Significance: A hexanucleotide repeat expansion in the first intron of the C9orf72 gene represents the most prominent form of heritable amyotrophic lateral sclerosis. Bidirectional transcription and ATG-independent translation of the expanded (GGGGCC) _n repeat specifies the production of toxic glycine:arginine (GR _n ) and proline:arginine (PR _n ) poly-dipeptides. The present study provides evidence that the PR _n poly-dipeptide binds directly to the central channel of nuclear pores, causing inhibition of both the import and export of macromolecules to and from the nucleus. Nuclear pore binding is shown to be mediated via direct interaction between the toxic PR _n poly-dipeptide and polymeric forms of nuclear pore proteins enriched in phenylalanine:glycine repeats.

Progress in development of biophysical analytic approaches has recently crossed paths with macromolecule condensates in cells. These cell condensates, typically termed liquid-like droplets, are formed by liquid-liquid phase separation (LLPS). More and more cell biologists now recognize that many of the membrane-less organelles observed in cells are formed by LLPS caused by interactions between proteins and nucleic acids. However, the detailed biophysical processes within the cell that lead to these assemblies remain largely unexplored. In this review, we evaluate recent discoveries related to biological phase separation including stress granule formation, chromatin regulation, and processes in the origin and evolution of life. We also discuss the potential issues and technical advancements required to properly study biological phase separation.

To identify critical events associated with heat-induced cell killing, we examined foci formation of γH2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of γH2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5°C to 45.5°C, we observed a linear increase with time in the number of γH2AX foci. An inflection point at 42.5°C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of γH2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of γH2AX foci formation. We also found that thermotolerance was due to a depression in the number of γH2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.