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Visualization of lipid raft membrane compartmentalization in living RN46A neuronal cells using single quantum dot tracking.

ABSTRACT: Lipid rafts are cholesterol-enriched subdomains in the plasma membrane that have been reported to act as a platform to facilitate neuronal signaling; however, they are suspected to have a very short lifetime, up to only a few seconds, which calls into question their roles in biological signaling. To better understand their diffusion dynamics and membrane compartmentalization, we labeled lipid raft constituent ganglioside GM1 with single quantum dots through the connection of cholera toxin B subunit, a protein that binds specifically to GM1. Diffusion measurements revealed that single quantum dot-labeled GM1 ganglioside complexes undergo slow, confined lateral diffusion with a diffusion coefficient of ?7.87 × 10(-2) ?m(2)/s and a confinement domain about 200 nm in size. Further analysis of their trajectories showed lateral confinement persisting on the order of tens of seconds, comparable to the time scales of the majority of cellular signaling and biological reactions. Hence, our results provide further evidence in support of the putative function of lipid rafts as signaling platforms.

SUBMITTER: Chang JC

PROVIDER: S-EPMC3474263 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Visualization of lipid raft membrane compartmentalization in living RN46A neuronal cells using single quantum dot tracking.

Chang Jerry C JC Rosenthal Sandra J SJ

ACS chemical neuroscience 20120801 10

Lipid rafts are cholesterol-enriched subdomains in the plasma membrane that have been reported to act as a platform to facilitate neuronal signaling; however, they are suspected to have a very short lifetime, up to only a few seconds, which calls into question their roles in biological signaling. To better understand their diffusion dynamics and membrane compartmentalization, we labeled lipid raft constituent ganglioside GM1 with single quantum dots through the connection of cholera toxin B subu ...[more]

PMID: 23077717

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Project description:We report the development of a quantum dot (QD)-peptide-fullerene (C60) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C60 electron acceptor. The photoexcited QD donor engages in ET with the C60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor-acceptor pair. When introduced to cells, the amphiphilic QD-peptide-C60 bioconjugate labels the plasma membrane by insertion of the peptide-C60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F0 of 2-20% depending on the QD-C60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD-peptide-C60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD-peptide-C60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD-peptide-C60 construct displays 20- to 40-fold greater ΔF/F0 than VSDs. The tractable nature of the QD-peptide-C60 system offers the advantages of ease of assembly, large ΔF/F0, enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

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Dataset Information

Visualization of lipid raft membrane compartmentalization in living RN46A neuronal cells using single quantum dot tracking.

Publications

Visualization of lipid raft membrane compartmentalization in living RN46A neuronal cells using single quantum dot tracking.

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