Intermolecular reorganisation of single-component condensates during ageing promotes multiphase architectures.

Intracellular proteins can undergo phase separation to form liquid-like biomolecular condensates with a multitude of functional roles. Liquid condensates can, however, further age and progressively rigidify. In addition to single-phase systems, multiphase condensates are increasingly identified commonly within multi-component systems, where the different molecular components present sufficient physicochemical diversity to sustain separate phases. Here, we develop a multiscale modeling approach that predicts conditions under which multiphase architectures can arise also within single-component protein condensates. Such single-component condensates are initially homogeneous but become heterogeneous over time due to the gradual enhancement of interprotein interactions. We find that such enhancement could originate, for instance, from intermolecular disorder-to-order transitions within low-complexity aromatic-rich kinked segments in the prion-like domain of FUS. Our model reveals that as increasing numbers of molecules undergo a disorder-to-order transition over time, single-component protein condensates convert into either gel-core/liquid-shell or liquid-core/gel-shell multiphase structures, depending on the relative surface tension of the liquid and gel phases. Despite being formed by proteins that are chemically-identical, the different liquid and gel phases present diverse surface tensions due to their fundamentally different molecular organization. Our study highlights the regulatory role of prion-like domains in tuning condensate behavior and, more generally, suggests a new route by which multilayered compartments or hierarchically organized condensate structures can emerge.

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and operation of a three-color prismTIRF microscope which can be utilized for the study of macromolecular complexes, including the multi-component protein–DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology.

Construction of a 3-color prism-based TIRF microscope to study the interactions and dynamics of macromolecules

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for realtime visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice. Here, we provide an easy-to-follow guide for the design, assembly, and oper-ation of a 3-color prismTIRF microscope which can be utilized for the study macromolecular complexes, including the multi-component protein-DNA complexes responsible for DNA repair, replication, and transcription. Our hope is that this article can assist laboratories that aspire to implement single-molecule TIRF techniques, and consequently expand the application of this technology to a broader spectrum of scientific questions.

Effect of Promoter Methylation on the Expression of Porcine MUC2 Gene and Resistance to PEDV Infection

Integrity of the intestinal mucosal barrier is closely related to the occurrence of diarrhea. As an important component protein of the intestinal mucosal barrier, Mucin 2 (MUC2) plays a critical role in preventing the invasion of pathogens, toxins, and foreign bodies. In the present study, we preliminary verified the function of the porcine MUC2 gene in resisting porcine epidemic diarrhea virus (PEDV) infection and investigated the effect of DNA methylation in the promoter region on MUC2 gene expression. The results showed that after PEDV infection, the intestinal mucosal barrier was damaged. Moreover, MUC2 expression was significantly higher in PEDV-infected piglets than in healthy piglets (P < 0.01). The mRNA expression of MUC2 was significantly higher in PEDV-infected IPEC-J2 cells than in non-infected IPEC-J2 cells (P < 0.05). Methylation of the mC-5 site in the MUC2 promoter inhibited the binding of Yin Yang 1 (YY1) to the promoter, down regulated the expression of MUC2 and increased the susceptibility of piglets to PEDV. In conclusion, this study suggests that MUC2 plays an essential regulatory role in PEDV infection. High MUC2 expression improves the resistance of pigs to PEDV infection. The binding of YY1 to the MUC2 promoter is hindered by the methylation of the mC-5 site, which downregulates MUC2 expression and ultimately affects the resistance of pigs to PEDV infection.

Denaturation Behavior and Kinetics of Single- and Multi-Component Protein Systems at Extrusion-Like Conditions

In this study, the influence of defined extrusion-like treatment conditions on the denaturation behavior and kinetics of single- and multi-component protein model systems at a protein concentration of 70% (w/w) was investigated. α-Lactalbumin (αLA) and β-Lactoglobulin (βLG), and whey protein isolate (WPI) were selected as single- and multi-component protein model systems, respectively. To apply defined extrusion-like conditions, treatment temperatures in the range of 60 and 100 °C, shear rates from 0.06 to 50 s⁻1, and treatment times up to 90 s were chosen. While an aggregation onset temperature was determined at approximately 73 °C for WPI systems at a shear rate of 0.06 s⁻1, two significantly different onset temperatures were determined when the shear rate was increased to 25 and 50 s⁻1. These two different onset temperatures could be related to the main fractions present in whey protein (βLG and αLA), suggesting shear-induced phase separation. Application of additional mechanical treatment resulted in an increase in reaction rates for all the investigated systems. Denaturation was found to follow 2.262 and 1.865 order kinetics for αLA and WPI, respectively. The reaction order of WPI might have resulted from a combination of a lower reaction order in the unsheared system (i.e., fractional first order) and higher reaction order for sheared systems, probably due to phase separation, leading to isolated behavior of each fraction at the local level (i.e., fractional second order).

The N-terminal amino-latch region of Hlg2 component of staphylococcal bi-component γ-haemolysin is dispensable for prestem release to form β-barrel pores

The contribution of N-terminal regions of staphylococcal bi-component γ-haemolysin toxin components to haemolytic activity towards human erythrocyte cells was investigated in this study. A deletion construct of N-terminal amino acids 1–10 of Hlg2 (Hlg2 ΔN10), which is the S-component protein of γ-haemolysin, had little effect on its haemolytic activity, whereas N-terminal 1–11 amino acid deletion (Hlg2 ΔN11) significantly delayed haemolysis. Moreover, a deletion of N-terminal amino acids 1–17 of LukF, which is the F-component protein of γ-haemolysin, increased its haemolytic activity in combination with either the wild-type or Hlg2 ΔN10. Unlike the N-terminal amino-latch region of staphylococcal α-haemolysin, which is a single component β-barrel pore-forming toxin, the N-terminal regions present in γ-haemolysin components are dispensable for the haemolytic activity of the bi-component toxin. These results strengthen our recent proposal that staphylococcal bi-component γ-haemolysin toxin uses an N-terminal amino-latch independent molecular switch for prestem release during the formation of β-barrel pores.