Academic Staff

Journal articles

• Piña Olmos S, Díaz Torres R, Elbakrawy E, Hughes L, Mckenna J, Hill MA, Kadhim M, Ramírez Noguera P, Bolanos-Garcia VM, 'Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.'
  Biomolecules 9 (5) (2019)
  ISSN: ISSN 2218-273X
  Abstract

  Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.
  

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• Gumber HK, McKenna JF, Tolmie AF, Jalovec AM, Kartick AC, Graumann K, Bass HW, 'MLKS2 is an ARM domain and F-actin-associated KASH protein that functions in stomatal complex development and meiotic chromosome segregation'
  Nucleus 10 (1) (2019)
  ISSN: 1949-1034 eISSN: 1949-1042
  Abstract

  The linker of nucleoskeleton and cytoskeleton (LINC) complex is an essential multi-protein structure spanning the eukaryotic nuclear envelope. The LINC complex functions to maintain nuclear architecture, positioning, and mobility, along with specialized functions in meiotic prophase and chromosome segregation. Members of the LINC complex were recently identified in maize, an important scientific and agricultural grass species. Here we characterized Maize LINC KASH AtSINE-like2, MLKS2, which encodes a highly conserved SINE-group plant KASH protein with characteristic N-terminal armadillo repeats (ARM). Using a heterologous expression system, we showed that actively expressed GFP-MLKS2 is targeted to the nuclear periphery and colocalizes with F-actin and the endoplasmic reticulum, but not microtubules in the cell cortex. Expression of GFP-MLKS2, but not GFP-MLKS2ΔARM, resulted in nuclear anchoring. Genetic analysis of transposon-insertion mutations, mlks2-1 and mlks2-2, showed that the mutant phenotypes were pleiotropic, affecting root hair nuclear morphology, stomatal complex development, multiple aspects of meiosis, and pollen viability. In male meiosis, the mutants showed defects for bouquet-stage telomere clustering, nuclear repositioning, perinuclear actin accumulation, dispersal of late prophase bivalents, and meiotic chromosome segregation. These findings support a model in which the nucleus is connected to cytoskeletal F-actin through the ARM-domain, predicted alpha solenoid structure of MLKS2. Functional conservation of MLKS2 was demonstrated through genetic rescue of the misshapen nuclear phenotype of an Arabidopsis (triple-WIP) KASH mutant. This study establishes a role for the SINE-type KASH proteins in affecting the dynamic nuclear phenomena required for normal plant growth and fertility.
  

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• McKenna JF, Rolfe DJ, Webb SED, Tolmie AF, Botchway SW, Martin-Fernandez ML, Hawes C, Runions J, 'The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.'
  Proceedings of the National Academy of Sciences (2019)
  ISSN: 0027-8424 eISSN: 1091-6490
  Abstract

  Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. 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.  Single  particle  tracking  reveals that these  proteins  move  at   different  rates   within  the   membrane.  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 quantifying single particle dynamics of 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 regulation of protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.
  

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• Groves NR, McKenna JF, Evans DE, Graumann K, Meier I, 'A nuclear localisation signal targets tail-anchored proteins to the inner nuclear envelope in plants.'
  Journal of Cell Science (2019)
  ISSN: 0021-9533 eISSN: 1477-9137
  Abstract

  Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism is transport-factor mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore if this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the NE of leaf epidermal cells. Airyscan sub-diffraction limited confocal microscopy showed that it displays localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tail-anchored membrane protein, were all sufficient for NE enrichment and both monopartite or bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery.
  

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• Groves NR, McKenna JF, Evans DE, Graumann K, Meier I, 'A nuclear localization signal targets tail-anchored membrane proteins to the inner nuclear envelope in plants'
  Journal of Cell Science 132 (2019)
  ISSN: 0021-9533 eISSN: 1477-9137
  Abstract

  Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, and meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism for INM targeting is transport factor-mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore whether this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the nuclear envelope (NE) of leaf epidermal cells. Airyscan subdiffraction limited confocal microscopy showed that this protein displays a localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tail-anchored membrane protein, were all sufficient for NE enrichment, and both monopartite and bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery.
  

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• Gumber HK, McKenna JF, Estrada AL, Tolmie AF, Graumann K, Bass HW, 'Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays.'
  Journal of Cell Science (2019)
  ISSN: 0021-9533 eISSN: 1477-9137
  Abstract

  The LINC (Linker of Nucleoskeleton to Cytoskeleton) complex is an essential multi protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture, and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. Using bioinformatic, biochemical, and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with ZmSUN2 in vivo. FRAP experiments using domain-deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research. 
  

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• Engelsdorf T, Gigli-Bisceglia N, Veerabagu M, McKenna JF, Vaahtera L, Augstein F, Van der Does D, Zipfel C, Hamann T, 'The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana'
  Science Signaling 11 (536) (2018)
  ISSN: 1945-0877 eISSN: 1945-0877
  Abstract Cell walls surround all plant cells, and their composition and structure are modified in a tightly-controlled, adaptive manner to meet sometimes opposing functional requirements during growth and development. The plant cell wall integrity (CWI) maintenance mechanism controls these functional modifications as well as responses to cell wall damage (CWD). We investigated how the CWI system mediates responses to CWD in Arabidopsis thaliana. CWD induced by cell wall–degrading enzymes or an inhibitor of cellulose biosynthesis elicited similar, turgor-sensitive stress responses. Phenotypic clustering with 27 genotypes identified a core group of receptor-like kinases (RLKs) and ion channels required for activation of CWD responses. A genetic analysis showed that the RLK FEI2 and the plasma membrane–localized mechanosensitive Ca2+ channel MCA1 functioned downstream of the RLK THE1 in CWD perception. In contrast, pattern-triggered immunity (PTI) signaling components, including the receptors for plant elicitor peptides (AtPeps) PEPR1 and PEPR2, repressed responses to CWD. CWD induced the expression of PROPEP1 and PROPEP3, which encode the precursors of AtPep1 and AtPep3, and the release of PROPEP3 into the growth medium. Application of AtPep1 and AtPep3 repressed CWD-induced phytohormone accumulation in a concentration-dependent manner. These results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses controlled by the CWI mechanism. This suppression was alleviated when PTI signaling downstream of PEPR1 and PEPR2 was impaired. Defense responses controlled by the CWI maintenance mechanism might thus compensate to some extent for loss of PTI signaling elements.Website 
• D'Abrantes S, Gratton S, Reynolds P, Kriechbaumer V, McKenna J, Barnard S, Clarke D, Botchway SW, 'Super-resolution nanoscopy imaging applied to DNA double strand breaks'
  Radiation Research 189 (1) (2017) pp.19-31
  ISSN: 0033-7587 eISSN: 1938-5404
  Abstract Genomic deoxyribonucleic acid (DNA) is continuously being damaged by endogenous processes such as metabolism or by exogenous events such as radiation. The specific phosphorylation of histone H2AX on serine residue 139, described as γ-H2AX, is an excellent indicator or marker of DNA double-strand breaks (DSBs). The yield of γ-H2AX (foci) is shown to have some correlation with the dose of radiation or other DSB-causing agents. However, there is some discrepancy in the DNA DSB foci yield among imaging and other methods such as gel electrophoresis. Super-resolution imaging techniques are now becoming widely used as essential tools in biology and medicine, after a slow uptake of their development almost two decades ago. Here we compare several super-resolution techniques used to image and determine the amount and spatial distribution of γ-H2AX foci formation after X-ray irradiation: stimulated emission depletion (STED), ground-state depletion microscopy followed by individual molecule return (GSDIM), structured illumination microscopy (SIM), as well as an improved confocal, Airyscan and HyVolution 2. We show that by using these super-resolution imaging techniques with as low as 30-nm resolution, each focus may be further resolved, thus increasing the number of foci per radiation dose compared to standard microscopy. Furthermore, the DNA repair proteins 53BP1 (after low-LET irradiations) and Ku70/Ku80 (from laser microbeam irradiation) do not always yield a significantly increased number of foci when imaged by the super-resolution techniques, suggesting that γ-H2AX, 53PB1 and Ku70/80 repair proteins do not fully co-localize on the units of higher order chromatin structure.Website 
• Tolmie AF, Poulet A, McKenna JF, Sassmann S, Graumann K, Deeks M, Runions J, 'The cell wall of Arabidopsis thaliana influences actin network dynamics'
  Journal of Experimental Botany 68 (16) (2017) pp.4517-4527
  ISSN: 0022-0957 eISSN: 1460-2431
  Abstract In plant cells, molecular connections link the cell wall–plasma membrane–actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)–fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP–Lifeact-labelled network that appeared much more sparse with thick bundles that underwent ‘simple movement’, in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, ‘pairwise image subtraction’, was applied to measurement of the more rapidly remodelling actin network labelled with GFP–FABD2, while the second, ‘cumulative fluorescence intensity’, was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP–Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall–plasma membrane–actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.Website 
• Van der Does D, Boutrot F, Engelsdorf T, Rhodes J, McKenna JF, Vernhettes S, Koevoets I, Tintor N, Veerabagu M, Miedes E, Segonzac C, Roux M, Breda AS, Hardtke CS, Molina A, Rep M, Testerink C, Mouille G, Höfte H, Hamann T, Zipfel C, 'The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses'
  PLoS Genetics 13 (6) (2017)
  ISSN: 1553-7390
  Abstract Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.Website 
• McKenna JF, Tolmie AF, Runions J, 'Across the great divide: the plant cell surface continuum'
  Current Opinion in Plant Biology (2014)
  ISSN: 1369-5266
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• Engl C, Waite CJ, McKenna JF, Bennett MH, Hamann T, Buck M., 'Chp8, a diguanylate cyclase from Pseudomonas syringae pv. Tomato DC3000, suppresses the pathogen-associated molecular pattern flagellin, increases extracellular polysaccharides, and promotes plant immune evasion.'
  mBio (2014)
  ISSN: 2150-7511
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• Wormit A, Butt SM, Chairam I, McKenna JF, Nunes-Nesi A, Kjaer L, O'Donnelly K, Fernie AR, Woscholski R, Barter MCL, Hamann T, 'Osmosensitive Changes of Carbohydrate Metabolism in Response to Cellulose Biosynthesis Inhibition'
  Plant Physiology (2012)
  ISSN: 0032-0889 eISSN: 1532-2548
  Website 
• Denness L, McKenna JF, Segonzac C, Wormit A, Madhou P, Bennett M, Mansfield J, Zipfel C, Hamann T., 'Cell Wall Damage-Induced Lignin Biosynthesis Is Regulated by a Reactive Oxygen Species- and Jasmonic Acid-Dependent Process in Arabidopsis'
  Plant Physiology (2011)
  ISSN: 0032-0889 eISSN: 1532-2548
  Website 

Other publications

• Barton KA, Schattat MH, Jakob T, Hause G, Wilhelm C, Mckenna JF, Mathe C, Runions J, Van Damme D, Mathur J, 'Epidermal Pavement Cells of Arabidopsis Have Chloroplasts'
  Plant Physiology 171 (2) (2016) pp.723-726ISSN: 0032-0889 eISSN: 1532-2548
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