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Principles Of Fluorescence Spectroscopy

: The unpolarized absorption and circular dichroism spectra of the fundamental vibrational transitions of the chiral molecule, 4-methyl-2-oxetanone, are calculated ab initio. Harmonic force fields are obtained using Density Functional Theory (DFT), MP2, and SCF methodologies and a 5S4P2D/3S2P (TZ2P) basis set. DFT calculations use the Local Spin Density Approximation (LSDA), BLYP, and Becke3LYP (B3LYP) density functionals. Mid-IR spectra predicted using LSDA, BLYP, and B3LYP force fields are of significantly different quality, the B3LYP force field yielding spectra in clearly superior, and overall excellent, agreement with experiment. The MP2 force field yields spectra in slightly worse agreement with experiment than the B3LYP force field. The SCF force field yields spectra in poor agreement with experiment.The basis set dependence of B3LYP force fields is also explored: the 6-31G* and TZ2P basis sets give very similar results while the 3-21G basis set yields spectra in substantially worse agreements with experiment. jg

Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields

Lipid Rafts: Elusive or Illusive?

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

The mystery of membrane organization: composition, regulation and roles of lipid rafts

Endocytic recycling is coordinated with endocytic uptake to control the composition of the plasma membrane. Although much of our understanding of endocytic recycling has come from studies on the transferrin receptor, a protein internalized through clathrin-dependent endocytosis, increased interest in clathrin-independent endocytosis has led to the discovery of new endocytic recycling systems. Recent insights into the regulatory mechanisms that control endocytic recycling have focused on recycling through tubular carriers and the return to the cell surface of cargo that enters cells through clathrin-independent mechanisms. Recent work emphasizes the importance of regulated recycling in such diverse processes as cytokinesis, cell adhesion and morphogenesis, cell fusion, and learning and memory.

Pathways and mechanisms of endocytic recycling.

Cholesterol is the single most abundant lipid species in mammalian cells. More than 2×109 years of evolution designed this molecule to perfectly fit into phospholipid bilayers regulating the fluidity, permeability and bending stiffness of biological membranes. Cholesterol also serves as a precursor of steroid hormones, bile acids and oxysterols, and its cellular synthesis is regulated by a complex machinery. While the molecular mechanisms underlying cholesterol synthesis are known in great detail, knowledge is rather sparse about the inter-compartment transport of cholesterol, including trafficking modes and kinetics, as well as control of endomembrane cholesterol content. This chapter provides an overview of our recent understanding of intracellular transport of cholesterol. It is aimed to create a link between the well characterized biophysical properties of cholesterol in model membranes and its behavior in living cells.

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Intracellular cholesterol transport

Vesicular and Non-vesicular Sterol Transport in Living Cells: THE ENDOCYTIC RECYCLING COMPARTMENT IS A MAJOR STEROL STORAGE ORGANELLE *

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly.

/pdf/analysis-of-cholesterol-trafficking-with-fluorescent-probes-2ycy55906h.pdf

Daniel Wüstner

Papers

Intracellular cholesterol transport

Vesicular and Non-vesicular Sterol Transport in Living Cells: THE ENDOCYTIC RECYCLING COMPARTMENT IS A MAJOR STEROL STORAGE ORGANELLE *

Analysis of cholesterol trafficking with fluorescent probes.

Fluorescent sterols as tools in membrane biophysics and cell biology.

Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins.