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Sheldon Penman - One of the best experts on this subject based on the ideXlab platform.
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RESINLESS SECTION ELECTRON MICROSCOPY REVEALS THE YEAST CYTOSKELETON
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Joshua Penman, Sheldon PenmanAbstract:The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective Detergent Extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow Detergent Extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or “soluble” proteins are distinct from the retained or “structural” proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters—5 nm and 15–20 nm—which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300–500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.
Joshua Penman - One of the best experts on this subject based on the ideXlab platform.
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RESINLESS SECTION ELECTRON MICROSCOPY REVEALS THE YEAST CYTOSKELETON
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Joshua Penman, Sheldon PenmanAbstract:The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective Detergent Extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow Detergent Extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or “soluble” proteins are distinct from the retained or “structural” proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters—5 nm and 15–20 nm—which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300–500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.
Thomas J. Mcintosh - One of the best experts on this subject based on the ideXlab platform.
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Stepping between Membrane Microdomains
Biophysical Journal, 2015Co-Authors: Thomas J. McintoshAbstract:Lateral membrane microdomains are postulated to be involved in a number of important functions in mammalian cells, such as membrane trafficking, exocytosis, endocytosis, signal transduction, and protein activity (1,2). These putative cell microdomains have been the subject of extensive research using a variety of biophysical and biochemical techniques, including Detergent Extraction (2), fluorescence microscopy (3), electron microscopy (4), fluorescence resonance energy transfer microscopy (5), fluorescence correlation spectroscopy (6), fluorescence recovery after photobleaching (7), and single-molecule tracking (8).
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Transbilayer Peptide Sorting between Raft and Nonraft Bilayers: Comparisons of Detergent Extraction and Confocal Microscopy
Biophysical journal, 2005Co-Authors: Adriana C. Vidal, Thomas J. McintoshAbstract:Membrane microdomains (“rafts”) that sequester specific proteins and lipids are often characterized by their resistance to Detergent Extraction. Because rafts are enriched in sphingomyelin and cholesterol, raft bilayers are thicker and have larger area compressibility moduli than nonraft bilayers. It has been postulated that rafts concentrate proteins with long transmembrane domains (TMDs) because of “hydrophobic matching” between the TMDs and the thick raft bilayers. However, previous Detergent Extraction experiments with bilayers containing raft and nonraft domains have shown that the peptides P-23 and P-29, designed to have single TMDs matching the hydrocarbon thicknesses of Detergent soluble membranes and Detergent resistant membranes, respectively, are both localized to Detergent soluble membranes. Those results imply that both peptides are preferentially located in nonraft domains. However, because the Detergent solubilizes part of the bilayer, it has been unclear whether or not Detergent Extraction experiments provide an accurate indication of the location of peptides in intact bilayers. Here we use confocal microscopy to examine the distribution of these same peptides in intact bilayers containing both raft and nonraft domains. At 20°C and 37°C, P-23 and P-29 were both primarily localized in fluorescently labeled nonraft domains. These confocal results validate the previous Detergent Extraction experiments and demonstrate the importance of bilayer cohesive properties, compared to hydrophobic mismatch, in the sorting of these peptides that contain a single TMD.
Sandro Keller - One of the best experts on this subject based on the ideXlab platform.
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Calorimetric Quantification of Cyclodextrin-Mediated Detergent Extraction for Membrane-Protein Reconstitution.
Methods in enzymology, 2015Co-Authors: Martin Textor, Sandro KellerAbstract:For many in vitro studies, purified membrane proteins need to be reconstituted from Detergent micelles into lipid bilayers to regain their native structures and functions. Stoichiometric complexation of Detergent by cyclodextrin provides a tightly controllable strategy for Detergent Extraction. Here, we describe a practical approach making use of isothermal titration calorimetry to obtain a complete set of thermodynamic parameters that allows for quantitative prediction of the transition from micelles to bilayer membranes during reconstitution. These parameters include the dissociation constant of the cyclodextrin/Detergent inclusion complex, the critical micellar concentration of the Detergent, and the phase boundaries of the lipid/Detergent phase diagram. The underlying theoretical framework involves linked equilibria among all pseudophases, as described previously (Textor, Vargas, & Keller, 2015). This chapter focuses on practical aspects of the approach and discusses caveats and calorimetry-specific details of data analysis. With the entire parameter set at hand, exploration of different reconstitution trajectories within the lipid/Detergent phase diagram is possible. Together with the straightforward control over the rate of Detergent Extraction offered by cyclodextrin complexation, this opens the possibility of systematically tuning and optimizing the reconstitution process of membrane proteins. Provided some particular precautions are taken, the approach can be adapted to many other combinations of proteins, lipids, Detergents, and cyclodextrins.
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Controlled Reconstitution of Integral Membrane Proteins by Detergent Extraction through Cyclodextrin Complexation
Biophysical Journal, 2014Co-Authors: Carolyn Vargas, Martin Textor, Natalia Markova, Sandro KellerAbstract:The reconstitution of purified membrane proteins from a Detergent-solubilized state into lipid bilayer membranes is a prerequisite for many in vitro studies on membrane channels and transporters. Among the diverse methods used for the removal of Detergent from ternary protein/lipid/Detergent mixtures, Detergent complexation with cyclodextrins offers a number of unique advantages. In particular, cyclodextrins sequester Detergents selectively and at defined stoichiometries, which would, in principle, allow for a tight control of the reconstitution process and facilitate the rational optimization of experimental protocols. However, no systematic, quantitative studies on the complex interactions among cyclodextrins, Detergents, and lipids have been reported to date. Thus, we adopted a microcalorimetric approach to thermodynamically characterize the complexation of a homologous series of alkyl maltoside Detergents by various substituted β-cyclodextrins.The binding affinity increased with alkyl chain length, as reflected by a Gibbs free energy increment of about 3 kJ/mol per methylene group. In contrast with many other complexation reactions involving cyclodextrins, Detergent binding did not reveal enthalpy−entropy compensation. Instead, the increase in affinity with chain length resulted from both a more favorable entropy term and a less unfavorable enthalpy change. These correlations can be ascribed to enhanced conformational flexibility and decreased repulsion between cyclodextrin and the Detergent headgroup, respectively, as the alkyl chain becomes longer. The thermodynamic data thus obtained were used to optimize the efficiency of Detergent Extraction from mixed micelles to form well-defined, unilamellar, and uniformly sized bilayer vesicles, and a quantitative model was established to simulate and analyze this phase transition. Finally, we combined the results from experiment and theory to develop new protocols for the online monitoring of the reconstitution process to aid the functional reconstitution of membrane proteins such as ion and water channels.
Marta I. Aveldaño - One of the best experts on this subject based on the ideXlab platform.
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Phospholipid solubilization during Detergent Extraction of rhodopsin from photoreceptor disk membranes.
Archives of biochemistry and biophysics, 1995Co-Authors: Marta I. AveldañoAbstract:Abstract The solubilization of rhodopsin and phospholipids from disks prepared from bovine retinal rods was studied using five different Detergents. The relative amounts of rhodopsin and lipid extracted during membrane solubilization differed dramatically with the nature of the surfactant; the two nonpolar Detergents, Emulphogene (polyoxyethylene-10 tridecylether) and octylglucoside, removed more protein than lipid; two bile salt-related Detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) and taurocholate, released relatively more lipid than protein; and digitonin, which shares characteristics with both groups of Detergents, extracted more lipid per mole of rhodopsin than the former two but less than the latter two. Solubilization was temperature-dependent with all five Detergents, though particularly so with octylglucoside: concentrations adequate for the total micellation of disks at 23°C were ineffectual at 4°C. In total solubilizates of disks, the amount of lipid recovered in rhodopsin–lipid–Detergent micelles showed a closer correlation with the critical micellar concentration (CMC) than with the chemical nature of the Detergent (octylglucoside > taurocholate > Chaps > digitonin > Emulphogene). The higher the CMC, the larger the amount of lipid associated to the solubilized rhodopsin and the larger the amount of lipid reassociated to rhodopsin upon surfactant dilution. For all five Detergents, the lipid progressively extracted from disks during solubilization was relatively richer in phosphatidylcholine (PC) than the lipid in the original membranes. The lipid which tended to be associated with rhodopsin in protein–lipid–Detergent mixed micelles was also consistently richer in PC than that present in lipid–Detergent micelles. Bleaching of solubilized rhodopsin decreased the amount of lipid in protein–lipid–Detergent micelles. Rhodopsin photolytic transitions were faster in nonionic than in bile salt-related Detergents.
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phospholipid solubilization during Detergent Extraction of rhodopsin from photoreceptor disk membranes
Archives of Biochemistry and Biophysics, 1995Co-Authors: Marta I. AveldañoAbstract:The solubilization of rhodopsin and phospholipids from disks prepared from bovine retinal rods was studied using five different Detergents. The relative amounts of rhodopsin and lipid extracted during membrane solubilization differed dramatically with the nature of the surfactant; the two nonpolar Detergents, Emulphogene (polyoxyethylene-10 tridecylether) and octylglucoside, removed more protein than lipid; two bile salt-related Detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) and taurocholate, released relatively more lipid than protein; and digitonin, which shares characteristics with both groups of Detergents, extracted more lipid per mole of rhodopsin than the former two but less than the latter two. Solubilization was temperature-dependent with all five Detergents, though particularly so with octylglucoside: concentrations adequate for the total micellation of disks at 23 degrees C were ineffectual at 4 degrees C. In total solubilizates of disks, the amount of lipid recovered in rhodopsin-lipid-Detergent micelles showed a closer correlation with the critical micellar concentration (CMC) than with the chemical nature of the Detergent (octylglucoside > taurocholate > Chaps > digitonin > Emulphogene). The higher the CMC, the larger the amount of lipid associated to the solubilized rhodopsin and the larger the amount of lipid reassociated to rhodopsin upon surfactant dilution. For all five Detergents, the lipid progressively extracted from disks during solubilization was relatively richer in phosphatidylcholine (PC) than the lipid in the original membranes. The lipid which tended to be associated with rhodopsin in protein-lipid-Detergent mixed micelles was also consistently richer in PC than that present in lipid-Detergent micelles. Bleaching of solubilized rhodopsin decreased the amount of lipid in protein-lipid-Detergent micelles. Rhodopsin photolytic transitions were faster in nonionic than in bile salt-related Detergents.