Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

Moonschi, Faruk H.; Effinger, Amy K.; Zhang, Xiaolu; Martin, William Marty; Fox, Ashley M.; Heidary, David K.; DeRouchey, Jason E.; Richards, Christopher I.

doi:10.1002/ange.201408707

Cited by 12 publications

(15 citation statements)

References 27 publications

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“…For imaging and immobilization, we fused the enhanced green fluorescent protein (EGFP) to the cytosolic N-terminus of the CNG channel TAX-4 (GFP-TAX-4). Cell-derived nanovesicles containing GFP-TAX-4 were generated as described for a study of nicotinic acetylcholine receptor stoichiometry 7–9,42 (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…However, their application to studies of ligand binding in membrane proteins is challenged by costly sample preparation, dysfunction in nonnative lipid or detergent environments [4][5][6] , lack of solution access to intracellular sites, and nonspecific dye adsorption to imaging surfaces. Here, we overcome these challenges by combining cell-derived nanovesicles [7][8][9] , microfluidics, micro-mirror total internal reflection fluorescence (mmTIRF) 10 , and colocalization SM spectroscopy 11 to optically track binding and unbinding of individual fluorescently-tagged ligands at single membrane proteins. Imaging a ~100 µm × 100 µm field of view much larger than a confocal spot enables high-throughput data acquisition of up to hundreds of molecules simultaneously.…”

Section: Introductionmentioning

confidence: 99%

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Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Patel

Salinas

et al. 2021

Preprint

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Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in nonnative lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. We further show that binding of the second ligand in the tetrameric channel is less cooperative than previously estimated from ensemble-averaged binding measures. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Patel

Salinas

et al. 2021

Preprint

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“…FCS tracks fluctuations in fluorescence as vesicles diffuse through the focal volume. 26,27 Both the diffusion time and the average number of molecules can be extracted from the autocorrelation curve (Figure 1B). The FCS focal volume was calibrated using commercial tetraspeck beads leading to a determination that our preparations yielded ∼4 × 10 11 vesicles per mL, which is approximately 1.3 × 10 11 vesicles per preparation (40 million cells).…”

Section: Resultsmentioning

confidence: 99%

Cell-Derived Vesicles for in Vitro and in Vivo Targeted Therapeutic Delivery

et al. 2019

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Efficient delivery of therapeutics across the cell membrane to the interior of the cell remains a challenge both in vitro and in vivo. Here, we demonstrate that vesicles derived from cellular membranes can be efficiently loaded with cargo that can then be delivered to the interior of the cell. These vesicles demonstrated cell-targeting specificity as well as the ability to deliver a wide range of different cargos. We utilized this approach to deliver both lipophilic and hydrophilic cargos including therapeutics and DNA in vitro. We further demonstrated in vivo targeting and delivery using fluorescently labeled vesicles to target tumor xenografts in an animal. Cell-derived vesicles can be generated in high yields and are easily loaded with a variety of cargos. The ability of these vesicles to specifically target the same cell type from which they originated provides an efficient means of delivering cargo, such as therapeutics, both in vitro and in vivo.

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“…Maturation efficiencies (including for mNeon) are typically above 0.5 [49][50][51][54][55][56][57][58] , suggesting that dimer, trimer, and hexamer or higher models are unlikely (Fig. 7d).…”

Section: Discussionmentioning

confidence: 99%

Oligomerization state of the functional bacterial twin arginine translocation (Tat) receptor complex

Sharma

Chowdhury

2021

Preprint

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The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargodependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable, but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typical low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

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Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

Cited by 12 publications

References 27 publications

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Cell-Derived Vesicles for in Vitro and in Vivo Targeted Therapeutic Delivery

Oligomerization state of the functional bacterial twin arginine translocation (Tat) receptor complex

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