single-molecule-localization-microscopy-membrane-proteins-using

Single-Molecule Localization Microscopy of Membrane Proteins using Single-Antibody Labeling

The plasma membrane defines the cell shape and serves as the interface that governs intercellular communication. Membrane proteins constitute a major ...

University of Illinois Chicago

Antibody-antigen interactions represent one of the most exploited biomolecular interactions in experimental biology. While numerous techniques harnessed immobilized antibodies for nanoscale fluorescence imaging, few utilized their reversible binding kinetics. Here, we investigated noncovalent interactions of the monoclonal hemagglutinin (HA) epitope tag antibody, 12CA5, in the fixed cellular environment. We observed that the use of a chaotropic agent, potassium thiocyanate (KSCN), promoted the dissociation of the 12CA5 antibody fragment (Fab), which already displayed faster dissociation compared to its immunoglobulin G (IgG) counterpart. Molecular dynamic simulations revealed notable root-mean-square deviations and destabilizations in the presence of KSCN, while the hydrogen-bonding network remained primarily unaffected at the antigen-binding site. The reversible interactions enabled us to achieve a superresolution molecular census of local populations of 3xHA tagged microtubule fibers with improved molecular quantification consistency compared to single-molecule localization microscopy (SMLM) techniques utilizing standard immunofluorescence staining for sample labeling. Our technique, termed superresolution census of molecular epitope tags (SR-COMET), highlights the utilization of reversible antibody-antigen interactions for SMLM-based quantitative superresolution imaging.

Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular Census.

As essential regulators of mitochondrial quality control, mitochondrial dynamics and mitophagy play key roles in maintenance of metabolic health and cellular homeostasis. Here we show that knockdown of the membrane-inserted scaffolding and structural protein caveolin-1 (Cav-1) and expression of tyrosine 14 phospho-defective Cav-1 mutant (Y14F), as opposed to phospho-mimicking Y14D, altered mitochondrial morphology, and increased mitochondrial matrix mixing, mitochondrial fusion and fission dynamics as well as mitophagy in MDA-MB-231 triple negative breast cancer cells. Further, we found that interaction of Cav-1 with mitochondrial fusion/fission machinery Mitofusin 2 (Mfn2) and Dynamin related protein 1 (Drp1) was enhanced by Y14D mutant indicating Cav-1 Y14 phosphorylation prevented Mfn2 and Drp1 translocation to mitochondria. Moreover, limiting mitochondrial recruitment of Mfn2 diminished formation of the PINK1/Mfn2/Parkin complex required for initiation of mitophagy resulting in accumulation of damaged mitochondria and ROS (mtROS). Thus, these studies indicate that phospho-Cav-1 may be an important switch mechanism in cancer cell survival which could lead to novel strategies for complementing cancer therapies.

Caveolin-1 controls mitochondrial damage and ROS production by regulating fission - fusion dynamics and mitophagy.

Correction to Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular Census.

In single-molecule localization microscopy (SMLM), immunofluorescence (IF) staining affects the quality of the reconstructed superresolution images. However, optimizing IF staining remains challenging because IF staining is a one-step, irreversible process. Sample labeling through reversible binding presents an alternative strategy, but such techniques require significant technological advancements to enhance the dissociation of labels without sacrificing their binding specificity. In this article, we introduce time-lapse imaging of single-antibody labeling. Our versatile technique utilizes commercially available dye-conjugated antibodies. The method controls the antibody concentrations to capture single-antibody labeling of subcellular targets, thereby achieving SMLM through the labeling process. We further demonstrate dual-color single-antibody labeling to enhance the sample labeling density. The new approach allows the evaluation of antibody binding at the single-antibody level and within the cellular environment. This comprehensive guide offers step-by-step instructions for time-lapse imaging of single-antibody labeling experiments and enables the application of the single-antibody labeling technique to a wide range of targets. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for single-antibody labeling Basic Protocol 2: Data acquisition for single-molecule localization microscopy Alternate Protocol: Dual-color single-antibody labeling using OptoSplit II equation Basic Protocol 3: Image analysis.

Single-Molecule Localization Microscopy Using Time-Lapse Imaging of Single-Antibody Labeling.

We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy (STORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.

Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy.

T cells coordinate intercellular communication through the meticulous regulation of cytokine secretion. Direct visualization of vesicular transport and intracellular distribution of cytokines provides valuable insights into the temporal and spatial mechanisms involved in regulation. Employing Jurkat E6-1 T cells and interleukin-2 (IL-2) as a model system, we investigated vesicular dynamics using single-particle tracking and the nanoscale distribution of intracellular IL-2 in fixed T cells using superresolution microscopy. Live-cell imaging revealed that in vitro activation resulted in increased vesicular dynamics. Direct stochastic optical reconstruction microscopy and 3D structured illumination microscopy revealed nanoscale clustering of IL-2. In vitro activation correlated with spatial accumulation of IL-2 nanoclusters into more pronounced and elongated clusters. These observations provide visual evidence that accelerated vesicular transport and spatial concatenation of IL-2 clusters at the nanoscale may constitute a potential mechanism for modulating cytokine release by Jurkat T cells.

Increased vesicular dynamics and nanoscale clustering of IL-2 after T cell activation.

We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane protrusions on the culture glass surface after lipopolysaccharide exposure. The concept of our work opens new possibilities for quantitative protein-specific PAINT using commercially available reagents.

Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin.

Chronic neuropathic pain is an increasingly prevalent societal issue that responds poorly to existing therapeutic strategies. The α9α10 nicotinic acetylcholine receptor (nAChR) has emerged as a potential target to treat neuropathic pain. However, challenges in expressing functional α9α10 nAChRs in mammalian cell lines have slowed the discovery of α9α10 ligands and studies into the relationship between α9α10 nAChRs and neuropathic pain. Here, we develop a cell line in the HEK293 background that stably expresses functional α9α10 nAChRs. By also developing cell lines expressing only α9 and α10 subunits, we identify distinct receptor pharmacology between homomeric α9 or α10 and heteromeric α9α10 nAChRs. Moreover, we demonstrate that incubation with nAChR ligands differentially regulates the expression of α9- or α10-containing nAChRs, suggesting a possible mechanism by which ligands may modify receptor composition and trafficking in α9- and α10-expressing cells. We then apply our α9α10 cell line in a screen of FDA-approved and investigational drugs to identify α9α10 ligands that provide new tools to probe α9α10 nAChR function. We demonstrate that one compound from this screen, diphenidol, possesses antinociceptive activity in a murine model of neuropathic pain. These results expand our understanding of α9α10 receptor pharmacology and provide new starting points for developing efficacious neuropathic pain treatments.

Discovery of Antinociceptive α9α10 Nicotinic Acetylcholine Receptor Antagonists by Stable Receptor Expression.

The transcription factor GATA-binding protein 3 (GATA-3) and the transcriptional program it regulates have emerged as oncogenic drivers across diverse T-cell lymphomas (TCLs), many of which are resistant to conventional chemotherapeutic agents and characterized by recurrent losses of key tumor suppressor genes, including TP53 and PTEN, both of which are clients of the nuclear export protein XPO1. Here, we demonstrated that XPO1 is highly expressed by malignant T cells expressing GATA-3 and by lymphoma-associated macrophages (LAMs) within their tumor microenvironment (TME). Using complementary genetically engineered mouse models, we demonstrated that TP53- and/or phosphate and tensin homolog (PTEN)-deficient TCLs, and LAMs within their TME, are sensitive to the selective exportin-1 (XPO1) antagonist selinexor. In an effort to identify TP53- and PTEN-independent mechanisms, we used complementary and orthogonal approaches to investigate the role of eIF4E and XPO1-dependent messenger RNA nuclear export in these TCLs. We identified a novel role for eIF4E/XPO1 in exporting GATA-3 and GATA-3-dependent transcripts from the nucleus in TCLs, and in the export of therapeutically relevant transcripts, including colony-stimulating factor-1 receptor, from LAMs. Therefore, XPO1 antagonism, by impairing oncogenic transcriptional programs in TCLs and depleting LAMs from their TME, is a novel approach to target 2 independent dependencies in a group of therapeutically challenging TCLs.

The GATA-3-dependent transcriptome and tumor microenvironment are regulated by eIF4E and XPO1 in T-cell lymphomas.

Dynamic protein distribution within and across the plasma membrane is pivotal in regulating cell communication. However, rapid, high-density labeling methods for multiplexed live imaging across diverse cell types remain scarce. Here, we demonstrate N-hydroxysuccinimide (NHS)-ester-based amine crosslinking of fluorescent dyes to uniformly label live mammalian cell surface proteins. Using model cell systems, we capture previously elusive membrane topology and cell-cell interactions. Live imaging shows transient membrane protein accumulation at cell-cell contacts and bidirectional migration patterns guided by membrane fibers in DC2.4 dendritic cells. Multiplexed superresolution imaging reveals the biogenesis of membrane tunneling nanotubes that facilitate intercellular transfer in DC2.4 cells, and caveolin 1-dependent endocytosis of insulin receptors in HEK293T cells. 3D superresolution imaging reveals membrane topology remodeling in response to stimulation, generation of microvesicles, and phagocytic activities in Jurkat T cells. Furthermore, NHS-labeling remains stable in vivo, enabling visualization of intercellular transfer among splenocytes using a T cell lymphoma mouse model.

Unveiling cellular communications through rapid pan-membrane-protein labeling.

Visualizing subcellular distributions of proteins and assessing their colocalization patterns are central to understanding the organization and interactions of molecular assemblies. In this issue of Structure, Kiuchi et al. introduce protein cluster coloring to map complex association patterns among eight endogenous proteins at the periphery of the clathrin-coated structure, revealing their multilayered complex associations upon epidermal growth factor stimulation.

Color mapping of multitarget protein clusters.

Rapid and uniform labeling of plasma membrane proteins is essential for high-resolution imaging of dynamic membrane topologies and intercellular communication in live mammalian cells. Existing strategies for labeling live cell membranes, such as fluorescent fusion proteins, enzyme-mediated tags, metabolic bioorthogonal labeling, and lipophilic dyes, face trade-offs in the requirement of genetic manipulation, the presence of non-uniform labeling, the need for extensive preparation times, and limited choices of fluorophores. Here, we present a streamlined protocol that leverages N-hydroxysuccinimide (NHS)-ester chemistry to achieve rapid (≤5 min), covalent conjugation of synthetic small-molecule dyes to surface-exposed primary amines, enabling pan-membrane-protein labeling. This workflow covers dye stock preparation, labeling for suspension and adherent cells, multiplex live-cell imaging, fusion protein co-staining (including insulin-triggered receptor endocytosis), 3D membrane visualization, and in vivo assays for visualizing membrane-derived material transfers between donor and recipient cells using a lymphoma T-cell mouse model. This high-density labeling approach is compatible with various cell types across diverse imaging platforms. Its speed, versatility, and stability make it a broadly applicable tool for studying plasma membrane dynamics and intercellular membrane trafficking. Key features • Rapid high-density membrane labeling with small-molecule fluorescent dyes. • Enables live-cell multiplexed imaging, amenable to primary cells and cells expressing fluorescent fusion proteins, and supports in vivo studies of membrane-associated cell-cell communications. • Compatible with various fluorescence imaging modalities.

Rapid and Uniform NHS-Ester-Based Membrane Protein Labeling of Live Mammalian Cells.

Vascular endothelial growth factor (VEGF) induces angiogenesis and vascular leakage by binding to cell surface receptor VEGFR2. KIF13B, a kinesin 3 family cellular motor, regulates VEGF/VEGFR2 signaling by bringing VEGFR2 to the cell surface. Regulating the trafficking of VEGFR2 could be a potential therapeutic target in pathogenesis originating from abnormal angiogenesis. However, the regulation mechanism of VEGFR2 trafficking is not fully understood. Here, we show that protein kinase D (PKD) phosphorylates KIF13B. The phosphorylation of KIF13B by PKD is required for KIF13B-VEGFR2 interaction and KIF13B-microtubule interaction. Live cell imaging using VEGFR2-mCherry revealed that the PKD inhibitor reduced the mean speed and total distance of VEGFR2 vesicular trafficking. Finally, inhibition of PKD also mitigated VEGF-induced permeability measured by trans-endothelial electrical resistance (TEER). Our results demonstrate how VEGFR2 trafficking is regulated by the phosphorylation of kinesin motor KIF13B by protein kinase PKD. We anticipate that elucidating the underlying mechanism of VEGFR2 trafficking can help progress the therapy development of the target diseases related to abnormal angiogenesis.

Article	Year
Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular Census. ACS nano\| PubMed ID: 34797055	2022
Caveolin-1 controls mitochondrial damage and ROS production by regulating fission - fusion dynamics and mitophagy. Redox biology\| PubMed ID: 35413643	2022
Correction to Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular Census. ACS nano\| PubMed ID: 36417787	2022
Single-Molecule Localization Microscopy Using Time-Lapse Imaging of Single-Antibody Labeling. Current protocols\| PubMed ID: 37830764	2023
Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy. bioRxiv : the preprint server for biology\| PubMed ID: 38496456	2024
Increased vesicular dynamics and nanoscale clustering of IL-2 after T cell activation. Biophysical journal\| PubMed ID: 38532626	2024
Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin. Biophysical journal\| PubMed ID: 38961624	2024
Discovery of Antinociceptive α9α10 Nicotinic Acetylcholine Receptor Antagonists by Stable Receptor Expression. ACS chemical biology\| PubMed ID: 39396195	2024
The GATA-3-dependent transcriptome and tumor microenvironment are regulated by eIF4E and XPO1 in T-cell lymphomas. Blood\| PubMed ID: 39652777	2025
Unveiling cellular communications through rapid pan-membrane-protein labeling. Nature communications\| PubMed ID: 40234465	2025
Color mapping of multitarget protein clusters. Structure (London, England : 1993)\| PubMed ID: 40480191	2025
Rapid and Uniform NHS-Ester-Based Membrane Protein Labeling of Live Mammalian Cells. Bio-protocol\| PubMed ID: 41080442	2025
Protein Kinase D Regulates Kinesin-Mediated VEGFR2 Trafficking and Influences Retinal Endothelial Permeability. Traffic (Copenhagen, Denmark)\| PubMed ID: 41681080	2026

Hirushi Gunasekara

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