Single‐particle tracking of immunoglobulin E receptors (FcεRI) in micron‐sized clusters and receptor patches

Spendier, Kathrin; Lidke, Keith A.; Lidke, Diane S.; Thomas, James L.

doi:10.1016/j.febslet.2012.01.013

Cited by 31 publications

(34 citation statements)

References 27 publications

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“…Similar profiles can be found for the diffusion of viruses [306], receptors [307], polyplexes [284] and lipoplexes [286]. Giving the Stokes-Einstein relation, Brownian motion depends on the viscosity of the medium and the size (radius) of the particles.…”

Section: Gene Electrotransfersupporting

confidence: 53%

Gene Electrotransfer: A Mechanistic Perspective

Rosazza¹,

Meglič²,

Zumbusch³

et al. 2016

CGT

177

142

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Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

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Section: Gene Electrotransfersupporting

confidence: 53%

Gene Electrotransfer: A Mechanistic Perspective

Rosazza¹,

Meglič²,

Zumbusch³

et al. 2016

CGT

177

142

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“…In live cells, single-particle tracking (1)(2)(3) and fluorescence correlation spectroscopy (FCS) with subdiffraction detection volumes (achieved by stimulated emission depletion microscopy)(4) have revealed the plasma membrane to be heterogeneous on tens of nanometers, with correspondingly heterogeneous dynamics. Taken together, these results suggest a hierarchical membrane organization, with the cytoskeleton influencing transport above 80 nm lengthscales, (5) and lipid-protein interactions operating below this lengthscale (6).…”

Section: Introductionmentioning

confidence: 99%

Toward Hydrodynamics with Solvent Free Lipid Models: STRD Martini

Zgorski

Lyman

2016

Biophysical Journal

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Solvent hydrodynamics are incorporated into simulations of the solvent-free Dry Martini model. The solvent hydrodynamics are modeled with the stochastic rotation dynamics (SRD) algorithm, a particle-based method for resolving fluid hydrodynamics. SRD does not require calculation of particle-particle distances in the solvent, and so is scalable to arbitrary volumes of solvent with minimal additional computational overhead. The viscosity of the solvent is easily tuned via parameters of the algorithm to span an order of magnitude in viscosity around the viscosity of water at room temperature. The combination ''Stochastic Thermostatted Rotation Dynamics (STRD) with Martini'' was implemented in Gromacs v.5.01. Simulations of an SRD/palmitoyloleoylphosphatidylcholine membrane demonstrate that the solvent may be included without reparametrizing the lipid model, with minimal perturbation to the thermodynamics. A recent generalization of Saffman-Delbruck theory to periodic geometries by Camley and Brown indicates that lipid dynamics are contaminated by a finite-size effect in typical molecular dynamics (MD) simulations, and that very large systems are required for quantitative simulation of dynamics. Analysis of lipid translational diffusion in this work shows good agreement with the theory, and with explicitly solvated simulations. This indicates that STRD Martini is a viable approach for quantitative simulation of membrane dynamics and does not require massive computational overhead to model the solvent.

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“…Due to the ability of RBL-2H3 cells to release histamine in an IgE-dependent manner and the expression of high-affinity FcεRI receptors, the RBL-2H3 cell line derived from basophils has been considered to model mast cells and has therefore been used extensively and successfully to study IgE-dependent degranulation [7][8][9][10]. To investigate FcεRI receptor aggregation and the formation of the FcεRI-centric synapse, FcεRI receptors on RBL-2H3 cells are typically fully loaded with fluorescently-labeled anti-dinitrophenyl (anti-DNP) IgE and then deposited onto DNP-coated surfaces and observed using total internal reflection fluorescence (TIRF) microscopy [11][12][13][14][15]. TIRF microscopy is widely used to study the motion of cell surface receptors [11,[16][17][18][19][20][21][22][23][24][25].…”

Section: Discussionmentioning

confidence: 99%

TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

Drawbond

Spendier

2019

Data

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Total internal reflection fluorescence (TIRF) microscope image sequences are commonly used to study receptors in live cells. The dataset presented herein facilitates the study of the IgE-FcεRI receptor signaling complex (IgE-RC) in rat basophilic leukemia (RBL-2H3) cells coming into contact with a supported lipid bilayer with 25 mol% N-dinitrophenyl-aminocaproyl phosphatidylethanolamine, modeling an immunological synapse. TIRF microscopy was used to image IgE-RCs within this FcεRI-centric synapse by loading RBL-2H3 cells with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) in suspension for 24 h. Fluorescent anti-DNP IgE (IgE 488 ) concentrations of this suspension increased from 10% to 100% and corresponding non-fluorescent anti-DNP IgE concentrations decreased from 90% to 0%. After the removal of unbound anti-DNP IgE, multiple image sequences were taken for each of these ten conditions. Prior to imaging, anti-DNP IgE-primed RBL-2H3 cells were either kept for a few minutes, for about 30 min, or for about one hour in Hanks buffer. The dataset contains 482 RBL-2H3 model synapse image stacks, dark images to correct for background intensity, and TIRF illumination profile images to correct for non-uniform TIRF illumination. After background subtraction, non-uniform illumination correction, and conversion of pixel units from analog-to-digital units to photo electrons, the average pixel intensity was calculated. The average pixel intensity within FcεRI-centric synapses for all three Hanks buffer conditions increased linearly at a rate of 0.42 ± 0.02 photo electrons per pixel per % IgE 488 in suspension. RBL-2H3 cell degranulation was tested by detecting β-hexosaminidase activity. Prolonged RBL-2H3 cell exposure to Hanks buffer inhibited exocytosis in RBL-2H3 cells.Dataset: http://dx.

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Single‐particle tracking of immunoglobulin E receptors (FcεRI) in micron‐sized clusters and receptor patches

Cited by 31 publications

References 27 publications

Gene Electrotransfer: A Mechanistic Perspective

Gene Electrotransfer: A Mechanistic Perspective

Toward Hydrodynamics with Solvent Free Lipid Models: STRD Martini

TIRF Microscope Image Sequences of Fluorescent IgE-FcεRI Receptor Complexes inside a FcεRI-Centric Synapse in RBL-2H3 Cells

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