Statistical Analysis of High-Resolution Light Microscope Images Reveals Effects of Cytoskeleton-Disrupting Drugs on the Membrane Organization of the Nicotinic Acetylcholine Receptor

Wenz, Jorge J.; Borroni, Virginia; Barrantes, Francisco J.

doi:10.1007/s00232-010-9261-6

Cited by 5 publications

(4 citation statements)

References 24 publications

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“…AChR is present at the plasma membrane of CHO-K1/A5 cells in the form of clusters of~55 nm only resolved by superresolution microscopy (9,10). Under control conditions at 4 C and in the absence of any external influence the mean GP of the membrane associated with AChR clusters did not differ from the mean GP of the membrane without AChR clusters.…”

Section: Discussionmentioning

confidence: 97%

“…Using stimulated emission depletion superresolution microscopy (nanoscopy) and antibody labeling, the diffraction-limited puncta could be resolved into nanoclusters of~55 nm (9). The organization and amount of these antibody-induced AChR nanoclusters, as well as their interrelationship, were found to be cholesterol (Chol)-and cytoskeleton-dependent (5,9,10). This is remarkably similar to the case of myotubes in culture (11)(12)(13)(14), where association of AChR with Chol-rich domains is required for the formation and maintenance of AChR clusters.…”

Section: Introductionmentioning

confidence: 99%

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Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

Kamerbeek

Borroni

Pediconi

et al. 2013

Biophysical Journal

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The distribution of nicotinic acetylcholine receptor (AChR) clusters at the cell membrane was studied in CHO-K1/A5 cells using fluorescence microscopy. Di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered (Lo) and liquid-disordered (Ld) phases in model membranes, was used in combination with monoclonal anti-AChR antibody labeling of live cells, which induces AChR clustering. The so-called generalized polarization (GP) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of antibody-induced AChR clusters, respectively. AChR clusters were almost equally distributed between Lo and Ld domains, independently of receptor surface levels and agonist (carbamoylcholine and nicotine) or antagonist (α-bungarotoxin) binding. Cholesterol depletion diminished the cell membrane mean di-4-ANEPPDHQ GP and the number of AChR clusters associated with Ld membrane domains increased concomitantly. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A had the opposite effect, with more AChR clusters associated with Lo domains. AChR internalized via small vesicles having lower GP and lower cholesterol content than the surface membrane. Upon cholesterol depletion, only 12% of the AChR-containing vesicles costained with the fluorescent cholesterol analog fPEG-cholesterol, i.e., AChR endocytosis was essentially dissociated from that of cholesterol. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane appears to be regulated by cholesterol content and cytoskeleton integrity.

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

Kamerbeek

Borroni

Pediconi

et al. 2013

Biophysical Journal

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“…Disruption of the cytoskeleton or the microtubule networks with Latrunculin A or nocodazole, respectively, affected the mobility of the neuronal α7 nAChR but not its ability to form clusters, as we have observed in muscle-type nAChRs using superresolution microscopy (Wenz et al, 2010). The exact mechanisms of nAChR immobilization in CNS synapses and in particular the role of the cytoskeleton or other diffusional traps merit further investigation.…”

Section: Diffusional Modulation and Confinement Of Nachr Assemblies Bmentioning

confidence: 59%

Cell-surface translational dynamics of nicotinic acetylcholine receptors

Barrantes¹

2014

Front. Synaptic Neurosci.

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Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain.

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“…Disruption of the cytoskeleton or the microtubule networks with Latrunculin A or nocodazole, respectively, affected the mobility of the neuronal α7 nAChR but not its ability to form clusters, as we have observed in muscle-type nAChRs using superresolution microscopy [105]. The exact mechanisms of nAChR immobilization in CNS synapses and in particular the role of the cytoskeleton or other diffusional traps merit further investigation.…”

Section: Aq4mentioning

confidence: 71%

Spatiotemporal Dynamics of Nicotinic Acetylcholine Receptors and Lipid Platforms

Barrantes¹

2017

Springer Series in Biophysics

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The relationships between neurotransmitter receptors and their membrane environment are complex, mutual (bidirectional) and physiologically important. Some of these relationships are established with subsets of the membrane lipid population, in the form of lipid platforms, lateral heterogeneities of the bilayer lipid having a dynamic chemical composition distinct from that of the bulk membrane. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion, clustering and anchorage of receptors at the lipid platforms play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir non-synaptic membranes and the synapse predominantly by thermally driven Brownian motion, and become immobilized at the perisynaptic region or the synapse proper by various mechanisms. These comprise: (a) clustering mediated by homotropic inter-molecular receptor-receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional "trapping", and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. Preceded by a brief introduction on the currently used methods to study protein lateral mobility in membranes, this review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells-the nicotinic acetylcholine receptor (nAChR). The translational mobility of nAChRs at these two cell surfaces differs in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. Neuronal α7 nAChRs exhibit diffusion coefficients similar to those of other neurotransmitter receptors and spend part of their lifetime confined to the perisynaptic region of glutamatergic (excitatory) and GABAergic (inhibitory) synapses; they may also be involved in the regulation of the dynamic equilibrium between excitation and inhibition in brain. Abbreviations αBTX α-Bungarotoxin FCS Fluorescence correlation spectroscopy FRAP Fluorescence recovery after photobleaching MSD Mean-square displacement nAChR Nicotinic acetylcholine receptor SPT Single particle tracking TIRF Total internal reflection fluorescence 9.1. Introduction 1✉ 1

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Statistical Analysis of High-Resolution Light Microscope Images Reveals Effects of Cytoskeleton-Disrupting Drugs on the Membrane Organization of the Nicotinic Acetylcholine Receptor

Cited by 5 publications

References 24 publications

Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

Cell-surface translational dynamics of nicotinic acetylcholine receptors

Spatiotemporal Dynamics of Nicotinic Acetylcholine Receptors and Lipid Platforms

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