In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge. Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid–liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.

Biomolecular condensates: organizers of cellular biochemistry

BACKGROUND Living cells contain distinct subcompartments to facilitate spatiotemporal regulation of biological reactions. In addition to canonical membrane-bound organelles such as secretory vesicles and endoplasmic reticulum, there are many organelles that do not have an enclosing membrane yet remain coherent structures that can compartmentalize and concentrate specific sets of molecules. Examples include assemblies in the nucleus such as the nucleolus, Cajal bodies, and nuclear speckles and also cytoplasmic structures such as stress granules, P-bodies, and germ granules. These structures play diverse roles in various biological processes and are also increasingly implicated in protein aggregation diseases. ADVANCES A number of studies have shown that membrane-less assemblies exhibit remarkable liquid-like features. As with conventional liquids, they typically adopt round morphologies and coalesce into a single droplet upon contact with one another and also wet intracellular surfaces such as the nuclear envelope. Moreover, component molecules exhibit dynamic exchange with the surrounding nucleoplasm and cytoplasm. These findings together suggest that these structures represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process. Liquid phase condensation increasingly appears to be a fundamental mechanism for organizing intracellular space. Consistent with this concept, several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation. At the molecular level, weak, transient interactions between molecules with multivalent domains or intrinsically disordered regions (IDRs) are a driving force for phase separation. In cells, condensation of liquid-phase assemblies can be regulated by active processes, including transcription and various posttranslational modifications. The simplest physical picture of a homogeneous liquid phase is often not enough to capture the full complexity of intracellular condensates, which frequently exhibit heterogeneous multilayered structures with partially solid-like characters. However, recent studies have shown that multiple distinct liquid phases can coexist and give rise to richly structured droplet architectures determined by the relative liquid surface tensions. Moreover, solid-like phases can emerge from metastable liquid condensates via multiple routes of potentially both kinetic and thermodynamic origins, which has important implications for the role of intracellular liquids in protein aggregation pathologies. OUTLOOK The list of intracellular assemblies driven by liquid phase condensation is growing rapidly, but our understanding of their sequence-encoded biological function and dysfunction lags behind. Moreover, unlike equilibrium phases of nonliving matter, living cells are far from equilibrium, with intracellular condensates subject to various posttranslational regulation and other adenosine triphosphate–dependent biological activity. Efforts using in vitro reconstitution, combined with traditional cell biology approaches and quantitative biophysical tools, are required to elucidate how such nonequilibrium features of living cells control intracellular phase behavior. The functional consequences of forming liquid condensates are likely multifaceted and may include facilitated reaction, sequestration of specific factors, and organization of associated intracellular structures. Liquid phase condensation is particularly interesting in the nucleus, given the growing interest in the impact of nuclear phase behavior on the flow of genetic information; nuclear condensates range from micrometer-sized bodies such as the nucleolus to submicrometer structures such as transcriptional assemblies, all of which directly interact with and regulate the genome. Deepening our understanding of these intracellular states of matter not only will shed light on the basic biology of cellular organization but also may enable therapeutic intervention in protein aggregation disease by targeting intracellular phase behavior.

Liquid phase condensation in cell physiology and disease.

Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.

Coexisting Liquid Phases Underlie Nucleolar Subcompartments

Protein Phase Separation: A New Phase in Cell Biology.

https://www.cell.com/molecular-cell/pdf/S1097-2765(15)00737-6.pdf

RNA Controls PolyQ Protein Phase Transitions

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape.

/pdf/micron-scale-plasma-membrane-curvature-is-recognized-by-the-tzutgaert8.pdf

Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.

Septin assemblies form by diffusion-driven annealing on membranes

http://www.jbc.org/content/290/28/17173.full.pdf

Septin Form and Function at the Cell Cortex

Bacterial biofilms represent a basic form of multicellular organization that confers survival advantages to constituent cells. The sequential stages of cell ordering during biofilm development have been studied in the pathogen and model biofilm-former Vibrio cholerae It is unknown how spatial trajectories of individual cells and the collective motions of many cells drive biofilm expansion. We developed dual-view light-sheet microscopy to investigate the dynamics of biofilm development from a founder cell to a mature three-dimensional community. Tracking of individual cells revealed two distinct fates: one set of biofilm cells expanded ballistically outward, while the other became trapped at the substrate. A collective fountain-like flow transported cells to the biofilm front, bypassing members trapped at the substrate and facilitating lateral biofilm expansion. This collective flow pattern was quantitatively captured by a continuum model of biofilm growth against substrate friction. Coordinated cell movement required the matrix protein RbmA, without which cells expanded erratically. Thus, tracking cell lineages and trajectories in space and time revealed how multicellular structures form from a single founder cell.

Andrew A. Bridges

Papers

RNA Controls PolyQ Protein Phase Transitions

Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton

Septin assemblies form by diffusion-driven annealing on membranes

Septin Form and Function at the Cell Cortex

Cell position fates and collective fountain flow in bacterial biofilms revealed by light-sheet microscopy.