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Department of Biological and Medical Sciences
Faculty of Health and Life Sciences
Sinclair Annex SNA1.01
Vice Chancellor's Research Fellow in Biology
I am a plant cell biologist and protein biochemist at Oxford Brookes University with expertise in the structure and function of the plant endoplasmic reticulum (ER), membrane proteins and auxin biosynthesis using biochemical techniques as well as high-resolution live cell imaging.
Areas of Expertise:
Over the last 10 years I have developed a research pathway in auxin biosynthesis going back to my degree and PhD work at the Technical University of Munich where I studied the nitrilase pathway in maize auxin biosynthesis and maize tryptophan synthase complex.
A short-term position at Oxford Brookes just after my PhD allowed me to expand my expertise to ER and Golgi as well as acquiring skills in live cell imaging.
I further investigated membrane proteins and the targeting of tail-anchored proteins at Sheffield Hallam University. Here I pursued my scientific interests in subcellular protein localisation, bioinformatics, and mathematical modelling. My additional independent research on ER localisation and splicing in auxin biosynthesis showed for the first time ER-localisation for an auxin biosynthetic protein. Based on this work I won a fellowship from the Korean Federation of Science and Technology Societies to investigate the subcellular localisation of maize auxin biosynthesis at Dankook University in Seoul which lead to a publication that showed for the first time that both steps of the TAA/YUC pathway of auxin biosynthesis can be ER-localised. I am committed to interdisciplinary research, and an example of the successes gained from this approach is the project with Prof A Nabok (Engineering Sheffield Hallam University) using total internal reflection ellipsometry to quantify protein-membrane interactions on native plant membranes and human cell lines.
I took up a position at Oxford Brookes University in 2012 investigating the role of reticulon proteins in ER tubulation and viral trafficking in order to develop my international reputation in ER research and advanced imaging. I published the first report of plant ER reticulon protein interactors by Co-IP and FRET-FLIM. Through this I established important collaborative links with physicists at the STFC Lasers for Science Facility at the Harwell Campus.
As I have a strong interest in translational research I wrote and lead a Leverhulme research grant (“pMMO in plants”) with senior Brookes staff (Dr Deborah Pearce) and in collaboration with Prof Tom Smith from Sheffield Hallam University. Here I aim to engineer plants to convert methane into carbon dioxide. Due to my interest in linking academia and industry I am part of the Faculty Innovation Team at Oxford Brookes and Innovation Forum Oxford as an Oxford Brookes representative. I am also a member of BBSRC Networks in Industrial Biotechnology and Bioenergy: C1Net, Plant to Product Network, and High Value Chemicals from Plants Network.
My current research aims to link the structure and function of the plant endoplasmic reticulum with auxin biosynthesis.
-Leverhulme Trust “pMMO in plants” with D. Pearce and C. Hawes (Dec 2015 to Nov 2017).
-STFC Harwell access grant with C. Hawes (Jan to March 2017).
-Fellowship Korean Federation of Science and Technology Societies (March to June 2013).
-Engineering for life Feasibility Study Grant with A. Nabok, D. Smith and B. Abell 2011 “Visualising the interaction of proteins in biological membranes for diagnosis of diseases”.
-Engineering for life pump prime funding with A. Nabok and B. Abell 2010 “Visualising the interaction of proteins in biological membranes for diagnosis of diseases”.
In Pflanzen ist die Aminosäure Tryptophan zusätzlich zu ihrer Rolle in der Proteinbiosynthese Ausgangspunkt für die Synthese zahlreicher Phytohormone und Sekundärmetabolite. Im Rahmen dieser Arbeit wurden verschiedene potentielle Kandidatengene auf eine Beteiligung am Tryptophansynthase-Komplex in Mais hin untersucht. Dieser Komplex katalysiert die Bildung von Indol aus Indol-3-Glycerinphosphat und die Kondensation von Indol und Serin zu Tryptophan.Ein wichtiges Tryptophanderivat im Pflanzenreich ist das am häufigsten vorkommende Auxin Indol-3-Essigsäure (IAA). IAA ist an zahlreichen Prozessen wie Apikaldominanz, Seitenwurzelbildung, Embryonalentwicklung und Tropismen beteiligt. Als Kandidatengene der Auxinbiosynthese in Mais (Zea mays) wurden neben einer Amidase (ZmAmi1) auch zwei Nitrilasegene (ZmNit1 und ZmNit2) isoliert. Expression von ZmNIT2, Enzymparameter und Mutantenphänotyp machen eine Beteiligung dieser Nitrilase an der Auxinbiosynthese im Korn und jungen Primärwurzeln wahrscheinlich. In vitro bilden ZmNIT1 und ZmNIT2 hochmolekulare heteromere Komplexe, die neben IAN auch beta-Cyanoalanin, das als Intermediat der Cyanid-Detoxifikation diskutiert wird, hydrolysieren.
Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role in the interface between host and parasite. Glycans can modify immune responses by binding to carbohydrate-binding receptors on immune cells and are able to elicit potent antibody responses. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids, but little is known on how these complex glycans contribute to parasitism. Up to 20 different fucosyltransferase (FucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. In order to unravel the synthesis of highly complex fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes successfully localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs that are involved in core α1,3- and α1,6-fucosylation, the synthesis of antennary Lewis X, LDN-F and F-LDN-F. This knowledge can now be used to specifically knock-out the synthesis of specific N-glycan structures in the parasite to investigate the role of N-glycans during parasitism. The functionally characterized SmFucTs can also directly be applied to synthesize complex helminth N-glycan structures on recombinant proteins in order to study their contribution to immunomodulation. Furthermore, this expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.
The plant Golgi apparatus is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Golgi matrix components, such as golgins, have been identified and suggested to function as putative tethering factors to mediate the physical connections between Golgi bodies and the ER network. Golgins are proteins anchored to the Golgi membrane by the C-terminus either through transmembrane domains (TMDs) or interaction with small regulatory GTPases. The golgin N-terminus contains long coiled coil domains which consist of a number of α-helices wrapped around each other to form a structure similar to a rope being made from several strands, reaching into the cytoplasm. In animal cells golgins are also implicated in specific recognition of cargo at the Golgi. Here we investigate the plant golgin Atgolgin-84A for its subcellular localisation and potential role as a tethering factor at the ER-Golgi interface. For this, fluorescent fusions of Atgolgin-84A and an Atgolgin-84A truncation lacking the coiled-coil domains (Atgolgin-84AΔ1-557) were transiently expressed in tobacco leaf epidermal cells and imaged using high-resolution confocal microscopy. We show that Atgolgin-84A localises to a pre-cis-Golgi compartment that is also labelled by one of the COPII proteins as well as by the tether protein AtCASP. Upon overexpression of Atgolgin-84A or its deletion mutant, transport between the ER and Golgi bodies is impaired and cargo proteins are redirected to the vacuole.
The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as ‘organised smooth endoplasmic reticulum’ (OSER) consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialised tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow.
In this review we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating the evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.
The endoplasmic reticulum (ER) is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. The ER structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review, we report on the findings that contributed toward the understanding of the ER morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes.
Distributing proteins to the correct sub-cellular compartments and organelles is crucial for the proper functionality of the proteins as well as for the general function of eukaryotic cells. Cellular targeting is best understood in the case of endoplasmic reticulum (ER) proteins targeted co-translationally via the signal recognition particle (SRP)-mediated pathway but various targeting mechanisms, signals and pathways are in place depending on organism, organelle and protein types to allow for specificity and efficiency of protein targeting.
This review aims to give an overview on membrane targeting sequences taking into account the connected targeting mechanism and co-factors. It focusses on primary targeting to membranes of the endoplasmic reticulum, chloroplast, mitochondrion, peroxisome, nucleus and tonoplast.
The availability of quantification methods for sub-cellular organelle dynamic analysis has increased rapidly over the last 20 years. The application of these techniques to contiguous sub-cellular structures that exhibit dynamic re-modelling over a range of scales and orientations is challenging as quantification of ‘movement’ rarely corresponds to traditional, qualitative classifications of types of organelle movement. The plant endoplasmic reticulum represents a particular challenge for dynamic quantification as it itself is an entirely contiguous organelle that is in a constant state of flux and gross remodelling, controlled by the actinomyosin cytoskeleton.
Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway by formation of an enzyme complex. To identify such protein interactions, two independent untargeted co49 immunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B1 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were shown to co-purify. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, interaction of CYP71A13 and Arabidopsis P450 Reductase 1 (ATR1) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating allosteric interaction. Camalexin biosynthesis involves glutathionylation of an intermediary indole-3-cyanohydrin, synthesized by CYP71A12 and especially CYP71A13. It was demonstrated by FRET-FLIM and co-IP, that the glutathione transferase GSTU4, which is co-expressed with tryptophan- and camalexin-specific enzymes, was physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knock-out and reduced in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a role in a competing mechanism.
The Arabidopsis ER-α-mannosidase I (MNS3) generates an oligomannosidic N-glycan structure that is characteristically found on ER-resident glycoproteins. The enzyme itself has so far not been detected in the ER. Here, we provide evidence that in plants MNS3 exclusively resides in the Golgi apparatus at steady-state. Notably, MNS3 remains on dispersed punctate structures when subjected to different approaches that commonly result in the relocation of Golgi enzymes to the ER. Responsible for this rare behavior is a novel amino acid signal motif (LPYS) within the cytoplasmic tail of MNS3 that acts as a specific Golgi retention signal. This retention is a means to spatially separate MNS3 from ER-localized mannose trimming steps that generate the glycan signal required for flagging terminally misfolded glycoproteins for ERAD. The physiological importance of the very specific MNS3 localization is demonstrated here by means of a structurally impaired variant of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1.
The plant hormone auxin is essential for plant growth and development, controlling both organ development and overall plant architecture. Auxin homeostasis is regulated by coordination of biosynthesis, transport, conjugation, sequestration/storage, and catabolism to optimize concentration-dependent growth responses and adaptive responses to temperature, water stress, herbivory and pathogens. At present, the best defined pathway of auxin biosynthesis is the TAA/YUC route, in which the tryptophan aminotransferases TAA and TAR and YUCCA flavin-dependent monooxygenases produce the auxin indole-3-acetic acid from tryptophan. This review highlights recent advances in our knowledge of TAA/YUC-dependent auxin biosynthesis focussing on membrane localisation of auxin biosynthetic enzymes, differential regulation in root and shoot tissue, and auxin biosynthesis during abiotic stress.
The endoplasmic reticulum (ER) is a highly dynamic polygonal membrane network composed of interconnected tubules and sheets (cisternae) that forms the first compartment in the secretory pathway involved in protein translocation, folding, glycosylation, quality control, lipid synthesis, calcium signalling, and metabolon formation. Despite its central role in this plethora of biosynthetic, metabolic and physiological processes, there is little quantitative information on ER structure, morphology or dynamics. Here we describe a software package (AnalyzER) to automatically extract ER tubules and cisternae from multi-dimensional fluorescence images of plant ER. The structure, topology, protein-localisation patterns, and dynamics are automatically quantified using spatial, intensity and graph-theoretic metrics. We validate the method against manually-traced ground-truth networks, and calibrate the sub-resolution width estimates against ER profiles identified in serial block-face SEM images. We apply the approach to quantify the effects on ER morphology of drug treatments, abiotic stress and over-expression of ER tubule-shaping and cisternal-modifying proteins.
The ER is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three way junctions, and back to tubules. Plant reticulon (RTNLB) proteins tubulate the ER by dimer- and oligomerization, creating localised ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmal (PD) proteome (Fernandez-Calvino et al., 2011) and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD (Knox et al., 2015). Here we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis (Knox et al., 2015). Co-immunoprecipitation of GFP-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localised proteins as well as plasma-membrane proteins with putative membrane anchoring roles. FRET-FLIM assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modelling, may play important roles in linking the cortical ER to the plasma membrane.
Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over more than sixty years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction and a variety of biosynthetic pathways in various species, tissues and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here we show that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localised to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the endoplasmic reticulum could be necessary to bring auxin biosynthesis in closer proximity to ER-localised factors for transport, conjugation and signalling and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore it might provide a link to ethylene action and be a factor in hormonal crosstalk as all five ethylene receptors are ER-localised.
Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell-plate formation. Here we show that members of the reticulon (RTNLB) family of ER-tubulating proteins may play a role in formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant co-localisation at PD with the viral movement protein of tobacco mosaic virus while super-resolution imaging (3D-SIM) of primary PD revealed the central desmotubule to be labelled by RTNLB6. FRAP studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its centre where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation
The endoplasmic reticulum forms the first compartment in a series of organelles which comprise the secretory pathway. It takes the form of an extremely dynamic and pleomorphic membrane bounded network of tubules and cisternae which have numerous different cellular functions. In this review we discuss the nature of endoplasmic reticulum structure and dynamics, its relationship with closely associated organelles, and its possible function as a highway for the distribution and delivery of a diverse range of structures from metabolic complexes to viral particles.
Background: Certain members of the Camelidae family produce a special type of antibody with only one heavy chain. The antigen binding domains are the smallest functional fragments of these heavy-chain only antibodies and as a consequence have been termed nanobodies. Discovery of these nanobodies has allowed the development of a number of therapeutic proteins and tools.In this study a class of nanobodies fused to fluorescent proteins (chromobodies), and therefore allowing antigen-binding and visualisation by fluorescence, have been used. Such chromobodies can be expressed in living cells and used as genetically encoded immunocytochemical markers.
Results: Here a modified version of the commercially available Actin-Chromobody® as a novel tool for visualising actin dynamics in tobacco leaf cells was tested. The actin-chromobody binds to actin in a specific manner. Treatment with latrunculin B, a drug which disrupts the actin cytoskeleton through inhibition of polymerisation results in loss of fluorescence after less than 30 min but this can be rapidly restored by washing out latrunculin B and thereby allowing the actin filaments to repolymerise.
To test the effect of the actin-chromobody on actin dynamics and compare it to one of the conventional labelling probes, Lifeact, the effect of both probes on Golgi movement was studied as the motility of Golgi bodies is largely dependent on the actin cytoskeleton. With the actin-chromobody expressed in cells, Golgi body movement was slowed down but the manner of movement rather than speed was affected less than with Lifeact.
Conclusions: The actin-chromobody technique presented in this study provides a novel option for in vivo labelling ofthe actin cytoskeleton in comparison to conventionally used probes that are based on actin binding proteins.
The actin-chromobody is particularly beneficial to study actin dynamics in plant cells as it does label actin without impairing dynamic movement and polymerisation of the actin filaments.
This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.
Tail-anchored (TA) proteins function in key cellular processes in eukaryotic cells, such as vesicle trafficking, protein translocation and regulation of transcription. They anchor to internal cell membranes by a C-terminal transmembrane domain, which also serves as a targeting sequence. Targeting occurs post-translationally, via pathways that are specific to the precursor, which makes TA proteins a model system for investigating post-translational protein targeting. Bioinformatics approaches have previously been used to identify potential TA proteins in yeast and humans, yet little is known about TA proteins in plants. The identification of plant TA proteins is important for extending the post-translational model system to plastids, in addition to general proteome characterization, and the identification of functional homologues characterized in other organisms. We identified 454 loci that potentially encode TA proteins in Arabidopsis, and combined published data with new localization experiments to assign localizations to 130 proteins, including 29 associated with plastids. By analysing the tail anchor sequences of characterized proteins, we have developed a tool for predicting localization and estimate that 138 TA proteins are localized to plastids.
Golgins are large coiled-coil proteins that play a role in tethering of vesicles to Golgi membranes and in maintaining the overall structure of the Golgi apparatus. Six Arabidopsis proteins with the structural characteristics of golgins were isolated and shown to locate to Golgi stacks when fused to GFP. Two of these golgin candidates (GC1 and GC2) possess C-terminal transmembrane (TM) domains with similarity to the TM domain of human golgin-84. The C-termini of two others (GC3/GDAP1 and GC4) contain conserved GRAB and GA1 domains that are also found in yeast Rud3p and human GMAP210. GC5 shares similarity with yeast Sgm1p and human TMF and GC6 with yeast Uso1p and human p115. When fused to GFP, the C-terminal domains of AtCASP and GC1 to GC6 localized to the Golgi, showing that they contain Golgi localization motifs. The N-termini, on the other hand, label the cytosol or nucleus. Immuno-gold labelling and co-expression with the cis Golgi Q-SNARE Memb11 resulted in a more detailed picture of the sub-Golgi location of some of these putative golgins. Using two independent assays it is further demonstrated that the interaction between GC5, the TMF homologue, and the Rab6 homologues is conserved in plants.
A number of pathways starting from tryptophan have been proposed for the biosynthesis of the auxin indole-3-acetic acid (IAA), a key regulator of plant development. Many aspects of auxin metabolism have been investigated in the model species Arabidopsis thaliana that, in addition to IAA, synthesizes tryptophan-derived indole glucosinolates and camalexin as defence compounds. This results in a complex metabolic network, which makes it difficult to assign specific enzymatic functions and biosynthetic genes to IAA biosynthesis. In this review, the
Arabidopsis system is compared to the maize system, where the branch point between IAA and secondary metabolite biosynthesis occurs prior to tryptophan.
The ER is a highly dynamic network of tubules and membrane sheets. Hence imaging this organelle in its native and mobile state is of great importance. Here we describe methods of labeling the native ER using fluorescent proteins and lipid dyes as well as methods for immunolabeling on plant tissue.
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