Adaptable P body physical states differentially regulate bicoid mRNA storage during early Drosophila development

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Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions

In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein—an essential viral protein required for viral replication—and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.     

Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets

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Regulation of the activity of the bacterial histidine kinase PleC by the scaffolding protein PodJ

Scaffolding proteins can customize the response of signaling networks to support cell development and behaviors. PleC is a bifunctional histidine kinase whose signaling activity coordinates asymmetric cell division to yield a motile swarmer cell and a stalked cell in the gram-negative bacterium Caulobacter crescentus. Past studies have shown that PleC’s switch in activity from kinase to phosphatase correlates with a change in its subcellular localization pattern from diffuse to localized at the new cell pole. Here we investigated how the bacterial scaffolding protein PodJ regulates the subcellular positioning and activity of PleC. We reconstituted the PleC-PodJ signaling complex through both heterologous expressions in Escherichia coli and in vitro studies. In vitro, PodJ phase separates as a biomolecular condensate that recruits PleC and inhibits its kinase activity. We also constructed an in vivo PleC-CcaS chimeric histidine kinase reporter assay and demonstrated using this method that PodJ leverages its intrinsically disordered region to bind to PleC’s PAS sensory domain and regulate PleC-CcaS signaling. Regulation of the PleC-CcaS was most robust when PodJ was concentrated at the cell poles and was dependent on the allosteric coupling between PleC-CcaS’s PAS sensory domain and its downstream histidine kinase domain. In conclusion, our in vitro biochemical studies suggest that PodJ phase separation may be coupled to changes in PleC enzymatic function. We propose that this coupling of phase separation and allosteric regulation may be a generalizable phenomenon among enzymes associated with biomolecular condensates.     

Get closer and make hotspots: liquid–liquid phase separation in plants

Liquid–liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus‐responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants.     

Quality Control of Membraneless Organelles

The formation of membraneless organelles (MLOs) by phase separation has emerged as a new way of organizing the cytoplasm and nucleoplasm of cells. Examples of MLOs forming via phase separation are nucleoli in the nucleus and stress granules in the cytoplasm. The main components of these MLOs are macromolecules such as RNAs and proteins. In order to assemble by phase separation, these proteins and RNAs have to undergo many cooperative interactions. These cooperative interactions are supported by specific molecular features within phase-separating proteins, such as multivalency and the presence of disordered domains that promote weak and transient interactions. However, these features also predispose phase-separating proteins to aberrant behavior. Indeed, evidence is emerging for a strong link between phase-separating proteins, MLOs, and age-related diseases. In this review, we discuss recent progress in understanding the formation, properties, and functions of MLOs. We pay special attention to the emerging link between MLOs and age-related diseases, and we explain how changes in the composition and physical properties of MLOs promote their conversion into an aberrant state. Furthermore, we discuss the key role of the protein quality control machinery in regulating the properties and functions of MLOs and thus in preventing age-related diseases.     

RGG/RG Motif Regions in RNA Binding and Phase Separation

RGG/RG motifs are RNA binding segments found in many proteins that can partition into membraneless organelles. They occur in the context of low-complexity disordered regions and often in multiple copies. Although short RGG/RG-containing regions can sometimes form high-affinity interactions with RNA structures, multiple RGG/RG repeats are generally required for high-affinity binding, suggestive of the dynamic, multivalent interactions that are thought to underlie phase separation in formation of cellular membraneless organelles. Arginine can interact with nucleotide bases via hydrogen bonding and π-stacking; thus, nucleotide conformers that provide access to the bases provide enhanced opportunities for RGG interactions. Methylation of RGG/RG regions, which is accomplished by protein arginine methyltransferase enzymes, occurs to different degrees in different cell types and may regulate the behavior of proteins containing these regions.     

The Role of RNA in Biological Phase Separations

Phase transitions that alter the physical state of ribonucleoprotein particles contribute to the spacial and temporal organization of the densely packed intracellular environment. This allows cells to organize biologically coupled processes as well as respond to environmental stimuli. RNA plays a key role in phase separation events that modulate various aspects of RNA metabolism. Here, we review the role that RNA plays in ribonucleoprotein phase separations.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Sam68 relocalization into stress granules in response to oxidative stress through complexing with TIA-1

Sam68 has been implicated in a variety of important cellular processes such as RNA metabolism and intracellular signaling. We have recently shown that Sam68 cytoplasmic mutants induce stress granules (SG) and inhibit HIV-1 nef mRNA translation [J. Henao-Mejia, Y. Liu, I.W. Park, J. Zhang, J. Sanford, J.J. He, Suppression of HIV-1 Nef translation by Sam68 mutant-induced stress granules and nef mRNA sequestration, Mol. Cell 33 (2009) 87-96]. These findings prompted us to investigate the possibility and the underlying mechanisms of the wild-type counterpart Sam68 SG recruitment. Herein, we revealed that Sam68 was significantly recruited into cytoplasmic SG under oxidative stress. We then demonstrated that domain aa269-321 and KH domain were both essential for this recruitment. Nevertheless, Sam68 knockdown had no effects on SG assembly, indicating that Sam68 is not a constitutive component of the SG. Moreover, we showed that Sam68 cytoplasmic mutant-induced SG formation was independent of eIF2α phosphorylation. Lastly, we demonstrated that Sam68 was complexed with T-cell intracellular antigen-1 (TIA-1), a core SG component, and that the complex formation was correlated with Sam68 SG recruitment. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1 in response to oxidative stress and suggest that cytoplasmic SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions.     

Who's In and Who's Out—Compositional Control of Biomolecular Condensates

Biomolecular condensates are two- and three-dimensional compartments in eukaryotic cells that concentrate specific collections of molecules without an encapsulating membrane. Many condensates behave as dynamic liquids and appear to form through liquid–liquid phase separation driven by weak, multivalent interactions between macromolecules. In this review, we discuss current models and data regarding the control of condensate composition, and we describe our current understanding of the composition of representative condensates including PML nuclear bodies, P-bodies, stress granules, the nucleolus, and two-dimensional membrane localized LAT and nephrin clusters. Specific interactions, such as interactions between modular binding domains, weaker interactions between intrinsically disorder regions and nucleic acid base pairing, and nonspecific interactions, such as electrostatic interactions and hydrophobic interactions, influence condensate composition. Understanding how specific condensate composition is determined is essential to understanding condensates as biochemical entities and ultimately discerning their cellular and organismic functions.     

Development of hybrid viral vectors for gene therapy

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

Liquid–Liquid Phase Separation at the Plasma Membrane–Cytosol Interface: Common Players in Adhesion,Motility, and Synaptic Function

Networks of scaffold proteins and enzymes assemble at the interface between the cytosol and specific sites of the plasma membrane, where these networks guide distinct cellular functions. Some of these plasma membrane–associated platforms (PMAPs) include shared core components that are able to establish specific protein–protein interactions, to produce distinct supramolecular assemblies regulating dynamic processes as diverse as cell adhesion and motility, or the formation and function of neuronal synapses. How cells organize such dynamic networks is still an open question. In this review we introduce molecular networks assembling at the edge of migrating cells, and at pre– and postsynaptic sites, which share molecular players that can drive the assembly of biomolecular condensates. Very recent experimental evidence has highlighted the emerging role of some of these multidomain/scaffold proteins belonging to the GIT, liprin-α and ELKS/ERC families as drivers of liquid–liquid phase separation (LLPS). The data point to an important role of LLPS: (i) in the formation of PMAPs at the edge of migrating cells, where LLPS appears to be involved in promoting protrusion and the turnover of integrin–mediated adhesions, to allow forward cell translocation; (ii) in the assembly of the presynaptic active zone and of the postsynaptic density deputed to the release and reception of neurotransmitter signals, respectively. The recent results indicate that LLPS at cytosol–membrane interfaces is suitable not only for the regulation of active cellular processes, but also for the continuous spatial rearrangements of the molecular interactions involved in these dynamic processes.