Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse.

Ligation of T cell receptor (TCR) to peptide-MHC (pMHC) complexes initiates signaling leading to T cell activation and TCR ubiquitination. Ubiquitinated TCR is then either internalized by the T cell or released toward the antigen-presenting cell (APC) in extracellular vesicles. How these distinct fates are orchestrated is unknown. Here, we show that clathrin is first recruited to TCR microclusters by HRS and STAM2 to initiate release of TCR in extracellular vesicles through clathrin- and ESCRT-mediated ectocytosis directly from the plasma membrane. Subsequently, EPN1 recruits clathrin to remaining TCR microclusters to enable trans-endocytosis of pMHC-TCR conjugates from the APC. With these results, we demonstrate how clathrin governs bidirectional membrane exchange at the immunological synapse through two topologically opposite processes coordinated by the sequential recruitment of ecto- and endocytic adaptors. This provides a scaffold for direct two-way communication between T cells and APCs.

FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P2-dependent switch.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Two-dimensional TIRF-SIM-traction force microscopy (2D TIRF-SIM-TFM).

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Astigmatic traction force microscopy (aTFM).

Quantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology. Traction force microscopy is one of the most broadly applied force probing technologies but ascertaining three-dimensional information typically necessitates slow, multi-frame z-stack acquisition with limited sensitivity. Here, by performing traction force microscopy using fast single-frame astigmatic imaging coupled with total internal reflection fluorescence microscopy we improve the temporal resolution of three-dimensional mechanical force quantification up to 10-fold compared to its related super-resolution modalities. 2.5D astigmatic traction force microscopy (aTFM) thus enables live-cell force measurements approaching physiological sensitivity.

Extended mechanical force measurements using structured illumination microscopy.

Quantifying cell generated mechanical forces is key to furthering our understanding of mechanobiology. Traction force microscopy (TFM) is one of the most broadly applied force probing technologies, but its sensitivity is strictly dependent on the spatio-temporal resolution of the underlying imaging system. In previous works, it was demonstrated that increased sampling densities of cell derived forces permitted by super-resolution fluorescence imaging enhanced the sensitivity of the TFM method. However, these recent advances to TFM based on super-resolution techniques were limited to slow acquisition speeds and high illumination powers. Here, we present three novel TFM approaches that, in combination with total internal reflection, structured illumination microscopy and astigmatism, improve the spatial and temporal performance in either two-dimensional or three-dimensional mechanical force quantification, while maintaining low illumination powers. These three techniques can be straightforwardly implemented on a single optical set-up offering a powerful platform to provide new insights into the physiological force generation in a wide range of biological studies. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

A dynamic CD2-rich compartment at the outer edge of the immunological synapse boosts and integrates signals.

The CD2-CD58 recognition system promotes adhesion and signaling and counters exhaustion in human T cells. We found that CD2 localized to the outer edge of the mature immunological synapse, with cellular or artificial APC, in a pattern we refer to as a 'CD2 corolla'. The corolla captured engaged CD28, ICOS, CD226 and SLAM-F1 co-stimulators. The corolla amplified active phosphorylated Src-family kinases (pSFK), LAT and PLC-γ over T cell receptor (TCR) alone. CD2-CD58 interactions in the corolla boosted signaling by 77% as compared with central CD2-CD58 interactions. Engaged PD-1 invaded the CD2 corolla and buffered CD2-mediated amplification of TCR signaling. CD2 numbers and motifs in its cytoplasmic tail controlled corolla formation. CD8+ tumor-infiltrating lymphocytes displayed low expression of CD2 in the majority of people with colorectal, endometrial or ovarian cancer. CD2 downregulation may attenuate antitumor T cell responses, with implications for checkpoint immunotherapies.

Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse.

A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4+ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.

Label-Free Optical Nanoscopy of Single-Layer Graphene.

The application of ultrafast pulsed laser sources and spectroscopic techniques enables label-free, deep-tissue optical microscopy. However, circumvention of the diffraction limit in this field is still an open challenge. Among such approaches, pump-probe microscopy is of increasing interest thanks to its highly specific nonfluorescent-based contrast mechanisms for the imaging of material and life science samples. In this paper, a custom femtosecond-pulsed near-infrared pump-probe microscope, which exploits transient absorption and stimulated Raman scattering interactions, is presented. The conventional pump-probe configuration is combined with a spatially shaped saturation pump beam, which allows for the reduction of the effective focal volume exploiting transient absorption saturation. By optimizing the acquisition parameters, such as power and temporal overlap of the saturation beam, we can image single-layer graphene deposited on a glass surface at the nanoscale and with increased layer sensitivity. These results suggest that saturation pump-probe nanoscopy is a promising tool for label-free high-resolution imaging.

Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells.

Planar supported lipid bilayers (PSLB) presenting T cell receptor (TCR) ligands and ICAM-1 induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but incorporation of other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. We demonstrate selective enrichment of CD40L and ICOS in SE in response to addition of CD40 and ICOSL, respectively, to SLB presenting TCR ligands and ICAM-1. SE are enriched in tetraspanins, BST-2, TCR signaling and ESCRT proteins. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2. CD40L+ SE retain the capacity to induce dendritic cell maturation and cytokine production.

Spatiotemporally Super-Resolved Volumetric Traction Force Microscopy.

Quantification of mechanical forces is a major challenge across biomedical sciences. Yet such measurements are essential to understanding the role of biomechanics in cell regulation and function. Traction force microscopy remains the most broadly applied force probing technology but typically restricts itself to single-plane two-dimensional quantifications with limited spatiotemporal resolution. Here, we introduce an enhanced force measurement technique combining 3D super-resolution fluorescence structural illumination microscopy and traction force microscopy (3D-SIM-TFM) offering increased spatiotemporal resolution, opening-up unprecedented insights into physiological three-dimensional force production in living cells.

Cytoskeletal actin patterns shape mast cell activation.

Activation of immune cells relies on a dynamic actin cytoskeleton. Despite detailed knowledge of molecular actin assembly, the exact processes governing actin organization during activation remain elusive. Using advanced microscopy, we here show that Rat Basophilic Leukemia (RBL) cells, a model mast cell line, employ an orchestrated series of reorganization events within the cortical actin network during activation. In response to IgE antigen-stimulation of FCε receptors (FCεR) at the RBL cell surface, we observed symmetry breaking of the F-actin network and subsequent rapid disassembly of the actin cortex. This was followed by a reassembly process that may be driven by the coordinated transformation of distinct nanoscale F-actin architectures, reminiscent of self-organizing actin patterns. Actin patterns co-localized with zones of Arp2/3 nucleation, while network reassembly was accompanied by myosin-II activity. Strikingly, cortical actin disassembly coincided with zones of granule secretion, suggesting that cytoskeletal actin patterns contribute to orchestrate RBL cell activation.

Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement.

We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used.

Plasmon dynamics in colloidal Au2Cd alloy-CdSe core/shell nanocrystals.

Metal-semiconductor nanocrystal heterostructures are model systems for understanding the interplay between the localized surface plasmon resonances in the metal domain and the relaxation of the excited carriers in the semiconductor domain. Here we report the synthesis of colloidal Au2Cd (core)/CdSe (shell) nanocrystal heterostructures, which were characterized extensively with several structural and optical techniques, including time-resolved fluorescence and broad-band transient absorption spectroscopy (both below and above the CdSe band gap). The dynamics of the transient plasmon peak was dominated by the relaxation of hot carriers in the metal core, its spectral shape was independent of the pump wavelength, and the bleaching lifetime was about half a picosecond, comparable with the value found in the AuCd seeds used for the synthesis.

Plasmon bleaching dynamics in colloidal gold-iron oxide nanocrystal heterodimers.

Colloidal nanocrystal heterodimers composed of a plasmonic and a magnetic domain have been widely studied as potential materials for various applications in nanomedicine, biology, and photocatalysis. One of the most popular nanocrystal heterodimers is represented by a structure made of a Au domain and a iron oxide domain joined together. Understanding the nature of the interface between the two domains in such type of dimer and how this influences the energy relaxation processes is a key issue. Here, we present the first broad-band transient absorption study on gold/iron oxide nanocrystal heterodimers that explains how the energy relaxation is affected by the presence of such interface. We found faster electron-electron and electron-phonon relaxation times for the gold "nested" in the iron oxide domain in the heterodimers with respect to gold "only" nanocrystals, that is, free-standing gold nanocrystals in solution. We relate this effect to the decreased electron screening caused by spill-out of the gold electron distribution at gold/iron oxide interface.

Plasmon dynamics in colloidal Cu2-xSe nanocrystals.

The optical response of metallic nanostructures after intense excitation with femtosecond-laser pulses has recently attracted increasing attention: such response is dominated by ultrafast electron-phonon coupling and offers the possibility to achieve optical modulation with unprecedented terahertz bandwidth. In addition to noble metal nanoparticles, efforts have been made in recent years to synthesize heavily doped semiconductor nanocrystals so as to achieve a plasmonic behavior with spectrally tunable features. In this work, we studied the dynamics of the localized plasmon resonance exhibited by colloidal Cu(2-x)Se nanocrystals of 13 nm in diameter and with x around 0.15, upon excitation by ultrafast laser pulses via pump-probe experiments in the near-infrared, with ∼200 fs resolution time. The experimental results were interpreted according to the two-temperature model and revealed the existence of strong nonlinearities in the plasmonic absorption due to the much lower carrier density of Cu(2-x)Se compared to noble metals, which led to ultrafast control of the probe signal with modulation depth exceeding 40% in transmission.

Optical and electrical properties of colloidal (spherical Au)-(spinel ferrite nanorod) heterostructures.

We report here a simple synthetic route to Au-Fe(x)O(y) heterostructures in which spinel ferrite (Fe(x)O(y)) grows as a nanorod on a spherical gold (Au) seed. The large red shift in the plasmon resonance in the heterostructures could be explained by a dielectric effect (although we could not entirely exclude a contribution due to electron transfer from Au to defect states at the Au-Fe(x)O(y) interface), while the magnetic properties of the Au-Fe(x)O(y) heterostructures were basically the same as those of the corresponding nanocrystals after Au leaching. In films of Au-Fe(x)O(y) heterostructures the electrical conductivity appeared to be mediated by the Au domains.