The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.DOI: http://dx.doi.org/10.7554/eLife.13571.001

The chain shape of polymers affects many aspects of their behavior and is governed by their intramolecular interactions. Delocalization of electrons along the backbone of conjugated polymers has been shown to lead to increased chain rigidity by encouraging a planar conformation. Poly(3-hexylthiophene) and other poly(3-alkylthiophenes) (P3ATs) are interesting for organic electronics applications, and it is clear that a hierarchy of structural features in these polymers controls charge transport. While other conjugated polymers are very rigid, the molecular structure of P3AT allows for two different planar conformations and a significant degree of torsion at room temperature. It is unclear, however, how their chain shape depends on variables such as side chain chemistry or regioregularity, both of which are key aspects in the molecular design of organic electronics. Small-angle neutron scattering from dilute polymer solutions indicates that the chains adopt a random coil geometry with a semiflexible backbone. The measured persistence length is shorter than the estimated conjugation length due to the two planar conformations that preserve conjugation but not backbone correlations. The persistence length of regioregular P3HT has been measured to be 3 nm at room temperature and decreases at higher temperatures. Changes in the regioregularity, side chain chemistry, or synthetic defects decrease the persistence length by 60–70%.

Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid-liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (Rmotifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes within NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.

Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the B. subtilis cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments—performed under biologically relevant conditions—answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.

Highlights d R-rich DPRs sequester NPM1 into large, soluble phaseseparation-inhibited complexes d NPM1 sequestration dissolves droplets in vitro and delocalizes nucleolar NPM1 in cells d poly(PR) entraps rRNA in static puncta in vitro and in nucleoli d poly(PR) interactions disrupt nucleolar organization in a length-dependent manner

The lipid raft hypothesis presents insights into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. As a result, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approaches with inelastic neutron scattering, we isolate the bending modulus of ∼13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. From additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.

1 This article will form part of a virtual special issue on advanced neutron scattering instrumentation, marking the 50th anniversary of the journal.Oak Ridge National Laboratory is home to the High Flux Isotope Reactor (HFIR), a high-flux research reactor, and the Spallation Neutron Source (SNS), the world's most intense source of pulsed neutron beams. The unique colocalization of these two sources provided an opportunity to develop a suite of complementary small-angle neutron scattering instruments for studies of largescale structures: the GP-SANS and Bio-SANS instruments at the HFIR and the EQ-SANS and TOF-USANS instruments at the SNS. This article provides an overview of the capabilities of the suite of instruments, with specific emphasis on how they complement each other. A description of the plans for future developments including greater integration of the suite into a single point of entry for neutron scattering studies of large-scale structures is also provided.

The flow and deformation of macromolecules is ubiquitous in nature and industry, and an understanding of this phenomenon at both macroscopic and microscopic length scales is of fundamental and practical importance. Here, we present the formulation of a general mathematical framework, which could be used to extract, from scattering experiments, the molecular relaxation of deformed polymers. By combining and modestly extending several key conceptual ingredients in the literature, we show how the anisotropic single-chain structure factor can be decomposed by spherical harmonics and experimentally reconstructed from its cross sections on the scattering planes. The resulting wave-number-dependent expansion coefficients constitute a characteristic fingerprint of the macromolecular deformation, permitting detailed examinations of polymer dynamics at the microscopic level. We apply this approach to survey a longstanding problem in polymer physics regarding the molecular relaxation in entangled polymers after a large step deformation. The classical tube theory of Doi and Edwards predicts a fast chain retraction process immediately after the deformation, followed by a slow orientation relaxation through the reptation mechanism. This chain retraction hypothesis, which is the keystone of the tube theory for macromolecular flow and deformation, is critically examined by analyzing the fine features of the two-dimensional anisotropic spectra from small-angle neutron scattering by entangled polystyrenes. We show that the unique scattering patterns associated with the chain retraction mechanism are not experimentally observed. This result calls for a fundamental revision of the current theoretical picture for nonlinear rheological behavior of entangled polymeric liquids.