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We report a method for the identification and quantification of glycerophospholipid molecular species that is based on the simultaneous automated acquisition and processing of 41 precursor ion spectra, specific for acyl anions of common fatty acids moieties and several lipid class-specific fragment ions. Absolute quantification of identified species was linear within a concentration range of 10 nM-100 [micro]M and was achieved by spiking into total lipid extracts a set of synthetic lipid standards with diheptadecanoyl (17:0/17:0) fatty acid moieties, representing six common classes of glycerophospholipids. The automated analysis of total lipid extracts was powered by a robotic nanoflow ion source and produced currently the most detailed description of the glycerophospholipidome.

Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of [beta]-amyloid (A[beta]) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The A[beta] peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by [beta]- and [gamma]-secretases. The endocytic system has been implicated in the cleavages leading to the formation of A[beta]. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated A[beta] is released into the extracellular milieu are not clear. Here, we show that [beta]-cleavage occurs in early endosomes followed by routing of A[beta] to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of A[beta] peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD. multivesicular bodies | rafts | amyloid precursor protein | [beta]-secretase | endocytosis

The yeast mating cell provides a simple paradigm for analyzing mechanisms underlying the generation of surface polarity. Endocytic recycling and slow diffusion on the plasma membrane were shown to facilitate polarized surface distribution of Snc1 p (Valdez-Taubas, J., and H.R. Pelham. 2003. Curr. Biol. 13:1636-1640). Here, we found that polarization of Fus1 p, a raft-associated type I transmembrane protein involved in cell fusion, does not depend on endocytosis. Instead, Fus1 p localization to the tip of the mating projection was determined by its cytosolic domain, which binds to peripheral proteins involved in mating tip polarization. Furthermore, we provide evidence that the lipid bilayer at the mating projection is more condensed than the plasma membrane enclosing the cell body, and that sphingolipids are required for this lipid organization.

Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrplp), known membrane traffic regulators (Kesl p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting. exocytosis | lipid rafts | Saccharomyces cerevisiae | sorting | Golgi

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.11.016 Byline: Kai Simons (1), Carol Featherstone (2) Abstract: Basic research in Europe is about to receive a shot in the arm with the creation of a European Research Council (ERC). This new agency will sustain fundamental investigation into all aspects of scientific knowledge and should drive up standards of scientific research across the continent. Author Affiliation: (1) President, European Life Scientist Organization (ELSO); Director, Max Planck Institute for Cell Biology and Genetics, Dresden 01307, Germany (2) Coordinator, ELSO Career Development Committee, Fourquevaux 31450, France

Formation of senile plaques containing the [beta]-amyloid peptide (A[beta]) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by [beta]-secretase or by [alpha]-secretase to initiate amyloidogenic (release of A[beta]) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A[beta] generation. Reducing cholesterol levels in N2a cells decreased A[beta] production. APP and the [beta]-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A[beta] in a cholesterol-dependent manner. A[beta] generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by anti-body cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by [beta]-secretase, APP outside rafts undergoes cleavage by [alpha]-secretase. Thus, access of [alpha]- and [beta]-secretase to APP, and therefore A[beta] generation, may be determined by dynamic interactions of APP with lipid rafts.

A hybrid quadrupole time-of-flight mass spectrometer featured with ion trapping capabilities was employed for quantitative profiling of total extracts of endogenous phospholipids. Simultaneous acquisition of precursor ion spectra of multiple fragment ions allowed detection of major classes of phospholipids in a single experiment. Relative changes in their concentration were monitored using a mixture of isotopically labeled endogenous lipids as a comprehensive internal standard. Precursor ion scanning spectra were acquired simultaneously for acyl anions of major fatty acids in negative ion mode and identified the fatty acid moieties and their relative position at the glycerol backbone in individual lipid species. Taken together, a combination of multiple precursor ion scans allowed quantitative monitoring of major perturbation in phospholipid composition and elucidating of molecular heterogeneity of individual lipid species.

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called [Beta]-amyloid (A[Beta]) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP ([APP.sub.sec]). It is A[Beta] that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-[Beta]-cyclodextrin, we show that the formation of A[Beta] is completely inhibited while the generation of [APP.sub.sec] is unperturbed. This inhibition of A[Beta] formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for A[Beta] formation to occur and imply a link between cholesterol, A[Beta], and Alzheimer's disease.

It is possible to demonstrate that proteins can be selectively included or excluded from the microdomains which arise as a result of preferential packing of sphingolipids and cholesterol into moving platforms, or rafts, Specific proteins attach to these rafts within the bilayer. It appears that the function of these microdomains is to act as rafts to transport particular membranes. It is also possible that they function as relay stations in intracellular signalling. It is still necessary to produce proof of the functional significance of lipid rafts in membrane trafficking and signalling.

All epithelial cells are characterized by specialized junctions, internal architecture and surface polarity which allow them to behave differently at their apical and basal sides. Epithelial cells have a polar cytoplasmic organization and a polar exterior and polarization is regulated by different mechanisms. The mechanism of polarization in four different epithelia is discussed.

Membrane transport of epithelial Madin-Darby canine kidney (MDCK) cells was inhibited by anti-N-ethylmaleimide-sensitive factor (NSF) in a permeabilized cell system, but was stimulated by alpha-soluble NSF attachment proteins. However, transport of cells from the trans-Golgi network to the apical cell surface was not affected by these proteins. Apical transport was also not affected by Rab-GDI and tetanus and botulinum neurotoxins which inhibit basolateral transport, suggesting that the Rab-NSF-SNAP-SNARE machinery only operates in basolateral transport.

The addition of N-glycan to proteins helps in protein folding and stabilizing of the protein structure after which the proteins can be transported out of the endoplasmic reticulum. N-glycan is also involved in the transfer of a GlcNAc phosphate to a mannose on the lysosome enzymes. However the proteins that do not undergo glycosylation or use a transport mechanism other than glycosylation have to use a sorting signal other than N-glycan. The transport of apical membrane proteins from the Golgi to the cell surface also involves N-glycan.

A newly developed polarized transport assay studies of the role of microtubules in permeabilized Madin-Darby canine kidney cells shows that transport of apical and basolateral vesicles in kidney epithelial cells is mediated by microtubule motors. Nocodazole treatment blocks apical and basolateral transport. Immunodepletion of cytosolic kinesin inhibits transport to basolateral surface, while dynein and kinesin are involved in apical transport.

Detergent-insoluble complexes in epithelial cells play an important role in protein sorting and delivery to the cell surface. A CHAPS (3-cholamindopropyl dimethylammonio-2-hydroxypropanesulfonate) insoluble complex containing the apical marker protein influenza hemaglutinin (HA) was studied in Madin-Darky canine kidney (MDCK) cells. The study showed that the CHAPS complex contains a small fraction of lipids and no GPI (glycosylphosphatidylinositol) linked proteins.