TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells.

The main function of T cells is to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data have indicated that global clustering of T cell antigen receptors (TCRs) occurs before 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 TCRs 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.

Monomeric TCRs drive T cell antigen recognition.

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.

Single-molecule optical absorption imaging by nanomechanical photothermal sensing.

Absorption microscopy is a promising alternative to fluorescence microscopy for single-molecule imaging. So far, molecular absorption has been probed optically via the attenuation of a probing laser or via photothermal effects. The sensitivity of optical probing is not only restricted by background scattering but it is fundamentally limited by laser shot noise, which minimizes the achievable single-molecule signal-to-noise ratio. Here, we present nanomechanical photothermal microscopy, which overcomes the scattering and shot-noise limit by detecting the photothermal heating of the sample directly with a temperature-sensitive substrate. We use nanomechanical silicon nitride drums, whose resonant frequency detunes with local heating. Individual Au nanoparticles with diameters from 10 to 200 nm and single molecules (Atto 633) are scanned with a heating laser with a peak irradiance of 354 ± 45 µW/µm2 using 50× long-working-distance objective. With a stress-optimized drum we reach a sensitivity of 16 fW/Hz1/2 at room temperature, resulting in a single-molecule signal-to-noise ratio of >70. The high sensitivity combined with the inherent wavelength independence of the nanomechanical sensor presents a competitive alternative to established tools for the analysis and localization of nonfluorescent single molecules and nanoparticles.

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms.

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents.

Diabetic foot complications-lessons learned from real-world data derived from a specialized Austrian hospital.

BACKGROUND: Diabetic foot complications, one of the most severe late complications of type 2 diabetes mellitus, are associated with a tremendous personal and financial burden. In order to drive the prevention of diabetic foot complications forward and facilitate early detection and personalized screening of high-risk patients, longitudinal studies are needed to identify risk factors associated with diabetic foot complications in large patient datasets. METHODS: This is a retrospective cohort study on 3002 patients with type 2 diabetes mellitus aged ≥ 18 years without prior foot complications. The data were collected between 2006 and 2017 in an Austrian hospital department specialized for diabetic patients. In addition to a univariate Cox regression analysis, multivariate Cox regression models were established to identify independent risk factors associated with diabetic foot complications and adjust for potential confounders. RESULTS: We observed a total of 61 diabetic foot complications in 3002 patients. In the multivariate Cox regression model, significant risk factors (hazard ratio, 95% confidence interval) for foot complications were age at diagnosis > 70 years (3.39, 1.33-8.67), male gender (2.55, 1.42-4.55), neuropathy (3.03, 1.74-5.27), peripheral arterial disease (3.04, 1.61-5.74), hypertension > 10 years after diagnosis (2.32, 1.09-4.93) and HbA1c > 9% (2.44, 1.02-5.83). CONCLUSION: The identified risk factors for diabetic foot complications suggest that personalized early detection of patients at high risk might be possible by taking the patient's clinical characteristics, medical history and comorbidities into account. Modifiable risk factors, such as hypertension and high levels of blood glucose might be tackled to reduce the risk for diabetic foot complications.

Risk factors for diabetic foot complications among patients with type 2 diabetes in Austria-A registry-based retrospective cohort study.

AIMS: Diabetic foot complications, a serious consequence of diabetes mellitus, are associated with a tremendous burden on both individual patients and health care systems. Since prevention strategies may reduce the incidence of this complication, identification of risk factors in large longitudinal studies is essential to optimize early detection and personalized screening of patients at increased risk. MATERIALS AND METHODS: We conducted a registry-based retrospective cohort study using data from 10,688 patients with type 2 diabetes mellitus aged ≥18 years. Cox regression models were used to identify risk factors for foot complications while adjusting for potential confounders. RESULTS: We observed 140 diabetic foot complications in our patient cohort. The multivariate Cox regression model revealed neuropathy, peripheral arterial disease and male gender as being positively associated with foot complications. The same effect was detected for nephropathy in the time >10 years after T2DM diagnosis. For higher age at diagnosis and use of insulin, however, a negative association was retrieved. CONCLUSION: Male gender and several diabetes-related comorbidities were identified as risk factors for subsequent initial foot complications in patients with type 2 diabetes mellitus. These findings suggest that personalized early detection of patients at increased risk might be feasible by using information on demographics, medical history and comorbidities.

Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy.

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.

What we talk about when we talk about nanoclusters.

Superresolution microscopy results have sparked the idea that many membrane proteins are not randomly distributed across the plasma membrane but are instead arranged in nanoclusters. Frequently, these new results seemed to confirm older data based on biochemical and electron microscopy experiments. Recently, however, it was recognized that multiple countings of the very same fluorescently labeled protein molecule can be easily confused with true protein clusters. Various strategies have been developed, which are intended to solve the problem of discriminating true protein clusters from imaging artifacts. We believe that there is currently no perfect algorithm for this problem; instead, different approaches have different strengths and weaknesses. In this review, we discuss single molecule localization microscopy in view of its ability to detect nanoclusters of membrane proteins. To capture the different views on nanoclustering, we chose an unconventional style for this article: we placed its scientific content in the setting of a fictive conference, where five researchers from different fields discuss the problem of detecting and quantifying nanoclusters. Using this style, we feel that the different approaches common for different research areas can be well illustrated. Similarities to a short story by Raymond Carver are not unintentional.

Direct observation of cargo transfer from HDL particles to the plasma membrane.

BACKGROUND AND AIMS: Exchange of cholesterol between high-density lipoprotein (HDL) particles and cells is a key process for maintaining cellular cholesterol homeostasis. Recently, we have shown that amphiphilic cargo derived from HDL can be transferred directly to lipid bilayers. Here we pursued this work using a fluorescence-based method to directly follow cargo transfer from HDL particles to the cell membrane. METHODS: HDL was either immobilized on surfaces or added directly to cells, while transfer of fluorescent cargo was visualized via fluorescence imaging. RESULTS: In Chinese hamster ovary (CHO) cells expressing the scavenger receptor class B type 1 (SR-B1), transfer of amphiphilic cargo from HDL particles to the plasma membrane was observed immediately after contact, whereas hydrophobic cargo remained associated with the particles; about 60% of the amphiphilic cargo of surface-bound HDL was transferred to the plasma membrane. Essentially no cargo transfer was observed in cells with low endogenous SR-B1 expression. Interestingly, transfer of fluorescently-labeled cholesterol was also facilitated by using an artificial linker to bind HDL to the cell surface. CONCLUSIONS: Our data hence indicate that the tethering function of SR-B1 is sufficient for efficient transfer of free cholesterol to the plasma membrane.