Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

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Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes of their conformation and biological functions. Disturbed LLPS homeostasis in MLOs cause fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes of the degree of crowding in MLOs. However, it has not been investigated well due to lack of an appropriate method. To address this, we developed a genetically-encoded molecular crowding sensor CRONOS that senses the degree of macromolecular crowding in MLOs using fluorescence resonance energy transfer (FRET) system. CRONOS is a very bright biosensor with wider dynamic range and detect changes in the macromolecular volume fraction better than the previously reported mCer-mCit sensor in solution. By fusing to scaffold protein of each MLO, we successfully delivered CRONOS to MLO of interest and detected previously undescribed difference of the degree of crowding in each MLO. If not tagged, CRONOS localized to interstitial space of MLOs, giving us the crowding information of inspace. CRONOS also detected changes of degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for determination of molecular crowding and detection of pathological changes in MLOs in live cells.

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