Formation of biological condensates via phase separation: Characteristics, analytical methods, and physiological implications

Abstract

Liquid-liquid phase separation (LLPS) facilitates the formation of condensed biological assemblies with well-delineated physical boundaries, but without lipid membrane barriers. LLPS is increasingly recognized as a common mechanism for cells to organize and maintain different cellular compartments in addition to classical membrane-delimited organelles. Membraneless condensates have many distinct features that are not present in membrane-delimited organelles and that are likely indispensable for the viability and function of living cells. Malformation of membraneless condensates is increasingly linked to human diseases. In this review, we summarize commonly used methods to investigate various forms of LLPS occurring both in 3D aqueous solution and on 2D membrane bilayers, such as LLPS condensates arising from intrinsically disordered proteins or structured modular protein domains. We then discuss, in the context of comparisons with membrane-delimited organelles, the potential functional implications of membraneless condensate formation in cells. We close by highlighting some challenges in the field devoted to studying LLPS-mediated membraneless condensate formation.

Keywords: Phase separation; biological condensates; cell biology; cell signaling; cellular regulation; protein/protein interaction; synapse.

Figure 1.

Types of multivalent interactions driven by intrinsically disordered elements in LLPS systems. A, phase diagram constructed by varying protein concentration and storage conditions such as buffer reagents and temperature. Solid line depicts the boundary at which molecules reach their solubility limit and become immiscible with the surrounding solution. Gray box highlights the confocal image showing the homogeneous solution state of the NR2B C-terminal tail (labeled with Alexa Cy3) in the absence of PSD scaffold proteins. Conditions within the spinodal curve (indicated as dashed line) are where spinodal decomposition occurs. Example of the fluorescence image, highlighted by the green box, shows that the membrane-tethered NR2B tail (labeled with Alexa Cy5) formed clusters on supported lipid bilayers upon the addition of major PSD scaffold proteins. Phase separation is only observed in the presence of a nucleation process when conditions lie in between the binodal (indicated as solid line) and spinodal curves. Representative image, highlighted by the yellow box, shows the clustered state of the NR2B tail (labeled with Alexa Cy3) in 3D solution in the presence of major PSD scaffold proteins (adapted from Ref. 45). Scale bar, 10 μm. B, aromatic residues in intrinsic disorder containing proteins are involved in π–π or cation–π interactions with positively charged residues such as Arg and Lys. RGG repeats are frequently found in LCRs. C, patterned charge distributions to facilitate electrostatic interactions between oppositely charged residues. D, secondary structural elements are involved in multivalent intermolecular interactions, such as the kinked cross–β-sheets formed by a segment of FUS LCR (PDB code 6BWZ).

Figure 2.

Types of multivalent interactions driven by modular domains in LLPS systems. A, interaction network of N-WASP, nephrin, and NCK. B, schematic representations showing the network of multivalent interactions involving major PSD proteins. Solid line indicates direct modular domain interactions. Dashed line indicates indirect recruitment of actin filaments via Shank3 and Homer proteins. C, schematic interaction network of presynaptic active-zone proteins RIM and RIM-binding protein together with the cytoplasmic tail of the N-type voltage-gated Ca²⁺ channel (NCav).

Figure 3.

Techniques for characterizing the condensed phase formed in 3D solution. A, DIC (left image) coupled with fluorescence imaging (middle and right images) of the phase droplets and multiple labeling of different components demonstrate their co-localization. B–D, dynamic properties of the condensed phase. E, fluorescence intensity–based absolute concentration estimation (adapted from Refs. 44, 45). A standard curve of fluorescence intensity to dye concentration is initially generated for calibration. Z direction scanning is performed to determine the proper focal plane for concentration estimation. At each Z stack, the fluorescence intensity distribution is plotted. Within the Z dimension of selected droplets, average fluorescence intensities are then compared across different layers. In a given system, the fluorescence intensity is constant regardless of the droplet size, and therefore the absolute protein concentration within a condensed phase can be calculated from the standard curve.

Figure 4.

Condensed phase formed on 2D supported lipid bilayer. A, schematic diagram of microdomain formation on 2D supported lipid bilayer. Membrane proteins homogeneously distribute on the supported lipid bilayer via tethering of the His-tag to Ni²⁺-NTA-decorated lipids. Protein clusters are observed on lipid bilayers after the addition of other components to drive phase separation. B, STORM analysis of membrane proteins, the cytoplasmic tail of NCav as an example, on supported lipid bilayer (adapted from Ref. 45). Image captured under TIRF microscopy mode first sketches the contours of the condensed phase, which turns out to perfectly overlap with the image reconstructed from STORM analysis. Trajectories of individual molecules are followed by single molecular tracking assay, both inside and outside the condensed phase. Direction of movement is marked by gradient color from black to red.

Figure 5.

Biological functions of LLPS-mediated membraneless compartments.