Wallace Lab

  • Dynamic label-free imaging of lipid nanodomains. de Wit G, Danial JS, Kukura P, Wallace MI. PNAS. 2015 Sep 23;

    Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220 ± 60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.

  • Probing channel, pump, and transporter function using single-molecule fluorescence. Weatherill EE, Danial JSH, Wallace MI. in Pumps Channels and Transporters: Methods of Functional Analysis. Wiley 2015;

    The ability of patch clamping to monitor individual ion channels revolutionized our approach to understanding the discrete functional states of these membrane proteins, including how ligands, point mutations, and the electrochemical potential affect channel function...

  • High-throughput optical sensing of nucleic acids in a nanopore array. Huang S, Romero-Ruiz M, Castell OK, Bayley H, Wallace MI. Nature Nano. 2015 Aug 31;

    Protein nanopores such as α-haemolysin and Mycobacterium smegmatis porin A (MspA) can be used to sequence long strands of DNA at low cost. To provide high-speed sequencing, large arrays of nanopores are required, but current nanopore sequencing methods rely on ionic current measurements from individually addressed pores and such methods are likely to prove difficult to scale up. Here we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid hybridization events can be parallelized. We make optical recordings at a density of ∼104 nanopores per mm2 in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2,500 bilayers with a micropatterned hydrogel chip, we are also able to load different samples into specific bilayers suitable for high-throughput nanopore recording.

  • Imaging potassium-flux through individual electropores in droplet interface bilayers. Szabo M, Wallace MI. Biochim Biophys Acta. 2015 Jul 22;

    Using total internal reflection fluorescence microscopy of droplet interface bilayers containing the potassium-sensitive fluorophore APG-4, we imaged the ionic flux through individual electropores. We are able to monitor up to 30 individual pores in parallel and show voltage depende...

  • The TatC component of the twin-arginine protein translocase functions as an obligate oligomer. Cleon F, Habersetzer J, Alcock F, Kneuper H, Stansfeld PJ, Basit H, Wallace MI, Berks BC, Palmer T. Mol Microbiol. 2015 Jun 25;

    The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is comprised of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild type tatC. Single substitutions that confer dominant negative TatC activity localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix five of TatC. Our findings show that TatC functions as an obligate oligomer. ...

  • Fluorescence-detected magnetic field effects on radical pair reactions from femtolitre volumes. Dodson CA, Wedge CJ, Murakami M, Maeda K, Wallace MI, Hore PJ. Chem Commun (Camb). 2015 May 11;51(38):8023-6

    We show that the effects of applied magnetic fields on radical pair reactions can be sensitively measured from sample volumes as low as ∼100 femtolitres using total internal reflection fluorescence microscopy. Development of a fluorescence-based microscope method is likely to be a key step in further miniaturisation that will allow detection of magnetic field effects on single molecules. ...

  • Combining single-molecule imaging and single-channel electrophysiology. Weatherill EE, Wallace MI. J Mol Biol. 2015 Jan 16;427(1):146-57

    Combining simultaneous single-molecule fluorescence measurements of ion channel conformational change with single-channel electrophysiology would enable a direct link between structure and function. Such methods would help us to create a truly molecular "movie" of how these important biomolecules work. Here we review past and recent progress toward this goal. ...

  • Membrane pore formation at protein-lipid interfaces. Gilbert RJ, Dalla Serra M, Froelich CJ, Wallace MI, Anderluh G. Trends Biochem Sci. 2014 Nov;39(11):510-6

    Pore-forming proteins (PFPs) interact with lipid bilayers to compromise membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate 'proteolipidic' pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal pore formation by peptides including melittin, protegrin, and Alzheimer's Aβ1-41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal pores might be investigated further. (jquery.zrssfeed.js, line 297)

  • Photobleaching reveals heterogeneous stoichiometry for equinatoxin II oligomers. Baker MA, Rojko N, Cronin B, Anderluh G, Wallace MI. Chembiochem. 2014 Sep 22;15(14):2139-45

    Equinatoxin II (EqtII), a sea anemone cytolysin, is known to oligomerize to form pores that spontaneously insert into membranes. Crystallographic and cryo-EM studies of structurally similar cytolysins offer contradictory evidence for pore stoichiometry. Here we used single-molecule photobleaching of fluorescently labeled EqtII to determine the stoichiometry of EqtII oligomers in supported lipid bilayers. A frequency analysis of photobleaching steps revealed a log-normal distribution of stoichiometries with a mean of 3.4±2.3 standard deviations. Comparison of our experimental data with simulations of fixed stoichiometries supports our observation of a heterogeneous distribution of EqtII oligomerization. These data are consistent with a model of EqtII stoichiometry where pores are on average tetrameric, but with large variation in the number of subunits in individual pores.

  • High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning. Spillane KM, Ortega-Arroyo J, de Wit G, Eggeling C, Ewers H, Wallace MI, Kukura P. Nano Lett. 2014 Sep 10;14(9):5390-7

    The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane "pickets" or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20-40 nm scattering gold beads with simultaneous < 2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.