Injection and transport of bacteria in nanotube-vesicle networks.

The microinjection of bacteria (the MG1655 strain of E. coli) into unilamellar lipid vesicles contained in surface-immobilized nanotube-vesicle networks is demonstrated. The density of bacteria can be controlled from a single bacterium up to several thousands of bacteria per injected vesicle. The bacteria retain flagellar motion and propulsion. The bacteria (approximately 2 × 0.8 µm) cannot escape from one vesicle to another as the size of the nanotubes is too small (∼200 nm in diameter) to allow for entry. Bacteria can, however, be moved from one vesicle to another in a nanotube-vesicle network by using Marangoni flows. Thus, single or several species can be transferred to a neighboring vesicle at will. The technique offers new possibilities for live matter functionalization into synthetic host networks, and may provide means for studying the effect of compartmentalization and perfusion of chemical species on a single bacterium. Furthermore, it may serve as an experimental model to study how vesicle-encapsulated bacteria evade destruction in macrophages or how bacteria surf along thin membrane nanotubes toward connected macrophage cell bodies.

Intercellular nanotubes: insights from imaging studies and beyond.

Cell-cell communication is critical to the development, maintenance, and function of multicellular organisms. Classical mechanisms for intercellular communication include secretion of molecules into the extracellular space and transport of small molecules through gap junctions. Recent reports suggest that cells also can communicate over long distances via a network of transient intercellular nanotubes. Such nanotubes have been shown to mediate intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, intercellular nanotubes have been observed in vivo and have been shown to enhance the transmission of pathogens such as human immunodeficiency virus (HIV)-1 and prions in vitro. These studies indicate that intercellular nanotubes may play a role both in normal physiology and in disease.

Imaging immune surveillance of individual natural killer cells confined in microwell arrays.

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

Electrophoretic transport in surfactant nanotube networks wired on microfabricated substrates.

Nanofluidic devices are rapidly emerging as tools uniquely suited to transport and interrogate single molecules. We present a simple method to rapidly obtain compact surfactant nanotube networks of controlled geometry and length. The nanotubes, 100-300 nm in diameter, are pulled from lipid vesicles using a micropipet technique, with multilamellar vesicles serving as reservoirs of surfactant material. In a second step, the nanotubes are wired around microfabricated SU-8 pillars. In contrast to unrestrained surfactant networks that minimize their surface free energy by minimizing nanotube path length, the technique presented here can produce nanotube networks of arbitrary geometries. For example, nanotubes can be mounted directly on support pillars, and long stretches of nanotubes can be arranged in zigzag patterns with turn angles of 180 degrees. The system is demonstrated to support electrophoretic transport of colloidal particles contained in the nanotubes down to the limit of single particles. We show that electrophoretic migration velocity is linearly dependent on the applied field strength and that a local narrowing of the nanotube diameter results from adhesion and bending around SU-8 pillars. The method presented here can aid in the fabrication of fully integrated and multiplexed nanofluidic devices that can operate with single molecules.

Zipper dynamics of surfactant nanotube Y junctions.

We investigate the formation of Y junctions in surfactant nanotubes connecting vesicles. Based on experimental observations of the surfactant flow on the nanotubes, we conclude that a Y junction propagates with a zipperlike mechanism. The surfactants from two nanotube branches undergo 1:1 mixing at the junction, and spontaneously form the extension of the third nanotube branch. Taking into account the tension driven surfactant flow, we develop a model for the Y junction dynamics that is in quantitative agreement with the experimental data.

Biomimetic nanoscale reactors and networks.

Methods based on self-assembly, self-organization, and forced shape transformations to form synthetic or semisynthetic enclosed lipid bilayer structures with several properties similar to biological nanocompartments are reviewed. The procedures offer unconventional micro- and nanofabrication routes to yield complex soft-matter devices for a variety of applications for example, in physical chemistry and nanotechnology. In particular, we describe novel micromanipulation methods for producing fluid-state lipid bilayer networks of nanotubes and surface-immobilized vesicles with controlled geometry, topology, membrane composition, and interior contents. Mass transport in nanotubes and materials exchange, for example, between conjugated containers, can be controlled by creating a surface tension gradient that gives rise to a moving boundary or by induced shape transformations. The network devices can operate with extremely small volume elements and low mass, to the limit of single molecules and particles at a length scale where a continuum mechanics approximation may break down. Thus, we also describe some concepts of anomalous fluctuation-dominated kinetics and anomalous diffusive behaviours, including hindered transport, as they might become important in studying chemistry and transport phenomena in these confined systems. The networks are suitable for initiating and controlling chemical reactions in confined biomimetic compartments for rationalizing, for example, enzyme behaviors, as well as for applications in nanofluidics, bioanalytical devices, and to construct computational and complex sensor systems with operations building on chemical kinetics, coupled reactions and controlled mass transport.