A primary aim of microbial ecology is to determine patterns and drivers of community distribution, interaction, and assembly amidst complexity and uncertainty. Microbial community composition has been shown to change across gradients of environment, geographic distance, salinity, temperature, oxygen, nutrients, pH, day length, and biotic factors 1-6 . These patterns have been identified mostly by focusing on one sample type and region at a time, with insights extra polated across environments and geography to produce generalized principles. To assess how microbes are distributed across environments globally-or whether microbial community dynamics follow funda mental ecological 'laws' at a planetary scale-requires either a massive monolithic cross environment survey or a practical methodology for coordinating many independent surveys. New studies of microbial environments are rapidly accumulating; however, our ability to extract meaningful information from across datasets is outstripped by the rate of data generation. Previous meta analyses have suggested robust gen eral trends in community composition, including the importance of salinity 1 and animal association 2 . These findings, although derived from relatively small and uncontrolled sample sets, support the util ity of meta analysis to reveal basic patterns of microbial diversity and suggest that a scalable and accessible analytical framework is needed.The Earth Microbiome Project (EMP, http://www.earthmicrobiome. org) was founded in 2010 to sample the Earth's microbial communities at an unprecedented scale in order to advance our understanding of the organizing biogeographic principles that govern microbial commu nity structure 7,8 . We recognized that open and collaborative science, including scientific crowdsourcing and standardized methods 8 , would help to reduce technical variation among individual studies, which can overwhelm biological variation and make general trends difficult to detect 9 . Comprising around 100 studies, over half of which have yielded peer reviewed publications (Supplementary Table 1), the EMP has now dwarfed by 100 fold the sampling and sequencing depth of earlier meta analysis efforts 1,2 ; concurrently, powerful analysis tools have been developed, opening a new and larger window into the distri bution of microbial diversity on Earth. In establishing a scalable frame work to catalogue microbiota globally, we provide both a resource for the exploration of myriad questions and a starting point for the guided acquisition of new data to answer them. As an example of using this Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of r...

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Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.

We describe a novel micropipet-assisted technique for the construction of complex, surface-immobilized two-dimensional microscopic networks of unilamellar phospholipid bilayer vesicles (1−50 μm in diameter, 10-15−10-12 L) interconnected by lipid nanotubes (100−300 nm in diameter). As starting material for the construction of networks, we used twinned vesicle pairs, one of which is multilamellar and functions as a membrane donor and the other unilamellar and functions as a membrane acceptor upon manipulation. By electromechanical insertion of a pipet tip into the unilamellar vesicle followed by lateral pulling of the micropipet away from the vesicle, a nanotube was formed. Buffer solution contained in the pipet was then injected into the nanotube orifice, forming a vesicle of controlled size that was immobilized on the surface. The networks have controlled connectivity and are well-defined with regard to the container size, angle between nanotube extensions, and nanotube length. The internal fluid composition of individual vesicles is defined during the formation of a network by selection of the solution contained in the micropipet.

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.

Exocytosis is the fundamental process underlying neuronal communication. This process involves fusion of a small neurotransmitter-containing vesicle with the plasma membrane of a cell to release minute amounts of transmitter molecules. Exocytosis is thought to go through an intermediate step involving formation of a small lipid nanotube or fusion pore, followed by expansion of the pore to the final stage of exocytosis. The process of exocytosis has been studied by various methods; however, when living cells are used it is difficult to discriminate between the molecular effects of membrane proteins relative to the mechanics of lipid-membranedriven processes and to manipulate system parameters (e.g., membrane composition, pH, ion concentration, temperature, etc.). We describe the use of liposome-lipid nanotube networks to create an artificial cell model that undergoes the later stages of exocytosis. This model shows that membrane mechanics, without protein intervention, can drive expansion of the fusion pore to the final stage of exocytosis and can affect the rate of transmitter release through the fusion pore.

We describe fluidic control in lipid nanotubes 50−150 nm in radius, conjugated with surface-immobilized unilamellar lipid bilayer vesicles (∼5−25 μm in diameter). Transport in nanotubes was induced by continuously increasing the surface tension of one of the conjugated vesicles, for example, by ellipsoidal shape deformation using a pair of carbon microfibers controlled by micromanipulators as tweezers. The shape deformation resulted in a flow of membrane lipids toward the vesicle with the higher membrane tension; this lipid flow in turn moved the liquid column inside the nanotube through viscous coupling. Thus, micrometer-sized vesicles are used as a handle for controlling fluid flow inside nanometer-sized channels. We show transport and trapping of a single 30-nm-diameter carboxylate-modified latex particle inside a ∼100-nm-radius nanotube. Fluidic control in nanometer-sized channels using a moving wall provides pluglike liquid flows, offers a means for efficient routing and trapping of small molecules, polymers, and colloids, and offers new opportunities to study chemistry in confined spaces. Networks of nanotubes and vesicles might serve as a platform to build nanofluidic devices operating with single molecules and nanoparticles.

We show how an electrolyte-filled capillary (EFC) coupled to a high-voltage power supply can be used as a versatile electroporation tool for the delivery of dyes, drugs, and biomolecules to the cytoplasm of single cells and cells in tissues. A large-voltage pulse applied across the EFC (fused silica, 30 cm long, 375-microm o.d., 30-microm i.d.) gives rise to a small electric field outside the terminus of the EFC, which causes pore formation in cell membranes and induces an electroosmotic flow of electrolyte. When the EFC contains cell-loading agents, then the electroosmotic flow delivers the agents at the site of pore formation. The combination of pore formation and delivery enables loading of materials into the cytoplasm. By patch-clamp and fluorescence microscopy, formation of pores was observed at estimated transmembrane voltages of <85 mV with half-maximum values around 206 mV. The electroporation protocol was demonstrated by introduction of fluorogenic dyes into single NG108-15 cells, cellular processes, and small populations of cells in organotypic hippocampal cultures. Preliminary results are shown in which this protocol was employed for in vivo electroporation of ventral mesencephalon in rat brains. The technique was also used to access organelle-based detection systems inside cells. As a demonstration, 1,4,5-inositoltriphosphate was added to the electrolyte and detected by intracellular organelles in electroporated cells.