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

Summary

This protocol describes single-antibody labeling (SAL) to resolve the nanoscale spatial organization of plasma membrane proteins. By leveraging cumulative antibody-epitope interactions at the single-molecule level, membrane SAL (mSAL) maps local epitope distributions while simultaneously capturing antibody binding behavior in the native cellular environment.

Abstract

The plasma membrane defines the cell shape and serves as the interface that governs intercellular communication. Membrane proteins constitute a major class of therapeutic targets; therefore, super-resolving the cell membrane through its constitutive proteins holds great promise in advancing cell biology and antibody therapeutics. In this regard, single-molecule localization microscopy (SMLM) enables nanoscale visualization of protein organizations on biological structures. Despite its importance, applying SMLM to plasma membrane proteins poses unique challenges. In this protocol, we present an effective approach using time-lapse single-antibody labeling (SAL) termed membrane SAL (mSAL). We provide detailed step-by-step instructions, including optimization of the antibody concentration, laser power density, duration of non-illumination intervals, image reconstruction, and density-based cluster analysis, to resolve nanoscale membrane protein distribution and membrane morphology. We use the tetraspanin protein CD81 as the model membrane protein to demonstrate the capability of mSAL on both adherent and suspension mammalian cells. In addition to super-resolving the cell membrane and distributions of membrane proteins, our technique enables the investigation of the pharmacodynamics of therapeutic antibodies interacting with their membrane targets in the native membrane environment.

Introduction

The plasma membrane contains a diverse array of proteins that regulate cell mobility, adhesion, sensing, and intercellular communication. Understanding the nanoscale distribution of plasma membrane proteins is important, as protein function is often governed not only by expression levels but also by their spatial organization on the membrane^1,2. Notably, many therapeutic antibodies target membrane proteins^3,4, making nanoscale investigations essential for understanding how antibody interactions are influenced by the local membrane protein organization.....

Protocol

This protocol uses the established Jurkat E6.1 and U2OS cell lines and does not involve primary human or live animal materials.

1. Cell seeding/landing and fixation

NOTE: The sample preparation follows standard immunofluorescence (IF) staining protocols, optimized to preserve the subcellular organization of plasma membrane proteins across different cell types. If an established immunostaining protocol exists for the target of interest, users may follow that protocol up to the sample-blocking step 1.2.14. Single-antibody labeling (SAL) relies on the cumulative detection of fluorescently labeled antibodies ....

Results

The mSAL technique described in this protocol enables visualization of single antibody binding events to their corresponding plasma membrane protein targets, producing a super-resolution map of membrane protein distribution. CD81 is used here as a representative example, though the approach is readily adaptable to other membrane proteins.

Immobilizing Jurkat T cells with sufficient spacing between cells ensures membrane epitope accessibility by antibodies and glass surface for fiducials to set.......

Discussion

The organization of plasma membrane proteins plays a vital role in regulating cell function. Conventional fluorescence microscopy techniques are limited by the diffraction limit of light, masking their nanoscale organization. SMLM overcomes this limitation by achieving spatial resolutions of 10-20 nm⁴⁷, enabling the characterization of their nanoscale assemblies^10,48,49. Clustering artifacts in SMLM arise.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-CD81 Alexa Fluor 488 Antibody	Thermo Fisher Scientific	MA5-44132	Imaging antibody
Benchtop centrifuge	Eppendorf	5424
Bovine serum albumin	MilliporeSigma	A7906-100G
Chambered cover glass, 8-well	Cellvis	C8-1-N
DMEM media	Thermo Fisher Scientific	11960069
Dulbecco's Phosphate Buffered Saline	Thermo Fisher Scientific	14190-144
Fetal bovine serum	MilliporeSigma	F0926-500ML
Glutaraldehyde, 10 %	Electron Microscopy Sciences	16120
Gold colloids, 100 nm	Ted Pella	15711−20	Fiducial marker
Immersion oil	Cargille Laboratories	16245
Inverted microscope equipped with TIRF module	Nikon Instruments	Eclipse Ti2-E	Microscope
Jurkat E6.1 cell line	MilliporeSigma	88042803-1VL	Suspension cells
Laser Launch, 6 line	Oxxius	L6CC-CS8-1511	Excitation laser
Lipofectamine 2000	Thermo Fisher Scientific	11668019	Transfection reagent
Microcentrifuge tube, 1.5 mL	VWR	89000-028
Microscopy imaging software	Nikon Instruments	NIS-Elements Advanced Research	Image acqisition software
Objective, 100x/1.49 CFI APO TIRF	Nikon Instruments	MRD01991	Microscope objective
Paraformaldehyde, 16 %	Electron Microscopy Sciences	15710
Penicillin-Streptomycin	Thermo Fisher Scientific	15140-122	Antibiotics
Poly-L-Lysine solution, 0.01%	MilliporeSigma	P4707-50ML
Prime 95B sCMOS Camera	Teledyne Vision Solutions	01-PRIME-95B-R-M-16-C	Camera
RPMI 1640 media	Thermo Fisher Scientific	11875-093	Media for suspension cells
Sodium azide	Thermo Fisher Scientific	19038-1000
Sodium borohydride	MilliporeSigma	213462-25G
T25 culture flasks	Thermo Fisher Scientific	169900
TetraSpeck microspheres			Fiducial marker
Tissue culture dish, 100 mm	Corning	353003
U2OS cell line	ATCC	HTB-96	Adherent cells