Probing ATPase Dependent Physical Properties of Biological Condensates

Biological condensates are membraneless organelles, formed through nonspecific or multivalent ribonucleoprotein interactions, which are thought to be involved in the biochemical organization of the cell. There is increasing evidence for the regulation of these biological condensates by DEAD-box RNA helicases, which canonically remodel RNA-protein and RNA-RNA interactions in an ATP-dependent manner. The specific relationship between helicase activity and condensate properties such as viscosity, elasticity, and network architecture has not been investigated to date. DEAD-box helicase LAF-1, a critical component of the P granule condensate of C. elegans, phase separates in vitro. The ability for LAF-1 to interact with RNA and the effect of RNA on its droplet fluidity have been previously studied, however the interplay between its enzymatic function and droplet properties has not been established. We analyze the diffusion of single-walled carbon nanotubes, which are infrared-fluorescent, photostable, passive probes, to analyze the physical properties of LAF-1 droplets. In studying how this model condensate system responds to base-pairing RNAs, ATP, and accessory proteins we will shed light on principles underlying energetic regulation of condensate fluidity.