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

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.

Tetraspanin proteins are closely associated with high-curvature membrane structures and play key roles in organizing membrane domains and regulating membrane signaling in immune cells. However, their specific roles in regulating T cell membrane signaling, particularly within the microvilli often characteristic of these cells, remain poorly understood. Here, we used Jurkat T cells as a model system and investigated CD81 as a member of the tetraspanin family. Using total internal reflection fluorescence (TIRF) microscopy and structured illumination microscopy (SIM), we identified an enrichment of the tetraspanin CD81 microdomains along the actin-rich membrane microvilli. At the distal end of the microvilli, SIM images revealed the spatial colocalization of CD81 with T cell receptors (TCR) and CD63, implying a potential role for CD81 in regulating TCR signaling in conjunction with CD63. Spatial analysis of CD81 and CD63 microdomains from the dual-color SIM data revealed their preference for associating with each other. Cluster analysis of direct stochastic optical reconstruction microscopy (STORM) data revealed that  T cell activation results in reduced domain sizes and increased domain separation of CD81. These findings provide visual evidence of the spatial organization and rearrangement of CD81 on the T cell microvilli, highlighting its potential role in signal regulation on specialized membrane protrusions.

Superresolution Imaging Reveals the Spatial Organization of CD81 Microdomains in Regulating Membrane Signaling on Jurkat T Cell Microvilli.

Proteolysis targeting chimeras (PROTACs) represent powerful tools to modulate the activity of classically "undruggable" proteins, but their application has been limited to known ligands and a few select protein classes. Herein, we present our chemoproteomic strategy for simultaneous  discovery of novel degraders and ligands for challenging and previously "undruggable" targets. Using comparative PROTAC versus ligand global proteomics analyses, we rapidly identify proteins selectively downregulated by several "untargeted" PROTACs containing a VHL E3 ligase recruiter and various covalent and noncovalent ligands. We showcase our approach by identifying a first-in-class PROTAC for metallothionein 2A (MT2A), a small, cysteine-rich, metal-binding protein implicated in heavy metal detoxification, zinc homeostasis, and cellular invasion. Notably, isoform-specific MT overexpression has been shown to augment cellular migration and invasion across several cancer cell lines, although the precise mechanisms are unknown due to insufficient tools to study MTs. We show that optimized PROTAC AA-BR-157 covalently binds conserved C44, degrades overexpressed MT2A with nanomolar potency, and reduces the migration and invasion of MDA-MB-231 cells. We further demonstrate a time-dependent increase in intracellular zinc levels following MT2A degradation as well as downregulation of protein diaphanous homolog 3 (DIAPH3), a positive regulator of actin and cell motility. Super-resolution imaging of MDA-MB-231 cells shows that the downregulation of MT2A and DIAPH3 inhibits cell polarization and thereby migration, suggesting that MT2A may regulate motility via DIAPH3-dependent cytoskeletal remodeling. In summary, our strategy enables the  discovery of PROTACs and ligands for novel disease-related targets and lays the groundwork for expansion of the druggable proteome.

Chemoproteomics-Enabled  Proteolysis Targeting Chimera Discovery Platform Identifies a Metallothionein Degrader to Probe Its Role in Cancer.

The diffraction limit of visible light governs the spatial resolution of optical microscopy to roughly half the wavelength of the light, typically around 200 nm in the lateral dimension and 500 nm in the axial dimension. However, many subcellular structures and processes occur on a much smaller length scale. Technological solutions beyond the optical diffraction limit are needed to study subcellular organizations of proteins and their related functions. Super-resolution microscopy (SRM) represents a family of techniques capable of resolving structures and objects below the optical diffraction limit. This chapter provides an overview of three super-resolution techniques, stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM), and their applications in investigating subcellular protein organizations and related functions in immune cells.

Article	Year
Increased vesicular dynamics and nanoscale clustering of IL-2 after T cell activation. Biophysical journal\| PubMed ID: 38532626	2024
Superresolution Imaging Reveals the Spatial Organization of CD81 Microdomains in Regulating Membrane Signaling on Jurkat T Cell Microvilli. bioRxiv : the preprint server for biology\| PubMed ID: 39677771	2024
Chemoproteomics-Enabled Proteolysis Targeting Chimera Discovery Platform Identifies a Metallothionein Degrader to Probe Its Role in Cancer. Journal of the American Chemical Society\| PubMed ID: 39989026	2025
Single-Molecule and Super-Resolution Techniques for Investigating Protein Organizations and Biological Functions of Immune Cells. Advances in experimental medicine and biology\| PubMed ID: 41652169	2026

Neal T. Ramseier

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