。
