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.
Highlights
For a very long time, biologists have observed cytoplasmic and nucleoplasmic bodies or compartments that did not have a lipid membrane
The purpose of this work was to draw the attention of researchers studying promyelocytic leukemia (PML) bodies to the question that neither the formation of S-S bonds nor SUMO/SUMO-interacting motif (SIM) interactions can be the primary cause of PML-bodies formation; to prove that polypeptide sequences of PML isoforms are prone to liquid-droplet formation; and to study the dynamic properties of canonical and ALT-associated PML-bodies and the effect of oxidative stress on them
This hypothesis was validated by the fluorescence recovery after photobleaching (FRAP)-based characterization of the canonical and ALT-associated PML-bodies and the analysis of their liquid-droplet properties under normal conditions and under the conditions of oxidative stress that promoted oxidative dimerization of PML monomers
Summary
For a very long time (starting from as early as the 17th century), biologists have observed cytoplasmic and nucleoplasmic bodies or compartments that did not have a lipid membrane. Only 10–15 years ago it became clear that the formation and functioning of these cell compartments are based on the reversible liquid-liquid phase transitions of biopolymers in the cellular milieu [1,2] Such compartments are called membrane-less organelles (MLOs) [2]. It was found that these organelles are dynamic liquid-droplet formations that constantly exchange their contents with the intracellular environment [3] These organelles demonstrate the characteristic properties of liquids: wetting, surface tension, fusion upon contact with each other [4]. The composition of such organelles is largely represented by intrinsically disordered proteins [5,6,7,8] and ribonucleic acids—biopolymers characterized by high conformational flexibility [9,10]. The MLOs arising in this case should be considered as open, non-linear dynamic systems existing at the edge of chaos [12], which determines the essential dependence of their properties on small changes in the external conditions and the action of various stimuli
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