**1. Introduction to Liquid–Liquid Phase Separation and Membraneless Organelles**

Cells contain crowded molecular environments hosting discrete functions that must be separated within time and space. Membrane-less compartments resulting from liquid–liquid phase separation (LLPS) are increasingly being recognized as mechanisms for organizing cellular activities. These distinct regions may be referred to as biomolecular condensates or membrane-less organelles (MLOs). As the names suggest, these organelles are not encapsulated in a membrane, yet contain enriched sets of specific macromolecules. Thus, LLPS is the biologically regulated process by which specific macromolecular components are concentrated into a specific MLO.

MLOs contain proteins, and frequently nucleic acids, and are dynamic in size (generally submicrometer), formation, and composition [1]. They behave like liquid droplets, capable of fusing, deforming, and rearranging [2]—all while being solvated in the larger aqueous environment of the cell. The macromolecular components of MLOs have a higher affinity for each other than for surrounding molecules, allowing for separation from the bulk solution by demixing, thus forming two co-existing liquid states with differing concentrations of particular solutes [3].

The network of multivalent interactions within an MLO is not ordered like a conventional protein complex [4–6]. The interactions are typically characterized as non-static and more dynamic, with less specificity and weaker binding than the forces that hold macromolecular complexes—such as the proteasome or ribosome–into rigid stoichiometric structures [2]. For example, a ribosome consists of large and small subunits with more-or-less specific quaternary arrangement of components that together form a large macromolecular machine. Interactions in MLOs are thought to be less specific, with greater fluctuation of molecular contacts and stoichiometry. The plasticity of interactions may permit these organelles to react more dynamically to specific cellular conditions.

Numerous distinct functional MLOs have been characterized, and their many unique protein constituents have been previously reviewed [7,8]. A recently developed database of nearly 3000 non-redundant LLPS-associated proteins suggests that many MLOs have yet to be fully characterized [9]. Of the MLOs that have been characterized, their diversity and ubiquity is remarkable. MLOs have been

observed in cytoplasm and nucleoplasm, and also in canonical membrane-enclosed organelles like mitochondria or chloroplasts [10]. Most commonly, MLOs are linked to specific functions involving ribonucleic acid, such as germ granules [11]. Pathological examples have also been proposed, such as the cytoplasmic inclusion bodies (IBs) within which measles viral RNA is replicated [12]. MLOs may exist transiently, like stress granules (SGs), which are stalled translation complexes that form upon cellular stress [13]. Alternatively, MLOs can have a more persistent presence, like the nucleolus, which is a constant site of ribosome production in the nucleus [7,14]. MLOs may also form in response to spatial necessity, such as neuronal RNA granules, which function in transport of mRNAs from dendrite bodies to distant synapses [15,16].

MLOs may also have roles in the pathogenesis of many diseases, particularly neurodegenerative disorders [17]. For example, many proteins linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) can undergo LLPS and accumulate within MLOs [18]. Mutations in these proteins not only cause disease but can alter LLPS and the physical properties of the phase-separated state [19,20]. It is hypothesized that aberrant irreversible phase transitions may result in proteinaceous neuronal inclusions that lead directly to cellular dysfunction [2].
