Table 1.
Summary of LLPS criteria that are matched or not by canonical PML NBs. In this table, we put forward the experimental evidence that sustains or not the involvement of LLPS in biogenesis of canonical PML NBs. Criteria listed here have been chosen based on the following reviews (4,8,32) and may not be all necessary/sufficient to prove LLPS. n.d. : non determined
| LLPS criterion | Criterion met by PML NBs | Experimental evidence | References |
|---|---|---|---|
| Spherical shape (roundness): liquid droplets have a spherical shape driven by surface tension | Yes | Super-resolution microscopy or transmission electron microscopy of PML NBs show sphericity of these nuclear bodies | (15,16,27,35) |
| Fusion/fission: like oil droplets in water, biomolecular condensates have the ability to fuse or drip | Yes | Time-lapse observations of PML NBs confirms their ability to undergo fusion/fission events during DNA replication or upon various stress conditions such as DNA damage, heat shock or physical pressure | (15,171–172) |
| Molecular mobility a: liquid condensates are characterized by a high mobility of proteins within them which is essentially depending on diffusion | Partially | FRAP experiments underlined fast recovery times for client proteins such as DAXX, CBP or BLM in the range of seconds, while PML isoforms exhibit slightly slower recovery times in the range of a few minutes compatible with the liquid-like nature of PML NBs. However, long recovery rates have been observed for specific isoforms such as PML V which may contribute to the structural integrity of nuclear bodies and could act as a stable scaffold for the recruitment of faster-exchanging molecules such as DAXX or CBP | (14–15,115) |
| Concentration buffering/dependence: LLPS is a function of concentration: past the critical concentration required for droplet formation, production of more protein increases droplet size but does not change concentration in either phase | Yes | Increase in PML intracellular concentration, as observed upon IFN-I treatment or senescence entry, results in an increased PML NBs size, while a decrease in PML protein concentration dissolves PML NBs in vivo | (38–43) |
| Interfacial boundary b: phase-separated proteins should preferentially move within the droplet. Presence of a phase boundary should reduce diffusion across the boundary | Partially | Diffusion coefficient for NLS-GFP was determined in nucleoplasm or in PML NBs by FCS. This demonstrated a 3-fold reduction in the diffusion coefficient inside the PML NBs as well as reduced exchanges of NLS-GFP between PML NBs and the nucleoplasm | (16,115) |
| Undergoes LLPS in vitro/in vivo | Partially | Not demonstrated for the PML protein itself. Yet, polySUMO-polySIM polymers form droplets in vitro and in vivo and recruit SUMO/SIM containing protein clients in vitro and in vivo | (6,33–34) |
| Temperature/ion strenght/pH dependance: measure of droplet formation in vivo should show dependance on temperature, ion concentration or pH | n.d. | n.d. | - |
| Sensitivity to 1,6-hexanediol: this chemical compound perturbs weak hydrophobic interactions that are involved in LLPS. Yet, sensitivity to 1,6 hexanediol is neither necessary nor sufficient to demonstate that a structure is formed by LLPS | n.d. | n.d. | - |
| Optodroplet assay: investigate whether expression of a fusion protein (protein of interest fused to a photolyase domain that can self-associate upon blue light) facilitates droplet formation in vivo upon blue light stimulation. Results should be interpreted with caution since these experiments rely on an artificial fusion protein system and should thus be combined with other experiments to prove LLPS in vivo | n.d. | n.d. | - |
aMolecular mobility is traditionally measured by Fluorescence Recovery After Photobleaching (FRAP). However, it should be noted that the use of recovery time as a marker of LLPS is insufficient per se since rapid recovery can result from a variety of mechanisms (32). One critical point is to demonstrate that the recovery rate is truely dominated by diffusion (rather than binding), which can be assessed by performing FRAP with various sizes of the bleach spot (32), which has not been performed yet in PML NBs.
bDiffusion across the boundary can be measured by fluorescence correlation spectroscopy (FCS) or single-molecule tracking (SMT). Alternatively, FRAP performed on half of the condensate, as performed in (45) provides an original and quantitative measure for the presence of a impermeable boundary, which could potentially be applied to PML NBs.