Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways

VLBI Monitoring Observations of Water Masers Around the Semi-Regular Variable Star R Crateris

We monitored water-vapor masers around the semi-regular variable star R Crateris with the Japanese VLBI Network (J-Net) at the 22 GHz band during four epochs with intervals of one month. The relative proper motions and Doppler-velocity drifts of twelve maser features were measured. Most of them existed for longer than 80 days. The 3-D kinematics of the features indicates a bipolar expanding flow. The major axis of the asymmetric flow was estimated to be at P.A. = 136 degrees. The existence of a bipolar outflow suggests that a Mira variable star had already formed a bipolar outflow. The water masers are in a region of apparent minimum radii of 1.3 x 10^12 m and maximum radii of 2.6 x 10^12 m, between which the expansion velocity ranges from 4.3 to 7.4 km/s. These values suggest that the water masers are radially accelerated, but still gravitationally bound, in the water-maser region. The most positive and negative velocity-drifting features were found relatively close to the systemic velocity of the star. We found that the blue-shifted features are apparently accelerated and the red-shifted apparently decelerated. The acceleration of only the blue-shifted features seems to be consistent with that of the expanding flow from the star.Comment: 15 pages, 5 figures, Accepted for publication in PASJ (2001), preprint can be obtained via WWW on http://www.nro.nao.ac.jp/library/report/list.htm

Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging

The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M-1 cm-1, mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths

Global analysis of neutrino masses, mixings and phases: entering the era of leptonic CP violation searches

We perform a global analysis of neutrino oscillation data, including high-precision measurements of the neutrino mixing angle theta_13 at reactor experiments, which have confirmed previous indications in favor of theta_13>0. Recent data presented at the Neutrino 2012 Conference are also included. We focus on the correlations between theta_13 and the mixing angle theta_23, as well as between theta_13 and the neutrino CP-violation phase delta. We find interesting indications for theta_23< pi/4 and possible hints for delta ~ pi, with no significant difference between normal and inverted mass hierarchy.Comment: Updated version, including recent data released at the Neutrino 2012 Conference. Some references adde

The 3-D kinematics of water masers around the semiregular variable RT Virginis

We report observations of water masers around the semiregular variable RT Virginis (RT Vir), which have been made with the Very Long Baseline Array (VLBA) of the National Radio Astronomy Observatory (NRAO) at five epochs, each separated by three weeks of time. We detected about 60 maser features at each epoch. Overall, 61 features, detected at least twice, were tracked by their radial velocities and proper motions. The 3-D maser kinematics exhibited a circumstellar envelope that is expanding roughly spherically with a velocity of about 8 km/s. Asymmetries in both the spatial and velocity distributions of the maser features were found in the envelope, but less significant than that found in other semiregular variables. Systematic radial-velocity drifts of individual maser features were found with amplitudes of <= 2 km/s/yr. For one maser feature, we found a quadratic position shift with time along a straight line on the sky. This apparent motion indicates an acceleration with an amplitude of 33 km/s/yr, implying the passage of a shock wave driven by the stellar pulsation of RT Vir. The acceleration motion is likely seen only on the sky plane because of a large velocity gradient formed in the accelerating maser region. We estimated the distance to RT Vir to be about 220 pc on the basis of both the statistical parallax and model-fitting methods for the maser kinematics.Comment: 18 pages, 8 figures. Accepted to appear in the Astrophysical Journa

Experimental Status of Neutrino Physics

After a fascinating phase of discoveries, neutrino physics still has a few mysteries such as the absolute mass scale, the mass hierarchy, the existence of CP violation in the lepton sector and the existence of right-handed neutrinos. It is also entering a phase of precision measurements. This is what motivates the NUFACT 11 conference which prepares the future of long baseline neutrino experiments. In this paper, we report the status of experimental neutrino physics. We focus mainly on absolute mass measurements, oscillation parameters and future plans for oscillation experiments