Jieqiong Lou

Correlated histone FLIM-FRET and SPT microscopy: a method to determine nuclear protein dynamics within the chromatin nanoscale landscape.

Jieqiong Lou, Ashleigh Solano, & Elizabeth Hinde

School of Physics & Department of Biochemistry and Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia.

The nanometre spacing between nucleosomes, which are folded together throughout live cell chromatin organisation, regulates DNA binding interaction and navigation of the nuclear landscape. This nanoscale feature of chromatin structure is however ‘invisible’ to diffraction limited light microscopy. Thus, direct observation of the regulatory impact nucleosome proximity has on live cell DNA binding protein dynamics has to date been a difficult task. In recent work we established the phasor approach to histone FLIM-FRET microscopy as a method to spatially map nucleosome spacing in a living cell (J Lou et al., 2019 and L Zhen et al., 2020), and here we couple this super-resolved readout of chromatin structure, with a single particle tracking (SPT) analysis of nuclear protein DNA target search behaviour (AJ McCann and J Lou et al., NAR, 2021). Collectively, this multiplexed microscopy approach provides direct insight into the reciprocal interplay between chromatin nanoscale architecture and nuclear protein DNA binding dynamics.

  1. Lou J, Scipioni L, Wright BK, Bartolec TJ, Zhang J, Masamsetti VP, Gaus K, Gratton E, Cesare AJ and Hinde E. 2019. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE. 116(15): 7323.

  2. Liang Z, Lou J, Scipioni L, Gratton E and Hinde E. 2020. DATA IN BRIEF. 30(105401).

  3. McCann A*, Lou J*, Moustaqil M, Blum A, Fontaine F, Liu H, Koopman P, Sierecki E, Gambin Y, Meunier FA, Liu Z, Hinde E and Francois M. 2021, Nucleic Acids Research. gkab820 * Co-first authors.

 

Richard Spinney

Bivalent Kinetics: Insights from Many Body Physics

Richard E. Spinney(1,2), Lawrence Lee(2) & Richard G. Morris(1,2)   

1. School of  Physics, University of New South Wales - Sydney  2052,  Australia

2. EMBL-Australia node in Single Molecule Science, School of Medical Sciences, University of New South Wales -  Sydney 2052, Australia

 

The kinetics of bivalent entities is important for a wide variety of topics in cell and molecular biology.  It is also increasingly relevant for the design and engineering of novel nano-scale soft systems, either bio-mimetic or otherwise.  Here, we draw upon classical techniques of statistical physics to revisit bivalent kinetics; in particular, the tenet that patterns of receptor sites can dictate the kinetic response to changes in bulk concentration.  Recasting the problem in terms of many-body coordination and geometrical frustration, we explore extended, translationally-invariant chains and lattices of receptor sites, and discuss their possible applications and/or realisations.  Our results enable us to distil core principles for the rational design of concentration-dependent kinetics in synthetic soft-systems, and also reveal the possibility of other tunable spatio-temporal features, such as correlation lengths, mean-squared displacements and percolation-like transitions.

 
 

James Brown

Multi-site competitive exchange of DNA and protein subunits on DNA origami

James Brown(1)^, Rokiah Alford(1)^, James Walsh(1), Richard Spinney(1), Stephanie Xu(1), Sophie Hertel(1), Jonathan F. Berengut(1,2),  Lisanne Spenkelink(3), Antoine Van Oijen(3), Till Böcking(1), Richard Morris(1) & Lawrence K. Lee(1,4)

1. EMBL Australia Node for Single Molecule Science, School of Medical Sciences, UNSW Sydney, 2052, Australia.

2. School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia

3. Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, New South Wales 2522, Australia

4. ARC Centre of Excellence in Synthetic Biology, University of New South Wales, Sydney, Australia

The rapid exchange of protein subunits in otherwise stable macromolecular complexes is a key feature of cellular regulation. The fundamental thermodynamic processes by which protein complexes can be formed through a number of weak interactions have been well defined. However, a general mathematical treatment of the implication of this multi-valency for rapid subunit exchange has only recently been described. Significantly, the presence of additional free binding sites within the complex greatly reduces the concentration of free protein required to increase the rate of exchange, in a model termed multi-site competitive exchange (MSCE). Here, we design and test the first experimental system based on this model and show that we can predictably exchange DNA and protein cargo on a DNA origami template, reducing the timescale of exchange within complexes from ~25 days to minutes. We show that individual kinetic parameters can be tuned in a controlled manner and can be fully parametrised independently. This precise nanometre positioning of molecules in space across timescales is broadly applicable in the field of nanotechnology and in particular for the concentration-dependent exchange of protein subunits on DNA origami without the need for covalent DNA modification.

Gurleen Kaur

Enzyme dynamics during lesion bypass at the e. coli replication fork

Gurleen Kaur(1,2), Jacob S. Lewis(1,2), Lisanne M. Spenkelink(1,2), Slobodan Jergic(1,2), Nicholas E. Dixon (1,2), & Antoine M. van Oijen(1,2)

1. School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia;

2. Illawarra Health and Medical Research Institute, Wollongong, New South Wales 2522, Australia

 

While duplicating DNA the replisome encounters a multitude of roadblocks, such as DNA lesions. The replisome needs to be able to efficiently bypass these lesions to ensure replication fidelity and genome stability. Conventional biochemistry methods have been used to study how lesions are bypassed on the template DNA by the replisome [1-3]. The proposed models of lesion bypass do not account for the inherent plasticity of the replisome where proteins can dynamically exchange into the replisome in a manner dependent on their concentration [4, 5]. We aim to study the effect of protein dynamics on lesion bypass using single-molecule fluorescence microscopy. Currently there are no suitable DNA substrates containing site-specific lesions suitable to study Escherichia coli DNA replication. To determine the molecular details of replisome bypass of template DNA lesions at the single-molecule level, a linear DNA template containing site-specific lesions is required. We have designed and constructed a modular linear DNA substrate containing a site-specific lesion that is readily visualized with single-molecule resolution. Moreover, using E. coli replisomes reconstituted from purified proteins we have observed rates of replication on this template consistent with previous studies. By using fluorescently labeled polymerases, we can simultaneously visualize DNA replication and polymerase dynamics upon lesion collision. For the first time, this single-molecule assay allows for investigation of the interplay between leading-strand lesion bypass and polymerase exchange dynamics.

  1. W. D. Rupp, P. Howard-Flanders, 1968, ‘Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation’, J. Mol. Biol. 31, 291–304.

  2. J. T. Yeeles, K. J. Marians, 2011 ‘The Escherichia coli replisome is inherently DNA damage tolerant’,  Science, 334, 235–238.

  3. J. T. Yeeles, K. J. Marians, 2013, ‘Dynamics of leading-strand lesion skipping by the replisome’, Mol. Cell, 52, 855–865.

  4. Beattie, T.R., Kapadia, N., Nicolas, E., Uphoff, S., Wollman, A.J., Leake, M.C. and Reyes-Lamothe, R., 2017, ‘Frequent exchange of the DNA polymerase during bacterial chromosome replication’, eLife, 6, e21763.

  5. Lewis, J.S., Spenkelink, L.M., Jergic, S., Wood, E.A., Monachino, E., Horan, N.P., Duderstadt, K.E., Cox, M.M., Robinson, A., Dixon, N.E., and van Oijen, A.M., 2017, ‘Single-molecule visualization of fast polymerase turnover in the bacterial replisome,’ eLife, 6, e23932.

 

Elaine Tao

Size and drug accessibility of fenestrations in voltage-gated sodium channel subtypes

Elaine Tao & Ben Corry

Research School of Biology, Australian National University, Canberra, Australia

 

Voltage-gated sodium channels (Nav) are critical membrane proteins that underlie the electrical activity of nerve and muscle cells. Humans have nine different subtypes of these channels, which are the targets of small molecule inhibitors commonly used to treat a range of conditions, such as pain, epilepsy and cardiac arrhythmias. Structural studies have identified four lateral fenestrations within the Nav pore module that have been shown to influence Nav pore blocker access during resting-state inhibition. However, the structural differences between the nine subtypes are still unclear. In particular, the dimensions of the four individual fenestrations across the Nav subtypes and their differential accessibility to pore blockers is yet to be characterised.

 

To address this, we applied classical molecular dynamics simulations to study the recently published structures of Nav1.1, Nav1.2, Nav1.4, Nav1.5 and Nav1.7. While there is significant variability in the bottleneck sizes of the Nav fenestrations, the subtypes follow a common pattern with wider DI-II and DIII-IV fenestrations, a more restricted DII-III fenestration and the most restricted DI-IV fenestration. We further identify the key bottleneck residues in each fenestration and show that the motions of aromatic residue sidechains govern the bottleneck radii. Well-tempered metadynamics simulations of Nav1.4 and Nav1.5 in the presence of the pore blocker lidocaine also support the DI-II fenestration being the most favourable access route for drugs. Our computational results provide a foundation for future in vitro experiments examining the route of drug access to sodium channels. Understanding the fenestrations and their accessibility to drugs are critical for future analyses of diseases mutations across different sodium channel subtypes, with the potential to inform pharmacological development of resting-state inhibitors and subtype-selective drug design.

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Ian Chin

Cardiac Cell Mechanosensation in 3D Stiffness Gradient Hydrogels

Ian Chin(1), Livia Hool(1,2) & Yu Suk Choi(1)

1. School of Human Sciences, The University of Western Australia, Perth, WA, Australia

2. Victor Chang Cardiac Research Institute, Sydney, NSW

 

During the development of the heart and the progression of heart disease, cardiomyocytes are exposed to a myriad of biomechanical and biophysical signals as the heart undergoes extracellular matrix (ECM) remodelling. Past studies have used 2D cell culture platforms to show that cardiomyocytes can sense and respond to these signals, resulting changes to cell morphology and to gene expression, and with the emergence of new biomaterials, we can begin to probe cardiac cell mechanosensation in a 3D microenvironment. To this end, we encapsulated and cultured H9C2 cardiac-derived cells in photopolymerized gelatin methacryloyl (GelMA) hydrogels. A photomask was used to pattern these hydrogels with a 3.7-19.1 kPa linear stiffness gradient, which our 2D studies had previously found was a critical range where stiffness could drive marked changes to H9C2 cell morphology and mechanosensitive protein expression. After 10 days of culture inside the hydrogels, we observed no significant changes in cell volume or shape across the stiffness gradient. Immunofluorescent image analysis was used to examine the expression of the mechanomarkers YAP, MRTF-A, and lamin-A in addition to the focal adhesion proteins paxillin and talin. YAP and MRTF-A were primarily localized to the nucleus at all stiffnesses and there were no detectable trends in lamin-A, paxillin or talin expression. These results are consistent with past observations from our own lab and from others, who have hypothesized that a cell’s capacity to expand is the major determinant of cell phenotype in a 3D microenvironment. Our findings illustrate an exciting opportunity to deepen our understanding of cardiomyocyte mechanobiology as we explore novel applications of emerging biomaterials.

ianchin.PNG
 

Michelle Xu

Diverse genetic background of individuals contribute to variable electrical phenotype to proarrhythmic drugs

Michelle J.O. Xu (1,2), Jamie I. Vandenberg (1,2), Matthew D. Perry (1,2,3) & Adam P. Hill (1,2)

1. Victor Chang Cardiac Research Institute (Sydney, Australia)
2. St. Vincent’s Clinical School, UNSW Sydney (Sydney, Australia)
3. School of Medical Sciences, UNSW Sydney (Sydney, Australia)

Background and Introduction: Over the past 30 years, a range of structurally unrelated therapeutics have been withdrawn from the market due to risk of fatal heart rhythm disturbances1. The underlying phenomenon, known as drug-induced long QT syndrome (diLQTS), occurs due to block of the human ether-a-go-go related gene (hERG) potassium channel in cardiomyocytes2,3. The pro-arrhythmic risk associated with diLQTS varies greatly between individuals taking the same drug, indicating a genetic contribution. Sex differences and variability in the expression of genes involved in electrical signaling in the heart (the ‘rhythmonome’) are thought to contribute, but the extent to which these modify the pro-arrhythmic risk hasn’t been fully explored. 

Aim and Methods: This study used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) to investigate the extent of variability in electrical phenotype and proarrhythmic risk observed between individuals in response to a panel of drugs with different pharmacological profiles. Electrical phenotypes of both spontaneous and paced data were assessed using a multi-electrode array (MEA). Additionally, RNA was collected to measure expression differences in the rhythmonome to observe any correlations between genetic background and electric phenotypic response.

Results: Measurements of field potential duration (FPDc) demonstrates that the electrical phenotype varies between hiPSC-CM lines, both at baseline and in response to drugs. hiPSC-CM from females showed prolonged FPDc compared to males. Functional responses to each drug were different between iPSC lines from different individuals and the extent of the differences varied depending on the pharmacological profile of the drug. In silico models showed that the phenotypic response to drugs that act as multi-channel blockers (i.e. hERG block + calcium channel block) were not simply additive when compared to single channel blockers.

Conclusion: Baseline phenotype did not predict drug response, most likely due to varying levels of rhythmonome genes between individuals. Variable inter-individual differences in response to single channel blockers and multi-channel blockers illustrates the importance of safety screening against a range of ion channels during drug development.

  1. Roden, D. M. (2004). New England Journal of Medicine, 350: 1013–1022 

  2. Perrin et al. (2008) Progress in Biophysics and Molecular Biology: 1–12

  3. Kannankeril et al. (2010). Pharmacological Reviews, 62: 760–781

michellexu.png

Figure 1: Example trace of electrical phenotype pre- (B) and post-drug (C1). Corrected FPD is calculated using Fredericia’s formula.

 

Yunduo Zhao

N-terminal autoinhibitory module of A1 domain in von Willebrand factor stabilizes the mechanosensor catch bond

Yunduo Charles Zhao(1,2)*, Haoqing Wang(1,3), Yao Wang(4) & Lining Arnold Ju(1,2,3,5)*†,

1. School of Biomedical Engineering, Faculty of Engineering, The University of Sydney, NSW 2006, Australia.

2. Charles Perkins Centre, The University of Sydney, NSW 2006, Australia

3. Heart Research Institute, Camperdown, NSW 2006, Australia

4. Cellular and Genetic Medicine Unit, School of Medical Sciences, University of

New South Wales, NSW 2052, Australia

5. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, United States

*These authors contribute equally.

†Corresponding author: Lining Arnold Ju. Email: arnold.ju@sydney.edu.au

 

Introduction: As the Jedi knight in blood plasma, von Willebrand factor (VWF) interacts with glycoprotein Ibα on platelets under elevated hemodynamic force to regulate haemostasis and thrombosis (light or dark side of the force) (1). Clinically, targeting VWF-A1–GPIbα axis represents a novel antithrombotic therapeutic strategy (1,2). Element regulating VWF biomechanical activation predominantly resides in autoinhibitory modules (AIM) flanking its A1 domain. However, how AIM sequences regulate VWF-A1–platelet binding remains controversial.

Methods: To address this, for the first time we performed molecular dynamics (MD) simulation to predict the N-terminal AIM (N-AIM; residues Q1238-E1260) and C-terminal AIM (C-AIM; residues p1467-N1493) structure. Furthermore, the biomembrane force probe and microfluidic perfusion assays were employed to experimentally investigate the role of N-AIM in molecular and cellular scale, respectively (Figure 1, top).

Results: In the MD simulation, the N-AIM cooperated with C-AIM to form a joint Rotini-like structure thereby partially autoinhibit VWF-A1–GPIbα interaction. Experimental results reflected that VWF-A1 containing N-AIM sequence (1238-A1) exhibits more stable binding states with stronger interaction to platelets over increasing force (catch bond), whereas short N-AIM A1s start at residue D1261 (1261-A1) displayed bi-variable force-strengthen and force-weaken interaction with platelets. Moreover, under harsh environments (i.e. -80°C low temperature or 9.6 high pH level), the N-AIM acted as the Jedi knight’s lightsaber to protect and stabilise the VWF-A1 structure (Figure 1, bottom). Taken together, our novel 3M multidisciplinary approaches provide new mechanobiology on how N-AIM regulates and stabilises VWF-A1 activities, which inspires future VWF-A1 dependent antithrombotic approaches.

1. Rana, A., Westein, E., Niego, B. & Hagemeyer, C. E. Shear-Dependent Platelet Aggregation: Mechanisms and Therapeutic Opportunities. Front Cardiovasc Med 6, 141, doi:10.3389/fcvm.2019.00141 (2019).

2. Wang, Q. et al. Structural and functional basis of SARS-CoV-2 entry by using human ACE2.  (2020).

3M multidisciplinary approaches reveal the structural and functional basis of VWF-A1 N-AIM.

yundaozhao.png
 

Natasha Freidman

SLC1A transporters can be pharmacologically trapped at varying stages of the transport cycle

Natasha Freidman*, Chelsea Briot*, Renae Ryan

School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, NSW, Australia

*Authors contributed equally to this work.

 

The Solute Carrier 1A (SLC1A) family comprises a group of membrane proteins that act as dual-function amino acid transporters and chloride channels. It includes the alanine serine cysteine transporters (ASCTs) and the human glutamate transporters known as excitatory amino acid transporters (EAATs). ASCT2 can transport a range of neutral amino acids into cells including glutamine. It is upregulated in a range of solid tumours and is regarded as a promising target for cancer therapy. L-γ-glutamyl-p-nitroanilide (GPNA) is a compound widely used in studies probing the role of ASCT2 in cancer biology. Here, we demonstrate that GPNA activates the chloride conductance of ASCT2 to the same extent as a transported substrate, whilst not undergoing the full transport cycle. This is a previously unreported phenomenon for compounds in the SLC1A family and corroborates a body of literature suggesting that the structural requirements for transport are distinct from those for chloride channel formation. We also show that in addition to its currently known targets, GPNA inhibits several of the glutamate transporters (EAATs). Together, these findings raise questions surrounding the true mechanisms of its anticancer effects.

 

Daryan Kempe

Dynein-mediated retrograde forces shape cytotoxic synapse topography to enhance target perforation

Matt A. Govendir(1)*, Daryan Kempe(1)*, Setareh Sianati(1), Kate Poole(1,2,3), Jessica Mazalo(1), Feyza Colakoglu(1) & Maté Biro(1,2) 

 1. EMBL Australia, Single Molecule Science node, School of Medical Sciences, University of New South Wales, Sydney, Australia

2. Cellular and Systems Physiology, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia

3. ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia

* These authors contributed equally

Cytotoxic T lymphocytes (CTLs) are able to eliminate virally infected or cancerous cells, a process critically depending on the disruption of the target cell membrane through the secretion of the pore former perforin(1). Perforin is stored in lytic granules which, upon CTL activation, polarize towards the cytotoxic immunological synapse (CIS)(2), a dynamic interface characterized by the circumferential assembly(3) and concurrent central disassembly(4) of F-actin. Here, using a dual-pipette aspiration assay in combination with fluorescence microscopy and computational image analysis, we explore the role of CTL-intrinsic forces in lytic granule delivery and target perforation at the CIS. We find that Rho-associated protein kinase (ROCK) dependent contractions of the cell rear trigger an increase in CTL surface tension that is correlated with the anterograde transport of cytolytic granules towards the nucleus. Despite the tension increase, simultaneous retrograde, dynein-mediated forces cause the CTL membrane to be concave within the F-actin-cleared zone of the synapse. In line with this, cytolytic granules are transported to pockets of negative membrane curvature that are opposite of positively curved bulges on the target cell membrane. Inhibition of dynein impairs degranulation pocket formation and target perforation. Using high-speed pressure clamp electrophysiology, we uncover a curvature bias in the action of perforin, which preferentially perforates convex tumour cell membrane patches. Together, our findings reveal an intricate coordination of cytoskeletal forces and modulation of synaptic topography to enhance target cell perforation by T cells.

 

1. Voskoboinik, I., Whisstock, J.C. & Trapani, J.A. Perforin and granzymes: function, dysfunction and human pathology. Nat Rev Immunol 15, 388-400 (2015).

2. Stinchcombe, J.C., Majorovits, E., Bossi, G., Fuller, S. & Griffiths, G.M. Centrosome polarization delivers secretory granules to the immunological synapse. Nature 443, 462-465 (2006).

3. Le Floc'h, A. et al. Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse. J Exp Med 210, 2721-2737 (2013).

4. Ritter, A.T. et al. Actin depletion initiates events leading to granule secretion at the immunological synapse. Immunity 42, 864-876 (2015).

 

Waleed Mirza

Active self-organization in cortex

Waleed Mirza(1), Alejandro Torres-Sanchez(2), Marco De Corato(3), Guillermo Vilanova(4), Marco Pensalfini(5) & Marino Arroyo(6)

 1. Barcelona Graduate School of Mathematics (BGSMath), Campus de Bellaterra, Edifici C 08193 Barcelona, Spain.

2,4,5,6. LaCaN, Universitat Politecnica de Catalunya BarcelonaTech, 08034 Barcelona, Spain.

3. Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology(BIST), Baldiri Reixac 10-12, 08028 Barcelona, Spain.

Actin networks exhibit a variety of architectures that contribute to different cellular functions. Notably, the actin cytoskeleton can adopt a nematic order, with aligned actin filaments of mixed-polarity associated with myosin-II and other actin-bundling proteins. These nematic actin bundles conform to a variety of contractile structures at the sub-cellular and supra-cellular levels. While biological literature emphasizes the morphological, dynamical, molecular and functional specificities of each of these families of bundles, observations across cell types also suggest that nematic strands emerge as a result of self-organization of the active actomyosin gel. To test this idea, we develop here a continuum active gel model accounting for orientational order, in which order is promoted by flow and active power input and controls anisotropic active tensions. By performing linear stability and fully nonlinear simulations, we show how activity can drive the formation of a variety of out-of-equilibrium patterns reminiscent of those observed in in vitro reconstituted systems and cells such as transverse and radial stress fibers, asters, nematic tactoids, sarcomeres and the cytokinetic ring during cell division. Finally, we verify the behavior of the proposed model against discrete network simulations.

 

Emelie Flood

 Molecular dynamics simulations to elucidate the origins of rapid inactivation in hERG

Emelie Flood(1), Carus Lau(2,3), Mark Hunter(2,3), Chai Ng(2,3), James Bouwer(4), Alastair Stewart(2,3), Eduardo Perozo(5), Jamie Vandenberg(2,3) & Toby Allen(1)

1. RMIT University

2. Victor Chang Cardiac research Institute

3. University of NSW

4. University of Wollongong

5. University of Chicago

 

The human ether á-go-go related gene (hERG) encodes the delayed rectifier potassium channel (Kv11.1), best known for mediation of the repolarising current in the cardiac action potential. Loss of function of hERG can lead to long QT-syndrome which causes cardiac arrhythmias and sudden cardiac arrest. hERG is susceptible to a wide range of drugs which can lead to drug induced long QT-syndrome. Here, we investigate the structural basis of hERG inactivation using a combination of free and enhanced simulations to analyse new cryo-EM structures of hERG, captured in a conductive and an inactivated state, which is characterised by flipped Val625 carbonyls. We show that hERG has a destabilised conductive state, as well as reduced free energy barrier for Val625 flipping, that is voltage dependent. Furthermore, multi and single ion occupancies are unable to keep the Val625 in a conductive state. It is this propensity for flipping that leads to the fast and voltage dependent inactivation in hERG. Furthermore, we show that the conductive state relies on a network of interactions behind the selectivity filter, where Ser620, unique to hERG, plays a key role in regulating the balance of conductive and inactivated states. When ions leave the outer selectivity filter, this balance is shifted, and the channel inactivates, explaining the sensitivity to extracellular K+ concentration. Together with mutagenesis experiments, these simulations provide molecular-level understanding of hERG inactivation, with considerable clinical and pharmacological potential.

 

Lucy Ham

Unravelling the correlation structure of noise in molecular pathways

Lucy Ham(1), Marcel Jackson(2) & Michael P. H. Stumpf(1)

 1. School of BioSciences and School of Mathematics and Statistics, University of Melbourne, Australia

2. School of Mathematics and Statistics, La Trobe University, Australia

 

Noise is ubiquitous at the molecular scale, and its presence has profoundly shaped cellular life.  Understanding the sources of noise, how it is propagated, amplified, and attenuated has therefore become a cornerstone of modern cellular biophysics. Often seen as a nuisance, we here show how the correlation structure of noise can be exploited to dissect the molecular machinery underlying cellular processes.

Noise leads to significant heterogeneity between cells subject to identical conditions, observable in “snapshot” distributions of molecule (e.g., transcript) numbers across ensembles of cells. Because temporal information is lost in such data, we are presented with significant challenges for inferring the molecular mechanisms underlying gene transcription, as well as the causes of cell-to-cell variability. In particular, we typically cannot separate dynamic variability from within cells (“intrinsic noise") from variability across the population (“extrinsic noise"). By developing stochastic models of gene expression that describe both intrinsic and extrinsic noise (Fig. A), we prove that it is in general impossible to identify the sources of variability, and consequently, the underlying transcription dynamics, from observed transcript abundance distributions alone. Systems with intrinsic noise alone can always present identically to similar systems with extrinsic noise.

 

Using these results, we identify new experimental set-ups that can assist in resolving this non-identifiability. We show that multiple generic reporters from the same biochemical pathways (e.g., mRNA and protein) can infer magnitudes of intrinsic and extrinsic transcriptional noise, identifying sources of heterogeneity (Fig. B). We validate this approach for restoring identifiability using synthetic data for genes with non-trivial gene expression dynamics; “pathway-reporter" approaches are remarkably robust to the details of the mRNA and protein synthesis dynamics. Stochastic simulations show further that pathway reporters compare favourably to the well-known, but often difficult to implement, dual-reporter methods. While experimental design has often played a subsidiary role in single-cell transcriptomics, our approach shows how the correlated structure of noise can be used to gain deeper mechanistic insights into molecular processes from snapshot data.

 

L. Ham, M. Jackson, M.P.H Stumpf, Pathway dynamics can delineate the sources of transcription noise in gene expression, eLife (to appear), 2021

lucyham.png
 

Ashley Rozario

Characterizing drug-induced microtubule filament dysfunction using super-resolution microscopy

Ashley M. Rozario(1), Sam Duwé(2), Cade Elliott(3), Riley B. Hargreaves(3), Gregory W. Moseley(1), Peter Dedecker(4), Donna R. Whelan(5) & Toby D. M. Bell(3)

1. Department of Microbiology, Monash Biomedicine Discovery Institute, Clayton, Australia

2. Department of Neurosciences, Hasselt University, Diepenbeek, Belgium

3. School of Chemistry, Monash University, Clayton, Australia

4. Department of Chemistry, KU Leuven, Leuven, Belgium

5. La Trobe Institute for Molecular Science, Bendigo, Australia

Microtubule (MT)-interacting drugs have been long known to disrupt cellular mitosis and therefore have been used to treat cancer. Recent studies have found that non-mitotic drug effects may also contribute to therapeutic success(1,2). Given the importance of MTs for overall cellular health, characterization of non-mitotic MT dysfunction could improve understanding of MT-interacting drug mechanisms for better dosing regimens in cancer therapy. Direct observation of MT filaments in situ can be achieved with super-resolution microscopies that surpass the diffraction limit (~200 nm). Single molecule localization microscopy dSTORM(3) reaches as good as 20 nm spatial resolution, ideal for visualizing individual MT filaments in fixed cells. Fluctuation correlation microscopy SOFI(4) employs milder imaging conditions (i.e. low laser power) ideal for interrogating live cells and enables time-lapse movies of MT filament dynamics with as good as 20 s temporal resolution.

 

Here we applied complementary super-resolution imaging techniques for a holistic perspective of MT dysfunction caused by low doses of antimitotic drug colcemid(5). Using dSTORM, we quantified MT filament curvatures increased with increasing colcemid concentrations from 7 - 80 nM. Aberrant filament curvature induced by 50 - 80 nM of colcemid reached up to 2 rad/µm, a value previously associated with MT filament breakage(6). Higher colcemid doses at 100 and 200 nM, interestingly, revealed short and few MT filaments, suggesting MT fragmentation as a possible drug mechanism. At lower colcemid doses where no prominent structural defects were observed from dSTORM, we investigated filament dynamics using live-cell SOFI and found 18 nM colcemid suppressed MT filament dynamics. Further work looks to adapt these super-resolution assays to characterize subcellular effects induced by other MT-interacting drugs.

 

1. Komlodi-Pasztor E, Sackett D, Wilkerson J, Fojo T. Mitosis is not a key target of microtubule agents in patient tumors. Nature Reviews Clinical Oncology. 2011;8(4):244-50.

2. Kaul R, Risinger AL, Mooberry SL. Microtubule-Targeting Drugs: More than Antimitotics. Journal of Natural Products. 2019;82(3):680-5.

3. Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, et al. Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes. Angewandte Chemie International Edition. 2008;47(33):6172-6.

4. Dedecker P, Mo GCH, Dertinger T, Zhang J. Widely accessible method for superresolution fluorescence imaging of living systems. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(27):10909-14.

5. Rozario AM, Duwé S, Elliott C, Hargreaves RB, Moseley GW, Dedecker P, Whelan DR, Bell TDM. Nanoscale Characterization of Drug-Induced Microtubule Filament Dysfunction using Super-Resolution Microscopy. BMC Biology (accepted 2021).

6. Odde DJ, Ma L, Briggs AH, DeMarco A, Kirschner MW. Microtubule bending and breaking in living fibroblast cells. Journal of Cell Science. 1999;112(19):3283.

 

Harrison York

EEA1 drives an emergent determinism, suppressing stochasticity in early endosomal maturation

Harrison M York(1), Kunal Joshi(2), Charlie Wright(2), Ullhas K Moorthi(1), Hetvi Gandhi(1), Abhishek Patil(1), Srividya Iyer-Biswas(2) & Senthil Arumugam(1)

1. Monash Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University & European Molecular Biological Laboratory Australia (EMBL Australia), Monash University, Clayton/ Melbourne, VIC 3800, Australia

2. Department of Physics and Astronomy, Purdue University, West Lafayette, IN 47907-2036, USA

 

Endosomal maturation is a major process in endosomal trafficking in which endosomes shed one specific protein and acquire another, resulting in an identity change. Individual processes that build up endosomal trafficking, including conversions, are interlinked and are inherently stochastic. While the general biochemical interactions have been well described, how all the events come together to overcome the inherent noise and stochasticity is much less explored.  Here, capitalising on the rapid volumetric imaging capability of Lattice-light sheet, we capture whole-cell volumes, enabling post-acquisition analysis of all conversions and their dynamic characteristics. We show that early endosome maturation is driven by endosomal collision-induced conversions. Using live-cell Förster Resonance Energy Transfer, we demonstrate that this is underpinned by cyclical conformational changes in EEA1, which promotes the biochemical maturation of these vesicles through its asymmetric binding capacity and clustering on the endosomal membranes. Using simulations, we recapture the experimentally observed characteristics in the reaction scheme and the activity of EEA1. Taken together, we describe an EEA1 based feed-forward mechanism that enables deterministic outcomes in ensemble endosomal conversions in an otherwise stochastic system. 

 

David Simpson

Bio-sensing and imaging with diamond quantum probes

David A. Simpson(1), Julia M. McCoey(1), Mirai Matsuoka(1), Robert W. de Gille(1), Liam T. Hall(2), Jeremy A. Shaw(3), J-P. Tetienne(4), David Kisailus(5) & Lloyd C. L. Hollenberg(1)

 1. School of Physics, University of Melbourne, Parkville, Victoria 3010, Australia.

2. School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia.

3. Center for Microscopy, Characterisation and Analysis, University of Western   Australia, Perth 6009, WA, Australia.

4. School of Science, RMIT, Melbourne, Victoria 3001, Australia.

5. Department of Chemical and Environmental Engineering and Materials Science and Engineering, University of California, Riverside, CA 92507, USA.

 

The nitrogen vacancy (NV) centre in diamond(1) has emerged as a promising system for nanoscale sensing and imaging due to its size and sensitivity to a range of physiological parameters including temperature(2), magnetic(3) and electric fields(4). The optical and quantum properties of the NV centre in diamond are ideal for biological imaging, the material itself is bio-compatible, the NV fluorescence is photo-stable, and the quantum properties of the NV centre can be manipulated and readout at room temperature. These attributes have driven application of these quantum probes into biological systems(5,6). Diamond quantum probes can be found in nanodiamonds <100 nm in size or engineered into 2D NV imaging arrays using single crystal diamond.

 

Here, I will describe our recent work exploiting 2D NV imaging arrays for magnetic microscopy. In particular, I will show how these magnetic imaging techniques can be used to non-invasively map the magnetic properties of iron-oxide complexes in biological systems at the sub-cellular scale(7, 8). I will also discuss the future possibilities of this technology and how it can be applied to address significant and outstanding questions in biology.

1.   M. W. Doherty, et al., Physics Reports 528 (1), 1-45 (2013).

2.   A. Jarmola, et al., Phys. Rev. Lett. 108 (19), 197601 (2012).

3.   G. Balasubramanian, et al., Nature 455 (7213), 648-651 (2008).

4.   F. Dolde, et al., Nat. Phys. 7 (6), 459-463 (2011).

5.   L. P. McGuinness, et al., Nat. Nanotech. 6 (6), 358-363 (2011).

6.   D. A. Simpson, et al., ACS Nano 11 (12), 12077-12086 (2017).

7.   J. M. McCoey, et al., Small Methods 4 (3), 1900754 (2020).

8.   J. M. McCoey, et al., Small 15 (18), 1805159 (2019).

 

Anna Wang

Quantifying properties of lipid bilayer vesicles with holographic microscopy

Siddharth Rawat(1,2) & Anna Wang(1,2)

1. School of Chemistry, UNSW Sydney, NSW 2052, Australia

2. Australian Centre for Astrobiology, UNSW Sydney, NSW 2052, Australia

  

Holograms are two-dimensional images that contain information about all three spatial dimensions. Capturing holograms can help recover information about the morphology of objects, as well as where they are localised in 3D, without the use of fluorescent or radioactive labels. Tracking can be as precise as 2 nm in all three dimensions, at fast-camera frame rates.

 

Ordinary microscopes can be modified to take holograms: when the light source in brightfield microscopy is replaced with a coherent light source, such as an LED or laser, the scattered and undiffracted light interfere to form holograms on the camera sensor.

 

Here we use the technique to image lipid bilayer vesicles (Fig. 1). Lipid bilayers are central to living organisms. Although approximately 5 nm in thick, they can still interact significantly with light when forming structures extending into the micrometre regime, or encapsulating material. There is thus potential to extract quantitative information from holograms of giant vesicles.

 

We take experimental holograms of vesicles, and also generate holograms using light scattering theory. We then compare the models to data to extract quantitative information of vesicles such as their internal solute content, size, location, and refractive index. We can also track probe particles inside vesicles to quantify properties about the internal environment.

  1. J.T. Kindt, J.W. Szostak, A. Wang, ACS Nano (2020)

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Figure 1. Left - Confocal microscopy image of oleic acid vesicles encapsulating 1 mM pyranine, taken from [1]. Centre – experimental hologram and Right – simulated hologram of a single vesicle. Image widths 15 μm.

 

Stefan Mueller

Rapid single-molecule characterisation of nucleic-acid enzymes

Stefan H. Mueller, Lisanne M. Spenkelink & Antoine M. van Oijen

Molecular Horizons, The University of Wollongong

 

Maintenance of DNA, involving replication, repair, and recombination is of utmost importance to all live forms. These processes require many different enzymes with a range of different activities. Development of information-rich biochemical assays that report on these activities is an important step towards our understanding of their molecular mechanisms of disease pathways such as anti-microbial resistance and cancer. We present a novel single-molecule assay to rapidly characterise proteins involved in DNA metabolism. In contrast to traditional biochemical methods our assay provides information on subpopulations, dynamic molecular mechanisms and intermediate reaction states.

As a proof of principle we characterised strand-displacement DNA synthesis by the bacteriophage phi29 DNA polymerase using the S.cerevisiae single-stranded binding protein RPA as a probe for single-stranded DNA.

stefanmueller.png

Figure 1 Phi29 DNA polymerase (DNAp) mediated strand-displacement synthesis of individual DNA molecules. (A) Schematic representation of the assay. First, DNA is tethered to the surface of a coverslide. Second, strand-displacement synthesis is initiated. Upon completion of the synthesis of the full template the daughter strand dissociates. (B) Montage of a TIRF microscopy movie. The dashed boxes mark individual DNA molecules that disappear over time (bottom row), while RPA intensity increases (top row). Scale bar = 2 µm. (C) Single-molecule trajectories (top two graphs), showing the intensity over time of DNA stained by SYTOX orange (black) and fluorescently labelled RPA (magenta). The trajectories are synchronized to the event of dissociation to produce a high-quality average trajectory (bottom graph).

 

Toby Bell

Mapping epigenetic histone modifications in T cells using single molecule super-resolution microscopy

Toby D M Bell(1), Alison Morey(2), Ashley M Rozario(1,2), Cade Elliott(1) & Stephen J Turner(2)

1. School of Chemistry, Monash University, Wellington Road, Clayton, Vic. 3800.

2. Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University, Wellington Road, Clayton, Vic. 3800.

 

T cells are an essential part of the adaptive immune system. In response to pathogen challenge, naïve T cells undergo rapid clonal expansion and differentiation into effector T cells that have the capacity to kill infected cells. Following clearance of the pathogen, the majority of the effector cells die and a small population remains as memory T cells, capable to respond rapidly to reinfection. Each stage of differentiation is defined by different transcriptional profiles and a key molecular mechanism that drives these differences is the post translational modification of histone proteins.(1) These modifications modulate chromatin conformation with repressive modifications leading to chromatin compaction and gene silencing, while active modifications induce chromatin loosening and gene activation. What is not clear, is the relationship between the spatial arrangement of histone modifications within the nucleus and their roles in gene regulation and cell differentiation.

We have applied single molecule super-resolution microscopy (SRM) to map the distribution of various histone modifications at different stages of T cell differentiation. (Figure 1) We have also established lamin as a suitable reference structure to quantify the spatial arrangement of histone modifications.(2) We find that histone modifications associated with gene repression are located mostly at the periphery of the nucleus whereas modifications that lead to gene expression are distributed through the interior of the nucleus. Current work on extending our SRM imaging to 3D mapping of entire nuclei and application of expansion microscopy will also be discussed.

1. BE Russ, M Olshanksy, HS Smallwood, J Li, AE Denton, JE Prier, AT Stock, HA Croom, JG Cullen, MLT Nguyen, S Rowe, MR Olson, DB Finkelstein, A Kelso, PG, Thomas TP, Speed, S Rao, SJ Turner, Immunity, 2014, 41, 853-865.

2. A Morey, AM Rozario, C Elliott, TDM Bell, SJ Turner, manuscript in preparation.

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Figure 1: (left) Two-colour SRM image of a T cell nucleus labelled for lamin (Alexa Fluor 532 - blue) and H3K4me3 (Alexa Fluor 647 - yellow); (right) 3D en bloc SRM image of a whole T cell nucleus labelled for lamin with Alex Fluor 647.

 

Sophie Hertel

The HIV capsid acts as a karyopherin to enter the nucleus

Claire Dickson, Sophie Hertel, Derrick Lau, Yann Gambin, Till Böcking & David Jacques

Single Molecule Science and ARC Centre of Excellence for Advanced Molecular Imaging, School of Medical Sciences, UNSW Sydney, Australia

 

The human nuclear pore complex (NPC) is a macromolecular complex comprised of 32 different nucleoporins (Nups) of varying stoichiometries. One third of the Nups possess regions of intrinsic disorder that are enriched for phenylalanine-glycine (‘FG’) repeats. These FG-Nups create a liquid-liquid phase separated ‘diffusion barrier’, which passively prevents the diffusion of molecules greater than ~30 kDa.

 

HIV-1’s ability to infect non-dividing cells maps to the capsid, which recently has been shown to subvert the NPC’s FG-Nup barrier intact, despite being over one thousand times greater in size than the passive diffusion limit. Transporters in the karyopherin family can carry cargo larger than the 30 kDa cut-off through the NPC by specifically interacting with the FG motifs. Interestingly, the capsid lattice also specifically interacts with an FG motif in the characterised cofactors Nup153 and CPSF6 through a shared binding site. We hypothesised that the HIV capsid emulates the activity of a karyopherin to achieve nuclear entry by using this binding site to directly engage with the FG motifs of a broad variety of Nups.

 

We have used a cell free expression system to produce the ten human FG-repeat nucleoporins as GFP fusion proteins. Using our recently published fluorescence fluctuation spectroscopy screen we show that seven of the ten FG-NUPs interact directly with the HIV-1 capsid. These novel capsid cofactors can be competed off the capsid by the drugs PF74 and GS6207, which are known to target the CPSF6/Nup153 binding site. In addition, binding to a panel of known capsid mutations further indicates specificity for this pocket.

 

To further demonstrate that the capsid not only binds but also diffuses though the NPC, we have produced recombinant Nup condensates with selective properties similar to the phase separated diffusion barrier. Using confocal microscopy, we show that the HIV-1 capsid partitions into the Nup condensate, while other globular proteins are excluded. This karyopherin like behaviour was also inhibited upon addition of PF74 or GS6207. These results demonstrate that the HIV capsid is able to mediate its own selective diffusion across the NPC and therefore acts as its own karyopherin.

 

Richard Spinks

Who is driving the DNA replication machine? - A single-molecule study

Richard R. Spinks(1,2), Lisanne M. Spenkelink(1,2), Slobodan Jergic(1,2), Jacob S. Lewis(1,2), Nicholas E. Dixon(1,2) & Antoine M. van Oijen(1,2)

 1. Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia

2. Illawarra Health & Medical Research Institute, Wollongong, NSW, Australia

Helicase enzymes exist to catalyse DNA unwinding. In the DNA replication machine, a dedicated helicase coordinates with the other replication proteins separate the chromosomal DNA and duplicate it into two identical molecules.

 

The last 50-60 years of research in this field was thought to have confidently established the helicase as the driving force of DNA replication given its capacity as an ATPase and position at the replication fork.

 

Recent innovations in single-molecule study of DNA replication have made it possible to observe real-time events of DNA replication in parallel. Using these single-molecule techniques, we sought to challenge the canonical model of replication by assembling the replication machine, but then depriving it of ATP.

 

The outcome these experiments were truly unexpected. We show that replication can proceed without ATP and that despite being an ATPase, the helicase does need this function during replication. Instead, we show the polymerase is main driver of the replication process [1].

1. Spenkelink, L.M., et al., The E. coli helicase does not use ATP during replication. bioRxiv, 2021, DOI: 10.1101/2021.07.07.451541

 

Arif Kabir

Scaling-up biomolecular motors using DNA nanotechnology

Arif Md. Rashedul Kabir(1), Akinori Kuzuya(2) & Akira Kakugo(1) 

1. Faculty of Science, Hokkaido University, Japan

2. Department of Chemistry and Materials Engineering, Kansai University, Japan

 

Nature provides elegant examples of molecular machines that work in living organisms with extreme sophistication, high energy efficiency, and broad functional diversity. Recently, there has been a surge in interest in creating molecular machines using synthetic molecules. However, the research has been still in its infancy since the synthetic molecular machines are far apart than the natural ones in terms of functional ability, energy efficiency, structural organization etc. A promising approach to advance the development of molecular machines has been to utilize the reconstructed natural machineries in engineered environments, which may also expand their applications in various fields, e.g., materials science, nanotechnology, biomedical engineering. Biomolecular motor proteins, such as kinesin or dynein and their associated cytoskeletal protein microtubule are prominent examples of such natural molecular machines. Due to the ability of biomolecular motors to produce force and perform works by consuming chemical energy, they have appeared potential candidates as molecular actuators and hold great prospects for applications in artificial environments. Consequently, in recent years, biomolecular motors have attracted much attention in fabricating artificial machines, molecular robots, or devices. Here, we would like to present our recent efforts aimed at scaling-up biomolecular motors in a programmable manner by utilizing the advantages of DNA nanotechnology, that is expected to benefit the emerging field of molecular robotics.

In our work, we employed single strand DNA (ssDNA) as an information processor to demonstrate active self-organization of microtubules where kinesin was used as a molecular actuator (Figure 1). Thousands of microtubule filaments self-organized into bundles or ring-shaped structures in response to duplex formation by the complementary ssDNA conjugated to the microtubules (1). Such self-organization of microtubules was reversibly regulated using azobenzene tethered ssDNA and ultraviolet light irradiation. In another approach, we have utilized DNA origami nanostructures to fabricate microtubule asters through their self-organization. Upon activation of kinesin actuators, a global network of the microtubule asters exhibited dynamic contraction like the smooth muscles in living organisms (2). Programmability of the self-organization of microtubules using DNA nanotechnology permitted us to utilize the biomolecular motor system for molecular computation. These results are expected to contribute to fabrication of molecular machines from biomaterials and would open a new paradigm in molecular robotics.

(1) Keya, J.J., Suzuki, R., Kabir, A.M.R., Inoue, D., Asanuma, H., Sada, K., Hess, H., Kuzuya, A. and Kakugo, A., Nature Communications, 2018, 9, 1-8.     

(2) Matsuda, K., Kabir, A.M.R., Akamatsu, N., Saito, A., Ishikawa, S., Matsuyama, T., Ditzer, O., Islam, M.S., Ohya, Y., Sada, K. and Konagaya, A., Nano Letters, 2019, 19, 3933-3938.

arifkabir.PNG

Figure 1: Dynamic contraction of a global microtubule network induced by DNA origami nanostructures. Scale bar: 500 µm.

 

Stephanie Xu

A novel method for probing interactions in the HIV-1 viral capsid using DNA scaffolds

Stephanie Xu(1), Sophie Hertel(1), Chu Wai Liew(2), Andrew Tuckwell(1) & Lawrence Lee(1)

1. EMBL Australia Node for Single Molecule Science, School of Medical Sciences, UNSW Sydney, 2052, Australia

2. School of Biotechnology and Biomolecule Sciences, UNSW Sydney, 2052, Australia

 

The HIV-1 viral capsid is a conical-shaped protein shell made up of ~1,500 identical copies of a single capsid (CA) protein, arranged into a lattice of hexamers and precisely 12 pentamers. As well as encapsulating the viral genome and associated replication machinery, the capsid has a functional role in almost every step of the early stage of the HIV viral cycle, before undergoing controlled disassembly inside the nucleus to release the genomic content. The dynamics that control this coordinated behaviour of CA subunits are complex and not well characterised, yet they remain critical to our understanding of the fundamental principles underlying viral self-assembly.

 

Capsid assembly and dynamics are necessarily driven by weak, non-covalent interactions, which are challenging to characterise experimentally. To address this, we synthesised a set of DNA nanostructures containing CA binding sites that enable control over the relative spatial positions of multiple subunits at low nanometre resolution. By increasing the local concentration of subunits, we were able to measure extremely weak and cooperative CA-CA interactions in a variety of different stoichiometric and spatial arrangements for the first time, using surface plasmon resonance. This use of DNA nanostructures for probing interactions within CA complexes provides new insight into the underlying kinetics of viral self-assembly. We believe this approach also has significant potential for broader application across different protein systems, and across different experimental platforms, including single-molecule techniques such as TIRF microscopy.

 

Eloisa Perez-Bennetts

Targeting brain regions with DNA nanorobots: An analytical and experimental study

Eloisa Perez-Bennetts(1,2), Jasleen Kaur Daljit Singh(3,4,5), Ben D. Fulcher(1) & Shelley Wickham(1,3,4)

1. School of Physics, The University of Sydney, 2006, NSW, Australia

2. Brain and Mind Centre, The University of Sydney, 94 Mallett St, 2050, NSW, Australia

3. School of Chemistry, The University of Sydney, 2006, NSW, Australia

4. Sydney Nano, The University of Sydney, 2006, NSW, Australia

3. School of Chemical & Biomolecular Engineering, The University of Sydney, 2006, NSW, Australia

 

One of the greatest limitations in current drug therapies is their lack of specificity. While therapeutic effects are often only required in one specific part of the body, the drugs are distributed everywhere, which can cause wide-ranging negative side-effects. In the context of Central Nervous System diseases, we proposed utilising emerging high-resolution brain maps in combination with recent advances in DNA nanotechnology to build nanorobots that can deploy a molecular payload to specific locations in the brain. However, we do not yet know how to design molecular sensors to optimally exploit the spatial information in whole-brain gene-expression maps.

 

In this work, we develop a novel machine-learning algorithm, called DART, which identifies brain regions using their distinctive gene-expression patterns. The algorithm leverages whole-brain gene-expression atlas data, such as the Allen Mouse Brain Atlas(1), to select genes as molecular markers for different brain regions. The theoretical targeting accuracy of a DNA nanorobot with sensors for one or more of those markers is also estimated. The marker selection corresponds to a series of simple sensing rules for the DNA nanorobot, which can be expressed as combinations of AND and OR statements implementable with DNA computing(2). We found that the DART algorithm identifies key genes known to play a role in the spatial organization of the cortex, including Ephb6 and Flrt2, suggesting the usefulness of this data-driven approach. We also show that navigation accuracy to a given brain region improves when a combination of markers is used in the sensing rules.

 

We also carried out preliminary DNA logic gate experiments in the laboratory to verify the feasibility of performing simple molecular computations with multiple gene sequences inputs. Gene-sensing logic gates were designed using the Nupack molecular simulation package(3), and subsequently fabricated and tested experimentally. A proof-of-principle for detecting gene sequences with DNA logic gates was thus demonstrated.

 

This research is the first demonstration of the feasibility of targeting specific brain regions using a combination of neuroinformatics, machine learning and nanoscience. We identify promising molecular targets for nanoscale sensing that could form the basis for a new generation of influential brain treatments that will continue to improve with advances in brain mapping and DNA nanoscience.

 

1. Q. Wang, S.-L. Ding, Y. Li, et al. The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas. Cell 181, 936 (2020).

2. T. Song, A. Eshra, S. Shah, et al. Fast and compact DNA logic circuits based on single-stranded gates using strand-displacing polymerase. Nature Nanotechnology 14, 1075 (2019). Number: 11 Publisher: Nature Publishing Group.

3. J. N. Zadeh, C. D. Steenberg, J. S. Bois, et al. NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry 32 (2011).

 

Nevena Todorova

Computational insights into DNA origami protection by sequence-defined peptoids

Nevena Todorova(1), Shih-Ting Wang(2) & Oleg Gang(2) 

1. School of Engineering, RMIT University, Melbourne, Australia

2. Center of Functional Nanomaterials, Brookhaven National Laboratory, NY, USA

 

DNA nanotechnology provides a structural toolkit for the fabrication of programmable DNA nano-constructs through specific Watson−Crick base-pairing and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery (1,2). However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications.

 

A class of structurally well-defined peptoids capable of protecting DNA origamis in ionic and bioactive conditions have been designed by the experimental partners at the Brookhaven National Laboratory (3). Using all-atom molecular dynamics simulations the effects of peptoid architecture and sequence dependency on DNA origami stability were systematically explored. Specifically, the role of multivalent interactions between different peptoid architectures and the phosphate backbone in stabilising a duplex DNA were elucidated. The work presents a systematic study on rationalising designs of peptoids for enabling a modular strategy of molecular coatings for DNA origamis.

1. P. Wang, T. A. Meyer, V. Pan, P. K. Dutta and Y. Ke “The Beauty and Utility of DNA Origami” Cell, Vol. 2, pp. 359 (2017)

2. W. Engelen and H. Dietz “Advancing Biophysics Using DNA Origami” Annu. Rev. Biophys., Vol. 50, pp. 469 (2021)

3. S. Wang, M. A. Gray, S. Xuan, Y. Lin, J. Byrnes, A. I. Nguyen, N. Todorova, M. M. Stevens, C. R. Bertozzi, R. N. Zuckermann and O. Gang “DNA Origami Protection and Molecular Interfacing Through Engineered Sequence-Defined Peptoids” PNAS, Vol. 117 (12), pp. 6339 (2020)

nevenatorodova.png

Peptoid Stabilized DNA Origami System

 

Dietmar Oelz

Protein friction and F-actin bending promote contraction of disordered actomyosin networks

Alex Tam(1), Alex Mogilner(2) & Dietmar Oelz(3)   

1. Queensland University of Technology (QUT)

2. New York University (NYU).

3. University of Queensland (UQ)

 

The origins of disordered actomyosin network contraction such as in the cellular cortex remain an active topic of research. We derive a mathematical model for the evolution of two-dimensional networks. A major advantage of our approach is that it enables direct calculation of the network stress tensor, which provides a quantitative measure of contractility. Exploiting this, we use simulations of disordered networks to confirm that both protein friction and actin filament bending are required for contraction. We also show that actin filament turnover is necessary to sustain contraction and prevent pattern formation.

 

We then consider a toy-model version of the model for only two filaments immersed in an actomyosin network. We find that bending induces a geometric asymmetry that enables motors to move faster close to filament plus-ends, inhibiting expansion. Our findings confirms the role of filament bending in giving rise to microscopic-scale asymmetry facilitating network-scale contraction.

 
 

Pietro Ridone

 Spontaneous adaptation of ion selectivity in a bacterial flagellar motor

Pietro Ridone(1), Tsubasa Ishida(2), Angela Lin(1), Yoshiyuki Sowa(2,3) & Matthew A. B. Baker(1,4)

1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.

2. Department of Frontier Bioscience, Hosei University, Tokyo, Japan.

3. Research Center for Micro-Nano Technology, Hosei University, Tokyo, Japan.

4. CSIRO Synthetic Biology Future Science Platform, Brisbane, Australia.

 

Motility provides a selective advantage to many bacterial species and is often achieved by rotation of flagella that propel the cell towards more favourable conditions. In most species, the rotation of the flagellum, driven by the Bacterial Flagellar Motor (BFM), is powered by H+ or Na+ ion transit through the torque-generating stator subunit of the motor complex. The ionic requirements for motility appear to have adapted to environmental changes throughout history but the molecular basis of this adaptation remains unknown. Here we used CRISPR engineering to replace the native E. coli H+-powered stator with Na+-powered stator genes and report the rapid and spontaneous adaptation of the motor to a low sodium environment. We characterize the biophysical characteristics of the transgenic motor such as speed, torque and switch frequency, and follow the evolution of its stator subunits during the cell's adaptation to a new power source using whole-genome sequencing and RNAseq to identify both the flagellar- and non-flagellar-associated genes involved. This work highlights the utility of the flagellar stator system for studying the molecular mechanisms underlying adaptation and demonstrates how environmental change can rapidly alter the function of an ion transporter.

Alice Hutchinson

Modelling thermophoresis at the molecular scale - which water model is best?

Alice Hutchinson(1), Ben Corry(1) & Juan Felipe Torres(2)

1. Research School of Biology, Australian National University, Canberra, ACT

2. Research School of Engineering, Australian National University, Canberra, ACT

Thermophoresis is the migration of particles due to a temperature gradient. It is a peculiar phenomenon that has particles moving towards either warmer or cooler areas, depending sensitively on the particles nature and environment, as well as the solution concentration and average temperature. Thermophoresis is taken advantage of by a suite of biologically relevant separation and accumulation technologies because of its extraordinary ability to separate particles with very small differences. For example, thermophoresis can be used as a substitute for electrophoresis when particles are functionally distinct but have similar size and charge. It can be used to separate vesicles that differ only by the head-group of the lipids they are comprised of. Thermophoresis can accumulate DNA in thermal traps and increase PCR replication rates, or locally accumulate buffer molecules to create stable pH gradients in vitro. Perhaps most exciting, thermophoresis has recently been used to quantify the binding affinity of a ligand to a protein.

 

Intriguingly, thermophoresis has no fully accepted physical explanation, even in solutions as simple as aqueous sodium chloride. Molecular dynamics (MD) simulations are a useful tool for exploring the rich behaviour of thermophoresis and can qualitatively recreate particle migration. However, MD models of thermophoresis in aqueous ionic solutions do not yet accurately predict the Soret coefficient, a value that characterises the magnitude and direction of particle migration under a temperature gradient.

 

In this work, we aim to improve the quantitative accuracy of MD thermophoresis models by exploring how well different water models can recreate thermophoresis. We expect the choice of water models to impact simulation results as thermophoresis is hypothesised to be strongly related to the enthalpic and entropic interactions between ions and water.

 

We tested four of the best available rigid non-polarisable water models (TIP3P-FB, TIP4P-FB, OPC3 and OPC) and the commonly used TIP3P water model for their ability to recreate thermophoresis in 0.5, 2, and 4 molar aqueous sodium chloride solutions. The ionic solution dynamics were modelled under a linear temperature gradient spanning 5-95◦C using the all-atom molecular dynamics software, NAMD. Resulting ion concentrations were measured and used to calculate the Soret coefficient. 

 

Each water model predicted a noticeably different ion distribution, demonstrating that water models influence the thermophoretic behaviour of ions. By comparing the modelled Soret coefficients to published experimental values, we determine the water model that best recreates thermophoresis and aid future works in selecting the most accurate water model for investigating thermophoresis through MD simulations.

 

Aaron Elbourne

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Figure 1. AFM and Molecular dynamics simulation investigation of the interaction of 5 nm AuNPs with a supported DOPC lipid bilayer formed at a mica surface.

Dynamics and behaviour of ultra-small gold nanoparticles at bio-membranes – Combining experiment with simulation

Rashad Kariuki(1), Saffron Bryant(1), Rebecca Orrell-Trigg(1), Vi Khanh Truong(1), Russell J. Crawford(1), Christopher F. McConville(1,2), Gary Bryant(1), Charlotte Conn(1), Kislon Voïtchovsky(3), Andrew Christofferson(1) & Aaron Elbourne(1) 

1. School of Science, RMIT University, Melbourne VIC 3001, Australia.

2. Deakin University, Geelong, Australia

3. University of Durham, Physics Department, Durham DH1 3LE, UK

 

Introduction: Nanomaterials - materials with nanoscale dimensions - are widely investigated, especially in many biological settings. This is due to their potential us as advanced nano-medicines and diagnostic technologies,[1-4] antimicrobials,[5] as cellular probes, and in cellular-imaging,[6] among other applications.[1, 4, 5, 7] The commonality between all applications is that they utilise the nanosized features of the material, specifically their departure from traditional bulk-like properties. In general, nanoparticle-based technologies must interact with, and often cross, a cellular membrane to be useful. Aim: To combine advanced experimental and computation studies to study the interaction of ultra-small gold nanoparticles (AuNP) at a synthetic bio-membrane. Methods: A combination of small-amplitude - atomic force microscopy (AM-AFM) and molecular dynamics (MD) simulations will be used to study the fundamental behaviour of the AuNPs at the bio-membrane-liquid interface. The system of interest is a model system consisting of a supported lipid bilayers (SLB) which act as an archetypal bio-membrane. The lipid used will be 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC) supported by muscovite (mica), an atomically smooth, phyllosilicate substrate. Results: We investigate the behaviour (dynamics, adsorption, translocation, and physical interactions) of 5 nanometre AuNPs with a SLB. The precise mechanism by which the AuNPs adsorb to the bio-membrane was elucidated, revealing several interesting behaviours: 1) initial adsorption, 2) nanoparticle incorporation within the bilayer, and 3) two-dimensional (2D) translocations within the upper-leaflet of the DOPC bilayer. Conclusion: These interactions are of broad scientific and medical interest because nanomaterials have recently become a viable method for manipulating matter at the cellular level, particularly for therapeutic and diagnostic applications.

1.Shi, J., et al., Nature Reviews Cancer 2017.

2. Rizvi, S. A. A., et al., Saudi Pharm J 2018.

3. Irvine, D. J., et al., Nature Reviews Immunology 2020.

4. Singh, P., et al., International journal of molecular sciences 2018.

5. Wang, L., et al., International journal of nanomedicine 2017.

6. Murphy, C. J., et al., Accounts of chemical research 2008.

7. Khan, I., et al., Arabian journal of chemistry 2019.

 

Kathleen Schell

Honey; Breaking down the wall to new antibiotics

Kathleen Schell(1), Nural Cokcetin(2) & Evelyne Deplazes(2)

1. University of Technology Sydney

2. University of Queensland

Honey has been used by many cultures since ancient times as an effective topical treatment for numerous diseases, predominantly due to its antimicrobial activity. Although its therapeutic use has declined since the discovery of modern antibiotics, there is renewed interest in complex natural products with antimicrobial activity, like honey, due to the rise in antimicrobial resistance – one of the biggest threats to human health globally.

The antibacterial activity of honey is attributed to many components such as high sugar, low pH, and the presence of hydrogen peroxide, methylglyoxal, bee derived antimicrobial peptides, and phenolic compounds. It is widely accepted that the antimicrobial activity in most honeys is driven by the in-situe production of hydrogen peroxide, or in the case of manuka-type (Leptospermum) honeys by the high levels of methylglyoxal. However, when tested in isolation these components do not account for the antibacterial effect of whole Leptospermum honey. Our previous work investigating the antibacterial mechanism of action of Leptospermum honey showed that the phenolic fraction (and in particular, phenolic acids) in this honey is a key contributor to its inhibitory and killing action against a model organism, Pseudomonas aeruginosa. Using a combination of whole cell studies and model membranes, the phenolic fraction and phenolic acids were shown to have a membrane damaging effect in bacteria, likely to be related to the phospholipid bilayer.

 

To fully discern the antibacterial activity of Leptospermum honey, we carried out susceptibility testing of the phenolic fraction, and individual phenolic acids against a Gram-negative Acinetobacter baumannii and two Gram-positive Staphylococcus aureus model bacteria. Leptospermum honey, the phenolic fraction of honey, and individual phenolic acids of interest as identified from the susceptibility assays were then investigated for their lipid-dependent membrane interactions using a model membrane approach, tethered bilayer lipid membrane electrical impedance spectroscopy (tBLM-EIS).

 

Whole Leptospermum honey, the phenolic fraction, and individual synthetic phenolic acids were found to be antibacterial at very low concentrations against all tested organisms. Whole Leptospermum honey was effective against S. aureus at 4.3-7.3% and at 9-10% against A. baumannii. The phenolic fraction was effective on S. aureus at 0.8-2.1%. Finally, the phenolic acids were inhibitory between concentrations ranging 0.022 –3.75%, depending on the acid and bacteria. The tBLM-EIS assays showed that whole Leptospermum honey and the phenolic fraction caused significant increase in membrane disruption of the model membrane. Similarly, of the X individual phenolic acids tested, caffeic acid, caffeic acid methyl ester, kojic acid, chlorogenic acid, and protocatechuic acid showed significant membrane disruption. Additionally, they also displayed a cumulative effect where after the treatment is washed out of the model membrane, the subsequent additions increase membrane disruption higher than the original peak. This cumulative effect is also thought to be the cause of the heightened efficacy of the phenolic fraction when compared to individual acids and may indicate that individual phenolic acids work in synergy. Further investigation into the effect of phenolic acids on specific Gram-negative and Gram-positive model membranes could help design antibiotic components in drugs that target specific bacterial infections. 

 

Suganeya Soundararajan

Susceptibility of leukemia cells to low cost low toxic agents

Suganeya Soundararajan, Martin Stewart, Andrew Care & Charles G Cranfield

School of Life Science, University of Technology Sydney, Ultimo, NSW 2007.

Leukemia is one of the most common cancers globally, especially amongst children where, it accounts for at least 35% of cases, with more than 50% of those relapsing in adults. In low-income countries, it has a less than 30% five-year survival rate. Current treatments are costly, and once the condition has reoccurred, previously used treatment methods are not as effective. Ascorbic acid, which has shown cancer-specific cell cytotoxicity in both cellular and pre-clinical animal models, has proven antioxidant properties and can be potentially combined with other low cost low toxicity (LCLT) compounds that target cancer cells(1,2). Here, using established viability assays, we investigated the strongest synergy combinations of a library of LCLT compounds with ascorbic acid on leukemia cell lines and primary human lymphocytes. These combinations were also tested on tethered bilayer lipid membranes (tBLMs) in conjunction with electrical impedance spectroscopy. Flow cytometric analysis revealed that leukemia cell lines are susceptible to 800 µM ascorbic acid when used in synergy with 4 µM curcumin, a potential anticancer compound derived from Curcuma longa (turmeric) (Figure 1). This compares to a 95% viability of primary lymphocytes exposed to the same ascorbic acid-curcumin regimen. tBLM experiments showed that these compounds did not cause any lasting damage to the in-vitro cell membrane model at the same concentrations, suggesting the mode-of-action is not as a result of membrane disruption. This is further supported by annexin-V assays that showed that cell death is as a result of induced apoptotic pathways.

 

1. Afroze, N., Pramodh, S., Hussain, A., Waleed, M. & Vakharia, K. A review on myricetin as a potential therapeutic candidate for cancer prevention. 3 Biotech 10, 211, doi:10.1007/s13205-020-02207-3 (2020).

2. Zhu, G. et al. Curcumin inhibited the growth and invasion of human monocytic leukaemia SHI-1 cells in vivo by altering MAPK and MMP signalling. Pharm Biol 58, 25-34, doi:10.1080/13880209.2019.1701042 (2020).

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Figure 1. Different concentrations of curcumin-ascorbic acid tested on Jurkat, a leukemia cell line, and against healthy lymphocytes

 

Gary Bryant

Characterisation of cell motility using differential dynamic microscopy (DDM)

Gary Bryant(1), Reece Nixon-Luke(1), Monerh Al-Shahrani(1) & Vincent Martinez(2)

1. Physics, School of Science, RMIT University, Melbourne, Australia

2. School of Physics and Astronomy, The University of Edinburgh, Edinburgh, UK.

The ability of cells to explore their environment is important for a range of biological systems, including bacteria, algae and spermatozoa. The independent motion of cells is known as cell motility, and allows for cells to seek out nutrients, follow chemical signals, and escape danger. In the case of bacteria, motility is involved in cell propagation and proliferation. The standard method of measuring motility involves microscopic tracking of individual cells, so only a finite number of cells can be tracked, and obtaining statistically reliable measurements of populations is time consuming and difficult.

 

Differential Dynamic Microscopy (DDM) [1] is a relatively new technique in which a time-evolving sample is imaged at fast frame rates, and the intensity fluctuations in each pixel are correlated. DDM complements dynamic light scattering and has been applied to a wide range of systems including characterisation of colloidal nanorods [3], as well as the measurement of motility in bacteria and algae [3-5], and allows the measurement of statistically valid velocity distributions, as shown in Figure 1.

 

We will explain the principles of DDM, illustrates with examples of the characterization of bacterial motility, and discuss the advantages and disadvantages of the technique.

1. Cerbino, R. and Trappe, V. Differential dynamic microscopy: Probing wave vector dependent dynamics with a microscope. Phys. Rev. Lett., 2008, 100, 188102.

2. Nixon-Luke, R., and Bryant, G. Differential dynamic microscopy to measure the translational diffusion coefficient of nanorods, J. Phys.: Condens. Matter 2020, 32, 115102.

3. Wilson, L.G., Martinez, V.A., Schwarz-Linek, J., Tailleur, J., Bryant, G., Pusey, P.N., Poon, W.C.K. Differential Dynamic Microscopy of Bacterial Motility. Phys. Rev. Lett. 2011, 106 (1), 018101.

4. Martinez, V.A., Besseling, R., Croze, O.A., Tailleur, J., Reufer, M., Schwarz-Linek, J., Wilson, L.G.. Bees, M.A.. Poon, W.C.K. Differential Dynamic Microscopy: A High-Throughput Method for Characterizing the Motility of Microorganisms. Biophys. J., 2012, 103, 1637–1647.

5. Hu, Y.X.; Zou, W.Y.; Julita, V.; Ramanathan, R.; Tabor, R.F.; Nixon-Luke, R.; Bryant, G.; Bansal, V.; Wilkinson, B. L., Chemical Science 2016, 7 (11), 6628-6634.

Figure 1: Velocity distributions for three bacterial species measured using DDM.

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Megan Coomer

Stochastic simulations of biological systems in fluctuating environments: The extrinsic chemical Langevin equation

Lucy Ham(1), Megan A. Coomer(2) and Michael P. H. Stumpf(1,2)

1. School of BioSciences, University of Melbourne, Parkville VIC 3010, Australia

2. School of Mathematics and Statistics, University of Melbourne, Parkville VIC 3010, Australia

 

Modelling and simulation of complex biochemical reaction networks form cornerstones of modern theoretical biophysics. Many of the approaches developed so far can capture temporal fluctuations due to the inherent stochasticity of the biophysical processes, referred to as intrinsic noise. Stochastic fluctuations, however, predominantly stem from the interplay of the network with many other — and mostly unknown — fluctuating processes, as well as with various random signals arising from the extracellular world; these sources contribute extrinsic noise. Here we provide a computational simulation method to probe the stochastic dynamics of biochemical systems subject to both intrinsic and extrinsic noise.  

 

Intrinsic stochasticity is captured by the Chemical Master Equation (CME). The CME can only be solved for a select few different systems. This has spurred the development of direct simulation methods, including Gillespie’s stochastic simulation algorithm (SSA). These methods, however, quickly become computationally infeasible as the system size increases; further difficulties arise when reactions occur at multiple timescales. Here we can often make progress by employing continuous approximation methods such as the Chemical Langevin Equation (CLE). On the flip side, there is no accepted framework to model extrinsic noise, and methods that combine both noise sources are scarce. SSA approaches can be adapted to include extrinsic sources of variability, but they suffer similar computational drawbacks; existing approximation methods make sweeping assumptions about the timescale regimes between the primary and extrinsic processes, ignoring the process dynamics of the exogenous noise source. Sometimes ad hoc, and often physically unfounded, extrinsic noise terms have been incorporated into CLE-like frameworks, but this can lead to spurious and physically unrealistic system dynamics.

 

Here we develop an Extrinsic Chemical Langevin Equation —a physically motivated extension of the CLE— to model intrinsically noisy reaction networks embedded in a stochastically fluctuating environment. The Extrinsic CLE is a continuous approximation to the CME with time-varying propensities. Here, noise is incorporated at the level of the CME, and can account for the full dynamics of the exogenous noise process, irrespective of timescales and their mismatches. We show that our method accurately captures the first two moments of the resulting probability density when compared with exact stochastic simulation methods, while reducing the computation runtime by several orders of magnitude. Our approach provides a method that is practical, computationally efficient and physically accurate to study systems that are simultaneously subject to a variety of noise sources.