Abstract
Intercellular fluids in living organisms contain high concentrations of macromolecules such as nucleic acid and protein. Over the past few decades, several studies have examined membraneless organelles in terms of liquid-liquid phase separation. These studies have investigated aggregation/attraction among a rich variety of biomolecules. Here, we studied the association between the polymerization/depolymerization of actin, interconversion between monomeric (G-actin) and filamentous states (F-actin), and water/water phase separation in a binary polymer solution using polyethylene glycol (PEG) and dextran (DEX). We found that actin, which is a representative cytoskeleton, changes its distribution in a PEG/DEX binary solution depending on its polymerization state: monomeric G-actin is distributed homogeneously throughout the solution, whereas polymerized F-actin is localized only within the DEX-rich phase. We extended our study by using fragmin, which is a representative actin-severing and -depolymerizing factor. It took hours to restore a homogeneous actin distribution from localization within the DEX-rich phase, even with the addition of fragmin in an amount that causes complete depolymerization. In contrast, when actin that had been depolymerized by fragmin in advance was added to a solution with microphase-separation, F-actin was found in DEX-rich phase droplets. The micro-droplets tended to deform into a non-spherical morphology under conditions where they contained F-actin. These findings suggest that microphase-separation is associated with the dynamics of polymerization and localization of the actin cytoskeleton. We discuss our observations by taking into consideration the polymer depletion effect.
Highlights
Inside living cells, various solutes and biological factors, including macromolecules such as nucleic acid and protein complexes, are present in high concentrations
By observing the behavior of actin that was added to a polyethylene glycol (PEG)/DEX binary solution together with a protein that helps regulate the polymerization/depolymerization of actin, we investigated whether the regulatory system functions as in the bulk solution even under conditions that lead to phase separation, i.e., even in crowded environments like the cell interior
To confirm that the addition of fragmin has no effect on liquid–liquid phase separation (LLPS), fragmin or only the buffer used to make the fragmin stock solution was added to the PEG/DEX binary solution in the absence of actin
Summary
Various solutes and biological factors, including macromolecules such as nucleic acid and protein complexes, are present in high concentrations. The mechanisms that maintain a variety of multi-step, but robust, regulatory systems that are often found in cells, trigger the onset of diseases caused by scitation.org/journal/jcp proteins essential for cells, or sense physical signals, such as temperature, have been difficult to explain solely in terms of individual biological molecules. These phenomena are understood in terms of the spatial sorting of biological factors by LLPS or the physicochemical dependency of LLPS.. Using an ATPS, researchers have attempted to construct a model of the cellular structure in a self-organized manner, which includes membraneless microcompartments in cells.
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