Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach

Evidence for complete and partial surface renewal at an air-water interface

A wind-wave flume is used to determine the extent to which the thermal boundary layer (TBL) at a wind-forced air-water interface is completely renewed from below. We measure skin temperature, Tskin, radiometrically, temperature immediately below the TBL, Tsubskin, using a temperature profiler, and net heat flux using the gradient flux technique. The Tskin probability density function, p(Tskin), and surface renewal time scale, τ, were measured using passive and active infrared imaging techniques, respectively. We find that the mean percentile rank of Tsubskin in p(Tskin) is 99.90, implying that complete surface renewal occurs. This result suggests an alternative to radiometric measurement of Tskin through the simple combination of an infrared camera and an in situ temperature sensor. Comparison of the temperature difference across the TBL to the expected cooling implies that a significant portion of events only partially renew the TBL. This result should impact efforts to improve air-sea transfer models

SLAC/CERN high gradient tests on an X-band accelerating section

High frequency linear collider schemes envisage the use of rather high accelerating gradients: 50 to 100 MV/m for X-band and 80 MV/m for CLIC. Because these gradients are well above those commonly used in accelerators, high gradient studies of high frequency structures have been initiated and test facilities have been constructed at KEK [1], SLAC [2] and CERN [3]. The studies seek to demonstrate that the above mentioned gradients are both achievable and practical. There is no well-defined criterion for the maximum acceptable level of dark current but it must be low enough not to generate unacceptable transverse wakefields, disturb beam position monitor readings or cause RF power losses. Because there are of the order of 10,000 accelerating sections in a high frequency linear collider, the conditioning process should not be too long or difficult. The test facilities have been instrumented to allow investigation of field emission and RF breakdown mechanisms. With an understanding of these effects, the high gradient performance of accelerating sections may be improved through modifications in geometry, fabrication methods and surface finish. These high gradient test facilities also allow the ultimate performance of high frequency/short pulse length accelerating structures to be probed. This report describes the high gradient test at SLAC of an X-band accelerating section built at CERN using technology developed for CLIC

Statistics of surface divergence and their relation to air-water gas transfer velocity

Air-sea gas fluxes are generally defined in terms of the air/water concentration difference of the gas and the gas transfer velocity,kL. Because it is difficult to measure kLin the ocean, it is often parameterized using more easily measured physical properties. Surface divergence theory suggests that infrared (IR) images of the water surface, which contain information concerning the movement of water very near the air-water interface, might be used to estimatekL. Therefore, a series of experiments testing whether IR imagery could provide a convenient means for estimating the surface divergence applicable to air-sea exchange were conducted in a synthetic jet array tank embedded in a wind tunnel. Gas transfer velocities were measured as a function of wind stress and mechanically generated turbulence; laser-induced fluorescence was used to measure the concentration of carbon dioxide in the top 300 μm of the water surface; IR imagery was used to measure the spatial and temporal distribution of the aqueous skin temperature; and particle image velocimetry was used to measure turbulence at a depth of 1 cm below the air-water interface. It is shown that an estimate of the surface divergence for both wind-shear driven turbulence and mechanically generated turbulence can be derived from the surface skin temperature. The estimates derived from the IR images are compared to velocity field divergences measured by the PIV and to independent estimates of the divergence made using the laser-induced fluorescence data. Divergence is shown to scale withkLvalues measured using gaseous tracers as predicted by conceptual models for both wind-driven and mechanically generated turbulence