Professor Dan Mulvihill

Article

• Palani, S., Köster, D., Hatano, T., Kamnev, A., Kanamaru, T., Brooker, H., Hernandez-Fernaud, J., Jones, A., Millar, J., Mulvihill, D. and Balasubramanian, M. (2019). Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement. Journal of Cell Biology [Online] 218:3548-3559. Available at: http://dx.doi.org/10.1083/jcb.201809089.
  Abstract View in KAR
  Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. Whereas, in muscle cells, tropomyosin is subject to calcium regulation, its regulation in non-muscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of non-muscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.
• Baker, K., Gyamfi, I., Mashanov, G., Molloy, J., Geeves, M. and Mulvihill, D. (2019). TORC2-Gad8 dependent myosin phosphorylation modulates regulation by calcium. eLife [Online] 8. Available at: https://dx.doi.org/10.7554/eLife.51150.
  Abstract View in KAR
  Cells respond to changes in their environment through signalling networks that modulate cytoskeleton and membrane organisation to coordinate cell cycle progression, polarised cell growth and multicellular development. Here, we define a novel regulatory mechanism by which the motor activity and function of the fission yeast type 1 myosin, Myo1, is modulated by TORC2 signalling dependent phosphorylation. Phosphorylation of the conserved serine at position 742 within the neck region changes both the conformation of the neck region and the interactions between Myo1 and its associating calmodulin light chains. S742 phosphorylation thereby couples calcium and TOR signalling networks in the modulation of myosin-1 dynamics to co-ordinate actin polymerisation and membrane reorganisation at sites of endocytosis and polarised cell growth in response to environmental and cell cycle cues.
• Tyuleva, S., Allen, N., White, L., Pepes, A., Shepherd, H., Saines, P., Ellaby, R., Mulvihill, D. and Hiscock, J. (2018). A Symbiotic Supramolecular Approach to the Design of Novel Amphiphiles with Antibacterial Properties Against MSRA. Chemical Communications [Online] 55. Available at: http://dx.doi.org/10.1039/C8CC08485H.
  Abstract View in KAR
  Herein, we identify Supramolecular Self-associating Amphiphiles (SSAs) as a novel class of antibacterials with activity towards Methicillin-resistant Staphylococcus aureus. Structure-activity relationships have been identified in the solid, solution and gas phases. Finally, we show that when supplied in combination, SSAs exhibit increased antibacterial efficacy against these clinically relevant microbes.
• Brooker, H., Gyamfi, I., Wieckowska, A., Brooks, N., Mulvihill, D. and Geeves, M. (2018). A novel live cell imaging system reveals a reversible hydrostatic pressure impact on cell cycle progression. Journal of Cell Science [Online] 131:jcs212167. Available at: http://dx.doi.org/10.1242/jcs.212167.
  Abstract View in KAR
  Life is dependent upon the ability of a cell to rapidly respond to changes in
  environment. Small perturbations in local environments change the ability of
  molecules to interact and hence communicate. Hydrostatic pressure provides
  a rapid non-invasive, fully-reversible method for modulating affinities between
  molecules both in vivo and in vitro. We have developed a simple fluorescence
  imaging chamber that allows intracellular protein dynamics and molecular
  events to be followed at pressures up to 200 bar in living cells. Using yeast we
  investigate the impact of hydrostatic pressure upon cell growth and cell cycle
  progression. While 100 bar has no affect upon viability, it induces a delay in
  chromosome segregation, resulting in the accumulation of long-undividedbent
  cells, consistent with disruption of the cytoskeletons. This delay is
  independent of stress signalling and induces synchronisation of cell-cycle
  progression. Equivalent affects were observed in Candida albicans, with
  pressure inducing a reversible cell-cycle delay and hyphal growth. We present
  a simple novel non-invasive fluorescence microscopy based approach to
  transiently impact molecular dynamics to visualise, dissect and study signalling pathways and cellular processes in living cells.
• Johnson, M. and Mulvihill, D. (2018). Dependency Relationships within the Fission Yeast Polarity Network. FEBS Letters [Online] 592:2543-2549. Available at: https://doi.org/10.1002/1873-3468.13180.
  Abstract View in KAR
  The ability to regulate polarised cell growth is crucial to maintain the viability
  of cells. Growth is modulated to facilitate essential cell functions and respond
  to the external environment. Failure to do so can lead to numerous
  developmental and disease states including cancer. We have undertaken a
  detailed analysis of the regulatory interplay between molecules involved in the
  regulation and maintenance of polarised cell growth within fission yeast.
  Internally controlled live cell imaging was used to examine interactions
  between 10 key polarity proteins. Analysis reveals: interplay between the
  microtubule and actin cytoskeletons; multiple novel dependency pathways
  and feedback networks between groups of proteins. This study provides
  important insights into the conserved regulation of polarised cell growth within
  eukaryotes.
• Dan Mulvihill, D., Seynnaeve, D., Vecchio, M., Fruhmann, G., Jimmy, B., Joke, V., Melody, C., Joris, W. and Vanessa, F. (2018). Recent insights on Alzheimer’s disease originating from yeast models. International Journal of Molecular Sciences [Online] 19:1947. Available at: http://dx.doi.org/10.3390/ijms19071947.
  Abstract View in KAR
  In this review article, yeast model-based research advances regarding the role of Amyloid-? (A?),
  Tau and frameshift Ubiquitin UBB+1 in Alzheimer’s disease (AD) are discussed. Despite having
  limitations with regard to intercellular and cognitive AD aspects, these models have clearly shown
  their added value as a complementary model for the study of the molecular aspects of these
  proteins, including their interplay with AD related cellular processes such as mitochondrial
  dysfunction and altered proteostasis. Moreover, these yeast models have also shown their
  importance in translational research, e.g. in compound screenings and for AD diagnostics
  development. In addition to well-established Saccharomyces cerevisiae models, new upcoming
  Schizosaccharomyces pombe, Candida glabrata and Kluyveromyces lactis yeast models for A? and Tau are
  briefly described. Finally, traditional and more innovative research methodologies, e.g. for studying
  protein oligomerization/aggregation, are highlighted.
• Johnson, C., Brooker, H., Gyamfi, I., O’Brien, J., Ashley, B., Brazier, J., Dean, A., Embling, J., Grimsey, E., Tomlinson, A., Wilson, E., Geeves, M. and Mulvihill, D. (2017). Temperature sensitive point mutations in fission yeast tropomyosin have long range effects on the stability and function of the actin- tropomyosin copolymer. Biochemical and Biophysical Research Communications [Online]. Available at: https://doi.org/10.1016/j.bbrc.2017.10.109.
  Abstract View in KAR
  The actin cytoskeleton is modulated by regulatory actin-binding proteins which fine- tune the dynamic properties of the actin polymer to regulate function. One such actin-binding protein is tropomyosin (Tpm), a highly-conserved alpha-helical dimer which stabilises actin and regulates interactions with other proteins. Temperature sensitive mutants of Tpm are invaluable tools in the study of actin filament dependent processes, critical to the viability of a cell. Here we investigated the molecular basis of the temperature sensitivity of fission yeast Tpm mutants which fail to undergo cytokinesis at the restrictive temperatures. Comparison of Contractile Actomyosin Ring (CAR) constriction as well as cell shape and size revealed the cdc8.110 or cdc8.27 mutant alleles displayed significant differences in their temperature sensitivity and impact upon actin dependent functions during the cell cycle. In vitro analysis revealed the mutant proteins displayed a different reduction in thermostability, and unexpectedly yield two discrete unfolding domains when acetylated on their amino-termini. Our findings demonstrate how subtle changes in structure (point mutations or acetylation) alter the stability not simply of discrete regions of this conserved cytoskeletal protein but of the whole molecule. This differentially impacts the stability and cellular organisation of this essential cytoskeletal protein.
• Mulvihill, D. (2017). Live Cell Imaging in Fission Yeast. Cold Spring Harbor Protocols [Online] 2017. Available at: http://dx.doi.org/10.1101/pdb.top090621.
  Abstract View in KAR
  Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast.
• Eastwood, T., Baker, K., Brooker, H., Frank, S. and Mulvihill, D. (2017). An enhanced recombinant amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affection alpha-synuclein oligomerisation. FEBS letters [Online] 591:833-841. Available at: http://dx.doi.org/10.1002/1873-3468.12597.
  Abstract View in KAR
  Amino terminal acetylation is a ubiquitous protein modification affecting the majority of eukaryote proteins to regulate stability and function. We describe an optimised recombinant expression system for rapid production of aminoterminal-acetylated proteins within bacteria. We go on to describe the
  system’s use in a fluorescence based in vivo assay for use in the highthroughput screen to identify drugs that impact amino-terminal acetylation
  dependent oligomerisation. These new tools and protocols will allow researchers to enhance routine recombinant protein production and identify
  new molecules for use in research and clinical applications.
• Brooker, H., Geeves, M. and Mulvihill, D. (2016). Analysis of biophysical and functional consequences of Tropomyosin - fluorescent protein fusions. FEBS letters [Online]:3111-3121. Available at: http://onlinelibrary.wiley.com/doi/10.1002/1873-3468.12346/full.
  Abstract View in KAR
  The dynamic nature of actin polymers is modulated to facilitate a diverse
  range of cellular processes. These dynamic properties are modulated by
  different isoforms of Tropomyosin, which are recruited to distinct subpopulations
  of actin polymers to differentially modulate their functional
  properties. This makes them an attractive target for labelling discrete actin
  populations. We have assessed the effect of different fluorescent labelling
  strategies for this protein. Although tropomyosin fluorescent fusions
  decorate actin in vivo, they are either non-functional or perturb regulation
  of actin nucleation and cell cycle timings. Thus conclusions and
  physiological relevance should be carefully evaluated when using
  tropomyosin fusions.
• Baker, K., Kirkham, S., Halova, L., Atkin, J., Franz-Wachtel, M., Cobley, D., Krug, K., Ma?ek, B., Mulvihill, D. and Petersen, J. (2016). TOR complex 2 localises to the cytokinetic actomyosin ring and controls the fidelity of cytokinesis. Journal of Cell Science [Online] 129. Available at: http://doi.org/10.1242/jcs.190124.
  Abstract View in KAR
  The timing of cell division is controlled by the coupled regulation of growth and division. The TOR signalling network synchronises these processes with the environmental setting. Here we describe a novel interaction of the fission yeast TOR Complex 2 (TORC2) with the Cytokinetic Actomyosin Ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that a myosin II, Myp2, and myosin V, Myo51, play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2 dependent phosphorylation of Acp1 (Actin Capping Protein, a known regulator of cytokinesis) controls CAR stability and the modulation of CAPZA/BAcp1/2 heterodimer formation and is essential for survival upon stress. Thus TORC2 localisation to the CAR and TORC2 dependent CAPZAAcp1 phosphorylation contributes to timely control and fidelity of cytokinesis and cell division.
• Manstein, D. and Mulvihill, D. (2016). Tropomyosin-mediated Regulation of Cytoplasmic Myosins. Traffic [Online] 17:872-877. Available at: http://www.dx.doi.org/10.1111/tra.12399.
  Abstract View in KAR
  The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic ?- or ?-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures.
• Gunning, P., Hardeman, E., Lappalainen, P. and Mulvihill, D. (2015). Tropomyosin - master regulator of actin filament function in the cytoskeleton. Journal of Cell Science [Online] 128:2965-2974. Available at: http://doi.org/10.1242/jcs.172502.
  Abstract View in KAR
  Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this ‘master regulator’ role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.
• Johnson, M., East, D. and Mulvihill, D. (2014). Formins Determine the Functional Properties of Actin Filaments in Yeast. Current Biology [Online] 24:1525-1530. Available at: http://dx.doi.org/10.1016/j.cub.2014.05.034.
  Abstract View in KAR
  The actin cytoskeleton executes a broad range of essential functions within a living cell. The dynamic nature of the actin polymer is modulated to facilitate specific cellular processes at discrete locations by actin-binding proteins (ABPs), including the formins and tropomyosins (Tms). Formins nucleate actin polymers, while Tms are conserved dimeric proteins that form polymers along the length of actin filaments. Cells possess different Tm isoforms, each capable of differentially regulating the dynamic and func- tional properties of the actin polymer. However, the mecha- nism by which a particular Tm localizes to a specific actin polymer is unknown. Here we show that specific formin family members dictate which Tm isoform will associate with a particular actin filament to modulate its dynamic and functional properties at specific cellular locations. Exchanging the localization of the fission yeast formins For3 and Cdc12 results in an exchange in localizations of Tm forms on actin polymers. This nucleator-driven switch in filament composition is reflected in a switch in actin dynamics, together with a corresponding change in the filament’s ability to regulate ABPs and myosin motor activity. These data establish a role for formins in dictating which specific Tm variant will associate with a growing actin filament and therefore specify the functional capacity of the actin filaments that they create.
• Lawrence, A., Frank, S., Newnham, S., Lee, M., Brown, I., Xue, W., Rowe, M., Mulvihill, D., Prentice, M., Howard, M. and Warren, M. (2014). Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor. ACS Synthetic Biology [Online] 3:454-465. Available at: http://dx.doi.org/10.1021/sb4001118.
  Abstract View in KAR
  Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.
• East, D., Mulvihill, D., Todd, M. and Bruce, I. (2011). QD-Antibody conjugates via carbodiimide-mediated coupling; a detailed study of the variables involved and a possible new mechanism for the coupling reaction under basic aqueous conditions. Langmuir [Online] 27:13888-13896. Available at: http://dx.doi.org/10.1021/la203273p.
  Abstract View in KAR
  A detailed study into the optimization of carbodiimide-mediated coupling of antibodies (Ab) and quantum dots (QD) for use in cellular imaging has been undertaken. This involved the grafting of commercially available carboxyl-modified QDs (Evident Technologies "Lake Placid Blue" Evitag and eBioscience's eflour nanocrystals) with anti-Cdc8 Abs to produce conjugates with specific affinity for fission yeast tropomyosin Cdc8 protein. The water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to activate the QDs prior to their incubation with antibody, and a range of QD-carboxyl/EDC/Ab mole ratios were used in the experiments in attempts to optimize fluorescence and bioaffinity of the conjugate products (EDC to QD-carboxyl-600 nmol/15pmol to 0.12 nmol/15 pmol and QD to Ab 120 pmol/24 pmol to 120 pmol/1.2 pmol). It was observed that a specific "optimum" ratio of the three reactants was required to produce the most fluorescent and biologically active product and that it was generated at alkaline pH 10.8. Increasing the ratio of Ab to QD produced conjugate which was less fluorescent while reducing the ratio of EDC to QD in the activation step led to increased fluorescence of product. Conjugates were tested for their possession of antibody by measurement of their absorption at OD(280 nm) and for their fluorescence by assay ?(max(em)) at 495 nm. A quantitative assay of the bioactivity of the conjugates was developed whereby a standardized amount of Cdc8 antigen was spotted onto nylon membranes and reacted with products from conjugation reactions in a sandwich-type colormetric assay The "best" conjugate was used in intracellular imaging of yeast Cdc8 protein and produced brighter, higher definition images of fixed yeast cell actin structure than a fluorescein-Ab conjugate routinely produced in our laboratory. The QD-Ab conjugate was also significantly more resistant to photobleaching than the fluorescein-Ab conjugate. Results from other experiments involving EDC, the water-soluble carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphonate (CMC), and EDC.HCl have suggested a new reaction mechanism for EDC coupling under basic aqueous conditions. In summary, a robust understanding of commercial QD-COOH surface chemistry and the variables involved in the materials' efficient conjugation with a bioligand using carbidiimide has been obtained along with an optimized approach for Ab-QD conjugate production. A novel assay has been developed for bioassay of QD-Ab conjugates and a new mechanism for EDC coupling under basic aqueous conditions is proposed.
• East, D., Sousa, D., Martin, S., Edwards, T., Lehman, W. and Mulvihill, D. (2011). Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind cooperatively to actin and regulate myosin. Biochemical Journal [Online] 438:265-273. Available at: http://dx.doi.org/10.1042/BJ20101316.
  Abstract View in KAR
  Tropomyosin (Tm) is an evolutionarily conserved ?-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilising actin-filaments and regulate myosin function. The fission yeast, Schizosaccharomyces pombe, possesses a single essential Tm, Cdc8, which can be acetylated on its amino terminal methionine to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with amino terminal substitutions to explore how stability of the Cdc8-polymer overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown the stability of the amino terminal of the Cdc8 ?-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased amino-terminal stability, dimers of which are capable of forming Tm-polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild type, and was unable to regulate actomyosin interactions. The data presented here are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin-filament, consistent with a cooperative model for Tm binding to actin.
• East, D. and Mulvihill, D. (2011). Regulation and function of the fission yeast myosins. Journal of Cell Science [Online] 124:1383-1390. Available at: http://dx.doi.org/10.1242/jcs.078527.
  Abstract View in KAR
  It is now quarter of a century since the actin cytoskeleton was first described in the fission yeast, Schizosaccharomyces pombe. Since then, a substantial body of research has been undertaken on this tractable model organism, extending our knowledge of the organisation and function of the actomyosin cytoskeleton in fission yeast and eukaryotes in general. Yeast represents one of the simplest eukaryotic model systems that has been characterised to date, and its genome encodes genes for homologues of the majority of actin regulators and actin-binding proteins found in metazoan cells. The ease with which diverse methodologies can be used, together with the small number of myosins, makes fission yeast an attractive model system for actomyosin research and provides the opportunity to fully understand the biochemical and functional characteristics of all myosins within a single cell type. In this Commentary, we examine the differences between the five S. pombe myosins, and focus on how these reflect the diversity of their functions. We go on to examine the role that the actin cytoskeleton plays in regulating the myosin motor activity and function, and finally explore how research in this simple unicellular organism is providing insights into the substantial impacts these motors can have on development and viability in multicellular higher-order eukaryotes.
• Johnson, M., Couton, A., Geeves, M. and Mulvihill, D. (2010). Targeted amino-terminal acetylation of recombinant proteins in E. coli. PLoS ONE [Online] 5:e15801. Available at: http://dx.plos.org/10.1371/journal.pone.0015801.
  Abstract View in KAR
  One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co- expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.
• Coulton, A., East, D., Galinska-Rakoczy, A., Lehman, W. and Mulvihill, D. (2010). The recruitment of acetylated and unacetylated tropomyosin to distinct actin polymers permits the discrete regulation of specific myosins in fission yeast. Journal of Cell Science [Online] 123:3235-3243. Available at: http://dx.doi.org/10.1242/jcs.069971.
  Abstract View in KAR
  Tropomyosin (Tm) is a conserved dimeric coiled-coil protein, which forms polymers that curl around actin filaments in order to regulate actomyosin function. Acetylation of the Tm N-terminal methionine strengthens end-to-end bonds, which enhances actin binding as well as the ability of Tm to regulate myosin motor activity in both muscle and non-muscle cells. In this study we explore the function of each Tm form within fission yeast cells. Electron microscopy and live cell imaging revealed that acetylated and unacetylated Tm associate with distinct actin structures within the cell, and that each form has a profound effect upon the shape and integrity of the polymeric actin filament. We show that, whereas Tm acetylation is required to regulate the in vivo motility of class II myosins, acetylated Tm had no effect on the motility of class I and V myosins. These findings illustrate a novel Tm-acetylation-state-dependent mechanism for regulating specific actomyosin cytoskeletal interactions.
• Doyle, A., Martín-García, R., Coulton, A., Bagley, S. and Mulvihill, D. (2009). Fission yeast Myo51 is a meiotic spindle pole body component with discrete roles during cell fusion and spore formation. Journal of Cell Science [Online] 122:4330-4340. Available at: http://dx.doi.org/10.1242/jcs.055202.
  Abstract View in KAR
  Class V myosins are dimeric actin-associated motor proteins that deliver cellular cargoes to discrete cellular locations. Fission yeast possess two class V myosins, Myo51 and Myo52. Although Myo52 has been shown to have roles in vacuole distribution, cytokinesis and cell growth, Myo51 has no as yet discernible function in the vegetative life cycle. Here, we uncover distinct functions for this motor protein during mating and meiosis. Not only does Myo51 transiently localise to a foci at the site of cell fusion upon conjugation, but overexpression of the Myo51 globular tail also leads to disruption of cell fusion. Upon completion of meiotic prophase Myo51 localises to the outside of the spindle pole bodies (SPBs), where it remains until completion of meiosis II. Association of Myo51 with SPBs is not dependent upon actin or the septation initiation network (SIN); however, it is dependent on a stable microtubule cytoskeleton and the presence of the Cdc2-CyclinB complex. We observe a rapid and dynamic exchange of Myo51 at the SPB during meiosis I but not meiosis II. Finally, we show that Myo51 has an important role in regulating spore formation upon completion of meiosis.
• Attanapola, S., Alexander, C. and Mulvihill, D. (2009). Ste20-kinase-dependent TEDS-site phosphorylation modulates the dynamic localisation and endocytic function of the fission yeast class I myosin, Myo1. Journal of Cell Science [Online] 122:3856-3861. Available at: http://dx.doi.org/10.1242/jcs.053959.
  Abstract View in KAR
  Type I myosins are monomeric motors involved in a range of motile and sensory activities in different cell types. In simple unicellular eukaryotes, motor activity of class I myosins is regulated by phosphorylation of a conserved 'TEDS site' residue within the motor domain. The mechanism by which this phosphorylation event affects the cellular function of each myosin I remains unclear. The fission yeast myosin I, Myo1, activates Arp2/3-dependent polymerisation of cortical actin patches and also regulates endocytosis. Using mutants and Myo1-specific antibodies, we show that the phosphorylation of the Myo1 TEDS site (serine 361) plays a crucial role in regulating this protein's dynamic localisation and cellular function. We conclude that although phosphorylation of serine 361 does not affect the ability of this motor protein to promote actin polymerisation, it is required for Myo1 to recruit to sites of endocytosis and function during this process.
• Martín-García, R. and Mulvihill, D. (2009). Myosin V spatially regulates microtubule dynamics and promotes the ubiquitin-dependent degradation of the fission yeast CLIP-170 homologue, Tip1. Journal of Cell Science [Online] 122:3862-3872. Available at: http://dx.doi.org/10.1242/jcs.054460.
  Abstract View in KAR
  Coordination between microtubule and actin cytoskeletons plays a crucial role during the establishment of cell polarity. In fission yeast, the microtubule cytoskeleton regulates the distribution of actin assembly at the new growing end during the monopolar-to-bipolar growth transition. Here, we describe a novel mechanism in which a myosin V modulates the spatial coordination of proteolysis and microtubule dynamics. In cells lacking a functional copy of the class V myosin, Myo52, the plus ends of microtubules fail to undergo catastrophe on contacting the cell end and continue to grow, curling around the end of the cell. We show that this actin-associated motor regulates the efficient ubiquitin-dependent proteolysis of the Schizosaccharomyces pombe CLIP-170 homologue, Tip1. Myo52 facilitates microtubule catastrophe by enhancing Tip1 removal from the plus end of growing microtubules at the cell tips. There, Myo52 and the ubiquitin receptor, Dph1, work in concert to target Tip1 for degradation.
• Grallert, A., Martín-García, R., Bagley, S. and Mulvihill, D. (2007). In vivo movement of the type V myosin Myo52 requires dimerisation but is independent of the neck domain. Journal of Cell Science [Online] 120:4093-4098. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18003699.
  Abstract View in KAR
  Intracellular movement is a fundamental property of all cell types. Many organelles and molecules are actively transported throughout the cytoplasm by molecular motors, such as the dimeric type V myosins. These possess a long neck, which contains an IQ motif, that allow it to make 36-nm steps along the actin polymer. Live cell imaging of the fission yeast type V myosin Myo52 reveals that the protein moves rapidly throughout the cytoplasm. Here, we describe analysis of this movement and have established that Myo52 moves long distances on actin filaments in an ATP-dependent manner at approximately 0.5 mum/second. Myo51 and the microtubule cytoskeleton have no discernable role in modulating Myo52 movements, whereas rigour mutations in Myo52 abrogated its movement. We go on to show that, although dimerisation is required for Myo52 movement, deleting its neck has no discernable affect on Myo52 function or velocity in vivo.
• Coulton, A., Skoumpla, K., Lehman, W., Geeves, M. and Mulvihill, D. (2007). Acetylation regulates tropomyosin function in the fission yeast Schizosaccharomyces pombe. Journal of Cell Science [Online] 120:1635-1645. Available at: http://dx.doi.org/10.1242/jcs.001115.
  Abstract View in KAR
  Tropomyosin is an evolutionarily conserved alpha-helical coiled-coil protein that promotes and maintains actin filaments. In yeast, Tropomyosin-stabilised filaments are used by molecular motors to transport cargoes or to generate motile forces by altering the dynamics of filament growth and shrinkage. The Schizosaccharomyces pombe tropomyosin Cdc8 localises to the cytokinetic actomyosin ring during mitosis and is absolutely required for its formation and function. We show that Cdc8 associates with actin filaments throughout the cell cycle and is subjected to post-translational modification that does not vary with cell cycle progression. At any given point in the cell cycle 80% of Cdc8 molecules are acetylated, which significantly enhances their affinity for actin. Reconstructions of electron microscopic images of actin-Cdc8 filaments establish that the majority of Cdc8 strands sit in the 'closed' position on actin filaments, suggesting a role in the regulation of myosin binding. We show that Cdc8 regulates the equilibrium binding of myosin to actin without affecting the rate of myosin binding. Unacetylated Cdc8 isoforms bind actin, but have a reduced ability to regulate myosin binding to actin. We conclude that although acetylation of Cdc8 is not essential, it provides a regulatory mechanism for modulating actin filament integrity and myosin function.
• Mulvihill, D., Edwards, S. and Hyams, J. (2006). A critical role for the type V myosin, Myo52, in septum deposition and cell fission during cytokinesis in Schizosaccharomyces pombe. Cell Motility and the Cytoskeleton [Online] 63:149-161. Available at: http://dx.doi.org/10.1002/cm.20113.
  Abstract View in KAR
  Cytokinesis in fission yeast involves the coordination of septum deposition with the contraction of a cytokinetic actomyosin ring. We have examined the role of the type V myosin Myo52 in the coupling of these two events by the construction of a series of deletion mutants of the Myo52 tail and a further mutant within the ATP binding domain of the head. Each mutant protein was ectopically expressed in fission yeast cells. Each truncation was assayed for the ability to localize to the cell poles and septum (the normal cellular locations of Myo52) and to rescue the morphology defects and temperature sensitivity of a myo52Delta strain. A region within the Myo52 tail (amino acids 1320-1503), with a high degree of similarity to the vesicle-binding domain of the budding yeast type V myosin Myo2p, was essential for Myo52's role in the maintenance of growth polarity and cell division. A separate region (amino acids 1180-1320) was required for Myo52 foci to move throughout the cytoplasm; however, constructs lacking this region, but which retained the ability to dimerize still associated with actin at sites of cell growth. Not all of the Myo52 truncations which localized rescued the morphological defects of myo52Delta, demonstrating that loss of function was not simply brought about by an inability of mutant proteins to target the correct cellular location. By contrast, Myo52 motor activity was required for both localization and cellular function. myo52Delta cells were unable to efficiently localize the beta-1,3-glucan synthase, Bgs1, either at the cell poles or at the division septum, regions of cell wall deposition. Bgs1 and Myo52 localized to vesicle-like dots at the poles in interphase and these moved together to the septum at division. These data have led to the formulation of a model in which Myo52 is responsible for the delivery of Bgs1 and associated molecules to polar cell growth regions during interphase. On the commencement of septum formation, Myo52 transports Bgs1 to the cell equator, thus ensuring the accurate deposition of beta-1,3-glucan at the leading edge of the primary septum.
• Mulvihill, D. and Hyams, J. (2003). Role of the two type II myosins, Myo2 and Myp2, in cytokinetic actomyosin ring formation and function in fission yeast. Cell Motility and the Cytoskeleton [Online] 54:208-216. Available at: http://dx.doi.org/10.1002/cm.10093.
  Abstract View in KAR
  The formation and contraction of a cytokinetic actomyosin ring (CAR) is essential for the execution of cytokinesis in fission yeast. Unlike most organisms in which its composition has been investigated, the fission yeast CAR contains two type II myosins encoded by the genes myo2(+) and myp2(+). myo2(+) is an essential gene whilst myp2(+) is dispensable under normal growth conditions. Myo2 is hence the major contractile protein of the CAR whilst Myp2 plays a more subtle and, as yet, incompletely documented role. Using a fission yeast strain in which the chromosomal copy of the myo2(+) gene is fused to the gene encoding green fluorescent protein (GFP), we analysed CAR formation and function in the presence and absence of Myp2. No change in the rate of CAR contraction was observed when Myp2 was absent although the CAR persisted longer in the contracted state and was occasionally observed to split into two discrete rings. This was also observed in myp2Delta cells following actin depolymerisation with latrunculin. CAR contraction in the absence of Myp2 was completely abolished in the presence of elevated levels of chloride ions. Thus, Myp2 appears to contribute to the stability of the CAR, in particular at a late stage of CAR contraction, and to be a component of the signalling pathway that regulates cytokinesis in response to elevated levels of chloride. To determine whether the presence of two type II myosins was a feature of cytokinesis in other fungi that divide by septation, we searched the genomes of two filamentous fungi, Aspergillus fumigatus and Neurospora crassa, for myosin genes. As in fission yeast, both A. fumigatus and N. crassa contained myosins of classes I, II, and V. Unlike fission yeast, both contained a single type II myosin gene that, on the basis of its tail structure, was more reminiscent of Myp2 than Myo2. The significance of these observations to our understanding of septum to formation and cleavage is discussed.
• Mulvihill, D. and Hyams, J. (2003). Myosin-cell wall interactions during cytokinesis in fission yeast: a framework for understanding plant cytokinesis?. Cell Biology International [Online] 27:239-40. Available at: http://dx.doi.org/10.1016/S1065-6995(02)00311-6.
  View in KAR
• Mulvihill, D. and Hyams, J. (2002). Cytokinetic actomyosin ring formation and septation in fission yeast are dependent on the full recruitment of the polo-like kinase Plo1 to the spindle pole body and a functional spindle assembly checkpoint. Journal of Cell Science [Online] 115:3575-3586. Available at: http://jcs.biologists.org/cgi/content/abstract/115/18/3575.
  Abstract View in KAR
  In dividing cells, the assembly and contraction of the cytokinetic actomyosin ring (CAR) is precisely coordinated with spindle formation and chromosome segregation. Despite having a cell wall, the fission yeast Schizosaccharomyces pombe forms a CAR reminiscent of the structure responsible for the cleavage of cells with flexible boundaries. We used the myo2-gc fission yeast strain in which the chromosomal copy of the type II myosin gene, myo2(+), is fused to the gene encoding green fluorescent protein (GFP) to investigate the dynamics of Myo2 recruitment to the cytokinetic actomyosin ring in living cells. Analysis of CAR formation in relation to spindle pole body (SPB) and centromere separation enabled us to pinpoint the timing of Myo2 recruitment into a stable CAR structure to the onset of anaphase A. Depolymerisation of actin with latrunculin B did not affect the timing of Myo2 accumulation at the cell equator (although Myo2 no longer formed a ring), whereas depolymerisation of microtubules with either thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC) resulted in a delay of up to 90 minutes in CAR formation. Microtubule depolymerisation also delayed the localisation of other CAR components such as actin and Mid1/Dmf1. The delay of cytokinesis in response to loss of microtubule integrity was abolished in cells lacking the spindle assembly checkpoint protein Mad2 or containing non-functional Cdc16, a component of the fission yeast septation initiation network (SIN). The delay was also abolished in cells lacking Zfs1, a component of the previously described S. pombe cytokinesis checkpoint. Recruitment of the polo-related kinase, Plo1, a key regulator of CAR formation, to the SPBs was substantially reduced in TBZ in a Mad2-dependent manner. Loading of Cdc7, a component of the SIN and downstream of Plo1 in the cytokinesis pathway, onto the the SPBs was also delayed in TBZ to the same extent as CAR formation. We conclude that CAR formation is subject to regulation by the spindle assembly checkpoint via the loading of Plo1 onto the SPBs and the consequent activation of the SIN.
• Win, T., Mulvihill, D. and Hyams, J. (2002). Take five: a myosin class act in fission yeast. Cell Motility and the Cytoskeleton [Online] 51:53-6. Available at: http://dx.doi.org/10.1002/cm.10021.
  View in KAR
• Mulvihill, D., Barretto, C. and Hyams, J. (2001). Localization of fission yeast type II myosin, Myo2, to the cytokinetic actin ring is regulated by phosphorylation of a C-terminal coiled-coil domain and requires a functional septation initiation network. Molecular Biology of the Cell [Online] 12:4044-4053. Available at: http://dx.doi.org.10.1091/mbc.12.12.4044.
  Abstract View in KAR
  Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR). The localization domain contained two predicted phosphorylation sites. Mutation of serine 1518 to alanine (S(1518)A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S(1518)E) localized to the CAR. GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25 degrees C but not at 36 degrees C. GFP-Myo2S(1518)E rings persisted at 36 degrees C in cdc7-24 but not in another SIN kinase mutant, sid2-250. To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy of myo2(+) was fused to GFP (strain myo2-gc). Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature. In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36 degrees C. We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction. Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail. Other kinases and/or phosphatases may control ring contraction.
• Mulvihill, D., Pollard, P., Win, T. and Hyams, J. (2001). Myosin V-mediated vacuole distribution and fusion in fission yeast. Current Biology [Online] 11:1124-1127. Available at: http://dx.doi.org/10.1016/S0960-9822(01)00322-0.
  Abstract View in KAR
  The class V myosins are actin-based motors that move a variety of cellular cargoes [1]. In budding yeast, their activity includes the relocation of a portion of the vacuole from the mother cell to the bud [2, 3]. Fission yeast cells contain numerous (approximately 80) small vacuoles. When S. pombe cells are placed in water, vacuoles fuse in response to osmotic stress [4]. Fission yeast possess two type V myosin genes, myo51(+) and myo52(+) [5]. In a myo51Delta strain, vacuoles were distributed throughout the cell, and mean vacuole diameter was identical to that seen in wild-type cells. When myo51Delta and wild-type cells were placed in water, vacuoles enlarged by fusion. In myo52Delta cells, by contrast, vacuoles were smaller and mostly clustered around the nucleus, and fusion in water was largely inhibited. When cells containing GFP-Myo52 were placed in water, Myo52 was seen to redistribute from the cell poles to the surface of the fusing vacuoles. Vacuole fusion in fission yeast was inhibited by the microtubule drug thiabendazole (TBZ) but not by the actin inhibitor latrunculin B. This is the first demonstration of the involvement of a type V myosin, possibly via an interaction with microtubules, in homotypic membrane fusion.
• Tanaka, K., Petersen, J., MacIver, F., Mulvihill, D., Glover, D. and Hagan, I. (2001). The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe. Embo Journal [Online] 20:1259-70. Available at: http://dx.doi.org/10.1093/emboj/20.6.1259.
  Abstract View in KAR
  Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatment abolished this activity. Mitotic Plo1 activation had a requirement for prior activation of M-phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment. A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1 Delta cells to septate and the prolific septation following plo1(+) overexpression. Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1-associated kinase activity. Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1-associated kinase activity. plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36 degrees C. Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum. The ability of plo1(+) overexpression to induce septation was severely compromised by SIN inactivation. We propose that Plo1 acts before the SIN to control septation.
• Mulvihill, D. and Hyams, J. (2001). Shedding a little light on light chains. Nature Cell Biology [Online] 3:E10-2. Available at: http://dx.doi.org/10.1038/35050634.
  Abstract View in KAR
  Myosin II regulatory light chains have an important role in the organization and function of the contractile machinery at cytokinesis. Two recent reports provide new insights into these important proteins.
• Win, T., Gachet, Y., Mulvihill, D., May, K. and Hyams, J. (2001). Two type V myosins with non-overlapping functions in the fission yeast Schizosaccharomyces pombe: Myo52 is concerned with growth polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring. Journal of Cell Science [Online] 114:69-79. Available at: http://jcs.biologists.org.chain.kent.ac.uk/cgi/reprint/114/1/69.
  Abstract View in KAR
  The fission yeast genome project has identified five myosin genes: one type I myosin, myo1(+), two type II myosins, myo2(+) and myp2(+), and two type V myosins, myo51(+) and myo52(+). Cells deleted for myo51(+) show normal morphology and growth rates whereas deletion of myo52(+) results in a partial loss of cell polarity, slow growth and cytokinetic defects. Combining both deletions in a single strain is phenotypically non-additive, myo52(delta) being epistatic to myo51(delta). Overproduction of Myo51 gives rise to elongated cells which fail to form functional septa whereas overproduction of Myo52 results in branched cells with aberrant septa that fail to cleave. Myo52 localises to the poles of growing cells but during cell division it relocalises to the cell equator as a bar that is bisected by the cytokinetic septum. Myo51 shows no obvious localisation during interphase but at cytokinesis it is associated with the contractile cytokinetic actin ring (CAR). Both myosins are dependent upon an intact actin cytoskeleton for localisation. Myo52 partially colocalises with the (alpha)-glucan synthase Mok1 at the cell tips and to a lesser extent at the septum. Mok1 is delocalised and upregulated in myo52(delta) and myo52(delta) cell walls are resistant to digestion by the cell wall degrading enzyme zymolyase. Thus myo52(+) appears to be involved in the local delivery or positioning of vesicles containing cell wall precursors at the cell tips and has a role in the maturation or cleavage of the septum. Myo51 has a non-essential role in cytokinesis as a component of the cytokinetic actin ring.
• Mulvihill, D., Win, T., Pack, T. and Hyams, J. (2000). Cytokinesis in fission yeast: a myosin pas de deux. Microscopy Research and Technique [Online] 49:152-60. Available at: http://dx.doi.org/10.1002/(SICI)1097-0029(20000415)49:2<152::AID-JEMT7>3.0.CO;2-7.
  Abstract View in KAR
  Cytokinesis in the fission yeast, Schizosaccharomyces pombe consists of two distinct but overlapping events: the assembly and constriction of a cytokinetic actomyosin ring (CAR) and the formation of a cross wall or septum. These two processes must be spatially and temporally coordinated both with each other and with other cell cycle events, most notably spindle formation and anaphase chromosome segregation. In fission yeast, the CAR contains two unusual type II myosins, Myo2, encoded by the gene myo2(+), and Myp2, encoded by myp2(+). The relationship of these two proteins to each other and their relative contribution to CAR assembly and contraction is largely unknown. Here we review what is known about the role of each myosin in cytokinesis and present some new information concerning their regulation and possible physical interaction.
• Mulvihill, D., Petersen, J., Ohkura, H., Glover, D. and Hagan, I. (1999). Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe. Molecular Biology of the Cell [Online] 10:2771-85. Available at: http://dx.doi.org/10.1091/mbc.10.8.2771.
  Abstract View in KAR
  Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis (Ohkura et al., 1995; Bahler et al., 1998). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 (Hudson et al., 1990). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression of plo1+ activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation.

Book section

• Mulvihill, D. (2017). Live Cell Imaging in Fission Yeast. In: Hagan, I. M., Carr, A. M., Grallert, A. and Nurse, P. eds. Fission Yeast: A Laboratory Manual. Long Island, New York, USA: Cold Spring Harbour Laboratory Press. Available at: http://cshprotocols.cshlp.org/content/early/2017/07/21/pdb.top090621.abstract?sid=45bc9a5c-f4f5-4989-a1e9-d64bcf4a8c56.
  Abstract View in KAR
  Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluo- rescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast.
• O’Brien, J., Hoque, S., Mulvihill, D. and Sirlantzis, K. (2017). Automated Cell Segmentation of Fission Yeast Phase Images - Segmenting Cells from Light Microscopy Images. In: Silveira, M., Fred, A., Gamboa, H. and Vaz, M. eds. Proceedings of the 10th International Joint Conference on Biomedical Engineering Systems and Technologies. Scitepress, pp. 92-99. Available at: http://dx.doi.org/10.5220/0006149100920099.
  Abstract View in KAR
  Robust image analysis is an important aspect of all cell biology studies. The geometrics of cells are critical for developing an understanding of biological processes. Time constraints placed on researchers lead to a narrower focus on what data are collected and recorded from an experiment, resulting in a loss of data. Currently, preprocessing of microscope images is followed by the utilisation and parameterisation of inbuilt functions of various softwares to obtain information. Using the fission yeast, Schizosaccharomyes pombe, we propose a novel, fully automated, segmentation software for cells with a significantly lower rate of segmentation errors than PombeX with the same dataset.
• Mulvihill, D. (2014). Using Fluorescence to Study Actomyosin in Yeasts. In: Toseland, C. P. and Fili, N. eds. Fluorescent Methods for Molecular Motors. Springer, pp. 277-298. Available at: http://dx.doi.org/10.1007/978-3-0348-0856-9_13.
  Abstract View in KAR
  This year marks the 30th anniversary of the first description of the cellular distribution of actin within a yeast cell. Since then advances in both molecular genetics and imaging technologies have ensured research within these simple model organisms has blazed a trail in the field of actomyosin research. Many yeast proteins and their functions are functionally conserved in human cells. This, combined with experimental speed, minimal cost and ease of use make the yeasts extremely attractive model organisms for researching diverse cellular processes, including those involving actomyosin. In this chapter, current state-of-the-art fluorescence methodologies being applied to yeast actomyosin research, together with an honest appraisal of their limitations, such as the pitfalls that should be considered when fluorescently labelling proteins interacting within a dynamic cytoskeleton, will be described. Papers describing the established techniques developed for yeast localisation studies will be highlighted. This will provide the reader with an informed overview of the arsenal of imaging techniques available to the yeast actomyosin researcher and encourage them to consider novel ways these simple unicellular eukaryotes could be used to address their own research questions
• Johnson, M., Geeves, M. and Mulvihill, D. (2013). Production of Amino-Terminally Acetylated Recombinant Proteins in E. coli. In: Hake, S. and Janzen, C. eds. Protein Acetylation. Humana Press, pp. 193-200. Available at: http://dx.doi.org/10.1007/978-1-62703-305-3_15.
  Abstract View in KAR
  The majority of proteins in eukaryote cells are subjected to amino-terminal acetylation. This co-translational modification can affect the stability of a protein and also regulate its biological function. Amino-terminally acetylated recombinant proteins cannot be produced using prokaryote expression systems, such as E. coli, as these cells lack the appropriate N-?-terminal acetyltransferase complexes. Here we describe a simple protocol that allows the recombinant expression and purification of NatB-dependent amino-terminally acetylated proteins from E. coli.

Thesis

• Brooker, H. (2017). Characterisation and Functional Analysis of Actin Cytoskeleton Components and Development of a Novel Hydrostatic Pressure Live Cell Imaging System.
  Abstract View in KAR
  The main aims of this project were to use a variety of techniques to study various components of the actin cytoskeleton using Caenorhabditis elegans and Schizosaccharomyces pombe, in addition to developing a novel hydrostatic pressure imaging system.
  The function and localisation of the C. elegans class I myosins: HUM-1 and HUM-5, were first explored using fluorescent protein fusions and phenotype analysis. The impact of a conserved phosphorylation event on myosin Ie function and localisation was then examined in C. elegans and S. pombe HUM-1 and Myo1 respectively. The results show HUM-1 and HUM-5 are non-essential, with HUM-1 expressed in a variety of tissues whereas HUM- 5 was expressed exclusively in the nervous system. Loss of HUM-1 reduces maximal brood size, inducing delayed embryo release. The conserved serine residue is required for the function and localisation of both HUM-1 and Myo1. Mutations of the residue altered HUM-1 localisation in a tissue specific manner and abolished Myo1 membrane association in addition to inducing spore formation.
  In vitro and in vivo studies were next undertaken to explore the impact of fluorescent protein fusions and temperature sensitive mutations upon the the stability and function of S. pombe tropomyosin. While both Ts mutants, Cdc8-27 and Cdc8-110, had a two-step thermal unfolding transition lower than that of wild-type Cdc8, fluorescent fusions did not impact stability. Amino- terminal fusions however, mimic acetylation whereas carboxyl terminal fusions abolished polymerisation, localisation and functionality. The final part of this project used a methodical approach to develop a hydrostatic imaging system that could be used to follow fluorescently labelled protein dynamics in a live cell context. Preliminary data revealed the application of 100 bar of pressure induced a cell cycle delay in both S. pombe and Candida albicans, however the current limitations of the system make it incompatible with fluorescence microscopy.
• Wilson, B. (2017). Assay Development and Efficacy Testing of Novel and Established Antimicrobials.
  Abstract View in KAR
  Over the past 25 years, new antimicrobial discovery has ground to a halt, this combined
  with a rise in antimicrobial resistance in pathogenic bacteria, is causing the number of
  effective treatments available to humankind decrease.
  For my thesis, I explored the testing methods for screening antimicrobial compounds and
  found them to be inefficient. One of the limiting factors for the discovery of new antimicrobial
  compounds are the efficacy testing assays and high-throughput screening methods used
  by researchers. These methods mainly measure the ability of a compound to diffuse
  through or across a medium, be it agar, filter papers or testing strips.
  Within this thesis I developed new testing assays for a family of novel antimicrobials, as
  their efficacy wasn't measurable by the standard procedures. I also measured the MIC50
  and MIC100 values of all the compounds in the group. They had measurable antimicrobial
  effect against Gram-Positive bacteria - comparable with established compounds like
  Vancomycin, and measurable interaction with Gram-Negative bacteria, coating the cells
  and inhibiting growth.
  I then further explored the coating interaction, adding the compounds to bacteria, but also
  adding solutions of established antimicrobials, I found that the compound boosted the
  efficacy of the established compounds and was also able to overcome resistance
  mechanisms developed by the bacteria.
  Finally, I developed a modular, 3D printed robot to standardise and increase the accuracy
  of the new testing methods.
• Baker, K. (2016). Signalling Dependent Regulation of the Fission Yeast Actomyosin Cytoskeleton.
  Abstract View in KAR
  The actomyosin cytoskeleton is a highly dynamic complex of proteins which facilitate diverse processes within the cell. It is regulated by a network of signalling pathways to coordinate growth and division in response to changes in the extracellular environment. A wide range of actin binding proteins control the organisation and dynamics of the actin network within eukaryotic cells. This study explored the function of signalling dependent phosphorylation events on two S. pombe actin binding proteins, the actin capping protein, Acp1, and the class I myosin, Myo1.
  
  Disruption of the TOR-signaling complex, TORC2, results in defects in the formation and constriction of the cytokinetic actomyosin ring (CAR). This study explores the localisation of TORC2 to the CAR, and the function of TORC2dependent phosphorylation of Acp1. Data shows that phosphorylation of Acp1 functions to regulate the stability and cellular organization of the actin cytoskeleton. This allows a cell to couple growth and division with changes in the environment.
  
  Similarly, a conserved serine residue in the IQ motif of the neck region of Myo1 was shown to be TORC2-dependent. Fission yeast express two calmodulin-like light chains, Cam1 and Cam2, which are capable of associating with the IQ domains of Myo1. The location of this phosphorylation site, between the two IQ motifs, provides a potential mechanism of determining which light chain associates with Myo1. This study provides evidence that this TORC2-dependent phosphorylation event is cell cycle dependent, and may result in a switch between Cam1 and Cam2 binding.
  
  These potentially conserved mechanisms of regulating the organization of the actomyosin cytoskeleton provide an important insight into the system of control networks that regulate cell growth and division in response to changes in the extracellular environment.
• Eastwood, T. (2016). Analysis of the Role of Amino-Terminal Acetylation in Modulating the Cellular Distribution and Physical Properties of the Parkinson’s Disease Associated Protein Alpha-Synuclein.
  Abstract View in KAR
  Alpha-Synuclein (aS) is a small, 140 amino acid, neuronal protein, found predominantly in the presynaptic terminals of neurons. Although the precise cellular function of ?S remains unknown, it is known to be critical in the pathology of a number of neurodegenerative disorders including Parkinson's Disease (PD) and other related dementias. A common symptomatic feature of PD and ?S associated disease states, is the aggregation of misfolded ?S into Lewy bodies. ?S is subject to a number of post-translational modifications, including amino-terminal (Nt) acetylation and is known to bind to membranes, both are believed to effect oligomer formation and the accumulation of ?S aggregates. In this project I am using different microbial model systems to examine the effect of Nt-acetylation on membrane binding and oligomer formation and their effect on the accumulation of ?S aggregates within the cell. The first model system used is the bacteria E. coli, in which we are making use of a novel Nt-acetylation system developed within this lab. The second model system being used is the fission yeast Schizosaccharomyces pombe, which allows us to use molecular genetic approaches to examine how the post-translational modification of Nt-acetylation effects ?S within a eukaryotic cell.
• Johnson, M. (2014). Regulation of the Actin Cytoskeleton and Polarized Growth in Schizosaccharomyces Pombe.
  Abstract View in KAR
  The fission yeast, Schizosaccharomyces pombe grows only in length in a highly polarized manner in a process tightly regulated by both the microtubule and actin cytoskeletons, which play key roles in the establishment of cell polarity and growth of the cell respectively. Previous studies have shown that within the microtubule cytoskeleton a large group of proteins accumulate at the cell poles, where they are believed to form a “polarisome” which coordinates the cell’s polarity. Whilst the main functions and key interactions between these proteins are known, many of the finer details remain unclear. The actin cytoskeleton is a highly dynamic network of different actin structures, with multiple changes occurring that are regulated by actin binding proteins (ABPs). One of the most important actin binding proteins is tropomyosin, which is responsible for stabilising interphase actin cables and the cytokinetic actomyosin ring (CAR). Within fission yeast tropomyosin is present in both acetylated and unacetylated forms which each localise to distinct actin structures.
  Interphase actin cables and the CAR are nucleated by two different formins; For3 and Cdc12 respectively. Within this study it has been shown that it is these formins that determine which tropomyosin isoform associates with specific actin structures. Exchanging the localisation of these formins results in a corresponding exchange of tropomyosin present on actin filaments. This exchange also has a subsequent effect on actin dynamics and the interaction of other ABPs. In addition, when investigating the regulation of cell polarity, it was shown that this is a very complex process. Proteins from both the actin and microtubule cytoskeletons playing key roles in the transport, recruitment, tethering and turnover of each other as shown by deletions of one polarity protein often having a significant effect on the localisation and abundance of others.

Forthcoming

• White, L., Boles, J., Allen, N., Alesbrook, L., Sutton, J., Hind, C., Hilton, K., Blackholly, L., Ellaby, R., Williams, G., Mulvihill, D. and Hiscock, J. (2020). Controllable hydrogen bonded self-association for the formation of multifunctional antimicrobial materials. Journal of Materials Chemistry B [Online]. Available at: http://dx.doi.org/10.1039/D0TB00875C.
  Abstract View in KAR
  SSAs are a class of Supramolecular Self-associating Amphiphilic salt, the anionic component of which contains a covalently bound hydrogen bond donor-acceptor motif. This results in a monomeric unit which can adopt multiple hydrogen bonding modes simultaneously. Previous investigations have shown examples of SSAs to act as antimicrobial agents against clinically relevant methicillin-resistant Staphylococcus aureus (MRSA). Herein, we report an intrinsically fluorescent SSA which can self-associate producing dimers, spherical aggregates and hydrogels dependent on solvent environment, while retaining antimicrobial activity against both model Gram-positive (MRSA) and Gram-negative (Escherichia coli) bacteria. Finally, we demonstrate the SSA supramolecular hydrogel to tolerate the inclusion of the antibiotic ampicillin, leading to the enhanced inhibition of growth with both model bacteria, and derive initial molecular structure-physicochemical property-antimicrobial activity relationships.