Synthetic Physiology

Optogenetic tools are DNA-encoded molecules that, when genetically targeted to cells, enable the control of specific physiological processes within those cells through exposure to light. These tools can pinpoint how these specific processes affect the emergent properties of a complex biological system, such as a mammalian organ or even an entire animal. They can also allow control of a biological system for therapeutic or bioengineering purposes. Many of the optical control tools explored to date are single-component reagents containing a photoactive signaling domain. An interesting question is raised by comparing optogenetics to synthetic biology. In the latter, interchangeable and modular DNA-encoded parts are assembled into complex biological circuits, thus enabling sophisticated logic and computation as well as the production of biologics and reagents (1, 2). Is it possible to devise strategies for the temporally precise cell-targeted optical control of complex engineered biological computational or chemical-synthetic pathways? Such a marriage of optogenetics and synthetic biology—which one might call synthetic physiology—would open up the ability to use optogenetics to trigger and regulate engineered synthetic biology systems, which in turn could execute computational and biological programs of great complexity (3). On page 1565 of this issue, Ye et al. (4) explore such a hybrid approach to controlling a biological system, as well as the bioengineering and preclinical capabilities opened up by such an approach

Resource determinants of strategy and performance: the case of British exporters

This study adopts the RBV of the firm in order to identify critical advantage-generating resources and capabilities with strong positive export strategy and performance implications. The proposed export performance model is tested using a structural equation modeling approach on a sample of 356 British exporters. We examine the individual as well as the concurrent (simultaneous) direct and indirect effects of five resource bundles on export performance. We find that four resources/capabilities: managerial, knowledge, planning, and technology, have a significant positive direct effect on export performance, while relational and physical resources exhibited no unique positive effect. We also find that the firm’s export strategy mediates the resource-performance nexus in the case of managerial and knowledge-based resources. The theoretical and methodological grounding of this study contributes to the advancement of export related research by providing better specification of the nature of the effects – direct or indirect – of particular resource factors on export performance

Ultrastructural and functional fate of recycled vesicles in hippocampal synapses

Efficient recycling of synaptic vesicles is thought to be critical for sustained information transfer at central terminals. However, the specific contribution that retrieved vesicles make to future transmission events remains unclear. Here we exploit fluorescence and time-stamped electron microscopy to track the functional and positional fate of vesicles endocytosed after readily releasable pool (RRP) stimulation in rat hippocampal synapses. We show that most vesicles are recovered near the active zone but subsequently take up random positions in the cluster, without preferential bias for future use. These vesicles non-selectively queue, advancing towards the release site with further stimulation in an actin-dependent manner. Nonetheless, the small subset of vesicles retrieved recently in the stimulus train persist nearer the active zone and exhibit more privileged use in the next RRP. Our findings reveal heterogeneity in vesicle fate based on nanoscale position and timing rules, providing new insights into the origins of future pool constitution

Optogenetics and deep brain stimulation neurotechnologies

Brain neural network is composed of densely packed, intricately wired neurons whose activity patterns ultimately give rise to every behavior, thought, or emotion that we experience. Over the past decade, a novel neurotechnique, optogenetics that combines light and genetic methods to control or monitor neural activity patterns, has proven to be revolutionary in understanding the functional role of specific neural circuits. We here briefly describe recent advance in optogenetics and compare optogenetics with deep brain stimulation technology that holds the promise for treating many neurological and psychiatric disorders

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots

LED Arrays as Cost Effective and Efficient Light Sources for Widefield Microscopy

New developments in fluorophores as well as in detection methods have fueled the rapid growth of optical imaging in the life sciences. Commercial widefield microscopes generally use arc lamps, excitation/emission filters and shutters for fluorescence imaging. These components can be expensive, difficult to maintain and preclude stable illumination. Here, we describe methods to construct inexpensive and easy-to-use light sources for optical microscopy using light-emitting diodes (LEDs). We also provide examples of its applicability to biological fluorescence imaging

Live cell fluorescence microscopy to study microbial pathogenesis

Advances in microscopy and fluorescent probes provide new insight into the nanometer-scale biochemistry governing the interactions between eukaryotic cells and pathogens. When combined with mathematical modelling, these new technologies hold the promise of qualitative, quantitative and predictive descriptions of these pathways. Using the light microscope to study the spatial and temporal relationships between pathogens, host cells and their respective biochemical machinery requires an appreciation for how fluorescent probes and imaging devices function. This review summarizes how live cell fluorescence microscopy with common instruments can provide quantitative insight into the cellular and molecular functions of hosts and pathogens.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72689/1/j.1462-5822.2009.01283.x.pd

Two glycerol uptake systems contribute to the high osmotolerance of Zygosaccharomyces rouxii

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. Here we show that the osmotolerant yeast Zygosaccharomyces rouxii has two genes, ZrSTL1 and ZrSTL2, encoding transporters mediating the active uptake of glycerol in symport with protons, contributing to cell osmotolerance and intracellular pH homeostasis. The growth of mutants lacking one or both transporters is affected depending on the growth medium, carbon source, strain auxotrophies, osmotic conditions and the presence of external glycerol. These transporters are localised in the plasma membrane, they transport glycerol with similar kinetic parameters and besides their expected involvement in the cell survival of hyperosmotic stress, they surprisingly both contribute to an efficient survival of hypoosmotic shock and to the maintenance of intracellular pH homeostasis under non-stressed conditions. Unlike STL1 in Sa. cerevisiae, the two Z. rouxii STL genes are not repressed by glucose, but their expression and activity are downregulated by fructose and upregulated by non-fermentable carbon sources, with ZrSTL1 being more influenced than ZrSTL2. In summary, both transporters are highly important, though Z. rouxii CBS 732(T) cells do not use external glycerol as a source of carbon.The help of Dr. P. Ergang with the real-time PCR experiments is gratefully acknowledged. We thank O. Zimmermannova for critical reading of the paper. This work was supported by the following grants: Grant Agency of the Czech Republic P503/ 10/0307, institutional concept RVO:6798582, Grant Agency of the Charles University 299611/2011/B-Bio/PrF, an Lifelong Learning Programme ERASMUS practical placement grant and by Fundo Europeu de Desenvolvimento Regional – Programa Operacional de Fatores de Competitividade – COMPETE and by national funds from Fundação para a Ciência e Tecnologia through the project PEstC/BIA/UI4050/ 2011.info:eu-repo/semantics/publishedVersio

Integrin Signaling Switches the Cytoskeletal and Exocytic Machinery that Drives Neuritogenesis

Neurons establish their unique morphology by elaborating multiple neurites that subsequently form axons and dendrites. Neurite initiation entails significant surface area expansion, necessitating addition to the plasma membrane. We report that regulated membrane delivery coordinated with the actin cytoskeleton is crucial for neuritogenesis, and identify two independent pathways that use distinct exocytic and cytoskeletal machinery to drive neuritogenesis. One pathway employs Ena/VASP-regulated actin dynamics coordinated with VAMP2-mediated exocytosis, and involves a novel role for Ena/VASP in exocytosis. A second mechanism occurs in the presence of laminin through integrin-dependent activation of FAK and src, and utilizes coordinated activity of the Arp2/3 complex and VAMP7-mediated exocytosis. We conclude that neuritogenesis can be driven by two distinct pathways that differentially coordinate cytoskeletal dynamics and exocytosis. These regulated changes and coordination of cytoskeletal and exocytic machinery may be utilized in other physiological contexts involving cell motility and morphogenesis.Jane Coffin Childs Memorial Fund for Medical ResearchNational Institutes of Health (U.S.) (NIH grant GM68678)Stanley Medical Research Institut