Publications by authors named "E G Allwood"

30 Publications

Phosphorylation of the WH2 domain in yeast Las17/WASP regulates G-actin binding and protein function during endocytosis.

Sci Rep 2021 May 6;11(1):9718. Epub 2021 May 6.

Department of Biomedical Science, Firth Court, University of Sheffield, Sheffield, S10 2TN, UK.

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue Las17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of Las17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on Las17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.
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http://dx.doi.org/10.1038/s41598-021-88826-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102491PMC
May 2021

The Pex3-Inp1 complex tethers yeast peroxisomes to the plasma membrane.

J Cell Biol 2020 10;219(10)

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, England, UK.

A subset of peroxisomes is retained at the mother cell cortex by the Pex3-Inp1 complex. We identify Inp1 as the first known plasma membrane-peroxisome (PM-PER) tether by demonstrating that Inp1 meets the predefined criteria that a contact site tether protein must adhere to. We show that Inp1 is present in the correct subcellular location to interact with both the plasma membrane and peroxisomal membrane and has the structural and functional capacity to be a PM-PER tether. Additionally, expression of artificial PM-PER tethers is sufficient to restore retention in inp1Δ cells. We show that Inp1 mediates peroxisome retention via an N-terminal domain that binds PI(4,5)P2 and a C-terminal Pex3-binding domain, forming a bridge between the peroxisomal membrane and the plasma membrane. We provide the first molecular characterization of the PM-PER tether and show it anchors peroxisomes at the mother cell cortex, suggesting a new model for peroxisome retention.
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http://dx.doi.org/10.1083/jcb.201906021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659723PMC
October 2020

Female genital mutilation and women's healthcare experiences with general practitioners in the Netherlands: A qualitative study.

PLoS One 2020 7;15(7):e0235867. Epub 2020 Jul 7.

Pharos, Dutch Centre of Expertise on Health Disparities, Utrecht, The Netherlands.

Objectives: While the general practitioner (GP) in the Netherlands is the first point of entry to and gatekeeper of the healthcare system, no study exists to explore the experiences of women with female genital mutilation or cutting (FGM/C) in general practice. Therefore, the aim of this study is to look into the experiences of women with FGM/C in Dutch general practice.

Methods: Semistructured interviews were held with 16 women with FGM/C. Sampling was purposeful. The interview guide and thematic analysis were based on the Illness Perception Model and Kleinman's Explanatory model. Interviews were held in English or Dutch. All data were anonymized, and recordings were transcribed verbatim. Transcripts were coded and thematically analyzed.

Results: The women considered FGM/C to be connected to a range of health problems, for which not all of them sought medical care. They had difficulty discussing such a sensitive topic with their GP, did not know their problems could be relieved or perceived GPs to have insufficient knowledge of FGM/C. Lack of time during consultations and overall dissatisfaction with Dutch GP care hampered trust. They strongly preferred the GP to be proactive and ask about FGM/C.

Conclusion: There is room for improvement as most women would like their GP to discuss their health problems related to FGM/C. GPs should take a proactive attitude and ask about FGM/C. In addition, to develop the trusted relationship needed to discuss sensitive topics and provide culturally sensitive person-centered care, sufficient time during consultations is needed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235867PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340277PMC
September 2020

Disruption of the Burkholderia pseudomallei two-component signal transduction system BbeR-BbeS leads to increased extracellular DNA secretion and altered biofilm formation.

Vet Microbiol 2020 Mar 8;242:108603. Epub 2020 Feb 8.

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia; Department of Microbiology, Monash University, Melbourne, Victoria, Australia. Electronic address:

Two-component signal transduction systems (TCSTS) are abundant among prokaryotes and regulate important functions, including drug resistance and virulence. The Gram-negative bacterium Burkholderia pseudomallei, which causes the severe infectious disease melioidosis, encodes 136 putative TCSTS components. In silico analyses of these TCSTS indicated that the predicted BbeR-BbeS system (BPSL1036-BPSL1037) displayed significant amino acid sequence similarity to the Shigella flexneri virulence-associated OmpR-EnvZ osmoregulator. To assess the function of the B. pseudomallei BbeR-BbeS system, we constructed by allelic exchange a ΔbbeRS double mutant strain lacking both genes, and single ΔbbeR and ΔbbeS mutants. All three mutant strains caused disease in the BALB/c acute melioidosis model at the same rate as the wild-type strain, displayed unchanged swarming motility on semi-solid medium, and were unaffected for viability on high-osmolarity media. However, when cultured at 37 °C for at least 14 days, ΔbbeS and ΔbbeR colonies developed a distinct, hypermucoid morphology absent in similarly-cultured wild-type colonies. At both 30 °C and 37 °C, these hypermucoid strains produced wild-type levels of type I capsule but released increased quantities of extracellular DNA (eDNA). Upon static growth in liquid medium, all B. pseudomallei strains produced pellicle biofilms that contained DNA in close association with bacterial cells; however, the ΔbbeS and ΔbbeR strains produced increased biofilms with altered microscopic architecture compared to the wild-type. Unusually, while the ΔbbeS and ΔbbeR single-deletion mutants displayed clear phenotypes, the ΔbbeRS double-deletion mutant was indistinguishable from the wild-type strain. We propose that BbeR-BbeS indirectly affects eDNA secretion and biofilm formation through cross-talk with one or more other TCSTS.
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http://dx.doi.org/10.1016/j.vetmic.2020.108603DOI Listing
March 2020

Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis.

PLoS One 2019 22;14(4):e0215102. Epub 2019 Apr 22.

Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

The yeast dynamin-like protein Vps1 has roles at multiple stages of membrane trafficking including Golgi to vacuole transport, endosomal recycling, endocytosis and in peroxisomal fission. While the majority of the Vps1 amino acid sequence shows a high level of identity with the classical mammalian dynamins, it does not contain a pleckstrin homology domain (PH domain). The Dyn1 PH domain has been shown to bind to lipids with a preference for PI(4,5)P2 and it is considered central to the function of Dyn1 in endocytosis. The lack of a PH domain in Vps1 has raised questions as to whether the protein can function directly in membrane fusion or fission events. Here we demonstrate that the region Insert B, located in a position equivalent to the dynamin PH domain, is able to bind directly to lipids and that mutation of three lysine residues reduces its capacity to interact with lipids, and in particular with PI(4,5)P2. The Vps1 KKK-AAA mutant shows more diffuse staining but does still show some localization to compartments adjacent to vacuoles and to endocytic sites suggesting that other factors are also involved in its recruitment. This mutant selectively blocks endocytosis, but is functional in other processes tested. While mutant Vps1 can localise to endocytic sites, the mutation results in a significant increase in the lifetime of the endocytic reporter Sla2 and a high proportion of defective scission events. Together our data indicate that the lipid binding capacity of the Insert B region of Vps1 contributes to the ability of the protein to associate with membranes and that its capacity to interact with PI(4,5)P2 is important in facilitating endocytic scission.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215102PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476499PMC
December 2019