(58c) Use of Coiled-Coil Motifs to Assemble Membraneless Organelles for Tunable Control Liquid Liquid Phase Separation

In eukaryotic cells proteins can undergo self-assembly and phase separate to form membraneless organelles such as nucleoli, Cajal bodies, P-bodies, and stress granules. Because of their potential for subcellular compartmentalization and channel-less cargo recruitment and release, membraneless organelles made from liquid-liquid phase separation (LLPS) can be excellent bio-building blocks and hubs for cellular communication. Here, we combine intrinsically disordered RGG domains from Laf-1 with small coiled coil orthogonal peptide domains to facilitate targeted phase separation at room temperature. We engineered orthogonal coiled coil domains P1-P6, developed by the Jarala lab, on both N and C termini of the RGG domains and utilize fluorescence microscopy using RGG-GFP-RGG as a fluorescence reporter, and turbidity assays to measure coacervation. We find when two RGG domains are linked via P3-P4 and P5-P6 pairs, coacervation is facilitated. However, the incorporation of the P1-P2 pairs did not facilitate coacervation. In general, we find N terminal attachment of coiled coil linkers enhance coacervation but C terminus attachment inhibit coacervation. We then demonstrate phase separation induced through coiled coil linkers is reversible and could be thermally cycled and reconstituted. With these system, we are now exploring client recruitment into membraneless organelles assembled in yeast. This dynamic, synthetic RGG coiled coil system offers a tunable approach and generalizable platform for controlling coacervation, and has broad applications such as cargo recruitment and cellular enzymatic control in both bio-engineered cells and protocells.