Interrogating cellular perception and decision making with optogenetic tools.

Optogenetics promises to deepen our understanding of how cells perceive and respond to complex and dynamic signals and how this perception regulates normal and abnormal function. In this study, we present our vision for how these nascent tools may transform our view of fundamental cell biological processes

Build life to understand it

Biologists and engineers should work together: synthetic biology reveals how organisms develop and function, argue Michael Elowitz and Wendell A. Lim

Genetic Sensor for Strong Methylating Compounds

Methylating chemicals are common in industry and agriculture and are often toxic, partly due to their propensity to methylate DNA. The Escherichia coli Ada protein detects methylating compounds by sensing aberrant methyl adducts on the phosphoester backbone of DNA. We characterize this system as a genetic sensor and engineer it to lower the detection threshold. By overexpressing Ada from a plasmid, we improve the sensor’s dynamic range to 350-fold induction and lower its detection threshold to 40 μM for methyl iodide. In eukaryotes, there is no known sensor of methyl adducts on the phosphoester backbone of DNA. By fusing the N-terminal domain of Ada to the Gal4 transcriptional activation domain, we built a functional sensor for methyl phosphotriester adducts in Saccharomyces cerevisiae. This sensor can be tuned to variable specifications by altering the expression level of the chimeric sensor and changing the number of Ada operators upstream of the Gal4-sensitive reporter promoter. These changes result in a detection threshold of 28 μM and 5.2-fold induction in response to methyl iodide. When the yeast sensor is exposed to different S[subscript N]1 and S[subscript N]2 alkylating compounds, its response profile is similar to that observed for the native Ada protein in E. coli, indicating that its native function is retained in yeast. Finally, we demonstrate that the specifications achieved for the yeast sensor are suitable for detecting methylating compounds at relevant concentrations in environmental samples. This work demonstrates the movement of a sensor from a prokaryotic to eukaryotic system and its rational tuning to achieve desired specifications.National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)United States. Office of Naval Research (N00014-13-1-0074)National Science Foundation (U.S.) (557686-2117)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210

Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

The Notch protein is one of the most mechanistically direct transmembrane receptors—the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues

Role of electrostatic interactions in PDZ domain ligand recognition

ABSTRACT: PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse R1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements. Cells rely on modular protein-protein recognition domains to assemble multiprotein signaling complexes. PDZ 1 (PSD-95, Dlg, ZO-1 homology) domains form a large family of such modules. PDZ domain-containing proteins play an important role in organizing signaling structures at cellcell signaling junctions such as synapses (1, 2). PDZ domains were first characterized for their ability to specifically recognize C-terminal peptide ligands, including the C-termini of receptors and channels Here we describe experiments aimed at more precisely evaluating the role of electrostatic interactions in PDZmediated peptide recognition using the Mus musculus R1-syntrophin PDZ domain as a model system. First, we have determined the sensitivity of various PDZ-mediated interactions to ionic strength, and second, we have determined the effects of mutating a conserved basic residue in the PDZ binding groove that has been proposed to play a role in electrostatic recognition. These results indicate that electrostatics do not play a significant role in recognition of the terminal carboxylate. In addition, we have examined a library of ligand variants and found that PDZ domains discriminate very tightly between ligands with minor alterations in the precise chemical structure of the C-terminus. Together, these results are consistent with a model in which steric and † This research was supported by grants from the National Institutes of Health and the David and Lucille Packard Foundation (to W.A.L.) and the Stewart Trust (to R.K.G. alcohol; Fmoc, 9-fluorenylmethoxycarbonyl; FPLC, fast performance liquid chromatography; HATU, 2-(1H-9-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HPLC, high-pressure liquid chromatography; IPTG, isopropyl -D-thiogalactopyranoside; MeOH, methanol or methyl alcohol; Ni-NTA, nickel nitriloacetic acid; nNOS, neuronal nitric oxide synthase; PCR, polymerase chain reaction; PDZ, PSD-95, Dlg, ZO-1 homology; TEV, tobacco etch virus; TFA, trifluoroacetic acid; TIS, triisopropylsilane

Three flavor neutrino oscillation analysis of atmospheric neutrinos in Super-Kamiokande

We report on the results of a three-flavor oscillation analysis using Super-Kamiokande~I atmospheric neutrino data, with the assumption of one mass scale dominance ($\Delta m_{12}^2$$=$0). No significant flux change due to matter effect, which occurs when neutrinos propagate inside the Earth for $\theta_{13}$$\neq$0, has been seen either in a multi-GeV $\nu_e$-rich sample or in a $\nu_\mu$-rich sample. Both normal and inverted mass hierarchy hypotheses are tested and both are consistent with observation. Using Super-Kamiokande data only, 2-dimensional 90 % confidence allowed regions are obtained: mixing angles are constrained to $\sin^2\theta_{13} < 0.14$ and $0.37 < \sin^2\theta_{23} < 0.65$ for the normal mass hierarchy. Weaker constraints, $\sin^2\theta_{13} < 0.27$ and $0.37 < \sin^2\theta_{23} < 0.69$, are obtained for the inverted mass hierarchy case.Comment: 7 figures, 3 table