Methods for Physical Characterization of Phase-Separated Bodies and Membrane-less Organelles

Abstract

Membrane-less organelles are cellular structures which arise through the phenomenon of phase separation. This process enables compartmentalization of specific sets of macromolecules (e.g., proteins, nucleic acids), thereby regulating cellular processes by increasing local concentration, and modulating the structure and dynamics of their constituents. Understanding the connection between structure, material properties and function of membrane-less organelles requires inter-disciplinary approaches, which address length and timescales that span several orders of magnitude (e.g., Ångstroms to micrometer, picoseconds to hours). In this review, we discuss the wide variety of methods that have been applied to characterize the morphology, rheology, structure and dynamics of membrane-less organelles and their components, in vitro and in live cells.

Heterochromatin Protein HP1α Gelation Dynamics Revealed by Solid‐State NMR Spectroscopy

“…We interpret the dramatic change in total CP intensity to arise from an expanding cross-linking network during gelation that restricts the motion of individual pHP1a dimers and shifts their overall dynamics to as lower timescale where dipolar-based polarization transfer mechanisms are more efficient. [16,22] Over the course of aw eek, the fluorescence recovery slowed down, eventually reaching af lat line.R eminiscent of the observations made at heterochromatin foci, these results align well with our NMR spectroscopy time course and provide further evidence that the motion of individual molecules within the gel state is severely limited. In the initial liquid droplet state,the recovery of fluorescence intensity occurs on at imescale of several seconds,c onsistent with observations in other dense liquid systems.…”

“…[3] These studies suggest that HP1a forms distinct dilute,d ense liquid and gel-like phases within the nucleus to partition heterochromatin from the transcriptional machinery by either phase incompatibility or aphysical sieving mechanism. [22] Some details have emerged from solution nuclear magnetic resonance (NMR) spectroscopy experiments of the dense liquid phase that point to the importance of intrinsically disordered protein regions and their transient interactions with other LLPS components. [19][20][21] Within this framework, however, specific molecular interactions and dynamics have been challenging to describe experimentally due to the dense and amorphous nature of biological condensates.…”

“…[18] Biophysical treatments of LLPS proteins,s uch as HP1a, suggest that the compaction state,i nteraction strength, and multivalencyo ft he biomolecule can shape the collective material properties. [22][23][24] Solution NMR spectroscopy,h owever,cannot capture the molecular picture of biomolecules in the gel state where the increased viscosity and cross-linking interactions reduce molecular tumbling and cause severe linebroadening in the NMR spectra. [22] Some details have emerged from solution nuclear magnetic resonance (NMR) spectroscopy experiments of the dense liquid phase that point to the importance of intrinsically disordered protein regions and their transient interactions with other LLPS components.…”

“…[19][20][21] Within this framework, however, specific molecular interactions and dynamics have been challenging to describe experimentally due to the dense and amorphous nature of biological condensates. [22] Some details have emerged from solution nuclear magnetic resonance (NMR) spectroscopy experiments of the dense liquid phase that point to the importance of intrinsically disordered protein regions and their transient interactions with other LLPS components. [22][23][24] Solution NMR spectroscopy,h owever,cannot capture the molecular picture of biomolecules in the gel state where the increased viscosity and cross-linking interactions reduce molecular tumbling and cause severe linebroadening in the NMR spectra.…”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…In contrast with most synthetic LLPS systems, biomolecular condensates involvem any different types of weak and multivalent inter-actions thata ffect the material properties of the droplets formed [43] (Figure 1C). [44] Recent studies have also aimed at describing thep hysics underlying phases eparationi nl ivingc ells by adaptingtheoriesusedtodescribe LLPSinsynthetic polymers. [44] Recent studies have also aimed at describing thep hysics underlying phases eparationi nl ivingc ells by adaptingtheoriesusedtodescribe LLPSinsynthetic polymers.…”