Mini-viral RNA linked to deadly influenza virus
Influenza virus is a pathogen that plagues society in the form of flu. 2018 marks 100 years since the ‘Spanish flu’ pandemic that, according to the World Health Organisation, infected a third of the human population worldwide and led to more deaths than World War I. The influenza virus contains its genetic information in the form of RNA instead of DNA and relies on infecting a host cell in order to replicate.
Researchers in the Fodor lab have identified a link between influenza virus infection and the infected cell response. This takes the form of mini-viral RNA (mvRNA) and is produced by dysregulated viral RNA replication. These mvRNA molecules bind to and activate retinoic acid-inducible gene 1 (RIG-1), which starts a signalling cascade resulting in cytokine production and cell death. The most harmful influenza virus strains, including the 1918 pandemic strain and the H5N1 bird flu, produce high levels of mvRNA. Understanding the mechanisms of mvRNA production and their contribution to virulence is important for developing ways to combat future viral strains.
Velthuis AJWt, Long JC, Bauer, DLV, Fan, RLY, Yen HL, Sharps J, Siegers JY, Killip MJ, French H, Oliva-Martín MJ, Randall RE, Wit Ed, Riel Dv, Poon LLM, Fodor E (2018) Mini viral RNAs act as innate immune agonists during influenza virus infection
Nature Microbiology 10.1038/s41564-018-0240-5
Determining the fate of cells during embryonic differentiation
During embryonic development, key signalling cues dictate cell fates and allow specification of different cell populations. Three of these cues are Nodal, an extracellular ligand of a family of growth factors (TGFb), and Smad2 and Smad3, the downstream effectors inside the cell responsible for determining the head-to-tail axis of the embryo. Previous genetic studies showed that Smad2/3 double-mutant mouse embryos die shortly after implantation, yet the underlying molecular mechanisms remained unknown.
Senft and colleagues in the Robertson and Bikoff labs have recently characterised the effects of Smad2/3-deficiency in mouse embryonic stem cells (ESCs) using in vitro differentiation protocols. They found that Smad2/3 signalling is required to maintain embryonic and extra-embryonic cell identities, and to undergo key cell fate allocation steps during embryonic germ layers differentiation, in the pre-implantation mammalian embryo. Additionally, the balance of signalling pathways during neural precursor differentiation was not properly maintained in these mutant ESCs, with Nodal target genes being downregulated and Bmp upregulated. This detailed molecular study of the signalling in early mammalian development widens our comprehension of the genetic programs involved in embryonic post-implantation development.
Senft AD, Costello I, King HW, Mould AW, Bikoff EK, Robertson EJ (2018) Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage
Cell Rep 24(8):1977-1985.e7.
Don’t stop the beat: helping parasites change direction
Flagella are tail-like structures that beat to propel single-celled organisms along. The moving flagellum resembles a wave. Some parasites, such as Trypanosoma and Leishmania, can change their direction by adjusting where this wave starts.
Researchers from the Gull Lab have found that this ability is afforded by breaking the flagellum’s symmetry. They showed that a class of proteins, known as ODA-docking complexes (DCs), are unevenly distributed along trypanosome flagella. Some (dDCs) sit at the flagellum tip, while others (pDCs) sit nearer the cell body. Mathematical modelling suggested that this pattern is achieved by a process called intraflagellar transport (IFT) that constantly moves pDC towards the base. This frees up binding sites at the opposite end, which dDCs can fill.
This asymmetry is important for wave initiation; the localisation of a novel protein (‘LC4-like’), which acts as a molecular switch to change where the wave starts from, is dependent on dDC. This study explores why parasites are such expert movers and shakers and may also have implications for human health; DC mutations can cause ciliary dyskinesia, a disease characterised by inefficient ciliary movement.
Edwards BFL, Wheeler RJ, Barker AR, Moreira-Leite FF, Gull K, Sunter JD Direction of flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry
PNAS 115(31): E7341-E7350
A common birthplace for distinct organelles
Membrane-bound subcellular compartments (or organelles) are the hallmark of eukaryotic cells. Lipid droplets (LDs) and peroxisomes are distinct organelles at the centre of cellular metabolism; their dysfunction is linked to devastating diseases in humans, including diabetes and neurodevelopmental disorders. Both are thought to ‘bud off’ from the endoplasmic reticulum (ER), the cell’s largest organelle. However, how their assembly sites are defined and the mechanism by which they bud from the ER remained mysterious.
A recent study by Sihui Wang from the Carvalho lab and colleagues has uncovered that these structurally distinct organelles share their birthplace at specific ER domains. Using lipidomics, microscopy and genetic strategies in yeast, Wang et al. explore the cooperation of the protein complex Seipin and the membrane-shaping protein Pex30. They show that these factors facilitate LD and peroxisome formation by organising membrane regions of a lipid composition that favour budding. Their data advocate a new model for LD and peroxisome biogenesis, which paves the way for understanding the mechanisms of organelle formation at the ER.
Wang, Idrissi FZ, Hermansson M, Grippa A, Ejsing CS, Carvalho P. Seipin and the membrane-shaping protein Pex30 cooperate in organelle budding from the endoplasmic reticulum
Nat Commun. 9(1):2939. doi: 10.1038/s41467-018-05278-2
Nucleic acids from within the cell can activate antiviral response
Mitochondria, which generate energy for the cell, are descendants of endosymbiotic bacteria and possess a bacteria-like genetic make-up—a dense, circular genome. Mitochondrial DNA is transcribed on both strands, potentially leading to the generation of double-stranded RNAs (dsRNAs).
In this collaborative study, Ashish Dhir and colleagues from the Proudfoot lab, together with researchers from the US, France, Poland and Scotland, established that mitochondria are major sites of dsRNA generation in human cells. They identified the key factors that regulate the amounts of these dsRNAs—SUV3 and PNPase. In the absence of PNPase, mitochondrial dsRNAs leak into the cytoplasm and trigger an interferon response which is typically turned on in response to viral and bacterial infections. Interestingly, they found that mitochondrial disease patients with mutations in the gene PNPT1, which encodes PNPase, have increased levels of mitochondrial dsRNA and markers of immune activation, and an activated interferon response. In combination these changes can lead to autoimmunity in humans.
This study provides insights into how the cell processes a damaging internal source of nucleic acids to prevent unnecessary immune system activation.
Dhir, A., Dhir, S., Borowski, L. S., Jimenez, L., Teitell, M., Rötig, A., … Proudfoot, N. J. (2018) Mitochondrial double-stranded RNA triggers antiviral signalling in humans
Nature 560(7717) 238–242 https://doi.org/10.1038/s41586-018-0363-0
A rapid and inexpensive method to measure DNA replication timing
DNA replication is a fundamental property of life, passing genetic information down generations of cells. The timing of replication in organisms with larger genomes follows a defined and reproducible programme. How this is coordinated is poorly understood, as changes to replication timing have been involved in various human disorders.
To measure replication timing, researchers use measurements of DNA copy number as a proxy. However, current methods are time-consuming and/or expensive—especially for organisms with larger genomes. Dzmitry Batrakou and colleagues in the Nieduszynski lab utilised droplet digital PCR (around 1nl in size) to rapidly measure DNA copy number with high throughput, bypassing the need for expensive microfluidics devices. Not only is this method comparable to the best spatial and temporal resolution in current methods, it is also applicable to cultured human cells. The work uses this method to rapidly screen mutants to study locus-specific perturbations of replication timing and identify mechanisms that regulate local genome replication. In addition, the method may also be used to study other aspects of chromosome biology, such as Homologous Recombination, making it a valuable tool for research.
Batrakou, D. G., Heron, E. D. & Nieduszynski, C. A. Rapid high-resolution measurement of DNA replication timing by droplet digital PCR
Nucleic Acids Res, doi:10.1093/nar/gky590 (2018).
A structural investigation into the workings of a Type III Secretion System
Type III secretion systems (T3SS) are believed to act as nanomachines, which reside in the bacterial cell envelope and allow the passage of proteins. They are critical for building bacterial motility organelles and injecting toxins into host cells. Three putative integral membrane proteins namely P, Q and R, are believed to form the pore in the cell envelope called the export gate of these machines, with their structure remaining unresolved till date.
Kuhlen and colleagues have employed a combination of biochemistry, native mass-spectrometry (nMS) and cryoelectron microscopy (cryo-EM) techniques to investigate the structure and assembly of the nanomachines. They report a 4.2A resolution Cryo-EM structure for the PQR complex, in which none of the components assume a predicted integral membrane conformation. Excitingly, common helix-turn-helix structural elements allow them to form a helical assembly above the membrane. Additionally, they propose a mechanism of how the export gate opens. Their structure not only reveals the molecular architecture of the core of T3SS, but also has more general implications for predictions of membrane protein structure-function relationships.
Kuhlen L, Abrusci P, Johnson S, Gault J, Deme J, Caesar J, Dietsche T, Tesfazgi Mebrhatu M, Ganief T, Macek B, Wagner S, Robinson CV, Lea SM (2018). Structure of the core of the type III secretion system export apparatus
Nature Structural & Molecular Biology 25: 583–590
Reshaping fluids – a new microfluidics technology for biomedicine
Microfluidics deals with very small volumes of liquids (under 1µL). While cell biologists often manipulate tiny volumes, they do not normally use microfluidics due to the cost and complexity of manufacture, concerns with biocompatibility and the inaccessibility of cells once introduced into such enclosed spaces.
In a collaborative study between the Cook lab at the Dunn School of Pathology and the Walsh lab at the Department of Engineering Science, Soitu and colleagues have developed a revolutionary method for cell biologists to create microfluidic arrangements containing sub-microlitre volumes. They reshaped fluids on a substrate and exploited interfacial forces (or surface tension – the elastic tendency of a fluid surface to acquire the least surface area possible) at the microscale level. In this platform, liquids are confined within fluid (not solid) walls formed by the interface between water and an immiscible liquid (a fluorocarbon). These walls spontaneously seal around inserted pipettes, and self-heal when they are withdrawn. This method provides an accurate, simple and customisable approach for fabrication of microfluidic devices using materials that are familiar to biologists.
Soitu C, Feuerborn A, Tan AN, Walker H, Walsh PA, Castrejón-Pita AA, Cook PR, Walsh EJ (2018). Microfluidic chambers using fluid walls for cell biology
Proc Natl Acad Sci U S A.115(26):E5926-E5933.
Deciphering the mode of action of the influenza virus transcriptase
Curtailment of the spread of influenza virus has long been a subject of great interest and research. The influenza virus RNA polymerase performs both transcription and replication of the viral RNA genome. For transcription, it associates with host RNA polymerase II (Pol II) and steals nascent host capped RNA fragments that it uses as primers. Recent work has shown that the viral polymerase can assume multiple conformations, corresponding to different functional states, but much less is known about the factors that regulate these states and its mode of action.
With the aid of x-ray crystallography, cryo–electron microscopy, in vitro activity and cell based minireplicon assays, Serna Martin, Hengrung and colleagues set out to understand the mechanistic details of the interaction between host Pol II and the viral polymerase. Their investigations revealed that the interaction is important for stabilizing the transcriptase conformation of the viral polymerase and uncovered a binding site on the viral polymerase for Pol II. This binding site is crucial for influenza virus polymerase function, thus making it an attractive target for future anti-viral small molecule inhibitor development.
Serna Martin I, Hengrung N, Renner M, Sharps J, Martínez-Alonso M, Masiulis S, Grimes JM, Fodor E (2018). A Mechanism for the Activation of the Influenza Virus Transcriptase
Mol Cell. 70(6):1101-1110.e4
A novel iRhom interactor controls release of an inflammatory signalling protein
Cell communication requires the controlled release of signalling proteins, which act as messages that can be sent and received by cells. These messages must be sent in adequate quantities and at appropriate times. A protein called ADAM17 aids in the release of many signalling proteins from the cell surface, most notably the inflammatory cytokine, TNFα. ADAM17 itself forms a complex with proteins known as iRhoms, which modulate its activity. However, little is known about how iRhoms are regulated.
Researchers from the Freeman lab carried out a proteomic screen to search for binding partners of iRhoms. Their top hit was FRMD8, which appears to stabilise the ADAM17/iRhom complex at the cell surface through its interaction with iRhom2. The absence of FRMD8 leads to the degradation of the complex. Künzel and colleagues functionally validated this phenomenon in macrophage cells, finding that FRMD8 KO macrophages release lower levels of TNFα. Improper TNFα release can result in inflammatory diseases like rheumatoid arthritis, thus suggesting that this study may guide future anti-inflammatory drug targets.
Künzel U, Grieve A G, Meng Y, Sieber B, Cowley SA, Freeman M (2018). FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex
eLife 2018;7:e35012 DOI: 10.7554/eLife.35012