Quantitative reactivity profiling predicts functional cysteines in proteomes

Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.     

Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.     

Fluctuating stripes at the onset of the pseudogap in the high-T(c) superconductor Bi(2)Sr(2)CaCu(2)O(8+x)

Doped Mott insulators have a strong propensity to form patterns of holes and spins often referred to as stripes. In copper oxides, doping also gives rise to the pseudogap state, which can be transformed into a high-temperature superconducting state with sufficient doping or by reducing the temperature. A long-standing issue has been the interplay between the pseudogap, which is generic to all hole-doped copper oxide superconductors, and stripes, whose static form occurs in only one family of copper oxides over a narrow range of the phase diagram. Here we report observations of the spatial reorganization of electronic states with the onset of the pseudogap state in the high-temperature superconductor Bi(2)Sr(2)CaCu(2)O(8+x), using spectroscopic mapping with a scanning tunnelling microscope. We find that the onset of the pseudogap phase coincides with the appearance of electronic patterns that have the predicted characteristics of fluctuating stripes. As expected, the stripe patterns are strongest when the hole concentration in the CuO(2) planes is close to 1/8 (per copper atom). Although they demonstrate that the fluctuating stripes emerge with the onset of the pseudogap state and occur over a large part of the phase diagram, our experiments indicate that the stripes are a consequence of pseudogap behaviour rather than its cause.     

Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice

Electrical microstimulation can establish causal links between the activity of groups of neurons and perceptual and cognitive functions. However, the number and identities of neurons microstimulated, as well as the number of action potentials evoked, are difficult to ascertain. To address these issues we introduced the light-gated algal channel channelrhodopsin-2 (ChR2) specifically into a small fraction of layer 2/3 neurons of the mouse primary somatosensory cortex. ChR2 photostimulation in vivo reliably generated stimulus-locked action potentials at frequencies up to 50 Hz. Here we show that naive mice readily learned to detect brief trains of action potentials (five light pulses, 1 ms, 20 Hz). After training, mice could detect a photostimulus firing a single action potential in approximately 300 neurons. Even fewer neurons (approximately 60) were required for longer stimuli (five action potentials, 250 ms). Our results show that perceptual decisions and learning can be driven by extremely brief epochs of cortical activity in a sparse subset of supragranular cortical pyramidal neurons.     

DNA-guided crystallization of colloidal nanoparticles

Many nanometre-sized building blocks will readily assemble into macroscopic structures. If the process is accompanied by effective control over the interactions between the blocks and all entropic effects, then the resultant structures will be ordered with a precision hard to achieve with other fabrication methods. But it remains challenging to use self-assembly to design systems comprised of different types of building blocks-to realize novel magnetic, plasmonic and photonic metamaterials, for example. A conceptually simple idea for overcoming this problem is the use of 'encodable' interactions between building blocks; this can in principle be straightforwardly implemented using biomolecules. Strategies that use DNA programmability to control the placement of nanoparticles in one and two dimensions have indeed been demonstrated. However, our theoretical understanding of how to extend this approach to three dimensions is limited, and most experiments have yielded amorphous aggregates and only occasionally crystallites of close-packed micrometre-sized particles. Here, we report the formation of three-dimensional crystalline assemblies of gold nanoparticles mediated by interactions between complementary DNA molecules attached to the nanoparticles' surface. We find that the nanoparticle crystals form reversibly during heating and cooling cycles. Moreover, the body-centred-cubic lattice structure is temperature-tuneable and structurally open, with particles occupying only approximately 4% of the unit cell volume. We expect that our DNA-mediated crystallization approach, and the insight into DNA design requirements it has provided, will facilitate both the creation of new classes of ordered multicomponent metamaterials and the exploration of the phase behaviour of hybrid systems with addressable interactions.     

Event-horizon-scale structure in the supermassive black hole candidate at the Galactic Centre

The cores of most galaxies are thought to harbour supermassive black holes, which power galactic nuclei by converting the gravitational energy of accreting matter into radiation. Sagittarius A* (Sgr A*), the compact source of radio, infrared and X-ray emission at the centre of the Milky Way, is the closest example of this phenomenon, with an estimated black hole mass that is 4,000,000 times that of the Sun. A long-standing astronomical goal is to resolve structures in the innermost accretion flow surrounding Sgr A*, where strong gravitational fields will distort the appearance of radiation emitted near the black hole. Radio observations at wavelengths of 3.5 mm and 7 mm have detected intrinsic structure in Sgr A*, but the spatial resolution of observations at these wavelengths is limited by interstellar scattering. Here we report observations at a wavelength of 1.3 mm that set a size of 37(+16)(-10) microarcseconds on the intrinsic diameter of Sgr A*. This is less than the expected apparent size of the event horizon of the presumed black hole, suggesting that the bulk of Sgr A* emission may not be centred on the black hole, but arises in the surrounding accretion flow.