Generation of interconnected vesicles in a liposomal cell model

Abstract

We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca²⁺ solution from the micropipette tip. IVs are rapidly and sequentially generated and inserted into the GUV interior and encapsulate portions of the micropipette fluid content. The IVs remain connected to the GUV membrane and are interlinked by short lipid nanotubes and resemble beads on a string. The vesicle chain-growth from the GUV membrane is maintained for as long as there is the supply of membrane material and Ca²⁺ solution, and the size of the individual IVs is controlled by the diameter of the micropipette tip. We also demonstrate that the IVs can be co-loaded with high concentrations of neurotransmitter and protein molecules and displaying a steep calcium ion concentration gradient across the membrane. These characteristics are analogous to native secretory vesicles and could, therefore, serve as a model system for studying secretory mechanisms in biological systems.

Figure 1

Schematic illustration of the sequential steps of micromanipulation and microinjection required for IV formation. (a) Placement of the micropipette tip into the bulk sample solution containing surface-attached GUV-MLVs. (b) Positioning of the micropipette tip near the GUV surface. (c) A positive pressure (+ p) is applied to the micropipette while putting the pipette into direct contact with the GUV membrane, which results in a flow of calcium ion solution and inflation of the first IV. (d) Applying a continuous injection of calcium ion solution results in synthesis of a multitude of IVs, which are filling the interior of the GUV. The illustrations were drawn in Adobe Illustrator CS6.

Figure 2

Fluorescence microscopy images illustrating calcium-ion assisted formation of IVs. (a) Formation of the IVs upon a 25 s injection of calcium ion solution, supplemented with 10 µM Alexa-488 for visualization when placing the micropipette tip in contact with the GUV surface. (b) IVs co-loaded with streptavidin − 488 (10 µM). Injection time is 12 s. (c) IVs co-loaded with a glutamate solution (10 µM with Alexa 488 NHS ester co-solute). Injection time is 3 s. The images were brightness/contrast-enhanced for improved visualization. The white lines outline the position of the micropipette tip in the experiments. The images were prepared using the NIH ImageJ software, VirtualDub 1.10.4, and Adobe Illustrator CS6. The scale bars represent 5 µm.

Figure 3

Fluorescence microscopy images demonstrating the formation of IVs and their connectivity. (a) By pulling the micropipette (0.3 µm in diameter) away from the GUV, the IVs attached to the micropipette tip were pulled out of the GUV interior and displayed the vesicle connectivity of “beads on a string”. (b) A 5 s injection of calcium ion solution using a micropipette with an inner tip diameter of 2 µm resulted in the formation of polydisperse IVs. The images were prepared using the NIH ImageJ software, VirtualDub 1.10.4, and Adobe Illustrator CS6. The scale bars represent 5 µm.