In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.DOI: http://dx.doi.org/10.7554/eLife.01434.001

We describe the design, operation, and applications of two microfluidic devices that generate series of concentrations of oxygen, [O(2)], by on-chip gas mixing. Both devices are made of polydimethylsiloxane (PDMS) and have two layers of channels, the flow layer and the gas layer. By using in-situ measurements of [O(2)] with an oxygen-sensitive fluorescent dye, we show that gas diffusion through PDMS leads to equilibration of [O(2)] in an aqueous solution in the flow layer with [O(2)] in a gas injected into the gas layer on a time scale of approximately 1 sec. Injection of carbon dioxide into the gas layer causes the pH in the flow layer to drop within approximately 0.5 sec. Gas-mixing channel networks of both devices generate series of 9 gas mixtures with different [O(2)] from two gases fed to the inlets, thus creating regions with 9 different [O(2)] in the flow layer. The first device generates nitrogen-oxygen mixtures with [O(2)] varying linearly between 0 and 100%. The second device generates nitrogen-air mixtures with [O(2)] varying exponentially between 0 and 20.9%. The flow layers of the devices are designed for culturing bacteria in semi-permeable microchambers, and the second device is used to measure growth curves of E. coli colonies at 9 different [O(2)] in a single experiment. The cell division rates at [O(2)] of 0, 0.2, and 0.5% are found to be significantly different, further validating the capacity of the device to set [O(2)] in the flow layer with high precision and resolution. The degree of control of [O(2)] achieved in the devices and the robustness with respect to oxygen consumption due to respiration would be difficult to match in a traditional large-scale culture. The proposed devices and technology can be used in research on bacteria and yeast under microaerobic conditions and on mammalian cells under hypoxia.

We present a system consisting of a microfluidic device made of gas-permeable polydimethylsiloxane (PDMS) with two layers of microchannels and a computer-controlled multichannel gas mixer. Concentrations of oxygen in the liquid-filled flow channels of the device are imposed by flowing gas mixtures with desired oxygen concentrations through gas channels directly above the flow channels. Oxygen gradients with different linear, exponential, and non-monotonic shapes are generated in the same liquid-filled microchannel and reconfigured in real time. The system can be used to study directed migration of cells and the development of cell and tissue cultures under gradients of oxygen.

Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson’s disease, with an improved sensitivity of >100,000-fold over bulk measurements.