Generation of interconnected vesicles in a liposomal cell model

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

A high-performance lab-on-a-chip liquid sensor employing surface acoustic wave resonance: part II

We recently introduced an in-liquid sensing concept based on surface acoustic resonance (SAR) in a lab-on-a-chip resonant device with one electrical port. The 185 MHz one-port SAR sensor has a sensitivity comparable to other surface acoustic wave (SAW) in-liquid sensors, while offering a high quality factor (Q) in water, low impedance, and fairly low susceptibility to viscous damping. In this work, we present significant design and performance enhancements of the original sensor presented in part I. A novel \u27lateral energy confinement\u27 (LEC) design is introduced, where the spatially varying reflectivity of the SAW reflectors enables strong SAW localization inside the sensing domain at resonance. An improvement in mass-sensitivity greater than 100% at resonance is achieved, while the measurement noise stays below 0.5 ppm. Sensing performance was evaluated through real-time measurements of the binding of 40 nm neutravidin-coated SiO2nanoparticles to a biotin-labeled lipid bilayer. Two complementary sensing parameters are studied, the shift of resonance frequency and the shift of conductance magnitude at resonance

Lipid Nanotube Networks: Shape Transitions and Insights into the Dynamics of Self-Organization

Nanotube-vesicle networks (NVNs) are simplified models of cell membrane tubular systems which are dynamic transportation routs for molecular cargoes in biological cells. The presented work describes dynamic properties of NVNs such as self-organization, shape and topology transformations; moreover, specific geometric properties of the networks are used for controlling enzymatic reactions.A nanotube-vesicle network is a network of surface-adhered lipid vesicles (5-25 m in radius) connected by suspended lipid nanotubes (100-200 nm in radius). Vesicle size, nanotube length, and connectivity of a network can be controlled with high precision. Initially, the network is trapped in a high free energy state. By proper means, it is possible to trigger network self-organization towards a lower free energy state.Network evolution begins with merging of two adjacent nanotubes, and formation of a single nanotube three-way junction. Based on experimental observations of fluorescently labeled nanotubes and a theoretical model, the nanotube three-way junction is shown to propagate with a zipper-like mechanism, described in Paper I of this thesis. Lipids from two merging branches flow through the junction and form an extension on the third nanotube branch. Depending on the starting arrangement of the nanotubes, a NVN can evolve towards entangled and knotted geometries; or it can form a system of branching nanotubes. Paper II describes the formation of knotted nanotubes. The estimated size of the knot is comparable with the radius of a lipid nanotube. It is also demonstrated that such a knot can be used as a mechanical tweezer to capture and transport submicrometer-sized objects. In the experiments described in Paper III, NVNs are shown to form tree-like structures. The nanotubes arrange into symmetric three-way junctions with angles of 120o between the nanotubes. Moreover, the process of self-organization in the networks reveals a strong similarity with some optimization problems, such as the Euclidian Steiner Tree Problem. Paper IV suggests a method to form circular lipid nanotubes. The presented method gives new opportunities for preparing, manipulating and studying shape transitions of vesicles with non-spherical topology.Finally in Paper V, the geometry of a NVN is used to control the dynamics of an enzymatic reaction. Here, the vesicles are used as containers for reacting molecules, and the nanotubes serve as transportation routes. The narrow nanotube entrances act as transport barriers for the enzyme molecules. In such a reaction-diffusion system, the reaction occurs as a cascade through the containers and displays wave-like behavior

Ca2+ Gradient Induces Membrane Bending and Formation of Nanotubes in Giant Lipid Vesicles

Reshaping and bending of the cell membrane is imperative in many processes such as cell division, filopodia formation, and endocytosis. Understanding these shape transitions, will help to elucidate the underlying mechanisms of these essential cellular processes. In our work, we investigate an interplay between cell membrane morphology and chemical stimulation by constructing a biomimetic model system. More specifically, giant lipid vesicles were exposed to a chemical gradient of Ca2+, which was established over the membrane surface

Formation and release of circular lipid nanotubes

A method for formation of circular lipid nanotubes based on manipulation of nanotube-vesicle networks is presented

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping

Contactless Stimulation and Control of Biomimetic Nanotubes by Calcium Ion Gradients

Membrane tubular structures are important communication pathways between cells and cellular compartments. Studying these structures in their native environment is challenging, due to the complexity of membranes and varying chemical conditions within and outside of the cells. This work demonstrates that a calcium ion gradient, applied to a synthetic lipid nanotube, triggers lipid flow directed toward the application site, resulting in the formation of a bulge aggregate. This bulge can be translated in a contactless manner by moving a calcium ion source along the lipid nanotube. Furthermore, entrapment of polystyrene nanobeads within the bulge does not tamper the bulge movement and allows transporting of the nanoparticle cargo along the lipid nanotube. In addition to the synthetic lipid nanotubes, the response of cell plasma membrane tethers to local calcium ion stimulation is investigated. The directed membrane transport in these tethers is observed, but with slower kinetics in comparison to the synthetic lipid nanotubes. The findings of this work demonstrate a novel and contactless mode of transport in lipid nanotubes, guided by local exposure to calcium ions. The observed lipid nanotube behavior can advance the current understanding of the cell membrane tubular structures, which are constantly reshaped during dynamic cellular processes