We present a method to robustly discriminate clustered from randomly distributed molecules detected with techniques based on single-molecule localization microscopy, such as PALM and STORM. The approach is based on deliberate variation of labeling density, such as titration of fluorescent antibody, combined with quantitative cluster analysis, and it thereby circumvents the problem of cluster artifacts generated by overcounting of blinking fluorophores. The method was used to analyze nanocluster formation in resting and activated immune cells.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

It is the main function of T cells to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data has indicated that global clustering of the T cell receptor (TCR) occurs prior to T cell activation. Such pre-activation clustering has been interpreted as representing a potential regulatory mechanism that fine-tunes the T cell response. We found here that apparent TCR nanoclustering could be attributed to overcounting artifacts inherent to single-molecule-localization microscopy. Using complementary super-resolution approaches and statistical image analysis, we found no indication of global nanoclustering of the TCR on antigen-experienced CD4 + T cells under non-activating conditions. We also used extensive simulations of super-resolution images to provide quantitative limits for the degree of randomness of the TCR distribution. Together, our results suggest that the distribution of TCRs on the plasma membrane is optimized for fast recognition of antigen in the first phase of T cell activation.

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.