Fusing Artificial Cell Compartments and Lipid Domains Using Optical Traps: A Tool to Modulate Membrane Composition and Phase Behaviour

Abstract

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.

Keywords: artificial cells; membrane biophysics; membranes; optical traps; phase separation; vesicles.

Figure 1

Schematic of the processes involved in laser-assisted vesicle adhesion and fusion. (a) Experimental setup. By moving a motorized stage and the objective itself, a vesicle could be manipulated in the x, y, and z direction relative to its surroundings. (b) Gold nanoparticles (AuNP) labelled vesicles of defined composition are brought together using an optical trap where they adhere due to the presence of NaCl in the external solution. Focussing the laser at the adhesion patch is used to initiate fusion, forming a unified structure with lipids originating from two previously distinct vesicles. Images of fluorescently labelled Giant Unilamellar Vesicles (GUVs) pre- and post-fusion, as shown. Scale bar = 5 µm.

Figure 2

Changing membrane phase state through delivery of new lipid material. (a) Two giant vesicles of different compositions and phase states are fused together using an optical trap. Adding a fully fluid vesicle to one showing gel/fluid domain coexistence (irregularly shaped domains) yielded a vesicle exhibiting liquid-ordered/liquid disordered domain coexistence (spherical domains). Scale bar = 5 µm. (b) A schematic demonstrating adding lipid material from vesicle (1) to vesicle (2) leading to a new vesicle (3) occupying a different part of the phase diagram. Note: this is not an empirical phase diagram; it is an approximate one used for illustrative purposes only.

Figure 3

Controlled manipulation of membrane domains using optical traps. (a) Schematic showing manipulation of liquid-disordered (L_d) domains within a liquid-ordered (L_o) matrix to induce domain fusion. (b) Fluorescence image of a portion of a vesicle membrane showing an L_d domain being dragged by an optical trap (yellow dot and arrow) towards an adjacent domain to induce fusion. Lipid composition: DOPC/EggSM/Chol 1:1:1. (c) Time-course image showing eight sequential fusion events that were user-defined through domain manipulation with an optical trap (yellow circle). Scale bars = 5 µm.

Figure 4

Vesicle morphology changes under different osmotic conditions. (a) If vesicles have excess membrane area and low tension, they may contain tubules (white arrow). Upon adhesion, the vesicle deform to a non-spherical geometry, which increases its tension and leads to a retraction of the tubule into the main vesicle body. (b) Images showing post-fusion morphologies under iso-osmotic (0.75 M sucrose internally; 0.35 M glucose, 0.2 M NaCl externally) and osmotically deflated (0.75 M sucrose internally; 0.4 M glucose, 0.2 M NaCl externally; low tension) conditions. In the former, a clean-merge is seen; in the latter the vesicle slowly relaxes to a spherical shape and many smaller internal vesicles are produced and retained inside (white arrow). Orange dot corresponds to area of laser focus. Scale bars = 5 µm.

Figure 5

Post-fusion morphologies and intermediate structures. Under osmotically balanced conditions (0.75 M sucrose internally; 0.35 M glucose and 0.2 M NaCl externally) several alternative non-clean fusion events were occasionally observed after application of the laser (red dot and blue cone), including: (a) A meta-stable pore of c. 3 µm diameter in the bilayer partition, which after c. 5s rearranged (dotted arrow) to reform the interface membrane. (b) Quick opening and closing of a pore upon continuous laser illumination, after each event a portion of the membrane of one vesicle was subsumed into the other, with the formation of a single vesicle at the end. (c) An exposed pendant interface membrane (white arrow), which rearranged and reconnected with itself to yield in an inner vesicle. Scale bar = 5 µm.