Direct manipulation of liquid ordered lipid membrane domains using optical traps

Multicomponent lipid bilayers can give rise to coexisting liquid domains that are thought to influence a host of cellular activities. There currently exists no method to directly manipulate such domains, hampering our understanding of their significance. Here we report a system that allows individual liquid ordered domains that exist in a liquid disordered matrix to be directly manipulated using optical tweezers. This allows us to drag domains across the membrane surface of giant vesicles that are adhered to a glass surface, enabling domain location to be defined with spatiotemporal control. We can also use the laser to select individual vesicles in a population to undergo mixing/demixing by locally heating the membrane through the miscibility transition, demonstrating a further layer of control. This technology has potential as a tool to shed light on domain biophysics, on their role in biology, and in sculpting membrane assemblies with user-defined membrane patterning

Sculpting and fusing biomimetic vesicle networks using optical tweezers

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-Triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD

Development of microfluidic technologies for the construction of Multi-Compartment Vesicles (MCVs) and their applications as artificial cells

In recent years there has been an increasing interest in using lipid vesicles and related membrane structures as (i) artificial cells that mimic biological processes and (ii) bio-inspired micro-machines that serve functional purposes. To date, vesicles have largely been single-compartment structures with homogenous interiors, which has impeded the fulfilment of these goals. This thesis details the development of technologies to address this. We develop droplet-based methods to controllably generate multi-compartment vesicles (MCVs) for the first time. The potential of these novel structures as artificial cells capable of hosting a range of biological and bio-mimetic processes is explored. Most notably, we introduce spatial segregation of function, thus mimicking eukaryotic organelles, and incorporate an artificial enzymatic signalling cascade to transmit chemical signals between distinct vesicle regions. We also construct microfluidic devices to generate related structures known as multisomes. Microfluidic technologies enable the size of these constructs to be scaled-down (approaching characteristic cellular sizes), and the production throughput to be scaled-up (hundreds of multisomes produced a minute). We demonstrate their use as programmable modular microdroplet ‘factories’ for in situ chemical synthesis in physiological environments, with potential relevance for therapeutic applications. The above technologies provide a platform for further developments in bottom-up synthetic biology and in microreactor technologies, and will pave the way for the fulfilment of some of the ambitious goals of these fields.  Open Acces

MICROFLUIDIC GENERATION OF NETWORKED DROPLET COLLECTIONS AND LIPID MEMBRANE CONSTRUCTS

ABSTRACT We report on microfluidic strategies to generate several multi-compartment membrane-based structures, including droplet interface bilayer networks and multi-compartment vesicles. These developments allow the current status quo-where microdroplets are used as isolated vessels-to be changed. By linking droplets together with lipid membranes, higher order systems can be generated, with particular ramifications for bottom-up synthetic biology and for functional droplet-based microreactors and biodevices

Novel technologies for the formation of 2-D and 3-D droplet interface bilayer networks

Droplet Interface Bilayer (DIB) networks have vast potential in the field of membrane biophysics, synthetic biology, and functional bio-electronics. However a technological bottleneck exists in network fabrication: existing methods are limited in terms of their automation, throughput, versatility, and ability to form well defined 3-D networks. We have developed a series of novel and low-cost methodologies which address these limitations. The first involves building DIB networks around the contours of a microfluidic chip. The second uses flow rate and droplet size control to influence droplet packing geometries within a microfluidic chamber. The latter method enables controlled formation of various 3-D network arrays consisting of thousands of interconnected symmetric and asymmetric lipid bilayers for the first time. Both approaches allow individual droplet position and composition to be controlled, paving the way for complex on-chip functional network synthesis.<br/

Fusing artificial cell compartments and lipid domains using optical traps: a tool to modulate membrane composition and phase behaviour

New technologies for manipulating biomembranes have vast potential to aid the understanding of biological phenomena, and as tools to sculpt novel artificial cell architectures for synthetic biology. The manipulation and fusion of vesicles using optical traps is amongst the most promising due to the level of spatiotemporal control it affords. Herein, we conduct a suite of feasibility studies to show the potential of optical trapping technologies to (i) modulate the lipid composition of a vesicle by delivering new membrane material through fusion events and (ii) manipulate and controllably fuse coexisting membrane domains for the first time. We also outline some noteworthy morphologies and transitions that the vesicle undergoes during fusion, which gives us insight into the mechanisms at play. These results will guide future exploitation of laser-assisted membrane manipulation methods and feed into a technology roadmap for this emerging technology