Abstract
Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liquid–liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. Here we report quantitative measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase separation conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase separation model, we discovered that the operational proximity of clients (measured from client–client interactions) could be over 16 times higher than the expected proximity from actual client concentrations inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase separation compartmentalization: (1) clients are selectively recruited into compartments, leading to concentration enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.
Highlights
Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells
Recent studies indicate that repeated folded protein domains[4] or intrinsically disordered proteins/regions (IDPs/IDRs)[5,6,7] can drive liquid–liquid phase separation (LLPS), and that this is the major formation principle of compartmentalized biomolecular condensates[2,8,9]
By employing IDR-based condensates and clients, as well as interaction-responsive fluorescent protein (FP) probes, we systematically investigated FP interaction dynamics inside and outside of compartments formed in living cells
Summary
Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Their formation mechanisms have been steadily elucidated via the classical concept of liquid–liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. In addition to conventional membrane-bound organelles such as the endoplasmic reticulum or Golgi, many membrane-less compartments, which are condensed with distinct sets of biomolecules without discrete lipid bilayer barriers ( termed biomolecular condensates), have been reported[1] Examples of these membrane-less organelles include stress granules, p-bodies, and nucleoli, which are known as essential hubs of cellular processes such as signal transduction, stress response, and gene expression[2]. We report the real-time quantitative measurements of enhanced interactions between client molecules upon recruitment into membrane-less IDR compartments in living cells. For subsequent control of client recruitment into condensates, different IDRs or IDR fragments were fused to clients, in order that IDRfused clients can be recruited to IDR condensates via IDR–IDR interactions
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