Exploring functional biomembranes: supported lipid bilayers, toxins and
light-harvesting proteins
Peter G. Adams1, Cvetelin Vasilev2, Gabriel A. Montaño3, C. Neil Hunter2, Matthew P. Johnson2
1
School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT, UK
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK
3
Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, NM, 87185,
USA
2
[email protected]
I am interested in understanding the biophysical properties and interactions of lipid membranes and
membrane-active biomolecules, including photosynthetic proteins and toxins. Current topics include:
(1) dynamic optical properties of natural photosynthetic proteins, (2) artificial light-harvesting
systems, (3) toxin-induced lipid bilayer damage. In the first stages of photosynthesis, light-harvesting
membrane protein complexes form an interconnected network, absorbing photons and transferring
energy as electronic excited states with high efficiency. Firstly, we have studied how the optical
properties of Light Harvesting Complex II (LHCII) depend upon protein-protein and lipid-protein
interactions. Microscale array patterns of either single- or multi-layers of LHCII were studied by atomic
force microscopy and fluorescence microscopy with spectral and lifetime imaging. Fluorescence
spectra confirm that the native chromophore organization of LHCII was maintained. Interestingly,
LHCII had lower fluorescence lifetimes in multi-layers, suggesting that increased LHCII-LHCII
interactions promote the quenched state. Secondly, we highlight research into (re)designing light
harvesting systems. LHC-II and various lipids are used as building blocks for designing new energytransfer systems with modulated optical properties. For example, non-native chromophores are
shown to absorb light and act as energy donors to LHCII, extending its absorption cross-section.
Thirdly, we study the interaction of supported lipid bilayers (SLBs) with lipopolysaccahride (LPS), a
potent human toxin. LPS spontaneously inserts into SLBs, causing major reorganization and dynamic
changes to the membrane depending on its local ionic environment. Lipid membrane tubules,
perforations and multi-layers are observed by fluorescence microscopy, atomic force microscopy and
quartz crystal microbalance measurements. These lipid-LPS interactions may have implications for LPS
toxicity.
Towards engineering nanoporous membranes with specific permselectivity
Nesrine Aissaoui1, Leire Díaz-Ventura1, María Genua1, Nico B. Eisele1,2, Andrey Chuvilin3,4, Steffen
Frey2, Dirk Görlich2, Ralf P. Richer1,5,6
1
Biosurfaces Lab, CIC biomaGUNE, Donostia-San Sebastian, 20009, Spain
Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, 37077,
Germany
3
CIC nanoGUNE, Donostia-San Sebastian, 20009, Spain
4
IKERBASQUE, Basque Foundation for Science, Bilbao, 48013, Spain
5
School of Biomedical Sciences and School of Physics and Astronomy, University of Leeds, Leeds, LS2
9JT UK
6
Laboratory of Interdisciplinary Physics, University Grenoble Alpes - CNRS, Grenoble, 38402, France
2
[email protected]; [email protected]
Controlled macromolecular transport into and out of the nucleus is essential to eukaryotic cell life. To
this end, the nuclear envelope is perforated with nuclear pore complexes (NPCs), complex assemblies
of proteins that define a transport channel of about 40 nm diameter. The nuclear pore permeability
barrier - a selective passageway for cargoes bound to nuclear transport receptors (NTRs), arises from
natively unfolded protein domains that are rich in phenylalanine-glycine dipeptide motifs (FG
domains) and densely grafted to the channel walls. The physical mechanisms underlying the selectivity
of the permeability barrier are not completely understood, but there is reason to think that simple
polymer physics can help explaining basic features of this process.
Through a bottom-up engineering approach, we aim to reconstitute the selective permeability barrier
of the nuclear pore complex in solid-state nanopores. Such a device will enable novel studies to test
our hypothesis and to better understand the mechanisms of selective permeability.
One technical requirement towards this goal is the preparation of nanoporous substrates. Membranes
with arrays of nanopores of well-defined diameter (comparable to the nuclear pore complex channel)
were successfully produced by focused ion beam. Another requirement is the selective
functionalization of the interior of solid-state nanopores with FG domains. To this end, we have
developed a method for the functionalization of silicon oxide and gold with coatings that selectively
capture polyhistidine tagged proteins and are inert to protein binding, respectively. Silicon oxide is
chemically modified with amines and subsequently ethylenediaminetetraacetic acid (EDTA) to
selectively attach his-tagged proteins in an oriented way. Gold is modified with poly(ethylene glycol)
to prevent non-specific adsorption of proteins. The performance of this orthogonal functionalization
method was characterized and validated on planar surfaces using quartz crystal microbalance (QCMD) and spectroscopic ellipsometry (SE). The method is applicable to surfaces of arbitrary shapes and
can thus be transferred to selectively functionalize nanopores (in silicon nitride) and passivate the
surrounding surfaces of nanoporous membranes (coated with gold).
In parallel, we have devised a microfluidic device to investigate translocation of fluorescent probes
across the nanoporous membranes. In this device, the membrane separates two compartments source and target – that can be addressed independently for liquid exchange. Currently, we are
developing a method for the time-resolved and quantitative analysis of macromolecular translocation
across the membranes using confocal fluorescence microscopy. These technological advances are
important towards the development of a solid-state biosynthetic system that reconstitutes the NPC
permeability barrier thus enabling new functional studies. They should also be of general interest for
the development of artificial sieving devices with unique selectivity properties.
Development of biomimetic nanopore platforms for investigating
nucleocyptoplasmic transport
Bernice T. Akpinar1,2, Binoy P. Nadappuram1, Bart W. Hoogenboom2 and Joshua B. Edel1
1
Department of Chemistry, Imperial College London, London, SW7 2AZ, UK
London Centre for Nanotechnology, University College London, London, WC1H 0AH, UK
2
[email protected]
The nuclear pore complex (NPC) is responsible for the control of macromolecular transport between
the cell nucleus and cytoplasm. The walls of this complex are lined with intrinsically disordered
proteins that give rise to its transport selectivity.1 However, the precise nature of this flexible barrier
and the mechanism by which it controls the passage of specific analytes is still debated. Artificial
nanopores offer a tunable platform to better understand controlled transport through this elaborate
structure.2,3 Herein we present a strategy for biomimetic nanopore assembly as an alternative
platform for investigating the macromolecular transport through the NPC. The controlled chemical
modification of solid-state nanopores often suffers from poor selectivity, we here overcome this by
the use of dual-barrel nanopipettes. These artificial nanopores were fabricated by the formation of a
smoked carbon electrode in one barrel and subsequent electrochemical modification at the tip of the
open barrel. Modification with amine groups facilitates a route for further modification with
biomolecules using cross-linking chemistry. Molecular transport through these ~100 nm pores was
studied by monitoring the electrical signal associated with the translocation of 48.5 kbp DNA under
the influence of an applied electric field.
1
Bestembayeva, A., Kramer, A., Labokha, A. A., Osmanović, D., Liashkovich, I., Orlova, E. V., Ford, I. J., Charras,
G., Fassati, A. & Hoogenboom, B. W. Nanoscale stiffness topography reveals structure and mechanics of the
transport barrier in intact nuclear pore complexes. Nat. Nanotechnol., 10, 60-64, (2015)
2
Jovanovic-Talisman, T., Tetenbaum-Novatt, J., McKenney, A. S., Zilman, A., Peters, R., Rout, M. P. & Chait, B.
T., Artificial nanopores that mimic the transport selectivity of the nuclear pore complex, Nature, 457, 10231027, (2009)
3
Kowalczyk, S. W., Kapinos, L., Blosser, T., Magalhães, T., Nies, P., Lim, R. Y. H. & Dekker, C. Single-molecule
transport across an individual biomimetic nuclear pore complex, Nat. Nanotechnol., 6, 433–438, (2011)
Mechanical properties of membrane-containing virus PRD1 studied by
atomic force microscopy
Stavros Azinas1,2, Fouzia Bano2,5, Dennis H. Bamford3, Gustavo A. Schwartz4, Hanna M. Oksanen3, Ralf
P. Richter2,5,6 and Nicola G.A. Abrescia1,7
1
Structural Biology Unit, CIC bioGUNE, CIBERehd, Derio, 48160, Spain
Biosurfaces Lab, CIC biomaGUNE, Donostia-San Sebastian, 20009, Spain
3
Institute of Biotechnology and Department of Biosciences, Viikki Biocenter, University of Helsinki,
P.O. Box 56, Viikinkaari 9B, 00014, Finland
4
Centro de Física de Materiales, (CSIC-UPV/EHU), Donostia International Physics Center, DonostiaSan Sebastián, 20018, Spain
5
School of Biomedical Sciences and School of Physics and Astronomy, University of Leeds, Leeds LS2
9JT, UK
6
Laboratoire Interdisciplinaire de Physique, University Grenoble Alpes-CNRS, St. Martin d'Hères,
38402, France
7
IKERBASQUE, Basque Foundation for Science, Bilbao, 48013, Spain
2
[email protected]
Certain viruses are assembled by using a lipid membrane as a genome container, which is in turn
protected by a proteinaceous shell. Icosahedral bacteriophage PRD1 is a model virus of this type,
whose hierarchical architecture plays a both a structural and a functional role during the viral cycle.
Here, we present data from a study using atomic force microscopy (AFM) to visualize immobilized
PRD1 particles, and to characterize the virus’ mechanical properties in solution. AFM allowed for the
identification of the orientation of individual immobilized particles, and visualization of sub-features
on the viral surface. More importantly, by nanoindentation of the particles, we were able to map the
mechanical properties of PRD1, as well as those of selected mutants, providing new insight into the
structure-function relationship of such type of membrane-containing viruses.
We demonstrate that the stiff and brittle proteinaceous shell coupled with the soft and compliant
membrane vesicle provide a tough container that relieves the packaged DNA from a structural role,
further protecting it against mechanical damage. We also propose that this ordered structure acts as
a composite material, similar to the membrane and shell of bird’s eggs, which has evolved in response
to the quest of adaptation, survival and multifunctionality. The data from this multidisciplinary study
will help biophysicists and nanotechnologists understand how natural assemblies adapt to their
environmental needs.
Cell-surface mimics and surface-based sensing to probe herpesglycosaminoglycan interactions with single particle sensitivity
N. Peerboom1, Eneas Schmidt1, N. Altgärde1, E. Trybala2, Hudson Pace1, T. Bergström2, M. Bally1
1
Department of Physics, Chalmers University of Technology, Göteborg, S-412 96, Sweden
2
Department of Clinical Virology, University of Göteborg, Göteborg, S-413 46, Sweden
[email protected]
A number of enveloped viruses, including herpes simplex viruses attach to susceptible host cells via
interaction between their glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial
recognition is crucial in the viruses’ life cycle as it leads to infection. Equally important is however, the
capability of the virus to overcome these interactions upon egress to ensure virus propagation.
In our work, we study the molecular and physical mechanisms modulating HSV binding and release
from the cell surface. Our approach is primarily based on the use of cell-membrane mimics of various
complexities: A minimal model of the cell’s carbohydrate coat based on the end-on immobilization of
GAG chains 1 (Figure a) makes it possible to study the details of virus-GAG interactions; as more
complex model consisting of a native-membrane-like supported lipid bilayer2 (Figure b) allows for a
more comprehensive study taking into account all biomolecules involved. Total internal reflection
fluorescence microscopy (TIRFM) allows for the quantitative analysis of affinities3 and diffusion
coefficients of surface-bound viruses on a single virion level (Figure c). With our surface-based
approach, we gain insight into the modulatory function of protein glycosylation and interrogate the
role of GAG sulfation in the process: We show that mucin-like regions found on the glycoproteins of
HSV-1 and HSV-2 play an important role in modulating the interaction, an observation further
supported by cell experiments. We further show that the diffusion of virions on the surface depends
on the type of GAGs and their degree of sulfation. Taken together, our research contributes to a better
understanding of the mechanisms underlying the interaction between a virus and the surface of its
host. Such insights will without doubt facilitate the design of more efficient antiviral drugs or vaccines.
Cell-surface mimics to probe
HSV
-glycosaminoglycan
interactions.
Schematic
illustration of a) a minimal
model consisting of end-on
immobilized GAGs and b) a
native- -like supported lipid
bilayer. c) TIRFM microscopy is
used to visualize individual
particles, to quantify the
binding and release of the
virions and to probe their
diffusion on the surface.
1
N. Altgarde, et al., J Biol Chem 2015, 290, 21473-21485.
H. Pace, et al., Anal Chem 2015, 87, 9194-9203.
3
M. Bally, et al., Phys Rev Lett 2011, 107.
2
Single molecule force probe assays for unravelling the distinct unbinding
mechanics of the glycosaminoglycan hyaluronan from its vascular and
lymphatic endothelial receptors CD44 and LYVE-1
Fouzia Bano1,5, Suneale Banerji2, Mark Howarth3, David G. Jackson2, Ralf P. Richter1,4,5
1
Biosurfaces Lab, CICbiomaGUNE, Donostia-San Sebastian, 20009, Spain
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford,
Oxford, OX39DS, UK
3
Department of Biochemistry, University of Oxford, Oxford, OX13QU, UK
4
Université Grenoble Alpes - CNRS, Laboratoire Interdisciplinaire de Physique (LIPhy), 38402 Saint
Martin d’Hères, France
5
School of Biomedical Sciences and School of Physics and Astronomy, University of Leeds, Leeds,
LS29JT, UK
2
[email protected]
The interactions of extracellular polysaccharides from the glycosaminoglycan (GAG) family with cell
surface receptors are important for the correct communication of cells with their environment. It is
now also well established that mechanical stimuli are important for cellular communication. However,
limited methods are currently available to probe,,the properties of GAG•protein bonds under
mechanical stress (e.g. shear flow in blood vessels) on the level of individual GAG chains. Here, we
have devised such a method, by combining purpose-designed surfaces that allow immobilization of
GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy1,2.
We have applied the method to study the interaction of the GAG hyaluronan (HA) with CD44, its
receptor in vascular endothelium2, and LYVE-1, its counterpart receptor in lymphatic endothelium,
and to compare the binding behaviour in these two very different vessel systems.
Despite their structural similarity and comparable affinities for HA, CD44 and LYVE-13,4 exhibit
significantly different binding characteristics under force. Individual bonds between HA and CD44
form slowly and are remarkably resistant to rupture in spite of their low binding affinity whereas the
forces required to rupture individual HA-LYVE1 bonds were below the detection limit (1 pN) under
comparable conditions. Multiple HA•CD44 bonds rupture sequentially and independently under load2.
Interestingly, multiple LYVE-1 molecules unbind HA collectively, with a force response that is not
stochastic but deterministic and highly sensitive to the receptor surface density and ionic strength.
We propose that these differences reflect an adaptation of the receptors to the high and low shear
stress conditions within the lumen of blood vessels (CD44) and within the interstitium and lumen of
lymphatic vessels (LYVE1), respectively.
References:
[1]
[2]
[3]
[4]
N. B. Baranova et. al., J. Biol. Chem. 289, 25675 (2011).
F. Bano et. al., Sci. Rep. 6, 34176 (2016).
S. Banerji et. al., J. Biol. Chem. 285, 10724 (2010).
S. Banerji et al J. Biol. Chem, EPubl Oct 12 (2016)
Controlling Transmembrane Protein Concentration in Supported Lipid
Bilayers
Peng Bao1, Michael L. Cartron2, Khizar H. Sheikh3, Benjamin R. G. Johnson1, C. Neil Hunter2, Stephen
D. Evans1*
1
School of Physics & Astronomy, University of Leeds, LS2 9JT, UK
Department of Molecular Biology & Biotechnology, University of Sheffield, S10 2TH, UK
3
School of Biomedical Science, University of Leeds, LS2 9JT, UK
2
[email protected]
Supported lipid bilayers (SLBs) are attractive model systems for studying lipid behaviour,
membrane proteins and for the recapitulation of minimal membrane associated processes
and structures. Their formation via vesicle adsorption, rupture and fusion, restricts the
concentration of membrane proteins that can be incorporated whilst still permitting high
quality bilayer formation. Therefore, there is significant interest in routes to increase the
protein concentration post bilayer formation. Here we demonstrate that the trans-membrane
protein proteorhodopsin can be incorporated into such bilayers and retain its diffusive
mobility, D ~0.39 µm2s-1. In-plane electric fields can be used to move these proteins, within
the SLB, through electrophoresis. Using a combination of electric fields and patterning of the
SLB we have been able to manipulate the proteorhodopsin in a controlled manner to yield a
> 25-fold increase in local concentration, to ~1300 proteins µm-2. The build-up of protein was
monitored by fluorescence microscopy and atomic force microscopy.
Biofilms as tools to study supramolecular structures of the extracellular
matrix.
Aïseta Baradji1, 2, Edwin Yates1, Ralf Richter2, 3, 4 , David Fernig1
1
Biochemistry department, Institute of Integrative Biology, Liverpool, L69 7ZB, UK
2
Biosurfaces Unit, CIC biomaGUNE, Donostia-San Sebastian, 20009, Spain
3
School of Biomedical Sciences and School of Physics and Astronomy, Leeds, LS2 9JT, UK
4
Laboratory of Interdisciplinary Physics, University Grenoble Alpes-CNRS, Grenoble, 38402, France
[email protected]
The extracellular matrix is a complex compartment that contains many proteins and
glycosaminoglycans that are interacting with each other as a network. FGFs contain one or several
evolutionary conserved heparin binding sites (HBS1, 2, 3 and 4), and their interactions with heparan
sulfate (HS) control their diffusion in the ECM and their signalling through FGF receptors. We designed
a simple biomimetic model of the pericellular matrix using heparan sulfate (HS) brushes. The biofilms
were assembled layer by layer by grafting biotinylated HS polysaccharides on a supported lipid bilayer
via a streptavidin layer. These were challenged with FGFs and the interactions were followed by state
of the art techniques, quartz crystal microbalance with dissipation monitoring (QCM-D), spectroscopic
ellipsometry (SE) and fluorescence recovery after photobleaching (FRAP), to analyse the dynamics
occurring at the nanoscale. Upon binding to the HS brushes, multivalent FGFs rigidified by cross-linking
and immobilised HS brushes. Rigidification of soft and highly hydrated films can be assessed by QCMD whereas optical techniques such as SE quantify the biomolecules ate the surface and FRAP assess
dynamic of the biofilm.
Well defined biomimetic model for experimental use.
Experimental model supported by a SiO2 sensor (in QCM-D and SE apparatus) or a glass cover slip (in FRAP assays). The lipid
bilayer is formed by disruption upon coverage, by adsorption of biotinylated SUVs. The SAv or SAv-atto layer is specifically
formed on the bilayer and further exposed to biotinylated HS. Cytokines are presented to the model and changes upon
binding are monitored by QMD, SE and FRAP imaging.
Enhanced functional durability of membrane proteins in hybrid lipid – block
copolymer vesicles
Sanobar Khan1, Stephen P. Muench2, Lars J.C. Jeuken2, Paul A. Beales1
1
School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds,
LS2 9JT, UK
2
School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of
Leeds, Leeds, LS2 9JT, UK
[email protected]
Membrane proteins carry out a wide-range of functions at the interface between cellular
compartments that provide motivation and opportunity for their repurposing in bionanotechnology
applications. Unlike globular proteins, membrane proteins present greater challenges for in vitro
handling due to their instability in water. Membrane proteins require stabilising agents that mimic the
amphiphilic solvation of native membranes, often achieved by reconstitution within micelle or vesicle
architectures. Vesicles are advantageous for many applications as they naturally mimic biological
compartmentalisation due to their closed morphology that encloses a distinct aqueous environment
from the bulk media. Liposomes can provide the natural biocompatibility and biofunctionality
comparable to native biomembranes. However, technology applications usually require long term
stability and functionality that often isn’t provided by these systems. More robust and durable vesicles
can be created from synthetic amphiphilic block copolymers, yet these polymersomes lack the natural
biofunctionality of their lipid counterparts. Here we demonstrate that a model membrane protein,
cytochrome bo3, can be functionally reconstituted in a hybrid vesicle that combines the
biofunctionality of lipids with the mechanical stability of polymers. Importantly, these hybrid vesicles
stabilise the functional activity of the protein such that it is maintained over much longer periods of
time (>70% initial activity after 6 weeks) compared with traditional proteoliposomes (inactive after 34 weeks). This suggests hybrid vesicles are a highly promising paradigm for durable applications of
membrane proteins in bionanotechnology.
Evaluation of cDICE method for producing giant lipid vesicles
Matthew C. Blosser1,2, Benjamin G. Horst3, Sarah L. Keller4
1
Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK
Department of Chemistry, King’s College London, London, SE1 1DB, UK
3
Department of Chemistry, University of California, Berkeley, Berkeley, 94720, USA
4
Departments of Chemistry and Physics, University of Washington, Seattle, 98105, USA
2
[email protected]
Giant unilamellar vesicles are a powerful and common tool employed in biophysical studies of lipid
membranes. Limitations within some techniques of fabricating GUVs render important regions of
parameter space challenging to achieve. We evaluate a recently introduced method of vesicle
formation, “continuous droplet interface crossing encapsulation” (cDICE). This method produces
monodisperse giant unilamellar vesicles of controlled sizes and high encapsulation efficiencies, using
readily available instrumentation. We find that mixtures of phospholipids within vesicle membranes
produced by cDICE undergo phase separation at the same characteristic temperatures as lipids in
vesicles formed by a complementary technique, indicative that the composition and physical
properties are consistent. We also find that the cDICE method is effective both when vesicles are
produced from charged lipids and when the surrounding buffer contains a high concentration of salt.
A shortcoming of the technique is that cholesterol is not substantially incorporated into vesicle
membranes.
Anomalous Diffusion in Artificial Membranes
Helena L.E. Coker1,2, Matthew R. Cheetham1,2, Mark I. Wallace2
1
Chemistry Research Laboratory, 12 Mansfield Road, University of Oxford, OX1 3TA, UK
2
Department of Chemistry, 7 Trinity Street, King’s College London, SE1 1DB, UK
[email protected]
[email protected]
In contrast to membrane protein diffusion in artificial bilayers, the diffusion of proteins in cells is both
slower and non-ergodic. Anomalous diffusion in cells has been attributed to molecular pinning by
cytoskeletal-membrane interactions and to molecular crowding within the membrane. A hierarchy of
compartments and obstacles is thought to hinder diffusion over a wider range of time and length
scales. Here we present our preliminary efforts to produce an explanatory model involving membrane
crowding with PEG-lipids, validated using Monte-Carlo simulations. We change the nature of the
bilayer by varying the membrane composition, and observe the onset of anomalous diffusion. Using a
combination of single-molecule fluorescence tracking and interferometric scattering microscopy, we
are able to observe the diffusive behaviour at relevant timescales from microseconds to seconds.
Our single particle tracking routines were tested using a discrete-time random walk model to simulate
particle tracks comparable to experimental videos. When subjected to the same analysis procedure
as the experiments, we observe that the tracking precision is heavily dependent on spot size and
background noise levels. Falsely anomalous diffusion may occur at very short timescales, where the
error in localization precision dominates. We use this knowledge to carefully tailor experimental
conditions to achieve reliable results.
From the experimentation, we observe a reduction in tracer diffusion rates as membrane crowding is
increased, in addition to an increase in anomalous behaviour. The anomalous exponent exhibited a
sigmoidal behaviour with inflection points at 2.70 mol% (PEG2K) and 1.75 mol% (PEG5K). This is in
good agreement with predicted percolation thresholds showing that we can carefully tailor anomalous
behaviour using membrane composition. This behaviour is confirmed with a Monte-Carlo simulation
of hindered diffusion. The model consists of a square lattice of immobile obstacles of variable radius,
with a controllable spacing between obstacles. By simulating a random walk of tracer particles in this
system, we are able to show that immobile obstacles (or equivalently missing patches) in a supported
membrane give rise to anomalous diffusion. This is suggestive that our supported membranes with
PEG-lipids are “patchy” with patch sizes typically on the order of µm.
Cell homing at the blood vessel wall – biomechanical vs biochemical controls
Heather S. Davies1,2, Carlo Pifferi2, Olivier Renaudet2, Liliane Coche-Guerente2, Claude Verdier1,
Lionel Bureau1, Delphine Débarre1 and Ralf P. Richter1,3,4
1
Laboratory of Interdisciplinary Physics, University Grenoble Alpes-CNRS, Grenoble, 38402, France
2
Department of Molecular Chemistry, University Grenoble Alpes, Grenoble-CNRS, 38402, France
3
Biosurfaces Lab, CIC biomaGUNE, Donostia-San Sebastian, 20009, Spain
4
School of Biomedical Sciences and School of Physics and Astronomy, University of Leeds, Leeds, LS2
9JT UK
[email protected]
Circulating cells, such as immune cells and cancer cells, migrate to tissues via the endothelial cell layer.
Cells achieve this by attaching to, rolling on and migrating through the endothelium (Fig. 1). How do
cells initially attach to the blood vessel wall? The gate-keeper of cell entry to the blood vessel wall is
the endothelial glycocalyx, which extends into the blood vessel lumen by around 500 nm, and is made
up of glycosaminoglycans, such as hyaluronan (HA). HA has many binding sites for the cell receptor
CD44, and HA-CD44 interactions are crucial for the homing of various circulating cells to the
endothelium. Additionally, HA provides the glycocalyx with physical properties of softness and
deformability, which may be key to its role in regulating cell homing to the endothelium but, thus far,
have not been explored in this context. Furthermore, HA-CD44 interactions occur despite the
hydrodynamic force of blood flow; which raises the question - are HA-CD44 interactions strengthened
under flow?
Here, we use a multidisciplinary approach in order to uncouple the biochemical (affinity and avidity)
and biomechanical (softness, deformability, wall shear rate) factors that drive HA-CD44 mediated cell
homing. Using strategies from organic synthesis, surface science and physical chemistry, we have
developed well-defined biomimetic nanoscale surfaces that mimic the HA-rich endothelial glycocalyx.
Such models are tuneable and incorporate HA brushes with different properties, including HA
polymers of different size and novel HA-conjugates, generated here by chemical synthesis, which
possess similar softness and deformability of HA polymers but have only one CD44 binding site.
Integration of these model surfaces into flow chambers and examination of the rolling behaviour of
CD44-coated microbeads by high speed videomicroscopy enables us to study selected components of
the endothelial glycocalyx with controllable flow.
Fig. 1. Cell trafficking at the ESL. (A) Scheme of sequential steps involved in the cascade of adhesion of circulating
cells to the endothelium. (B) Scheme of the endothelial glycocalyx and its principal components.
Age-related changes in the outer blood-retinal barrier may contribute to the
pathology of a major form of blindness
Anthony J. Day1,2, Alex Langford-Smith1,2, Viranga Tilakaratna1,2, Larisa Logunova1,2, Paul R.Lythgoe3,
Simon J. Clark2 and Paul N. Bishop2
1
Wellcome Trust Centre for Cell-Matrix Research,
Faculty of Biology, Medicine & Health, University of Manchester, M13 9PT, UK
3
School of Earth & Atmospheric Sciences, University of Manchester, Manchester, M13 9PL, UK
2
[email protected]
A major hallmark of age-related macular degeneration (AMD), the leading cause of blindness in the
developed world, is the presence of particulate material (drusen) in the central retina. This
accumulates at the interface between Bruch’s membrane (BM), a multi-laminar extracellular matrix,
and the retinal pigment epithelium (RPE). Together the Bruch’s membrane and RPE form the outer
blood-retinal barrier, e.g. providing a filter for nutrients supplied to the neurosensory retina by blood
vessels in the choroid while preventing migration of immune cells into this immune privileged site; in
addition the RPE has an essential role in the phagocytic removal of photoreceptor outer segments that
are shed on a daily basis. Multiple genetic and environmental risk factors have been identified for
AMD, however, the role of ageing is less well characterised. We have found previously that there is a
reduction during normal ageing in the amount of heparan sulphate within the Bruch’s membrane,
where this may contribute to impaired complement regulation (part of innate immunity) leading to
the promotion of drusen formation due to local chronic inflammation.
Recently we have investigated the age-related accumulation of metal ions in the Bruch’s membrane
and how this affects the function of the adjacent RPE. We analysed eyes from human donors without
known AMD (aged 11-88 years) and quantified the level of 14 metal ions in Bruch’s membrane, by
inductively coupled plasma mass spectrometry (ICP-MS) (n=131). We determined gene expression
changes in the adjacent RPE cells by quantitative PCR (n=81) and RNA-seq (n=24), and performed
histological analyses on the macula regions from the contralateral eye (n=45).
ICP-MS identified a significant linear increase with age in the levels of cadmium and cobalt ions, and
a decrease in zinc ions, within the Bruch’s membrane; furthermore, there was a population of older
donors with high levels of aluminium within this matrix. Transcriptomic analyses revealed that the
age-related accumulation of these metal ions in the Bruch’s membrane are associated (differentially)
with gene expression changes in the RPE, i.e. in multiple pathways implicated in AMD pathology;
including complement, endoplasmic reticulum stress, inflammation, lipid biosynthesis, mitochondrial
dysfunction, oxidative stress and vascular pathways associated with angiogenesis. Age alone was
found to correlate with changes in cell viability, eumelanin biosynthesis and proteasomal activity, all
of which have been linked with RPE dysfunction; age also correlated with a histological increase in
carboxymethyl lysine, a maker of oxidative stress, within the Bruch’s membrane.
Thus we have identified age-related changes at an important biological interface within the human
eye, which may contribute directly to the initiation and progression of a major form of blindness.
Differential Structural Remodeling of Heparan Sulfate by Chemokines: the
Role of Chemokine Oligomerization
Douglas P. Dyer1,2, Catherina L. Salanga1, Elisa Migliorini3,4, Dhruv Thakar4, T. Kawamura1, Ralf P.
Richter3,4,5 and Tracy M. Handel1
1
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La
Jolla, California 92093-0684, USA
2
Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, G12 8TA, UK
3
Biosurfaces Lab, CIC biomaGUNE, Donostia-San Sebastian, 20009, Spain
4
Département de Chimie Moléculaire, Université Grenoble Alpes - CNRS, 38041 Grenoble Cedex 9,
38402, France
5
School of Biomedical Sciences and School of Physics and Astronomy, University of Leeds, Leeds, LS2
9JT, UK
[email protected]
Chemoattractant cytokines (chemokines) are integral in recruitment of leukocytes to inflammatory
sites and associated inflammatory pathologies, e.g. autoimmune diseases. Chemokine function is
dependent on their ability to bind to, and be presented on, cell surface glycoasminoglycans (GAGs),
mediating the formation of chemotactic gradients, enabling presentation and signaling through
receptors on leukocytes. GAGs are integral extracellular matrix components that not only support
chemokine localisation but also help to form the glycocalyx, which acts as a physical barrier to
leukocyte adhesion and subsequent migration. Chemokine oligomerization is essential to in vivo
chemokine mediated leukocyte recruitment (e.g. CCL2 and CCL5), however, monomeric chemokine is
largely sufficient for receptor signaling, leaving the mechanistic importance of oligomerization
unresolved.
We have utilized surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation
(QCMD) alongside endothelial binding to examine the interaction of chemokines, and mutants, with
different isolated GAGs and the cell surface.
These results demonstrate that the majority of chemokines bind to heparin and heparan sulfate (HS)
with a range of affinities, and more selectively with chondroitin sulfate-A. Inhibition of chemokine
oligomerization suggests that this process mediates the nature, strength and selectivity of some
chemokine-GAG interactions. Our data also suggests that chemokines have different abilities to crosslink individual HS chains on a biosensor. Importantly, this process may provide a novel mechanism
that enables retention of chemokines on the cell surface and subsequent modification of the
glycocalyx. Chemokine mediated HS cross-linking also appears to be dependent upon oligomerization,
in the case of CCL2, CCL5 and CXCL4. Therefore, there appears to be an overlap between GAG-binding,
HS chain cross-linking and chemokine oligomerization. We conclude that oligomerization enables
binding to, and modification of, GAG chains, enhancing retention on cell surfaces whilst also enabling
chemokine mediated GAG re-organization. This enables chemokine cell-surface localization under
flow and thus leukocyte recruitment in vivo and potentially suggests a novel function of chemokines
in physical re-organization of the glycocalyx.
Probing diffusion of biomolecules in nanoscale confined spaces
– The case of nuclear transport
Rickard Frost1, Nesrine Aissaoui2, Leire Díaz-Ventura2 and Ralf P. Richter1, 2, 3
1
School of Biomedical Science and School of Physics and Astronomy, University of Leeds, Leeds, LS2
9JT, UK
2
Biosurfaces Lab, CIC biomaGUNE, Donostia-San Sebastián, 20009, Spain
3
Laboratory of Interdisciplinary Physics, University Grenoble Alpes, Grenoble, 38402, France
[email protected]
The nuclear pore complex (NPC) controls exchange of biomolecules between the nucleus and the
cytoplasm.1 Through selective transport of RNA and proteins, NPCs in the nuclear envelope enable the
spatial separation between transcription (cell nucleus) and translation (cytoplasm), which provides a
powerful mechanism to control gene expression. The NPC channel is filled with a meshwork of natively
unfolded proteins (nucleoporins), acting as a selective permeation barrier that is effectively
permeated by nuclear transport receptors (NTRs) with their cargo.2 While the entry and release of
NTRs to/from the NPC channel have been studied in detail and are now rather well understood, much
less is known about the diffusion through the pore. The limited knowledge of the diffusion process is
largely due to limitations of existing analytical techniques. To address this issue, we have set out to
develop an in vitro methodology, based on fluorescence microscopy, which probes diffusion of
macromolecules in spaces confined at the nanoscale. Briefly, the confined space between a plane and
a millimeter-sized sphere, close to their touching point, is probed by fluorescence recovery after
photobleaching (FRAP). By functionalization of both the planar surface and the sphere, using
established biomimetic model systems,3, 4 the confinement is filled with a biomimetic protein
meshwork (e.g. nucleoporins) in which diffusion of biomolecules (e.g. NTRs) may be probed. The
methodology will generate a more detailed understanding of the permselectivity of transport of NTRs
through the nuclear pore, with possible implications in drug delivery and virology.
Figure 1. Schematic figures of (a) the nuclear pore complex (adapted from Ref 2), (b) the biomimetic
protein meshwork at a substrate surface, and (c) the confined space between a planar surface and a
sphere filled with the protein meshwork in which diffusion of NTRs will be probed.
1.
2.
3.
4.
Stewart, M. Nature Reviews Molecular Cell Biology 2007, 8, (3), 195-208.
Patel, S. S.; Belmont, B. J.; Sante, J. M.; Rexach, M. F. Cell 2007, 129, (1), 83-96.
Eisele, N. B.; Frey, S.; Piehler, J.; Goerlich, D.; Richter, R. P. EMBO Rep. 2010, 11, (5), 366-372.
Zahn, R.; Osmanovic, D.; Ehret, S.; Callis, C. A.; Frey, S.; Stewart, M.; You, C.; Goerlich, D.;
Hoogenboom, B. W.; Richter, R. P. eLife 2016, 5, e14119.
Layer-by-Layer Assembly and Characterization of Functional Lipid Multilayers
George R. Heath1, Valentin Radu1, Julea N. Butt2 and Lars J. C. Jeuken1
1
2
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK
Centre for Molecular and Structural Biochemistry, University of East Anglia, Norwich NR4 7TJ, UK
[email protected]
Multilayer lipid membranes perform many important functions in biology, such as electrical isolation
(myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and
mitochondrial cristae), and sequential processing (golgi cisternae). The power of multilayered
membranes lies in the ability to sequence processes, compartmentalize molecules or greatly increase
surface area and hence membrane protein concentration. Here we create a simple layer-by-layer
methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs)
using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the
macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic
force microscopy (AFM) for up to six lipid bilayers. We further develop this supramolecular assembly
to multilayer natural membranes at electrodes to enhance electrochemical catalysis by increasing
membrane enzyme content on the surface. We use membrane extracts overexpressing either, an
ubiquinol oxidase (cytochrome bo3 from Escherichia coli) or an oxygen tolerant hydrogenase (the
membrane bound hydrogenase from Ralstonia eutropha). Cyclic voltammetry (CV) reveals a linear
increase in biocatalytic activity with each additional membrane layer for both enzyme systems.
Electron transfer between these proteins and the electrode is mediated by the quinol pool in the lipid
phase. We deduce by atomic force microscopy, CV and fluorescence microscopy that the quinones are
able to diffuse between the lipid bilayer via defect sites where the lipid bilayers are interconnected.
This assembly is akin to that of interconnected thylakoids membranes or the folded lamella of
mitochondria and thus opens development of membrane protein multilayers to furthering our
understanding of in vivo systems. Additionally, these systems have significant potential for mimicry in
biotechnology applications such as energy production or biosensing.
Atomic Force Microscopy of membrane pore formation in pathogen attack
and immune response
Adrian W. Hodel1,2, Carl Leung2,3 Natalya V. Dudkina1,3, Natalya Lukoyanova1,3 Amelia J. Brennan4, Ilia
Voskoboinik4, Alan R. Lowe1,2, Helen R. Saibil1,3 & Bart W. Hoogenboom2,5
1
Institute of Structural and Molecular Biology, University College London and Birkbeck College,
London, UK
2
London Centre for Nanotechnology, University College London, London, WC1E 6BT, UK
3
Department of Crystallography, Birkbeck College, London, WC1E 7HX, UK
4
Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia
5
Department of Physics and Astronomy, University College London, London, WC1E 6BT, UK
[email protected]
Pore forming proteins are widely used by bacteria as weapons to attack and destroy host cells in the
course of their infection. In addition, the vertebrate immune system employs structurally related
proteins to defend a host from pathogens, and dispose of dysfunctional body cells. Our goal is to
elucidate the mechanisms of pore formation in both bacterial attack and immune defence. This may
enable us to identify new ways of intervening in diseases caused by either the function or misfunction
of pore forming proteins. We study suilysin, a bacterial toxin commonly related to meningitis in pigs,
and perforin, a protein employed by immune cells to kill cancerous or virus infected cells in humans.
We can investigate their pore forming action by atomic force microscopy (AFM), which enables us to
study functional proteins in liquid on a supported lipid membrane. The spatial and temporal resolution
of AFM is high enough to resolve membrane pore formation at molecular resolution in real time.
ARBRE: Integrating biophysical approaches for biology and healthcare
Thomas Jowitt
Faculty of Biology, Medicine and Health, University Of Manchester, Manchester, M13 9PT, UK
[email protected]
ARBRE (Association of Resource for Biophysics Research in Europe) is a pan-European network that
was funded by the COST association in 2016. The network is aimed at those who study biological
macromolecules and assemblies using biophysical approaches with the overarching aim of creating an
optimal environment for the development of innovative integrative biophysical approaches, at the
level of data acquisition, analysis and modelling, as well as for the design of unprecedented and
ambitious combinations of methodologies, to decipher more efficiently crucial biological phenomena
and to overcome significant biomedical challenges. To-date there are ~150 members from across
Europe.
Supramolecular assembly of brain extracellular matrix controls plasticity in
the ageing brain
Sujeong Yang1, Simona Foscarin1, Jessica C.F. Kwok2
1
John van Geest Centre for Brain Repair, University of Cambridge, Cambridge CB2 0PY, UK
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK
2
[email protected]
Perineuronal nets (PNNs) are special extracellular matrix structures wrapped on the surface of
neurons in the central nervous system, and are involved in the controlling developmental plasticity.
PNNs appear at the end of critical period in the sensory systems, terminating plasticity for adaptation
to environmental changes. We have mapped the molecular composition of PNNs – it is a hierarchical
assembly of 1) hyaluronan (HA) which is anchored on neuronal surface by the HA synthases; 2)
chondroitin sulphate proteoglycans (CSPGs) which binds to the HA chain, 3) members in the link
protein family (HAPLNs) which stabilises this interaction and 4) trimeric tenascin-R which binds to the
N-termini of the CSPGs. With the use of HAPLN1 knockout mice, we demonstrate enhanced plasticity
for functional recovery in ocular dominance plasticity model and after spinal cord injury. It is important
to note that HAPLN1 knockout mice showed attenuated PNNs while the concentrations of all other
PNN components remain the same in the extracellular matrix. This suggests that the observed
plasticity enhancement in the HAPLN1 knockout mice is due to the aggregation of matrix molecules
into PNNs. Recently, we have shown that the HAPLN knockout mice demonstrated superior memory
retention than the wildtype littermates in novel object recognition test. This observation prompts us
to ask if memory loss in ageing brains is related to changes in PNNs. Composition analysis from the
extracted PNN glycans reveals specific downregulation of chondroitin 6-sulphate (C6S), a chondroitin
sulphate subtype, in 18-month old aged brains when compared to young brains (3-months).
Transgenic mice with C6S downregulation showed early signs of memory loss. Enzymatic removal of
PNNs rescued age-related memory loss in 18-months old mice. These results demonstrate that
supramolecular assembly of ECM molecules into PNNs are crucial in controlling plasticity for memory
compensation in aged brains.
The purification of an improved recombinant form of CymA points to a
transient nature of interaction with its functional partner FccA during
fumarate reduction in Shewanella oneidensis MR-1
Theodoros Laftsoglou1, Julea N. Butt2, Lars J. C. Jeuken1
1
School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK
School of Biological Sciences and School of Chemistry, University of East Anglia, Norwich, NR4 7TJ,
UK
2
[email protected]
The dissimilatory metal-reducing Shewanella oneidensis MR-1 has become an important organism for
microbial electrochemistry, which has a range of promising applications, from green energy
production to biosensor technology. At the centre of the anaerobic respiratory system of S. oneidensis
MR-1 lies CymA – a 25 kDa transmembrane tetrahaem c-type cytochrome of the NapC/NrfH family of
quinol dehydrogenases that is responsible for the re-oxidation of the menaquinone pool of the inner
membrane, mediating electrons to periplasmic electron mediators or terminal reductases, such as
fumarate reductase FccA. The formation of a stable CymA:FccA complex was previously observed with
a CymA construct that carried a V5-epitope and a His6 tag on the C-terminus. However, those tags
were at the vicinity of the interacting protein partners, which may have anchored FccA via electrostatic
and/or metal-coordination interactions and, thus, give rise to an artefact. We investigated this by
engineering an improved protein construct of CymA, harbouring only a His8 tag at the end of the single
N-terminal transmembrane α-helix. Our construct was successfully over-expressed in S. oneidensis
MR-1 and purified by nickel affinity chromatography, while maintaining identical spectroscopic
signatures, catalytic activity, and purity with the previous construct, but with an increased protein
yield at 2 mg per litre of bacterial culture. The recombinant CymA was spectrophotometrically shown
to be necessary for FccA-specific fumarate reduction in vitro. However, electrochemical investigations
in a tethered bilayer membrane system with incorporated CymA could not resolve this activity. Nor
the formation of a stable complex could be observed by quartz-crystal microbalance with dissipation
in a similar system. Isothermal titration calorimetric experiments with a soluble form of CymA and
native FccA indicated a low-affinity between the two proteins (Kd > 40 µM). Overall, we report a new
approach for the purification of high quality pure recombinant CymA that is more suitable for proteinprotein interactions studies, as well as that the CymA:FccA interaction may be of a weak transient
nature.
At the interface between Shewanella oneidensis MR-1 and electrodes
Joseph Oram1, Lars Jeuken1
1
School of biomedical sciences, Faculty of biological sciences, University of Leeds, Leeds, LS2 9JT, UK
[email protected]
Exoelectrogenic bacteria can couple their metabolism to extracellular electron acceptors, including
macroscopic electrodes, and this has applications in energy production, bioremediation and
biosensing. Optimisation of these technologies relies on a detailed understanding of the microbial
interaction with the electrode surface, including the extracellular electron transfer (EET) mechanisms,
and Shewanella oneidensis MR- 1 (MR-1) has become a model organism for such fundamental studies.
Here, cyclic voltammetry was used to determine the relationship between the surface chemistry of
electrodes (modified gold, ITO and carbon electrodes) and the EET mechanism. On ultra-smooth gold
electrodes modified with self-assembled monolayers containing carboxylic-acid-terminated thiols, an
EET pathway dominates with an oxidative catalytic onset at 0.1 V versus SHE. Addition of
iron(II)chloride enhances the catalytic current, whereas the siderophore deferoxamine abolishes this
signal, leading us to conclude that this pathway proceeds via an iron mediated electron transfer
mechanism. The same EET pathway is observed at other electrodes, but the onset potential is
dependent on the electrolyte composition and electrode surface chemistry. EET pathways with onset
potentials above 0.1 V versus SHE have previously been ascribed to direct electron-transfer (DET)
mechanisms through the surface exposed decaheme cytochromes (MtrC/OmcA) of MR-1. In light of
the results reported here, we propose that the previously identified DET mechanism of MR-1 needs
to be re-evaluated.
Understanding salivary mucin interactions with black tea theaflavins
Elena Owen1, Caroline E. Ridley1, Paul D. A. Pudney2, Ralf P. Richter3,4,5, David J. Thornton1
1
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University
of Manchester, UK
2
Unilever Discover R&D Colworth Laboratory, Shambrook, Bedford, MK44 1LQ, UK
3
Biosurfaces Lab, CIC biomaGUNE, Donostia-San Sebastian, 20009, Spain
4
School of Biomedical Sciences and School of Physics and Astronomy, University of Leeds, Leeds, LS2
9JT UK
5
Laboratory of Interdisciplinary Physics, University Grenoble Alpes - CNRS, Grenoble, 38402, France
[email protected]
Mucus is a viscous hydrogel that acts as physical barrier on epithelial surfaces within the body.
Responsible for maintaining mucus structure are the mucin glycoproteins, these are large polymers
capable of forming cross-linked networks or scaffolds. Recently, there has been considerable interest
in the interactions between dietary components and the mucus barrier in terms of the bioavailability
of beneficial molecules.
The first mucosal barrier encountered by ingested food is saliva within the oral cavity. Saliva is present
in two phases the fluid phase and the adherent phase known as the salivary pellicle of which the main
structural component is the mucin MUC5B.
Green tea catechins and black tea theaflavins are potent antioxidants and have anticancer properties
however, their bioavailability is low. Interactions between these polyphenols and various salivary
components, including MUC5B, have been shown to be responsible for the reduction in their
bioavailability. Previous studies have focused on the catechins, epicatechin (EC) and epigallocatechin
gallate (EGCG) and have shown that EGCG facilitates salivary mucin cross-linking resulting in their
aggregation in the fluid phase1,2. Additionally, these studies have shown that EC does not cause mucin
reorganisation to the same extent, concluding that the galloyl moiety is essential in the interaction
with mucin1. The theaflavins (denoted TF, TF1, TF2 and TF3) are well characterised3 however, their
effect on the mucus barrier has not yet been elucidated.
The influence of these polyphenolic compounds is investigated here. We expressed recombinant
protein domains of MUC5B, the N-terminal, C-terminal and cysteine-rich domain and exposed them
to the polyphenols. Sedimentation analyses indicated that the polyphenols interact with the protein
domains of MUC5B. We are currently investigating the interactions between these recombinant mucin
proteins and polyphenols by using QCM-D. We have immobilised the recombinant proteins via their
histidine tags onto SLBs formed on SiO2 sensors and have exposed them to tea polyphenols to
elucidate their effects on the adherent mucin layer.
1
Davies H. S., Pudney P. D. A., Georgiades P., Waigh T A., Hodson N. W., Ridley C. E., Blanch E. W. &
Thornton D. J. (2014) Reorganisation of the Salivary Mucin Network by Dietary Components: Insights
from Green Tea Polyphenols. PLOS One.
2
Georgiades P., Pudney P. D. A., Rogers S., Thornton D. J & Waigh T. A. (2014) Tea Derived
Galloylated Polyphenols Cross-Link Purified Gastrointestinal Mucins. PLOS One. Vol 9(8): p1-11.
3
Haslam E. (2003) Thoughts on thearubigins. Phytochemistry. Vol. 64: p61-73.
Disentangling the roles of cholesterol and CD59 in pore formation by
intermedilysin
Ed Parsons1, Courtney Boyd2, Richard Smith3, John Seddon4, Oscar Ces4 and Doryen Bubeck2
1
London Centre for Nanotechnology, University College London, London WC1H 0AH, UK
Department of Life Sciences, Sir Ernst Chain Building, Imperial College London, London SW7 2AZ, UK
3
MRC Centre for Transplantation, King's College London, 5th Floor Tower Wing, Guys' Hospital,
London SE1 9RT, UK
4
Department of Chemistry, Imperial College London, London SW7 2AZ, UK
2
[email protected]
Cholesterol-dependent cytolysins comprise a class of pore-forming toxins which are secreted from
gram-positive bacteria and bind to the target membrane via a cholesterol recognition motif. Their
assembly proceeds from a soluble, monomeric form and results in the formation of a large,
homooligomeric -barrel pore, leading to lysis and eventual cell death. This dramatic remodelling
process occurs via the collapse of a pre-pore state in which two -helices in each subunit refold into
a transmembrane hairpin (TMH), allowing for the concerted formation of a huge membrane pore in
the absence of any external energy source1.
Intermedilysin (ILY) is an atypical member of this family, whereby ILY hijacks the human CD59 receptor
rather than binding cholesterol2. However, as cholesterol remains a requirement for lysis, it not clear
at what stage on the pathway towards pore formation CD59 and cholesterol respectively play a role.
Here, we use a biophysical approach utilizing model membrane systems in which we have control over
the presence of both CD59 and cholesterol. Combining vesicle lysis assays with visualisation by
electron microscopy and atomic force microscopy allows us to reveal how CD59 coordinates the ILY
prepore and triggers the collapse towards the membrane, whilst the role of cholesterol is likely limited
to the insertion of a transmembrane hairpin. We are therefore able to elucidate a structural timeline
for pore formation by ILY, and suggest a pathway in which the collapse of the prepore and insertion
into the membrane represent distinct mechanistic steps.
1.
Lukoyanova, N., Hoogenboom, B. W. & Saibil, H. R. The membrane attack complex, perforin and cholesteroldependent cytolysin superfamily of pore-forming proteins. Journal of Cell Science 129, 2125–2133 (2016).
2.
Johnson, S., Brooks, N. J., Smith, R. A. G., Lea, S. M. & Bubeck, D. Structural basis for recognition of the pore-forming
toxin intermedilysin by human complement receptor CD59. Cell Reports 3, 1369–1377 (2013).
Electrocatalytic properties of the full heterotrimeric membrane bound [NiFe]
hydrogenase of Ralstonia eutropha
Valentin Radu1, Stefan Frielingsdorf,2 Oliver Lenz,2 and Lars J. C. Jeuken1
1
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK
Institut für Chemie, Sekretariat PC14, Technische Universität Berlin, Straße des 17. Juni 135, Berlin,
10623, Germany
2
[email protected]
Hydrogenases engage Ni and Fe cofactors in a unique way to produce or oxidize H2.1 The catalytic
properties of hydrogenases have been investigated under both aerobic and anaerobic conditions to
uncover aspects regarding their (in)activation mechanism and their potential practical applications.
The two most important classes, the [NiFe] and the [FeFe] hydrogenases, comprise enzymes that can
convert H2 in both aerobic and anaerobic conditions. Oxygen tolerant membrane-bound [NiFe]
hydrogenases (MBHs) can sustain a high level of H2 oxidation activity in the presence of air. Protein
film electrochemistry has successfully probed enzymes from this sub-class in the form of
heterodimeric sub-complexes comprising the two hydrophilic subunits (α and β) by adsorbing them
directly to electrode surfaces. In order to investigate the full heterotrimeric protein complex including
the membrane integral cytochrome subunit we have used tethered bilayer lipid membranes (tBLMs)
attached to electrodes. This approach enables the use of native membrane extracts as well as purified
proteins. The present work describes the investigations of the electro-catalytic properties of the full
heterotrimeric O2 tolerant MBH from Ralstonia eutropha in equilibrium with the quinone pool. The
full protein complex was incorporated into tBLMs via cytoplasmic membrane extracts and as a
crosslinked construct. The data show that under anaerobic oxidizing conditions the heterotrimeric
MBH from R. eutropha reactivates faster than the hydrophilic sub-complexes of O2 tolerant MBHs.2, 3
It was also shown to fully reactivate in the presence of O2 at high electrode potential without requiring
reducing potentials.2 By employing the crosslinked MBH we have recently been able to investigate the
catalytic properties under aerobic conditions in the absence of interfering ubiquinol oxidases present
in membrane extracts. The data indicates that the MBH maintains a higher extent of the H2 oxidation
activity than previously estimated from experiments conducted on membrane extracts (unpublished
data).
1
Lubitz, W., Ogata, H., Rüdiger, O., and Reijerse, E., (2014). Hydrogenases. Chem. Rev. 114, 4081-4148.
2
Radu, V., Frielingsdorf, S., Evans, S. D., Lenz, O., and Jeuken, L. J. C. (2014). Enhanced Oxygen-Tolerance of the
Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha. J. Am. Chem. Soc. 136,
8512−8515.
3
Radu, V., Frielingsdorf, S., Evans, S. D., Lenz, O., and Jeuken, L. J. C. (2016). Reactivation from the Ni–B state in
[NiFe] hydrogenase of Ralstonia eutropha is controlled by reduction of the superoxidized proximal cluster.
Chem. Commun. 52, 2632-2635.
Chiral vortex dynamics on membranes is an intrinsic property of FtsZ, driven
by GTP hydrolysis
Ana Raso Alonso*1,3, Diego Ramirez*1,2, Daniela A. García-Soriano*1,2, Mario Feingold4, Germán Rivas3
and Petra Schwille#1
1
Department of Cellular and Molecular Biophysics, Max Planck Institute for Biochemistry, Martinsried,
82152, Germany
2
Graduate School for Quantitative Biosciences (QBM), Ludwig-Maximillians-University, Munich,
80539, Germany
3
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid,
28006, Spain
4
Department of Physics, Ben Gurion University, Beer Sheva, 8499000, Israel
*Contributed equally #Corresponding author: Petra Schwille
[email protected]
The primary protein of the bacterial Z ring guiding cell division, FtsZ, has recently been shown to
engage in intriguing self-organization together with one of its natural membrane anchors, FtsA. When
co-reconstituted on flat supported membranes, these proteins assemble into dynamic chiral vortices
whose diameters resemble the cell circumference. These dynamics are due to treadmilling polar FtsZ
filaments, supposedly destabilized by the co-polymerizing membrane adaptor FtsA, thus catalysing
their turnover. Here we show that FtsA is in fact dispensable and that the phenomenon is an intrinsic
property of FtsZ alone when supplemented with a membrane anchor. The emergence of these chiral
dynamic patterns is critically dependent on GTP concentration and FtsZ surface densities, in
agreement with theoretical predictions. The interplay of membrane tethering, GTP binding, and
hydrolysis promotes both, the assembly and the destabilization of FtsZ polymers, leading to the
observed treadmilling dynamics. Notably, the vortex chirality is defined by the position of the
membrane targeting sequence (mts) and can be inverted when attaching it to the opposite end of
FtsZ. This reveals a so far unknown vectorial character of these cytomotive filaments, comprising three
orthogonal directions: Filament polarity, curvature, and membrane attachment.
Eukaryotic cell surface saccharide-redox modulation: Is this a new cell
signalling mechanism?
Frankie Rawson
Division of Regenerative Medicine and Cellular Therapies, School of Pharmacy, University of
Nottingham.
[email protected]
The development and innovation of technology capable of forming a biocompatible interface between
cells and materials in their environment is of great significance for an array of applications, from
utilization as a research tool to inform biological investigations through to cell culture to tissue
engineering. All cells communicate with their external environment by externally transporting
electrons from inside the cell to their surrounding environment. It was thought that the only
mechanism by which this occurs is via transplasma membrane electron transport systems.
Importantly, there are no known reported examples of mammalian cells electrically communicating
with the external environment directly from the cell membrane. Herein we fabricate a molecularly
tailored surface functionalised with a saccharide binding motif, a phenyl boronic acid. The
functionalised surface facilitated the transfer of electrons, via unique electronic interactions mediated
by the presence of the boronic acid, from a macrophage cell line. We hypothesis that is the first
example of eukaryotic cellular-electrical communication mediated by the binding of cells via their
cell–surface saccharide units.
Nanomechanical characterisation of nuclear pore complexes
George Stanley1, Aizhan Bestembayeva1, Dino Osmanovic1, Ariberto Fassati2, Bart Hoogenboom1,
1
London Centre for Nanotechnology, UCL, London, WC1H 0AH, UK
Wohl Virion Centre, Windeyer Institute, UCL Medical School, London, W1E 6BT, UK
2
[email protected]
The nuclear pore complex (NPC) is the gateway through which molecules transport between the
nucleus and the cytoplasm. It selectively mediates the exchange of materials between the two
compartments, such as mRNA, tRNA and ribosomal subunits (making the NPC implicit in the gene
expression pathway), as well as viruses. Whilst small molecules (diameter < 5 nm) can passively
diffuse through the NPC, larger molecules (up to ~ 39 nm) must bind to nuclear transport receptors
and undergo a facilitated transport process.
The architectural structure of the NPC has been well characterised by various crystallographic
techniques and cryo-electron microscopy, and has revealed a ring shaped structure (the scaffold)
with an eight-fold rotational symmetry, surrounding a central channel. However, the selective
transport barrier - which lies deep inside the central channel and is formed of disordered proteins,
known as phenylalanine-glycine nuclear pore proteins (FG-Nups) – has not been well characterised,
and is therefore poorly understood. It has been hypothesised that, inside the central channel, these
disordered FG-Nups either interact to form a cohesive meshwork (or hydrogel), which must be
dissolved by the nuclear transport receptors to facilitate the translocation of large molecules; or,
that the FG-Nups form a more dynamic, disordered polymer brush, thereby providing a
predominantly steric barrier to transport.
In this study, we use atomic force microscopy (AFM) in solution to probe the Xenopus Oocyte NPC in
vitro. We elucidate the nanomechanical properties of the NPC central channel, and generate a
stiffness cross-section of the NPC as a whole1. The results show greater stiffness at the ring (scaffold)
of the NPC, and inside its central channel, as compared to the rest of the NPC and the surrounding
membrane. Upon a quantitative comparison between polymer models and the stiffness curves
rendered from the NPC central channel, we find that the transport barrier is more cohesive, rather
than a loosely interacting polymer brush. This implies that the FG-Nups form a cross-linked polymer
network inside the NPC’s central channel, and that nuclear transport factors must modulate the
interactions between these FG-Nups to allow the facilitated transport of larger molecules, such as
virus capsids.
To facilitate the acquisition of such nanomechanical data under various conditions, we currently
employ higher-throughput nanomechanical characterisation methods for AFM, and validate them
against our previous results.
1.
Bestembayeva, A. et al. Nanoscale stiffness topography reveals structure and mechanics of
the transport barrier in intact nuclear pore complexes. Nat. Nanotechnol. 10, 60–64 (2014).
Microenvironments created by liquid-liquid phase transition control the
dynamic distribution of bacterial division FtsZ protein
Marta Sobrinos-Sanguino1, Begoña Monterroso1, Silvia Zorrilla1, Christine D. Keating2, Germán Rivas1
1
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Consejo
Superior de Investigaciones Científicas, (CSIC), Madrid, 28040, Spain
2
Department of Chemistry, Pennsylvania State University, Pennsylvania, 16802, USA
[email protected]
Cells are complex entities spatially organized into compartments in which proteins and other
metabolites perform specialized functions [1]. The existence of these microenvironments in both
eukaryotic and prokaryotic organisms is now widely recognized and, when membraneless,
hypothesized to be formed by phase separation. These subcompartments provide distinct
environments with particular physicochemical properties in which proteins and other biomolecules
may differentially partition. This can potentially affect their functionality [1-3] and therefore the
biological processes in which they are involved. One of such processes is bacterial cell division, where
the GTP-dependent assembly cycle of the essential protein FtsZ is crucial for the formation of the
division ring at midcell, highly regulated in time and space [4]. Here, liquid/liquid phase separation
(LLPS) systems were used to mimic intracellular environments to study their effects on polymerization
and localization of division proteins. We found that crowding-induced liquid/liquid phase separation
strongly influences the dynamic spatial organization of FtsZ, which accumulates in one of the phases
and/or at the interface between both components, depending on the system composition and on the
association state of the protein. These experiments were performed by fluorescence spectroscopy
and confocal microscopy, in bulk LLPS and in lipid-stabilized, phase-separated aqueous microdroplets
[5]. The results suggest that the existence of these dynamic compartments can alter the local
concentration and reactivity of FtsZ during the cell cycle acting as nonspecific modulating factors of
division.
[1] Holthuis, J.C.
Ungermann, C. Cellular microcompartments constitute general suborganellar functional units in cells. Biol Chem 394, 151161 (2013).
[2] Amster-Choder, O. The compartmentalized vessel: The bacterial cell as a model for subcellular organization (a tale of
two studies). Cell Logist 1, 77–81 (2011).
[3] Brangwynne, C. P. Phase transitions and size scaling of membrane-less organelles. J Cell Biol 203, 875–881 (2013).
[4] Haeusser, D.P. & Margolin, W. Splitsville: structural and functional insights into the dynamic bacterial Z ring. Nat Rev
Microbiol 14, 305-319 (2016).
[5] Monterroso, B., Zorrilla, S., Sobrinos-Sanguino, M., Keating, C.D., Rivas, G. Microenvironments created by liquid-liquid
phase transition control the dynamic distribution of bacterial división FtsZ protein. Sci Rep 6: 35140 (2016)
How does molecular composition affect biophysical features and functions of
perineuronal nets?
Luke Souter1, Richard M. Hall1, Ralf P. Richter2,3,4, Jessica C.F. Kwok5
1
School of mechanical engineering, University of Leeds, Leeds LS2 9JT, UK
Biosurfaces Lab, CICbiomaGUNE, Donostia-San Sebastian, 20009, Spain
3
Université Grenoble Alpes - CNRS, Laboratoire Interdisciplinaire de Physique (LIPhy), 38402 Saint
Martin d’Hères, France
4
School of Biomedical Sciences and School of Physics and Astronomy, University of Leeds, Leeds, LS2
9JT, UK
5
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK
2
[email protected]
Perineuronal nets, are molecular assemblies of extracellular matrix molecules tethered to the
surface of a defined population of neurons, regulating plasticity in the central nervous system (CNS).
Enzymatic removal of perineuronal nets reactivates plasticity which improves recovery from spinal
cord injury and memory retention in mouse model of Alzheimer’s disease. This makes them an
interesting therapeutic target for enhancing neuronal repair and regeneration.
This project aims to understand how the formation of perineuronal nets change the
microenvironment and local mechanics of the neuronal surface, therefore affecting neuronal
behaviour.
The structure of perineuronal nets are condense and compact, different to other matrices of similar
chemical composition that are considered softer. Interestingly the CNS is one of the softest organs in
the body and is sensitive to mechanical changes.
Initially, regional differences of perineuronal nets in the brain will be characterised using
immunohistochemistry, glycan isolation and size-exclusion chromatography. Then the biophysical
properties will be measured using neuronal cultures, atomic force microscopy and imaging. The
biophysical and biochemical analyses will then be correlated and used to create a biomimetic
surface that simulates perineuronal nets with tuneable features. This will allow neuronal behaviour
to be studied in relation to changes in biophysical properties at their surface.
A probe will also be designed to image perineuronal nets in vivo using MRI.
This work will help identify how biophysical properties affect the CNS, which may eventually be used
towards translational therapies for spinal cord injury and Alzheimer’s disease.
Engineering bicontinuous cubic phases approaching nature's lengthscales
Arwen I.I. Tyler1,2, Hanna M.G. Barriga1, Edward S. Parsons1,3, Nicola L.C. McCarthy1, Oscar Ces1,
Robert V. Law1, John M. Seddon1, Nicholas J. Brooks1.
1
Department of Chemistry, Imperial College London, London, SW7 2AZ, UK
School of Food Science and Nutrition, University of Leeds, LS2 9JT, Leeds, UK
3
London Centre for Nanotechnology, University College London, London, SW7 2AZ, UK
2
[email protected]
Lipid bicontinuous cubic phases have attracted enormous interest as biocompatible scaffolds for use
in a wide range of applications including membrane protein crystallisation, drug delivery and
biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an
inability to create targeted, highly swollen bicontinuous cubic structures with large and tunable pore
sizes. In contrast, cubic structures found in vivo have periodicities approaching the micron scale.
Electrostatic and fluctuation interactions can swell lamellar phases however much less is known about
the bicontinuous cubic phases. We have engineered highly swollen (up to 48 nm) bicontinuous cubic
phases by increasing the bilayer stiffness whilst still keeping it fluid by adding cholesterol, inducing
electrostatic interactions by adding charged lipids to monoolein and applying hydrostatic pressure. By
applying these techniques we have managed to exceed the predicted maximum lattice parameter
swelling from calculations which suggest that thermal fluctuations should destroy such phases for
lattice parameters larger than 30 nm and have generated lattice parameters 5 times larger than pure
monoolein and nearly twice the size of any lipidic cubic phase reported previously.
By being able to control the structural properties of these systems by varying the composition,
temperature and pressure as well as understanding the physicochemical properties and interactions
that underpin this unprecedented structural swelling we hope to pave the way to developing
engineering rules for generating highly swollen structures as well as for using them for a wide range
of biotechnical applications such as biocontainment and delivery, tuneable photonic devices, high
surface area catalysts and scaffolds for in-cubo crystallization of large membrane proteins.
Artificial Cyanobacteria
Will Shewanella play the role?
Anna Wroblewska-Wolna1, Andrew Harivie1, Kevin Critchley2 and Lars Jeuken 1,
1
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT, UK
2
School of Physics and Astronomy, University of Leeds, LS2 9JT, UK
E-mail: [email protected]
Induced photosynthesis in non-photosynthetic bacteria is an intriguing concept in biotechnology. Here
we aimed to couple Shewanella oneidensis MR-1 (MR-1), a Gram-negative, facultative anaerobic
bacterium, to light harvesting quantum dots (light emitting semiconductor nanoparticles, QD) with
the ultimate aim to inject light-excited electrons into MR-1 metabolism. By using set of cytochromecontaining proteins, MR-1 exports respiratory electrons extracellularly to insoluble electron acceptors
(e.g. hematite). By reversing this electron flow using a variety of QDs to stimulate MR-1 to use her
own hydrogenases for photosynthetically-driven production of hydrogen.
Interaction between QDs and MR-1 and potential toxicity of nanoparticles were determined up to
concentrations of up to 5 μM. Three types of QD: CuInS2/ZnS/PMAL, commercial and custom made
CdTe QD were studied. Nano-toxicology experiment showed that the CuInS2 QD are non-toxic to MR1, while the two Cd based QD show minor reduction in viability at concentration of 0.05 μM and
higher. Fluorescent microscopy was employed to monitor interaction between MR-1 and QDs.
Although almost no interaction is present with custom QD, strong interactions were recorded for the
CuInS2 and commercial CdTe nanoparticles. Further steps will include optimisation of QD surface
chemistry while the production of hydrogen will be evaluated by electrochemistry tools.