Analyzing Angiotensin II Receptor Type 1 Clustering in PC12 Cells in Response to Hypoxia Using Direct Stochastic Optical Reconstruction Microscopy (dSTORM).

Angiotensin II (Ang II) is a hormone that plays a major role in maintaining homeostasis. The Ang II receptor type 1 (ATR) is expressed in acute O sensitive cells, including carotid body (CB) type I cells and pheochromocytoma 12 (PC12) cells, and Ang II increases cell activity. While a functional role for Ang II and ATRs in increasing the activity of O sensitive cells has been established, the nanoscale distribution of ATRs has not.

A framework for evaluating the performance of SMLM cluster analysis algorithms.

Single-molecule localization microscopy (SMLM) generates data in the form of coordinates of localized fluorophores. Cluster analysis is an attractive route for extracting biologically meaningful information from such data and has been widely applied. Despite a range of cluster analysis algorithms, there exists no consensus framework for the evaluation of their performance.

Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus.

The T cell receptor displays lateral signal propagation involving non-engaged receptors.

T cells are highly sensitive to low levels of antigen, but how this sensitivity is achieved is currently unknown. Here, we imaged proximal TCR-CD3 signal propagation with single molecule localization microscopy (SMLM) in T cells activated with nanoscale clusters of TCR stimuli. We observed the formation of large TCR-CD3 clusters that exceeded the area of the ligand clusters, and required multivalent interactions facilitated by TCR-CD3 phosphorylation for assembly.

The Role of Protein and Lipid Clustering in Lymphocyte Activation.

Lymphocytes must strike a delicate balance between activating in response to signals from potentially pathogenic organisms and avoiding activation from stimuli emanating from the body's own cells. For cells, such as T or B cells, maximizing the efficiency and fidelity, whilst minimizing the crosstalk, of complex signaling pathways is crucial. One way of achieving this control is by carefully orchestrating the spatiotemporal organization of signaling molecules, thereby regulating the rates of protein-protein interactions.

Analysis methods for interrogating spatial organisation of single molecule localisation microscopy data.

Single-molecule localisation microscopy (SMLM) gives access to biological information below the diffraction limit, allowing nanoscale cellular structures to be probed. The data output is unlike that of conventional microscopy images, instead consisting of an array of molecular coordinates. These represent a spatial point pattern that attempts to approximate, as closely as possible, the underlying positions of the molecules of interest.

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells.

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane.

tagPAINT: covalent labelling of genetically encoded protein tags for DNA-PAINT imaging.

Recently, DNA-PAINT single-molecule localization microscopy (SMLM) has shown great promise for quantitative imaging; however, labelling strategies thus far have relied on multivalent and affinity-based approaches. Here, the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA-docking strands and application of SMLM via DNA-PAINT is demonstrated. tagPAINT is then used for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy.

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy.

Stoichiometric quantification of spatially dense assemblies with qPAINT.

Quantitative PAINT (qPAINT) is a useful method for counting well-separated molecules within nanoscale assemblies. But whether cross-reactivity in densely-packed arrangements perturbs measurements is unknown. Here we establish that qPAINT measurements are robust even when target molecules are separated by as little as 3 nm, sufficiently close that single-stranded DNA binding sites can interact.

How does T cell receptor clustering impact on signal transduction?

The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR-CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR-CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood.

DNA-Based Super-Resolution Microscopy: DNA-PAINT.

Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore.

Monolayer surface chemistry enables 2-colour single molecule localisation microscopy of adhesive ligands and adhesion proteins.

Nanofabricated and nanopatterned surfaces have revealed the sensitivity of cell adhesion to nanoscale variations in the spacing of adhesive ligands such as the tripeptide arginine-glycine-aspartic acid (RGD). To date, surface characterisation and cell adhesion are often examined in two separate experiments so that the localisation of ligands and adhesion proteins cannot be combined in the same image. Here we developed self-assembled monolayer chemistry for indium tin oxide (ITO) surfaces for single molecule localisation microscopy (SMLM).

Can single molecule localization microscopy be used to map closely spaced RGD nanodomains?

Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD).

Tailoring the surface charge of dextran-based polymer coated SPIONs for modulated stem cell uptake and MRI contrast.

Tracking stem cells in vivo using non-invasive techniques is critical to evaluate the efficacy and safety of stem cell therapies. Superparamagnetic iron oxide nanoparticles (SPIONs) enable cells to be tracked using magnetic resonance imaging (MRI), but to obtain detectable signal cells need to be labelled with a sufficient amount of iron oxide. For the majority of SPIONs, this can only be obtained with the use of transfection agents, which can adversely affect cell health.

Partial mitigation of gold nanoparticle interactions with human lymphocytes by surface functionalization with a 'mixed matrix'.

Aim: To investigate interactions of gold nanoparticles with primary human lymphocytes and determine if the addition of a self-assembled monolayer of 'mixed-matrix' ligands influenced these interactions.

Materials & Methods: The effect of gold nanoparticles was measured by exposure to peripheral blood mononuclear cells (PBMCs) from healthy volunteers with subsequent examination of cell proliferation, cytokine secretion and CD4(+) T-cell activation relative to controls.

Results: Capped and as-synthesized gold nanoparticles augmented PBMC proliferation in response to phytohemagglutinin and this effect was greater for as-synthesized than for capped gold nanoparticles.