Stephanie C. Weber

Getting RNA and Protein in Phase

Nonmembrane-bound organelles such as RNA granules behave like dynamic droplets, but the molecular details of their assembly are poorly understood. Several recent papers identify structural features that drive granule assembly, shedding light on how phase transitions functionally organize the cell and may lead to pathological protein aggregation.

Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci

Chromosomal loci jiggle in place between segregation events in prokaryotic cells and during interphase in eukaryotic nuclei. This motion seems random and is often attributed to Brownian motion. However, we show here that locus dynamics in live bacteria and yeast are sensitive to metabolic activity. When ATP synthesis is inhibited, the apparent diffusion coefficient decreases, whereas the subdiffusive scaling exponent remains constant. Furthermore, the magnitude of locus motion increases more steeply with temperature in untreated cells than in ATP-depleted cells. This “superthermal” response suggests that untreated cells have an additional source of molecular agitation, beyond thermal motion, that increases sharply with temperature. Such ATP-dependent fluctuations are likely mechanical, because the heat dissipated from metabolic processes is insufficient to account for the difference in locus motion between untreated and ATP-depleted cells. Our data indicate that ATP-dependent enzymatic activity, in addition to thermal fluctuations, contributes to the molecular agitation driving random (sub)diffusive motion in the living cell.

RNA transcription modulates phase transition-driven nuclear body assembly

Significance Living cells contain various membraneless organelles whose size and assembly appear to be governed by equilibrium thermodynamic phase separation. However, the dynamics of this process are poorly understood. Here, we quantify the assembly dynamics of liquid-phase nuclear bodies and find that they can be explained by classical models of phase separation and coarsening. In addition, active nonequilibrium processes, particularly rRNA transcription, can locally modulate thermodynamic parameters to stabilize nucleoli. Our findings demonstrate that the classical phase separation mechanisms long associated with nonliving condensed matter can mediate organelle assembly in living cells, whereas chemical activity may serve to regulate these processes in response to developmental or environmental conditions. Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid–liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of “extranucleolar droplets” (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism.

Analytical Tools To Distinguish the Effects of Localization Error, Confinement, and Medium Elasticity on the Velocity Autocorrelation Function

Single particle tracking is a powerful technique for investigating the dynamic behavior of biological molecules. However, many of the analytical tools are prone to generate results that can lead to mistaken interpretations of the underlying transport process. Here, we explore the effects of localization error and confinement on the velocity autocorrelation function, Cυ. We show that calculation of Cυ across a range of discretizations can distinguish the effects of localization error, confinement, and medium elasticity. Thus, under certain regimes, Cυ can be used as a diagnostic tool to identify the underlying mechanism of anomalous diffusion. Finally, we apply our analysis to experimental data sets of chromosomal loci and RNA-protein particles in Escherichia coli.

Mu gets in the loop

In this issue of Molecular Cell, Han and Mizuuchi present evidence for a possible Turing-like reaction-diffusion mechanism underlying target immunity by the bacteriophage Mu.