introduction
Multivesicular bodies (MVBs) are important intracellular organelles that play a key role in cellular quality control. MVB membranes are invaginated and cleaved to form intraluminal vesicles (ILVs) that are responsible for sorting protein cargo, a process that is understood today to require the ESCRT protein complex to consume ATP [1]. Recent studies have shown that biomolecular agglomerations are essential mesostructures for cells to perform their functions, and they function through a variety of mechanisms. The condensate can interact with the membrane to "wetting" and cause capillary [2]. However, the biological significance of wetting-related capillary forces in cellular processes is still poorly understood.
2024年10月9日，清华大学方晓峰实验室与合作者在Nature杂志在线发表了题为Biomolecular condensates mediate bending and scission of endosome membranes的研究论文，揭示了植物蛋白FREE1相分离形成凝聚体，通过润湿作用诱导内体膜的内陷和不稳定性，足以在不依赖ESCRT机器和ATP的情况下介导ILV的形成。 Nature杂志同期以Research Briefing的形式对该工作进行了题为Cell membranes shaped and cut by phase-separated liquid protein condensates的报道。
Xiaofeng Fang's lab focuses on the mechanism of condensates formed by phase separation in plant perception, response, and memory of environmental stresses (the lab is recruiting postdoctoral fellows interested in this direction). Using a previously established phase separation protein screening system in the laboratory [3], the researchers found that the plant ESCRT component FREE1 has strong phase separation ability in vivo and in vitro, and its N-terminal intrinsic disordered region (IDR) is the main element driving phase separation (Figure 1). In addition, FREE1 has an FYVE domain that binds to the membrane lipid phosphatidylinositol 3-phosphate (PI3P), thereby localizing to the MVB membrane. Further studies showed that the formation of condensates significantly enhanced the binding ability of FREE1 to the membrane, and the FREE1 condensate was used as a scaffold to recruit other components of ESCRT into the condensate and enhance their binding ability to the membrane. Importantly, the authors found that replacing the IDR of FREE1 with a completely different sequence of FUS-IDR can completely compensate for the lethal phenotype of the free1 mutant (Figure 1); However, FUS-IDRm, which has lost the ability of phase separation, cannot do so, indicating that the phase separation of FREE1 is a necessary and sufficient condition for its function. However, the authors found that although FUS-IDR-FREE1 can replace the functions of FREE1, it cannot interact with ESCRT to recruit them into its condensate, implying that the condensate of FREE1 can function independently of ESCRT.
图1. FREE1相分离是其发挥功能所必需的。 （a）不同形式FREE1蛋白的结构示意图；（b）不同形式FREE1体外相分离实验；（c）所示基因型植物的发育表型（Credit: Nature）
Furthermore, through in vitro reconstruction and computer simulation of the interaction between FREE1 condensate and membrane, the research team found that FREE1 condensate can bend and invert the membrane in a very short time. In vitro reconstitution experiments, the investigators observed that in the absence of the ESCRT complex, small membrane vacuoles filled with FREE1 condensates could form inside the large vacuole GUV and diffuse freely internally (Fig. 2), indicating that the FREE1 condensate itself is sufficient to mediate vesicle cleavage. Theoretical calculations in physics support this speculation. The authors further provide strong genetic evidence that overexpression of FUS-IDR-FREE1, which can form condensates, in ESCRT-deficient plants, has been found to largely enable ILV formation within MVB. Finally, the researchers also found that although FUS-IDR-FREE1 could meet the growth and development of plants under normal conditions, it could not meet the germination and survival rates of plants under osmotic stress caused by high salinity and drought, indicating that ESCRT protein machine and FREE1 condensate may be the double insurance mechanism preserved by the evolutionary process to realize MVB production, and can better cope with environmental changes. How these two mechanisms synergize with each other under stress conditions needs to be further studied in the future.
Figure 2. Multiple methods to reveal the interaction between FREE1 condensates and membranes. (a) Interaction of FREE1 droplets (purple) with GUV membrane (green) in vitro; (b) Computer simulation of the dynamic process of droplets (purple) infiltrating the invaginated membrane (green); (c) Physical theory to calculate the relationship between the force required for membrane neck cutting and the droplet size (Credit: Nature)
In summary, this study reveals a new mechanism of MVB production mediated by ATP consumption by ESCRT machines, which is different from the traditional understanding: the liquid condensate formed by the separation of the FREE1 phase drives the MVB membrane to invert through the capillary force it generates, causing the instability of the membrane neck and completing the shearing of the membrane to form ILV, which does not rely on the ESCRT machine and does not deplete ATP (Fig. 3). This study greatly broadens the dimension of functional studies of phase separation in biology and deepens our understanding of the remodeling of the intracellular membrane system.
Figure 3. A working model of condensate-mediated ILV formation. The left shows the traditional thought process of ILV production through the synergy of the ESCRT protein machine with ATPase. The right shows the process by which the condensate identified in this study achieves ILV production through capillary force invagination of the MVB membrane and cleavage of the membrane neck (Credit: Nature)
bibliography
1. Vietri, M., M. Radulovic, and H. Stenmark, The many functions of ESCRTs. Nat Rev Mol Cell Biol, 2020. 21(1): p. 25-42.2. Gouveia, B., et al., Capillary forces generated by biomolecular condensates. Nature, 2022. 609(7926): p. 255-264.3. Zhang, H., et al., Large-scale identification of potential phase separation proteins from plants using a cell-free system. Mol Plant, 2023. 16(2): p. 310-313.https://www.nature.com/articles/s41586-024-07990-0
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