Genetically engineered sensors of cell signaling

<p>Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.</p> <p>We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.</p>

Search for heavy neutral resonances in the dielectron channel with the CMS detector at the LHC

A search for dielectron decays of heavy neutral resonances has been performed using proton-proton collision data collected at √s = 7 TeV by the Compact Muon Solenoid (CMS) experiment at the Large Hadron Collider (LHC) in 2011. The data sample corresponds to an integrated luminosity of 5 fb^−1. The dielectron mass distribution is consistent with Standard Model (SM) predictions. An upper limit on the ratio of the cross section times branching fraction of new bosons, normalized to the cross section times branching fraction of the Z boson, is set at the 95 % confidence level. This result is translated into limits on the mass of new neutral particles at the level of 2120 GeV for the Z′ in the Sequential Standard Model, 1810 GeV for the superstring-inspired Z′_ψ resonance, and 1940 (1640) GeV for Kaluza-Klein gravitons with the coupling parameter k/M_Pl of 0.10 (0.05).

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

<p>The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.</p> <p>It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.</p> <p>Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.</p> <p>MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.</p>

Inelastic K+p reactions at incident momenta from 1.37 GeV/c to 2.17 GeV/c

<p>A number of recent experiments have suggested the possibility of a highly inelastic resonance in K⁺p scattering. To study the inelastic K⁺p reactions, a 400 K exposure has been taken at the L.R.L. 25 inch bubble chamber. The data are spread over seven K⁺ momenta between 1.37 and 2.17 GeV/c.</p> <p>Cross-sections have been measured for the reaction K⁺p → pK°π+ which is dominated by the quasi-two body channels K∆ and K*N. Both these channels are strongly peripheral, as at other momenta. The decay of the ∆ is in good agreement with the predictions of the rho-photon analogy of Stodolsky and Sakurai. The data on the K*p channel show evidence of both pseudo scalar and vector exchange.</p> <p>Cross-sections for the final state pK⁺Ï€+Ï€- shows a strong contribution from the quasi-two body channel K*∆. This reaction is also very peripheral even at threshold. The decay angular distributions indicate the reaction is dominated as at higher momenta by a pion exchange mechanism. The data are also in good agreement with the quark model predictions of Bialas and Zalewski for the K* and ∆ decay.</p>

Temporal Control of Ion Channel Activation

<p>Ion channels are a large class of integral membrane proteins that allow for the diffusion of ions across a cellular membrane and are found in all forms of life. Pentameric ligand-gated ion channels (pLGICs) comprise a large family of proteins that include the nicotinic acetylcholine receptor (nAChR) and the γ-aminobutyric acid (GABA) receptor. These ion channels are responsible for the fast synaptic transmission that occurs in humans and as a result are of fundamental biological importance. pLGICs bind ligands (neurotransmitters), and upon ligand-binding undergo activation. The activation event causes an ion channel to enter a new physical state that is able to conduct ions. Ion channels allow for the flux of ions across the membrane through a pore that is formed upon ion channel activation. For pLGICs to function properly both ligand-binding and ion channel activation must occur. The ligand-binding event has been studied extensively over the past few decades, and a detailed mechanism of binding has emerged. During activation the ion channel must undergo structural rearrangements that allow the protein to enter a conformation in which ions can flow through. Despite this great and ubiquitous importance, a fundamental understanding of the ion channel activation mechanism and kinetics, as well as concomitant structural arrangements, remains elusive.</p> <p>This dissertation describes efforts that have been made to temporally control the activation of ligand-gated ion channels. Temporal control of ion channel activation provides a means by which to activate ion channels when desired. The majority of this work examines the use of light to activate ion channels. Several photocages were examined in this thesis; photocages are molecules that release a ligand under irradiation, and, for the work described here, the released ligand then activates the ion channel. First, a new water-soluble photoacid was developed for the activation of proton-sensitive ion channels. Activation of acid-sensing ion channels, ASIC2a and GLIC, was observed only upon irradiation. Next, a variety of Ru²⁺ photocages were also developed for the release of amine ligands. The Ru²⁺ systems interacted in a deleterious manner with a representative subset of biologically essential ion channels. The rapid mixing of ion channels with agonist was also examined. A detection system was built to monitor ion channels activation in the rapid mixing experiments. I have shown that liposomes, and functionally-reconstituted ELIC, are not destroyed during the mixing process. The work presented here provides the means to deliver agonist to ligand-gated ion channels in a controlled fashion.</p>