The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics

Abstract

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of “small” (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as “seeds” of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies

“…The dynamics of these bodies gradually decreases with increasing the body size, which distinguishes such condensates from canonical PML bodies in wild-type cells and from structures formed by other PML isoforms in PML −/− HeLa cells. The characteristics of the dynamics of the exchange of other PML isoforms that are part of the compartments formed by these proteins in PML −/− HeLa cells correspond to the characteristics of the dynamics of the exchange of these PML isoforms with PML bodies in the wild-type cells [26]. It has been established (Figure 1) that the acute oxidative stress conditions arising from the treatment of PML −/− HeLa cells with hydrogen peroxide affect the dynamics of the exchange of PML isoforms that are part of the condensates formed by these proteins within the nucleoplasm, as well as the dynamics of the exchange of the corresponding isoforms with endogenous PML-bodies in the wild-type cells [26].…”

“…The characteristics of the dynamics of the exchange of other PML isoforms that are part of the compartments formed by these proteins in PML −/− HeLa cells correspond to the characteristics of the dynamics of the exchange of these PML isoforms with PML bodies in the wild-type cells [26]. It has been established (Figure 1) that the acute oxidative stress conditions arising from the treatment of PML −/− HeLa cells with hydrogen peroxide affect the dynamics of the exchange of PML isoforms that are part of the condensates formed by these proteins within the nucleoplasm, as well as the dynamics of the exchange of the corresponding isoforms with endogenous PML-bodies in the wild-type cells [26]. The results obtained indicate that the exogenous PML isoforms form condensates with the same liquid droplet properties as the endogenous PML bodies.…”

“…The bodies formed by nuclear isoforms PML-I-V in the endogenous PML knockout HeLa cells are dynamic liquid droplet-like compartments (Figure 1). As well as in the wildtype cells, at least two populations of bodies can be distinguished among the compartmens formed by the individual PML isoforms: large bodies with a predominantly toroidal morphology and small spherical bodies, which differ in the dynamics of exchange of their PML proteins with the nucleoplasm [26]. The PML-IV isoform forms compartments in the endogenous PML knockout HeLa cells (Figure 1).…”

“…Our previous analysis of the sequences of PML isoforms revealed [26] that a number of PML isoforms, primarily PML-II, are prone to phase separation due to the polyampholytic properties and disordered nature of their C-terminal domains. We found the existence of a population of "small" PML bodies that do not have a pronounced toroidal structure, whose properties, according to FRAP analysis, are close to liquid-droplet compartments [26].…”

Identification of Key Proteins from the Alternative Lengthening of Telomeres-Associated Promyelocytic Leukemia Nuclear Bodies Pathway

“…PML bodies are membrane-less organelles found in the cell nucleus, which contain small ubiquitin-like modification (SUMO) sites [ 7 ] and are formed by PML, Sp100, and SUMO-1/2/3 proteins. Additionally, they use more than 50 proteins such as RAD52, RAD51, RAD50, RPA, BLM, and BRCA1, among others, which are involved in different cellular functions, such as tumoral suppression, DNA replication, gene transcription, DNA damage response (DDR), senescence, and apoptosis [ 8 , 9 ]. In the course of APBs formation, all of the six subunits (TRF1, TRF2, POT1, TPP1, TIN2, and Rap1) that constitute the shelterin complex detach from the telomeric DNA and are incorporated into the APBs (SUMOylation of shelterin), creating a recombigenic microenvironment that contributes to ALT triggering [ 8 ].…”