Cell-free reconstitution of multi-condensate assemblies

Abstract

Biomolecular condensates (BCs) are intracellular condensates that form by phase separation of proteins and RNA from the nucleoplasm or cytoplasm. BCs often form complex assemblies where compositionally distinct condensates wet each other without mixing. In this chapter, we describe methods to reconstitute multi-condensate assemblies from purified components. We include protocols to express, purify, label, and analyze the dynamics of proteins and RNAs that drive multi-condensate assembly. Analysis of the condensation and wetting behaviors of condensates in cell-free reconstituted systems can be used to define the molecular interactions that regulate BCs in cells.

Keywords: FRAP; Fluorescence labeling; Image analysis; Inclusion bodies; MBP; Maltose binding protein; Phase separation; Protein purification; RNA condensate.

Fig. 1

Condensation reaction of fluorescently labeled MEG-3-Dylight488 (150nM), PGL-3-Alexa647 (5000nM), and nos-2 RNA-Alexa546 (20ng/μL) incubated for 30min and visualized on a thin chambered glass slide with a 40× objective. MEG-3 particles assemble on the surface of PGL-3 condensates, nos-2 RNA is recruited to both phases, but is enriched in the MEG-3 phase.

Fig. 2

Representative SDS-PAGE gel of MEG-3 protein (denaturing protocol) labeled with Alexa647 fluor and stained with SimplyBlue^™ SafeStain (left panel) or fluorescently imaged before staining (right panel).

Fig. 3

Schematic of the RNA labeling transcription reaction. Black lines represent RNA, blue lines represent DNA, blue arrows represent PCR and reverse transcription primers, green stars represent fluorescent UTP, and the purple circle represents the RNA polymerase.

Fig. 4

Schematic of the imaging analysis workflow.