Cyclodextrins but not Compactin Inhibit the Lateral Diffusion of Membrane Proteins Independent of Cholesterol

Shvartsman, Dmitry; Gutman, Orit; Tietz, Alisa; Henis, Yoav I.

doi:10.1111/j.1600-0854.2006.00437.x

Cited by 69 publications

(69 citation statements)

References 67 publications

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“…Nevertheless, this study also found that BACE1 processing of APP is inhibited with further loss of cholesterol (Ͼ35%), consistent with earlier studies (32,33). Nevertheless, given the pleiotropic effects of cholesterol depletion on membrane properties and vesicular trafficking of secretory and endocytic proteins (42)(43)(44)(45)(46)(47), unequivocal conclusions regarding BACE1 processing of APP in lipid rafts cannot be reached based on cholesterol depletion studies.…”

supporting

confidence: 32%

“…Moderate loss of cholesterol in hippocampal neurons (Ͻ25% loss) facilitated colocalization of APP and BACE1 and increased A␤ production by promoting BACE1 cleavage of APP. In addition to these apparent discrepancies, proper interpretation of cholesterol depletion studies are confounded by the pleiotropic effects of cholesterol depletion on endocytosis, Golgi morphology, and vesicular trafficking, as well as perturbation in lateral diffusion of raft and non-raft proteins due to alterations in membrane fluidity and curvature (42,43,(45)(46)(47). For example, as cholesterol-rich lipid microdomains are required for the biogenesis of secretory vesicles from the TGN (44), it is highly likely that secretory trafficking of APP, BACE1, and ␥-secretase are all compromised in cells depleted of cholesterol.…”

Section: Discussionmentioning

confidence: 99%

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Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Vetrivel

Meckler

Chen

et al. 2009

Journal of Biological Chemistry

143

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Alzheimer disease ␤-amyloid (A␤) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and ␥-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of A␤ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of A␤. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

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supporting

confidence: 32%

Section: Discussionmentioning

confidence: 99%

Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Vetrivel

Meckler

Chen

et al. 2009

Journal of Biological Chemistry

143

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“…8A-C) (Nishimura et al, 2006). However, a reduction in mobility of non-raft proteins following treatment with MbCD has also been observed Shvartsman et al, 2006;Zidovetzki and Levitan, 2007). These authors have drawn attention to additional effects mediated by MbCD that can include cytoskeletal disruption, depletion of cholesterol from non-raft regions of the PM and effects on other phospholipids.…”

Section: Ac8 Mobility Depends On Membrane Cholesterolmentioning

confidence: 99%

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

Ayling

Briddon

Halls

et al. 2012

Journal of Cell Science

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SummaryThe central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub.

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“…Reaction dominant Shvartsman et al 2006). TIR-FRAP was applied to calculate the rates of binding and unbinding of hormones to and from the cell surface (Hellen and Axelrod 1991;Fulbright and Axelrod 1993).…”

Section: Applicationsmentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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Cyclodextrins but not Compactin Inhibit the Lateral Diffusion of Membrane Proteins Independent of Cholesterol

Cited by 69 publications

References 67 publications

Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

Fluorescence Techniques to Study Lipid Dynamics

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