Hedtfeld et al., 2024_A validation strategy to assess the role of phase separation as determinant of macromolecular localization

Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive
the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through in
vitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays
remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic
LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes
LLPS in vitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity
in vitro emerged as an unreliable predictor of subcellular localization. In vitro CPC LLPS in aqueous
buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media
dissolved condensates of the CPC and of several additional proteins. We also show that
centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS
of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless
compartments.