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New Insights into Phase Separation Processes and Membraneless Condensates of EIN2.

ABSTRACT: Recent technological advances allow us to resolve molecular processes in living cells with high spatial and temporal resolution. Based on these technological advances, membraneless intracellular condensates formed by reversible functional aggregation and phase separation have been identified as important regulatory modules in diverse biological processes. Here, we present bioinformatic and cellular studies highlighting the possibility of the involvement of the central activator of ethylene responses EIN2 in such cellular condensates and phase separation processes. Our work provides insight into the molecular type (identity) of the observed EIN2 condensates and on potential intrinsic elements and sequence motifs in EIN2-C that may regulate condensate formation and dynamics.

SUBMITTER: Lu J

PROVIDER: S-EPMC9416524 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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New Insights into Phase Separation Processes and Membraneless Condensates of EIN2.

Lu Jian J Wen Chi-Kuang CK Groth Georg G

Plants (Basel, Switzerland) 20220818 16

Recent technological advances allow us to resolve molecular processes in living cells with high spatial and temporal resolution. Based on these technological advances, membraneless intracellular condensates formed by reversible functional aggregation and phase separation have been identified as important regulatory modules in diverse biological processes. Here, we present bioinformatic and cellular studies highlighting the possibility of the involvement of the central activator of ethylene respo ...[more]

PMID: 36015451

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Project description:Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g., stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membraneless metastable (shape-changing) condensates in the cytoplasm. In our studies, we used the same cell lines and methods as those used by previous investigators but concluded that wild-type MxA formed variably sized spherical or irregular bodies, filaments, and even a reticulum distinct from that of ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of green fluorescent protein (GFP)-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24 ± 0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase, respectively, in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature and provide evidence for membraneless MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation and further report that MxA forms membraneless metastable (shape-changing) condensates in the cytoplasm consisting of variably sized spherical or irregular bodies, filaments, and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1 to 3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.

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New Insights into Phase Separation Processes and Membraneless Condensates of EIN2.

Publications

New Insights into Phase Separation Processes and Membraneless Condensates of EIN2.

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