Research Highlights

Controlling centriole growth by a homeostatic clock

Centrioles are organelles that perform key functions during cell division -as part of centrosomes- and ciliogenesis. These small structures are duplicated in each cellular division via the growth of a daughter centriole from the side of its mother. While the regulation of centriole and centrosome number in cells has been well characterised, the mechanisms controlling centriole growth have remained elusive to date.

Aydogan and colleagues (Raff lab) have used live imaging of early fly embryos, coupled with quantitative tools, to report a novel function for Polo-like Kinase 4 (Plk-4) in regulating centriole size. They observed that Plk4 drives centriole growth by linearly promoting the addition of building blocks (Sas-6 molecules) onto the proximal side of daughter centrioles throughout early/mid S-phase and halting growth once centrioles have reached their correct size. This homeostatic clock role of Plk4 is attributed to its predicted suicidal activity – the more active the kinase is, the faster it triggers its own degradation. This study sheds light on an enzyme that acts as a clock to control centriole growth and proposes a novel mechanism for organelle size regulation.

Written by Sonia Muliyil (@Muliyilsonia)

Aydogan MG, Wainman A, Saurya S, Steinacker TL, Caballe A, Novak ZA, Baumbach J, Muschalik N, Raff JW. (2018).
A homeostatic clock sets daughter centriole size in flies.

J Cell Biol. 217(4):1233-1248.

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Autophagy prevents TNF-induced apoptotic cell death in inflamed intestines to maintain barrier epithelium

Maintaining Intestinal Epithelial Integrity During Inflammation Through Autophagy

Autophagy is an evolutionarily conserved “self-devouring” process by which cells may degrade intracellular components, and is particularly important for recycling intracellular materials during periods of stress or starvation. In addition, it is also important in other physiological roles, such as the intracellular immune response. Autophagy genes such as ATG16L1 have been associated with inflammatory bowel disease (IBD); however, it is not known how autophagy regulates tissue homeostasis in the inflamed intestine.

Dr. Johanna Pott and colleagues in the Maloy Lab use cell-specific knockouts of the ATG16L1 gene in mice and intestinal stem cell (IEC) organoids to discover that autophagy was required in IECs to limit TNF-induced apoptosis in the epithelium during inflammatory conditions. Their work highlights the important role of autophagy in maintaining barrier integrity, by limiting cell death to reduce inflammation, and reveals a link between apoptosis and autophagy in intestinal epithelial cells. Additionally, the study indicates the potential role of anti-TNF based treatments in IBD patients in boosting epithelial barrier integrity.

Written by Derek Xu @derekcxu

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A crystal structure of a chimeric antigen composed of a PorA extracellular loop grafted onto an fHbp molecular scaffold.

Structure-based design of chimeric antigens for multivalent protein vaccines

Vaccines against bacterial pathogens, mostly based on the pathogens’ toxins or capsules components, have been revolutionary for human kind. However, these approaches are not feasible for pathogens such as serogroup B Neisseria meningitidis – where vaccine development has been impeded by pathogen diversity, the inability to use capsule-based vaccines and technical difficulties of generating antigens from membrane-embedded proteins. A solution for this problem is critical due to the rise of multi-drug resistant bacteria.

Hollingshead and colleagues have begun to address this problem by engineering chimeric antigens (ChA) consisting of two different N. meningitidis proteins. ChAs contain the protein fHbp as a molecular scaffold on to which they have engineered a critical region, the VR2 loop, from the membrane-bound protein PorA. They found that the ChAs retain the architecture of fHbp and the VR2 loop. The ChAs were also able to successfully elicit protective immune responses in mice against both protein antigens.

This proof-in-principle study demonstrates that ChAs can present important parts of membrane-bound proteins to the immune system and initiate an immune response, opening the door to a new generation of multivalent vaccines.

 

Written by Lisa Gartenmann

Hollingshead S, Jongerius I, Exley RM, Johnson S, Lea SM and Tang CM (2018).
Structure-based design of chimeric antigens for multivalent protein vaccines

Nat. Commun. 13;9(1):1051. 

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Colocalisation of phosphorylated Dicer (green) and a DNA damage marker (red) in the nucleus of a mouse cell upon induction of DNA damage.

Nuclear re-localization of Dicer in primary mouse embryonic fibroblast nuclei following DNA damage

The endoribonuclease Dicer is an integral component of the RNA interference pathway. Dicer generates small RNA species that regulate post-transcriptional gene silencing in the cytoplasm. However, increasing evidence indicates that Dicer has additional, non-canonical roles in the nucleus. Previous localisation studies with over-expressed tagged Dicer, yield limited and inconclusive insight in to the regulatory principles of nuclear Dicer localisation. To address the controversy in the field, Burger and colleagues have begun to unravel the cellular mechanisms that determine Dicer’s localisation when Dicer levels are kept physiological.

This study, performed in mouse cells, ensured endogenous expression levels of tagged Dicer. Burger and colleagues firstly assessed the localisation of Dicer in unperturbed cells. They show that approximately 5% of Dicer molecules localise to the nucleus under physiological conditions. Upon induction of DNA damage, in particular double-strand breaks, they found a 2-3 fold increase in nuclear Dicer localization. Under these conditions, >90% of nuclear Dicer was phosphorylated in a PI3K-dependent manner. Their findings suggest that damage-induced PI3K-signaling establishes distinct populations of Dicer, hinting at novel functions and complex regulation of RNAi components.

Written by Lisa Gartenmann

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Serine identification by ADP-ribosylating enzymes leads to damaged DNA repair

A chemical on/off switch for DNA damage repair

Cells have a complex protein machinery that activates a repair response after detecting damaged DNA. One of the mechanisms used to regulate the function and localisation of these proteins is the addition of molecules onto their building blocks (amino acids) that modify their behaviour.

Work from Ivan Ahel’s lab, together with collaborators at the Max Planck Institute in Cologne (Germany), has revealed that serine is the preferred amino acid of a family of modifying enzymes responsible for ADP-ribosylation (ADPr) – a protein modification regulating cellular processes, including the vital activation of DNA repair responses at DNA breaks. Palazzo et al. used cell biology and biochemical approaches to show that serine residues of histones (proteins that package DNA) become the main ADPr sites after DNA damage. Together with their previous work identifying the ‘eraser’ of serine ADPr, which enables the process to be reversed, this shows how proteins involved in DNA damage repair can be switched on/off in different physiological and pathological conditions. This knowledge could be used to develop new cancer drugs regulating ADPr signals and, therefore, controlling DNA repair. 

Written by Anna Caballe (@caballe_anna)

Palazzo L, Leidecker O, Prokhorova E, Dauben H, Matic I, Ahel I. (2018).
Serine is the major residue for ADP-ribosylation upon DNA damage.

Elife;7. pii: e34334. doi: 10.7554/eLife.34334.

Getting Lipid Droplets in shape via Ldo proteins

Cells store excess of energy as fat in dedicated organelles called Lipid Droplets (LDs). How the size and number of these energy stores are regulated by metabolic signals remains largely unknown. Employing yeast as a model system, Teixeira et al, identify a group of  Lipid droplet organisation (Ldo) proteins that regulate key components of LD biogenesis machinery. They also demonstrate that the relative abundance of Ldo proteins responds to metabolic signals which in turn feed onto LD shape and composition. Interestingly, this relationship gains special prominence  during nutrient starvation, a condition that triggers consumption of energy stored in LDs. This study sheds light on a new dimension of LD behaviour and dynamics, which ultimately may help better understand various metabolic disorders such as diabetes and obesity.

Written by Sonia Muliyil (@Muliyilsonia)

Teixeira V, Johnsen L, Martínez-Montañés F, Grippa A, Buxó L, Idrissi FZ, Ejsing CS, Carvalho P (2018).
Regulation of lipid droplets by metabolically controlled Ldo isoforms.

J Cell Biol. 217(1):127-138

Kinase links transcription termination and mRNA export

Adam Volanakis and Kinga Kamieniarz-Gdula in the Proudfoot lab at the Sir William Dunn School of Pathology, have identified a new role for a kinase that connects transcription termination and mRNA export to the cytoplasm.

These two processes occur sequentially in the overall process of gene expression. Previously, connections between the mRNA export machinery and other processes have been shown, for example a physical link to the pre-mRNA splicing machinery is made through an interaction between UAP56 and Aly. The new findings by Volanakis et al identify a new connection between mRNA export and transcription termination, mediated by phosphorylation; a post-transcriptional modification regulated by a kinase.

The kinase in question, WNK1, is known for regulating ion homeostasis in the cytoplasm. However, Adam and Kinga have identified that upon nuclear localisation it phosphorylates a specific domain of a termination factor named PCF11. This domain is important for interacting with RNA polymerase II, which is the transcription motor. The phosphorylation of PCF11 weakens this interaction and promotes transcript release. Subsequently, the newly released mRNA is free to be exported to the cytoplasm, thus linking the transcription and mRNA export processes.

Written by Heather Jeffery

Volanakis A, Kamieniarz-Gdula K, Schlackow M, Proudfoot NJ. (2017).  
WNK1 kinase and the termination factor PCF11 connect nuclear mRNA export with transcription.

Genes Dev. 31(21):2175-2185. doi: 10.1101/gad.303677.117. Epub 2017 Dec 1.

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Assembly of a centriole using 3D-printed pieces, inspired by the high-precision localisation studies from Gartenmann et al. 2017

Protein localisation with nanometre-scale precision: uncovering organelle assembly

Centrioles are small organelles that sit at the base of two cellular structures: cilia (motile or sensory) and centrosomes (organise spindle microtubules during mitosis). There is increasing evidence linking centrosomes and cilia dysfunction to a plethora of human pathologies, including cancer, obesity, microcephaly and dwarfism. Understanding the molecular mechanisms that regulate the assembly of these organelles is important from both basic biological and clinical perspectives.

Recent work from the Raff lab, in collaboration with Micron Oxford, has uncovered the localisation of some of the core proteins that make up centrioles at nanometre-scale resolution –with » 4 nm precision. Gartenmann et al. combined two super-resolution microscopy techniques, 3D-structured-illumination (3D-SIM) and single molecule localisation microscopy (SMLM or STORM), to generate the first detailed map of how proteins are arranged across centrioles in Drosophila melanogaster. This method, together with a unique image analysis code, could be used to unveil the distribution of molecules in high precision within other cellular organelles, such as nuclear pores or cilia/flagella, and will allow scientists to better understand how key cellular organelles work in health and disease.

Written by Anna Caballe (@caballe_anna)

Gartenmann L, Wainman A, Qurashi M, Kaufmann R, Schubert S, Raff JW, Dobbie IM (2017)
A combined 3D-SIM/SMLM approach allows centriole proteins to be localized with a precision of ∼4-5 nm.

Curr Biol. 27(19):R1054-R1055. doi: 10.1016/j.cub.2017.08.009.

Genetic plasticity of the Shigella virulence plasmid is mediated by intra- and inter-molecular events between insertion sequences

Shigella is a major agent of diarrhoeal disease worldwide causing 188 million cases and 600,000 deaths annually. Shigella emerged from non-pathogenic Escherichia coli, by acquiring a large plasmid (pINV), an essential virulence factor. The plasmid contains a pathogenicity island (PAI) encoding aType III Secretion System (T3SS) - placing a large metabolic burden on the bacterium. Spontaneous expression loss of the PAI is observed in laboratory grown Shigella, raising the question: what are the molecular mechanisms that mediate this event?

Pilla and colleagues discovered that in Shigella flexneri repeated sequences, present on the plasmid and the chromosome, mediate deletion of the PAI or reversible integration of the plasmid into the chromosome. Following integration, the expression of T3SS is down regulated and excision of the plasmid is sufficient to restore virulence. Hence, integration provides a strategy for regulating T3SS expression; allowing the bacteria to stay competitive, yet maintain this taxing but essential element over time. This study allows researchers to understand the molecular mechanisms that govern virulence maintenance andhow changes inplasmid can influence the evolution of this important pathogen.

Written by Lisa Gartenmann

A novel co-culture model to study human microglia functions

Microglia are important phagocytic cells of the brain. These specialised cells not only function to remove debris, but are also widely associated with a high expression of genes implicated in various neurodegenerative diseases. Till quite recently, there was a paucity of in vitro human models which could facilitate the study of these brain resident cells. To circumvent this problem, Haenseler et al., developed an elegant technique which involves the co-culture of human Induced Pluripotent Stem cell (iPSC) derived macrophages with iPS-neurons to transform the former into microglia. The resulting microglia and neuronal co-cultures appear to be stable for many weeks. More importantly, microglia derived via this technique display all the relevant genetic and phenotypic expression markers, and produce a distinct signature of cytokines specific to a co-culture model. This technique appears to be far superior to monocultures of microglia or transformed microglia lines, known to rapidly lose their identity. This study  opens up the possibility of modelling various neurodegenerative diseases in a dish.

Written by Sonia Muliyil (@Muliyilsonia)

Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS, Nicholls FJ, Chintawar S, Schnell C, Antel JP, Allen ND, Cader MZ, Wade-Martins R, James WS, Cowley SA. (2017).
A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and In

Stem Cell Reports. 8(6):1727-1742. doi: 10.1016/j.stemcr.2017.05.017.