Physical observables to determine the nature of membrane-less cellular sub-compartments

Abstract

The spatial organization of complex biochemical reactions is essential for the regulation of cellular processes. Membrane-less structures called foci containing high concentrations of specific proteins have been reported in a variety of contexts, but the mechanism of their formation is not fully understood. Several competing mechanisms exist that are difficult to distinguish empirically, including liquid-liquid phase separation, and the trapping of molecules by multiple binding sites. Here we propose a theoretical framework and outline observables to differentiate between these scenarios from single molecule tracking experiments. In the binding site model, we derive relations between the distribution of proteins, their diffusion properties, and their radial displacement. We predict that protein search times can be reduced for targets inside a liquid droplet, but not in an aggregate of slowly moving binding sites. These results are applicable to future experiments and suggest different biological roles for liquid droplet and binding site foci.

Protein diffusion in Escherichia coli cytoplasm scales with the mass of the complexes and is location dependent

“…The recent discovery of phase-separated compartments in bacteria points toward LLPS as yet another mechanism by which the prokaryotic cytoplasm could be compartmentalized. An excellent review of known and possible hyperstructures and their role in cell physiology is available (23,24), with many more on protein mobility under physiological and stress conditions (25)(26)(27)(28).…”

Repair Foci as Liquid Phase Separation: Evidence and Limitations

“…The diffusive behavior of molecules at the condensate boundary can be measured by SPT. From the trajectories of individual molecules, it is possible to quantify the radial movement of all molecules as a function of their initial position relative to the center of the focus [ 40 , 58 ]. Concentrating on molecules close to the boundary, in LLPS, we expect to see a region of attraction with an average movement of molecules towards the center of the focus.…”

“…In the fast limit of binding/unbinding, there is a possibility that molecules exhibit confined motion within the condensate in the binding/PPPS model, in particular, if binding sites themselves diffuse. Indeed, theoretical work shows that the PPPS/binding model can be reduced to an effective description that is mathematically equivalent to the LPPS model, but with specific constraints linking its properties [ 58 ]. Thus, it can be difficult to discriminate the binding/PPPS models from an LLPS when monitoring only the exchange rate and internal mobility.…”

HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation

“…For instance, spontaneous protein segregation into liquid-like supra-molecular assemblies can be driven by weak, multivalent affinity interactions among structured protein domains or large low-complexity, intrinsically-disordered regions (IDRs), which are commonly observed in many eukaryotic proteins (7,9). However, the difficulty of quantitatively and unequivocally distinguishing LLPS from alternative self-organization processes in vivo has generated disputes about its biological relevance (10)(11)(12). A particularly notable debate has focused on the case of pericentromeric heterochromatin, which forms large, distinct nuclear compartments required for chromosome folding and segregation, as well as for transcriptional silencing of transposons and genes (13).…”

“…However, the difficulty of quantitatively and unequivocally distinguishing LLPS from alternative self-organization processes in vivo has generated disputes about its biological relevance (10–12). A particularly notable debate has focused on the case of pericentromeric heterochromatin, which forms large, distinct nuclear compartments required for chromosome folding and segregation, as well as for transcriptional silencing of transposons and genes (13).…”