Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%

Lipid Nanotubes as a Model for Highly Curved Cellular Membrane Structures

Cells and their organelles show a variety of membrane morphologies with multiplesubmicrometer features, for example, tubules, vesicles, folds and pores. The shape of thecellular membranes can dynamically change to support a variety of functions, such as cargotransport, transmission of signals between the cells, cell movement and division. Aconvenient route to understanding the complexity of cellular membranes is to studyartificially created lipid bilayer membrane systems. The work presented in this thesis isfocused on highly curved membrane structures in the form of lipid bilayer nanotubes.Firstly, the shape transformation mechanism for free floating lipid nanotubes wasinvestigated. Driven by their high curvature energy, nanotubes contract in length andeventually transform into tubular stomatocyte-like structures. Secondly, diffusion, electricfield and Marangoni-flow-driven modes of transport through lipid nanotubes are described.Then, an important improvement in the characterization of lipid nanotubes was achieved bydeveloping a new technique for the measurement of lipid nanotube radii. This technique isbased on monitoring the translocation of a photobleached tube region between twonanotube-connected vesicles during the growth of a receiving/daughter vesicle. The validityof this measurement technique was confirmed using super resolution microscopy. Inaddition, our technique has proven useful for tracking membrane bending rigidity changesin response to environmental and compositional alterations, both in cell plasma membranesand in model vesicle systems. Finally, a microfluidic pipette with a self-confining volume atthe tip was presented. It allows for selectively affecting a chosen cell and accessingmembranes on the single cell level

A multi-purpose microfluidic pipette for single-cell analysis

We report a multi-purpose microfluidic pipette, with a recirculating liquid tip. This device, made in poly(dimethylsiloxane), enables contamination-free manipulation and chemical stimulation of selected single cells in cell collectives or tissue slices. The pipette is capable of carrying out a variety of complex fluid processing functionalities, such as mixing, multiplexing, or gradient generation. The concept is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. We have applied the pipette in a fluorescence uptake assay, electrophysiology studies and for chemical induction of membrane protrusion from biological cells

A Microfluidic Pipette for Single-Cell Pharmacology

We report on a free-standing microfluidic pipette made in poly(dimethylsiloxane) having a circulating liquid tip that generates a self-con-fining volume in front of the outlet channels. The method is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. The pipette is capable of carrying out a variety of complex fluid processing operations, such as mixing, multiplexing, or gradient generation at selected cells in cell and tissue cultures. Using an uptake assay, we show that it is possible to generate dose response curves in situ from adherent Chinese hamster ovary cells expressing proton-activated human transient receptor potential vanilloid (hTRPV1) receptors. Using confined superfusion and cell stimulation, we could activate hTRPV1 receptors in single cells, measure the response by a patch-clamp pipette, and induce membrane bleb formation by exposing selected groups of cells to formaldehyde/dithiothreitol-containing solutions, respectively. In short, the microfluidic pipette allows for complex, contamination-free multiple-compound delivery for pharmacological screening of intact adherent cells

Generation of phospholipid vesicle-nanotube networks and transport of molecules therein

We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires similar to 90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60-120 min, depending on the transport regime

Generation of phospholipid vesicle-nanotube networks and transport of molecules therein

We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires similar to 90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60-120 min, depending on the transport regime

Spontaneous shape transformation of free-floating lipid membrane nanotubes

Freely floating lipid nanotubes, up to several hundred micrometers long, were found to spontaneously contract in length, and eventually transform into stomatocyte-like structures. This transformation was largely driven by the high curvature energy. The nanotubes equilibrate their membrane leaflet areas, by folding into tubular stomatocyte-like structures without any significant volume change, but require a substantial interleaflet lipid transport rate, estimated to be as high as 0.01-0.001 s(-1). The rate of transformation was dependent on the fluorescent membrane stain used, and nanotubes labelled with a water-soluble styryl dye, FM1-43, transformed approximately five-fold faster than nanotubes labelled with the phospholipid conjugated dye Texas Red DHPE