Citation: Perelman, R.T.; Schmidt, A.; Khan, U.; Walter, N.G. Spontaneous Confinement of mRNA Molecules at Biomolecular Condensate Boundaries. Cells 2023, 12, 2250. https://doi.org/10.3390/ cells12182250 Academic Editor: Vladimir V. Didenko Received: 17 August 2023 Revised: 1 September 2023 Accepted: 9 September 2023 Published: 11 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Spontaneous Confinement of mRNA Molecules at Biomolecular Condensate Boundaries Rebecca T. Perelman 1 , Andreas Schmidt 1 , Umar Khan 2 and Nils G. Walter 1,3, * 1 Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI 48109, USA; btperel@umich.edu (R.T.P.); andreasschmidt58@gmail.com (A.S.) 2 Center for Advanced Biomedical Imaging and Photonics, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA 02115, USA; ukhan1@bidmc.harvard.edu 3 Center for RNA Biomedicine, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA * Correspondence: nwalter@umich.edu Abstract: Cellular biomolecular condensates, termed ribonucleoprotein (RNP) granules, are often enriched in messenger RNA (mRNA) molecules relative to the surrounding cytoplasm. Yet, the spatial localization and diffusion of mRNAs in close proximity to phase separated RNP granules are not well understood. In this study, we performed single-molecule fluorescence imaging experiments of mRNAs in live cells in the presence of two types of RNP granules, stress granules (SGs) and processing bodies (PBs), which are distinct in their molecular composition and function. We devel- oped a photobleaching- and noise-corrected colocalization imaging algorithm that was employed to determine the accurate positions of individual mRNAs relative to the granule’s boundaries. We found that mRNAs are often localized at granule boundaries, an observation consistent with recently published data. We suggest that mRNA molecules become spontaneously confined at the RNP granule boundary similar to the adsorption of polymer molecules at liquid–liquid interfaces, which is observed in various technological and biological processes. We also suggest that this confinement could be due to a combination of intermolecular interactions associated with, first, the screening of a portion of the RNP granule interface by the polymer and, second, electrostatic interactions due to a strong electric field induced by a Donnan potential generated across the thin interface. Keywords: membraneless organelles; mRNA; HILO microscopy; liquid–liquid phase separation; biomolecular condensates; RNP granules; colocalization; intermolecular interactions; Donnan potential 1. Introduction Biomolecular condensates are membraneless compartments in eukaryotic cells that most often contain RNA-binding proteins and mRNA molecules in concentrations higher than the surrounding cytoplasm [1]. Ribonucleoprotein (RNP) granules, a class of biomolec- ular condensates containing significant portions of RNA-binding proteins [2], have been found to play critical roles in epigenetic and post-transcriptional regulation [3] and, con- sequently, have been associated with numerous diseases, such as tumor progression and neurodegeneration [4,5]. RNP granules form liquid- or solid-like compartments of RNA and proteins [6] through processes, including liquid–liquid phase separation (LLPS) and percolation [7]. RNP granules often contain messenger RNAs (mRNAs) in concentrations higher than the surrounding cytoplasm. At the same time, the spatial localization and dynamics of mRNAs in close proximity to RNP granules are not well understood. We here present imaging data obtained with low-background highly inclined and laminated optical (HILO) sheet microscopy in live cells containing two types of RNP granules: stress granules (SG) and P-bodies (PB). We developed a coregistration imaging algorithm to determine the exact positions of the mRNAs relative to the granule boundaries, which revealed that ~80% of colocalized mRNAs were located at the SG and PB boundaries. These observations are Cells 2023, 12, 2250. https://doi.org/10.3390/cells12182250 https://www.mdpi.com/journal/cells