In recent years, the functions of membraneless organelles in eukaryotic cells have been widely concerned.It is also well accepted that formation and dispersion of membraneless organelles are regulated processes. However, up to now, how the fusion and fission of membraneless organelles are regulated, and which cellular signals are involved, remain unknown.

In response to this question, the team of Academician Mingjie Zhang conducted research on the regulation of fusion and fission of membraneless organelles using the postsynaptic density (PSD) as a platform. On January 18, 2024, they published a research paper titled “Phosphorylation-dependent membraneless organelle fusion and fission illustrated by postsynaptic density condensates” in Molecular Cell, suggesting the molecular mechanism by which SAPAP phosphorylation regulates the fusion and separation of two membraneless organelles, PSD core and PSD pallium.

We are honored to invite Dr. Haowei Wu, one of the first authors of this article, for a conversation to gain an in-depth understanding of Dr. Wu’s latest research results and the story behind the research.

The team of Academician Zhang has always focused on the research of phase separation in synapses. As early as eight years ago, they first discovered the phenomenon of phase separation of PSD protein complexes, which provided an important theoretical basis for understanding the pathogenesis of various neurological diseases. The volume of PSD is small, and its structure can only be seen under an electron microscope, making the study of PSD plasticity very difficult. To this end, the team successfully reconstructed PSD in vitro in 2018, establishing a model for studying PSD plasticity. Based on this, the team has carried out a series of studies on the functions of proteins in PSD, such as Arc, Homer, SAPAP, etc. Among them, the research on SAPAP has been going on for more than ten years. Their early research found that SAPAP has a regulatory effect on the aggregates of PSD. The phosphorylation of SAPAP can increase the interaction between it and the upstream protein PSD-95 by 100 times, which means that the phosphorylation of SAPAP has an important physiological function. PSD can be divided into the high-density upper layer PSD core and the low-density lower layer PSD pallium, and SAPAP is located at the junction of PSD core and PSD pallium in the body. Therefore, Zhang team boldly proposed the hypothesis that the phosphorylation of SAPAP can serve as a molecular switch connecting the upper and lower layers of PSD, regulating the phase separation of the PSD complex.

The research found that in the reconstructed PSD in vitro, the fusion and fission between the reconstructed PSD core and PSD pallium can be regulated by the phosphorylation of SAPAP. When non-phosphorylated SAPAP was added to the reconstructed PSD in vitro, PSD would spontaneously form two separate aggregates. However, when phosphorylated SAPAP was added, the two separate aggregates would fuse together. This fusion phenomenon was reversed after dephosphorylation of phosphorylated SAPAP. Further in vivo experiments in mice found that the phosphorylation of SAPAP can regulate the distance between PSD core and PSD pallium. These results indicate that membraneless organelles formed by phase separation will also undergo adjustable fusion and fission like membrane-enclosed organelles. Unlike the fusion of membrane-based organelles, which all require SNARE as the core protein complex to participate, the fusion and fission of membraneless organelles may occur directly through molecular interactions between the two phases.

In conclusion, this article reveals the regulatory effects of SAPAP phosphorylation on the fusion and fission of membraneless organelles, PSD core and PSD pallium, implying that membraneless organelles can also undergo regulated fusion and fission analogous to membrane-based organelles. In the future, the team of Academician Zhang will further study the impact of SAPAP phosphorylation on mouse behavior and other regulatory mechanisms for the fusion and separation of membraneless organelles.

Although Zhang team had started related research topics a long time ago, they still encountered difficulties in this research process. When measuring the distance between PSD core and PSD pallium in mouse brain slices, stochastic optical reconstruction microscopy (STORM) was used, which required immunofluorescence staining of mouse brain slices. However, the structure of PSD is very dense, which led to difficulties in immunostaining the scaffold proteins in PSD. Finally, through discussions with collaborators and continuous attempts, the problem was solved by reducing the fixation time and thickness of the brain slices.

In this experiment, Isothermal Titration Calorimetry (ITC) and miniDAWN Multi-AngleLightScatter were mainly used to study the interaction between SAPAP and its upstream protein PSD-95. Confocal laser scanning microscopy was used to observe the phase transition process. Primary neuron culture was used to study the function of SAPAP in neurons. STORM was used to measure the distance between PSD core and PSD pallium in mouse brain slices.

RWD Minux^® FS800 cryostat was used to make mouse brain slices for its stable temperarture control, which made a great contribution to 5 μm very thin brain sections, said Dr. Haowei Wu. Although he had not been in contact with other brands of cryostats before, based on the great experience with other RWD star products, the first cryostat purchased by the laboratory in 2022 was chosen by RWD. After using it, it was found that the temperature of RWD cryostat is stable, the temperature switch is fast, and the after-sales response speed is also timely. He hopes that he will have the opportunity to use more excellent RWD products in future work.

Academician Zhang is an amazing scientist in the field of life sciences. He has published 150 high-quality papers in authoritative journals, many of which have been published in top magazines such as Cell and Science. Moreover, many of his students have established independent scientific research teams around the world. Dr. Haowei Wu mentioned that professor Zhang gave her a lot of help during her doctoral studies. It is very valuable that professor Zhang always discusses subjects with every student in the team and solves every difficulty encountered in the research. In the discussion, Dr. Zhang’s keen thinking and deep understanding of the field often make our subject come to life.

“It’s a warm laboratory with a strong scientific research atmosphere”, Dr. Haowei Wu described their team.

Steady progress leads to far-reaching achievements. RWD wishes Dr. Haowei Wu all the best on the road in pursuing academic dreams and fruitful results! It also wishes the team of Academician Zhang continuous innovation and further progress on the road of scientific exploration!