Biophysical characterization of aberrant condensates by quantitative microscopy.

Abstract

The size, composition, and dynamics of biomolecular condensates are impacted in human pathologies, including neurodegeneration and cancer. Essential biological functions such as ribosome biogenesis in nucleoli and splicing biogenesis in Cajal bodies may be compromised due to these aberrations. A further understanding of the relationship between condensate form and function may aid the development of condensate-directed therapies. Thus, we aimed to quantitatively measure the impact of disease-driving mutations in patient-derived cells with compromised Cajal bodies and nucleoli. In these cells, we surveyed classical condensate markers (e.g., Coilin and NPM1) for their ability to rescue defects in Cajal body and nucleolar size, composition, and function. To do this, a construct was employed which allowed us to independently measure the extent of overexpression and localization of the protein within the cell. Building on this, we extended the design to enable two constructs to be measured, opening the door to multicomponent phase diagrams and measuring binding in cells. Furthermore, when ALS-associated mutant proteins are over-expressed, we can measure the dosage-dependent consequences of mutant proteins in cells. Our findings suggest that at higher concentrations of disease mutant proteins essential condensate markers become mislocalized. These quantitative microscopy methods allow us to better understand the basic principles underlying the relationship between condensate form and function, which may aid therapeutic design targeting condensates.