Biology subjects

Botchway, S. W.

Publications and source records attributed to Botchway, S. W..

6 recordsLinked to original sources

bioRxiv2026

Peptide-Induced Formation of Extracellular Vesicles that are DistinctFrom Endogenous E. coli OMVs, and Provide an Enhanced Platformfor Protein Production and Purification.

Bacterial outer membrane vesicles (OMVs), are nano-sized, spherical structures released by Gram-negative bacteria that play diverse roles in bacterial physiology, including communication, nutrient acquisition, and host interactions. These vesicles bud from the bacterial outer membrane and contain lipopolysaccharides, periplasmic proteins, nucleotides, and other biomolecules. The Vesicle Nucleating peptide (VNp) is a short peptide tag that, when fused to the amino terminus of a protein of interest, promotes the formation of bespoke recombinant extracellular vesicles in Escherichia coli, enabling efficient production and simplified purification of recombinant proteins. Here, we characterise VNp-induced vesicles and compare their composition and organisation with naturally produced E. coli OMVs. While both vesicle types possess a single outer membrane-derived lipid bilayer, recombinant protein is highly enriched within the VNp vesicles compared to endogenous OMVs. VNp-fusions and periplasm-targeted recombinant proteins localize to distinct vesicle populations, with VNp-fusions showing markedly higher intra-vesicular concentrations and vesicular purity, compared to the OMV targeted protein. OmpX co-expression further enriched the VNp-fusion content of vesicles, further enhancing yield. The VNp-vesicle lumen is an oxidizing environment, thus supports formation of inter- and intra-molecular disulfide bonds within encapsulated proteins. Overall, VNp-induced vesicles represent a distinct class of recombinant extracellular vesicles that offer a simple and efficient route for producing and purifying concentrated, correctly folded recombinant proteins, expanding the utility of bacterial vesicle systems for biotechnological applications. Significance StatementBacterial extracellular vesicles are recognized as versatile tools for biotechnology yet engineering bacterial vesicle production in a controlled and efficient manner remains challenging. Here we describe how a short Vesicle Nucleating Peptide (VNp) tag, fused to a protein of interest, that can be used to program Escherichia coli to produce recombinant extracellular vesicles that are compositionally and structurally distinct from natural bacterial outer membrane vesicles (OMVs). VNp-induced vesicles are more homogeneous, and more highly enriched in target fusion proteins, providing a simple and efficient route for protein production and purification. The oxidizing lumen of these vesicles supports disulfide bond formation, and rapid compartmentalisation enables expression of otherwise challenging or toxic proteins. This work characterises a distinct class of recombinant bacterial vesicles and establishes a practical platform for producing correctly folded, concentrated, partially purified proteins in a self-packaged form, expanding the potential applications of bacterial extracellular vesicles in biotechnology and synthetic biology.

bioengineering↗

bioRxiv2023

Near infrared-light treatment alters mitochondrial homeostasis to induce senescence in breast cancer cells

The application of near infrared (NIR)-light to living systems has been suggested as a potential method to enhance tissue repair, decrease inflammation, and possibly mitigate cancer therapy-associated side effects. In this study, we examined the effect of exposing three cell lines: breast cancer (MCF7), non-cancer breast cells (MCF10A), and lung fibroblasts (IMR-90), to 734 nm NIR-light for 20 minutes per day for six days, and measuring changes in cellular senescence. Positive senescent populations were induced using doxorubicin. Flow cytometry was used to assess relative levels of senescence together with mitochondria-related variables. Exposure to NIR-light significantly increased the level of senescence in MCF7 cells (13.5%; P<0.01), with no observable effects on MCF10A or IMR-90 cell lines. NIR-induced senescence was associated with significant changes in mitochondria homeostasis, including raised ROS level (36.0%; P<0.05) and mitochondrial membrane potential (14.9%; P<0.05), with no changes in mitochondrial Ca2+. These results suggest that NIR-light exposure can significantly arrest the proliferation of breast cancer cells via inducing senescence, while leaving non-cancerous cell lines unaffected.

cell biology↗

bioRxiv2022

Exceptional yield vesicle packaged recombinant protein production from E. coli.

We describe a novel system that exports diverse recombinant proteins in extracellular vesicles from E. coli. The vesicles not only compartmentalise toxic, insoluble and disulphide bond containing proteins in a soluble and functional form (e.g. DNaseI, nanobodies and IgG-fusions), but the continued release of the inducible vesicle packaged proteins into the media supports continuous isolation of protein from active culture within a micro-environment allowing stable long-term storage. This technology results in unprecedented yields of vesicle packaged functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.

molecular biology↗

bioRxiv2019

Chloroplasts navigate towards the pathogen interface to counteract infection by the Irish potato famine pathogen

Upon immune activation, chloroplasts switch off photosynthesis, produce anti-microbial compounds, and associate with the nucleus through tubular extensions called stromules. Although it is well-established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplasts movement in response to pathogen attack. Here, we report that chloroplasts accumulate at the pathogen interface during infection by the Irish potato famine pathogen Phytophthora infestans, associating with the specialized membrane that engulfs the pathogen haustorium. Chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at the pathogen haustoria, suggesting this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and interconnecting chloroplasts, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector mediated suppression of BAK1 mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

plant biology↗

bioRxiv2018

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. Recent models suggest that for these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signalling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using sub-diffraction-limited Airyscan confocal microscopy. In addition, single particle tracking reveals that these proteins move at different rates within the membrane. We show that the actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by affecting single particle dynamics of two proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for differentially regulating protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.\n\nOne sentence summarySize and mobility of protein nanodomains in the plant plasma-membrane are regulated by interaction with the cell wall extracellular matrix.\n\nSignificance statementThe plant plasma membrane acts as the front line for cellular perception of the environment. As such, a large number of signalling and transport proteins which perceive or transport environmental signals, developmental cues and nutrients are located within it. Recently, a number of studies have revealed that proteins located within the plasma membrane do not simply freely diffuse within its plane. Rather, proteins are localized in nanometer sized structures called nanodomains. In addition to the plasma-membrane, plant cells also have an extracellular matrix, the cell wall. Here we have shown that the cell wall has a role in regulating the dynamics and size of plasma membrane nanodomains for proteins involved in morphogenesis (PIN3) and pathogen perception (FLS2).

cell biology↗

bioRxiv2017

The Odd One Out: Aabidopsis Reticulon 20 Has A Role In Lipid Biosynthesis

The family of reticulon proteins has been shown to be involved in a variety of functions in eukaryotic cells including tubulation of the endoplasmic reticulum (ER), formation of cell plates and primary plasmodesmata. Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the reticulons sitting in the membrane in a W-topology. Here we report on a subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. We show that reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue reticulon 19 labels the whole ER. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in the roots indicating a role in lipid biosynthesis or regulation. A third homologue in this family - 3BETAHSD/D1- is localised to ER exit sites resulting in an intriguing location difference for the three proteins.

plant biology↗