Research Highlights

MPS1 kinase regulation by PP2A-B56 is vital for successful mitosis

The ultimate goal of mitosis is to produce two daughter cells with the same genetic make-up as the parent cell. It consists of several steps, kept in check by proteins such as kinases and phosphates and deregulation of which is associated with human pathologies such as cancer.

One important kinase in this process is Mono-Polar Spindle 1 (MPS1), which monitors the correct formation of microtubule-kinetochore attachments and initiates spindle assembly checkpoint signalling in case of errors. MPS1 is also actively involved in resolving incorrect kinetochore attachments by phosphorylating outer kinetochore proteins. Accurate regulation of MPS1 kinase activity, via autophosphorylation of the MPS1 T-loop, is therefore critical for faithful chromosome segregation.

Hayward and colleagues from the Gruneberg lab have now shown that a kinetochore-associated pool of the PP2A-B56 phosphatase regulates the T-loop autophosphorylation of MPS1 and hence its kinase activity. Expression of a constitutively active form of MPS1 refractory to PP2A-B56 dephosphorylation results in exaggerated MPS1-mediated error correction, mitotic delays and impaired cell cycle progression, stressing the importance of balanced kinase and phosphatase activities for successful mitosis.

Written by Iqbal Dulloo

Hayward D, Bancroft J, Mangat D, Alfonso-Pérez T, Dugdale S, McCarthy J, Barr FA, Gruneberg U. (2019)
Checkpoint signalling and error correction require regulation of the MPS1 T-loop by PP2A-B56.

http://doi.org/10.1083/jcb.201905026

First structures of the genome replication machinery of influenza A virus

Influenza A viruses are the most common cause of seasonal flu in humans and also infect animals, representing significant public health and economic burdens. During infection, the virus invades host cells and makes many copies of its RNA genome to produce new virus particles. RNA-dependent RNA polymerase is the enzyme responsible for this genome replication.

A collaboration between the Fodor group and the group of Jonathan Grimes (Division of Structural Biology, University of Oxford) published in Nature has revealed how influenza A virus RNA polymerase replicates the genome. Using cryo-electron microscopy and X-ray crystallography, they have produced the first high-resolution structures of human and avian influenza A virus RNA polymerases. Their results show that the influenza A virus RNA polymerase forms a dimeric complex, where one of the polymerases activates the other to copy RNA. However, if dimerisation is blocked viral genome replication is inhibited, meaning the virus cannot propagate. Therefore, the dimerisation interface is an attractive target for antiviral drug development, made possible by these first structures.

 

Written by Isabella Maudlin

Fan H, Walker AP, Carrique L, Keown JR, Serna Martin I, Karia D, Sharps J, Hengrung N, Pardon E, Steyaert J, Grimes JM, Fodor E (2019).
Influenza A virus RNA polymerase structures provide insights into viral genome replication

https://doi.org/10.1038/s41586-019-1530-7

Neisseria: friend or foe

The bacterial genus Neisseria comprises commensal and pathogenic species. Pathogenic Neisseria can be found in human nasal passages where the bacteria attach to the surface using special filaments, called Type IV pili. These are required both during harmless colonisation of the upper airway, and invasive disease.

Mariya Lobanovska and colleagues studied Neisseria meningitidis, a pathogenic member of the Neisseria species, which causes meningitis and septic shock. They studied the components of the Type IV pili and found that one version of a component called PilE is particularly common in strains which cause epidemic disease. Interestingly, this version is conserved and shares homology with PilE from commensal Neisseria. To understand how this version can be present in both pathogenic and commensal bacteria, the transcriptional regulation was investigated. This new paper shows that the transcriptional regulation differs between the pathogenic and commensal bacteria. This new understanding of PilE transcription provides insight into the divergent mechanisms of Type IV pili regulation in commensal and pathogenic Neisseria.

 

Written by Heather Jeffery @HeatherMJeffery

Lobanovska M, Tang CM, Exley RM.
The contribution of σ70 and σN factors to expression of class II pilE in Neisseria meningitidis

J. Bacteriol pii: JB.00170-19. doi: 10.1128/JB.00170-19. [Epub ahead of print]

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Figure legend: To study parasite swimming behaviour in vitro, the swimming speed and directionality of 100 Leishmania knockout lines was measured individually (data plot). A selection of these mutant lines (coloured symbols) was passaged though sand flies

Leishmania’s tail crucial for sand fly colonisation

Leishmaniais a parasite that causes leishmaniasis, a disease that affects millions of people in Mexico and Central America. The parasite is passed between mammals by the bite of sand flies. Leishmaniahas a flagellum, a long tail-like organelle primarily used for direction and speed of motion, that is remodelled depending on the phase of its life cycle.

Tom Beneke and his colleagues (Gluenz Lab) systematically studied the function of flagellar proteins of Leishmania mexicana by using proteomics and CRISPR gene editing technologies. They first identified the proteins that constitute Leishmania’s flagellum by mass spectrometry. With this knowledge, they generated a library of 100 knockout mutants, each of which lacked a flagellar protein. They showed that many mutants swam slower than normal Leishmania,while some could swim faster; the rest were completely immobilised. They later showed that two mutants, one immobile (PF16 knockdown) and one uncoordinated swimmer (MBO2 knockdown), failed to fully colonise sand flies. This comprehensive study sheds light on the importance of directional swimming mediated by flagellum for Leishmaniainfection of sand flies, which in turn infect humans.

Written by Sheng Kai Pong

Beneke T, Demay F, Hookway E, Ashman N, Jeffery H, Smith J, Valli J, Becvar T, Myskova J, Lestinova T, Shafiq S, Sadlova J, Volf P, Wheeler RJ, Gluenz E. Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections. PLoS Pathog. 2019 Jun 26;15(6):e1007828. doi: 10.1371/journal.ppat.100782



Conservation of bacterial protein export system

Certain bacteria use a protein complex called the injectisome to deliver proteins into host cells, effectively increasing their pathogenic potential. The injectisome “needle” grows from a protein scaffold around its base called the basal body, which is related to the basal body of the bacterial flagellum. Bacterial proteins destined for export are picked up by an export apparatus complex (EA) inside the basal body. EA then exports the proteins across bacterial envelope into the flagellum or injectisome. In the flagellum, three of the EA proteins form an export gate core complex (FliPQR) and assemble into a helical structure that forms the start of the channel which culminates in the flagellum.

Research led by Steven Johnson and Lucas Kuhlen from Susan Lea’s lab was aimed at investigating the structure of the core export gate complex in the bacterial injectisome (SctRST). Single-particle cryo-electron microscopy of the export gate purified from human pathogen Shigella revealed a structure with striking similarity to the flagellar export complex gate. This finding further solidifies the conservation of the apparatus supporting the function of bacterial flagella and injectisomes.

Written by Lucie Kafkova

Johnson S, Kuhlen L, Deme JC, Abrusci P, Lea SM.
The Structure of an Injectisome Export Gate Demonstrates Conservation of Architecture in the Core Export Gate between Flagellar

mBio 10 (3) e00818-19; DOI: 10.1128/mBio.00818-19

RNA as a target for ADP-ribosylation

The human genome encodes several poly(ADP-ribose) polymerases (PARPs), which are enzymes involved in ADP-ribosylation, a reversible chemical modification, of macromolecules such as proteins and DNA. ADP-ribosylation occurs in both prokaryotes and eukaryotes and is linked to vital cellular processes namely stress responses, DNA repair, host-virus interactions, etc. This modification is mostly associated with proteins and to some extent, DNA. It was recently proposed that RNA molecules could also be cellular targets of ADP-ribosylation.

Deeksha Munnur and colleagues from Ivan Ahel’s lab, have now shown that ADP-ribosylation of RNA molecules indeed happens and is a more common phenomenon than previously believed. They found that ADP-ribosylation occurs at the terminal phosphate of RNA forming a non-canonical RNA cap and can be regulated by both PARP-like proteins from bacteria and by some human PARPs. Furthermore, they provide the first evidence that ADP-ribosylation of RNA is a reversible process that can be controlled by ADP-ribosylhydrolases from human as well as VEEV and SARS viruses. This study shines a light on a new regulatory mechanism of RNA molecules with potential therapeutic relevance.

Written by Iqbal Dulloo

Munnur D, Bartlett E, Mikolčević P, Kirby IT, Matthias Rack JG, Mikoč A, Cohen MS, Ahel I.
Reversible ADP-ribosylation of RNA.

Nucleic Acids Res. 47(11): 5658-5669

Understanding Pandemic Flu

Influenza A viruses are responsible for both seasonal outbreaks of respiratory disease and occasional pandemics. Their genome is made of eight RNA segments. If two different virus strains of infect the same cell, they can “shuffle” their segments instead of packaging just their own, and generate a new strain. This can result in the generation of a pandemic, since people may lack the immunity necessary to defeat the new virus. This happened in 1957 and 1968, when pandemic viruses killed millions worldwide. While we can detect this genomic “shuffling”, little is known about how they occur. Therefore, predicting them is still a challenge.

Research led by David Bauer in Ervin Fodor’s group addressed the question of the structure of the viral RNA genome. Collaborating with groups in Australia (Lorena Brown) and in the US (Alain Laederach), they employed high-throughput analysis techniques to reveal specific interactions that control packaging of RNA segments. Rarely, these interactions facilitate inter-strain “shuffling”. This work sheds light on influenza virus biology and is a major step in our understanding of influenza pandemic generation.

Written by Shaked Ashkanazi

Dadonaite B, Gilbertson B, Knight ML, Trifkovic S, Rockman S, Laederach A, Brown LE, Fodor E, Bauer DLV.
The structure of the influenza A virus genome.

Nature Microbiology (2019).

The role of sugars in bacterial adhesion

Bacteria display molecules on their outside to facilitate adhesion and interaction with surfaces. This is particularly important during the initial stages of infection and biofilm formation for pathogenic bacteria. This adhesive ability allows bacteria to move on surfaces, both living and non-living, and eventually adhere irreversibly to form biofilms.

The Bharat lab uses a commonly studied bacterium, Caulobacter crescentus, to investigate this adhesion process. There are two main methods of adhesion, via proteins or polysaccharides (sugars). The focus of this paper is polysaccharide-mediated adhesion, in which a specialized sugar called holdfast plays a key role. In this paper, Sulkowski et al use a range of structural biology techniques, including cryo-electron microscopy, to identify the proteins which anchor and display holdfast on the cell surface. By looking at the mechanism of positioning holdfast at the membrane and maintenance of its cellular position, the role of sugars in bacterial adhesion can be better understood.

Written by Heather Jeffery @HeatherMJeffery

Sulkowski NI, Hardy GG, Brun YV, Bharat TAM
A multi-protein complex anchors adhesive holdfast at the outer membrane of Caulobacter crescentus.

J. Bacteriol pii: JB.00112-19. doi: 10.1128/JB.00112-19. [Epub ahead of print]

DNA replication unfazed by disruption of cohesin-dependent DNA organisation

DNA replication is a process fundamental to all known organisms on earth. This highly regulated process is linked to chromatin structure − the way DNA is organised in the cell. It has been hypothesised that chromatin structure determines the accessibility of DNA by the replication machinery and hence dictates DNA replication timing.

Cohesin, well-known for its function in separating sister chromatids during cell division, has been shown to help form loop-based chromatin structures called topologically associating domains (TADs). Increasing evidence showed that TADs correlate with replication domains, but a causative relationship has not been established.

Phoebe Oldach, a DPhil student from the Nieuduzynski Lab, studied the relationship between TADs and DNA replication timing by using a system that can rapidly cut down levels of cohesin. After confirming the efficiency and efficacy of the system, she showed that replication timing patterns are not impacted by the absence of cohesin in either G1 or S phases of the cell cycle, during which the cell prepares for and executes DNA replication, respectively. This indicates that replication timing is not driven by cohesin-dependent TADs.

Written by Sheng Pong

Oldach P. and Nieduszynski C. A. (2019).
Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains.

Genes, 10(3). pii: E196. doi: 10.3390/genes10030196.

Balancing act — How Nodal and Bmp signaling impact the emergence of primordial germ cells in mice

Multicellular organisms consist of two fundamental types of cells: somatic cells and germ cells. While somatic cells make up most of the organism, germ cells are a small group of cells in the gonads that undergo meiosis to produce haploid gametes—either the oocyte or the spermatocyte. These cells are capable of generating a new organism after fertilization, thus transmitting genetic information from one generation to another. Early in development, the primordial germ cells (PGCs) are set aside as the precursors of sperm and eggs. We still do not have a complete understanding of exactly how these cell-fate decisions are made in development.

Dr. Anna Senft and colleagues in the Robertson lab have used transgenic mouse embryos to determine that two signaling pathways—Nodal and Bmp—and their downstream effectors, the Smad proteins, cooperate to regulate the numbers and development of PGCs. The balance of Nodal/Bmp signaling in embryonic and extra-embryonic tissues of the early embryo provide early instructions for the specification of the PGC lineage, while protecting PGCs from a somatic fate.

Written by Derek Xu @derekcxu

Senft AD, Bikoff EK, Robertson EJ, Costello I.
Genetic dissection of Nodal and Bmp signalling requirements during primordial germ cell development in mouse.

Nat Commun10(1):1089. doi: 10.1038/s41467-019-09052-w.

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