Linker For Activation Research Articles

A sustainable approach towards utilization of plastic waste for an efficient electrode in microbial fuel cell applications

Microbial fuel cells (MFC) are a novel technique for power generation from wastewater. A number of approaches for the modification of physical as well as chemical properties of the electrodes can be employed to attain the maximum output power density and high power electricity. The use of an active organic linker, extracted from waste residue (plastic), for the synthesis of porous nanostructured materials would be beneficial in the fabrication of electrodes for MFC. Herein, terephthalic acid monomer (t) derived from plastic waste was successfully applied as an electrochemically active linking unit to form an iron-based metal-organic framework (Fe-t-MOF: MIL-53(Fe)). The synthesized Fe-t-MOF was further modified with conducting polymer (polyaniline (PANI)). The produced nanocomposite (Fe-t-MOF/PANI) was coated on stainless steel (SS) disk (as a current collector) for use as an electrode component of the MFC system. The power density, open circuit potential (OCP), and a limiting current density of the MFC are 680 mW/m2, 0.67 V, and 3500mA/m2, respectively. The technique opted here should help search a novel, efficient, sustainable, and cost-effective route for the modification of the plastic waste into an MFC electrode to achieve bioenergy production through wastewater treatment.

Positive feedback between the Tcell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch.

SUMMARYProtein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.

Adapting T Cell Receptor Ligand Discrimination Capability via LAT.

Self- and non-self ligand discrimination is a core principle underlying T cell-mediated immunity. Mature αβ T cells can respond to a foreign peptide ligand presented by major histocompatibility complex molecules (pMHCs) on antigen presenting cells, on a background of continuously sensed self–pMHCs. How αβ T cells can properly balance high sensitivity and high specificity to foreign pMHCs, while surrounded by a sea of self-peptide ligands is not well understood. Such discrimination cannot be explained solely by the affinity parameters of T cell antigen receptor (TCR) and pMHC interaction. In this review, we will discuss how T cell ligand discrimination may be molecularly defined by events downstream of the TCR–pMHC interaction. We will discuss new evidence in support of the kinetic proofreading model of TCR ligand discrimination, and in particular how the kinetics of specific phosphorylation sites within the adaptor protein linker for activation of T cells (LAT) determine the outcome of TCR signaling. In addition, we will discuss emerging data regarding how some kinases, including ZAP-70 and LCK, may possess scaffolding functions to more efficiently direct their kinase activities.

The pronounced lung lesions developing in LATY136F knock-in mice mimic human IgG4-related lung disease.

Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a novel clinical disease entity characterized by an elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4-positive plasma cells. Pathological changes are most frequently seen in the pancreas, lacrimal glands, and salivary glands, but pathological changes in the lung also exist. Linker for activation of T cell (LAT)Y136F knock-in mice show Th2-dominant immunoreactions with elevated serum IgG1 levels, corresponding to human IgG4. We have reported that LATY136F knock-in mice display several characteristic features of IgG4-RD and concluded that they constitute an appropriate model of human IgG4-RD in salivary glands, pancreas, and kidney lesions. The aim of this study is to evaluate whether lung lesions in LATY136F knock-in mice can be a model of IgG4-related lung disease. Lung tissue samples from LATY136F knock-in mice (LAT) and wild-type mice (WT) were immunostained for IgG1 and obtained for pathological evaluation, and cell fractions and cytokine levels in broncho-alveolar lavage fluid (BALF) were analyzed. In the LAT group, IgG1-positive inflammatory cells increased starting at 4 weeks of age and peaked at 10 weeks of age. The total cell count and percentage of lymphocytes increased significantly in BALF in the LAT group compared to the WT group. In BALF, Th2-dominant cytokines and transforming growth factor-β were also increased. In the LAT group, marked inflammation around broncho-vascular bundles peaked at 10 weeks of age. After 10 weeks, fibrosis around broncho-vascular bundles and bronchiectasis were observed in LATY136F knock-in mice but not WT mice. LATY136F knock-in mice constitute an appropriate model of lung lesions in IgG4-RD.

Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM).

Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary data are available at Bioinformatics online.

A LAT-Based Signaling Complex in the Immunological Synapse as Determined with Live Cell Imaging Is Less Stable in T Cells with Regulatory Capability.

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.

The role of prolines and glycine in the transmembrane domain of LAT.

Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane, is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using insilico analysis, confocal and super-resolution imaging and flow cytometry, we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics are further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of nonpalmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

Timing to Membrane-Associated Protein Condensation Controls Antigen Discrimination in T Cells

T cells discriminate between self and agonist ligands by requiring long dwelling binding interactions between peptide-MHC (pMHC) and the T Cell Receptor (TCR). A consequence of successful signal transduction is the formation of a multi-molecular protein assembly composed of downstream adaptor molecules crosslinking the phosphorylated scaffold protein Linker for Activation of T cells (LAT). While it is known that after ligand binding Zap70 kinase recruits to the TCR and begins phosphorylating LAT, the early molecular events controlling precipitation of LAT condensates and antigen discrimination in relation to a single ligated TCR is unknown.

Tuning the excited-state intramolecular proton transfer (ESIPT)-based luminescence of metal-organic frameworks by metal nodes toward versatile photoluminescent applications.

Here, using three metal cations (Mg2+, Al3+, and Zr4+) and an excited-state intramolecular proton transfer (ESIPT) active linker, 2,5-dihydroxyterephthalic acid (H2DHT), three luminescent metal-organic frameworks (LMOFs) were obtained. Importantly, their ESIPT-based luminescence originated from the linker was systematically tuned in emission profiles including intensity, emission color, and quantum efficiency in the solution as well as in the solid state, which is largely dependent on the composition and structural characteristics of these three LMOFs. Similar to the free linker, the Mg-based MOF possesses a relatively strong luminescence, the Al-based MOF has moderate luminescence due to the breathing effect, and the Zr-based MOF is very weakly luminescent, mainly caused by the LMCT process. Benefiting from unique emission behaviors of these three LMOFs, we further modulated their ESIPT-based luminescence through the interplay between guest species and components of LMOFs by combining with various photophysical processes, and successfully explored their potential applications as versatile photoluminescent platforms for target-triggered sensory materials, responsive fluorescent hydrogels, and white-light-emitting phosphors.

The Expression of NTAL and Its Protein Interactors Is Associated With Clinical Outcomes in Acute Myeloid Leukemia

Non–T cell activation linker (NTAL) membrane protein depletion from lipid rafts by alkylphospholipids or downregulation by shRNA knockdown decreases cell viability through regulation of the Akt/PI3K pathway in mantle cell lymphoma and acute promyelocytic leukemia cells. Here, we confirmed that the knockdown of NTAL in acute myeloid leukemia (AML) cell lines was associated with decreased cell proliferation and survival. Similarly, a xenograft model using AML cells transduced with NTAL–shRNA and transplanted into immunodeficient mice led to a 1.8-fold decrease in tumor burden. Using immunoprecipitation, LC–MS/MS analysis, and label-free protein quantification, we identified interactors of NTAL in two AML cell lines. By evaluating the gene expression signatures of the NTAL protein interactors using the PREdiction of Clinical Outcomes from Genomic Profiles database, we found that 12 NTAL interactors could predict overall survival in AML, in at least two independent cohorts. In addition, patients with AML exhibiting a high expression of NTAL and its interactors were associated with a leukemic granulocyte–macrophage progenitor–like state. Taken together, our data provide evidence that NTAL and its protein interactors are relevant to AML cell proliferation and survival and represent potential therapeutic targets for granulocyte–macrophage progenitor–like leukemias.

Coupled membrane lipid miscibility and phosphotyrosine-driven protein condensation phase transitions

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.

New 3-D Mn(II) coordination polymer with redox active oxalate linker; an efficient and robust electrocatalyst for oxygen evolution reaction

A novel three dimensional coordination polymer {[Mn(ox)3/2][bap].(H2O)}n, (1) (ox = oxalate, bap = N-benzyl-4-aminopyridinium) is reported in this work as an efficient electrocatalyst for oxygen evolution reaction (OER). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) based dispersion condition as well as interactions between the adjacent layers of anionic coordination polymer and N-benzyl-4-aminopyridinium were found necessary for the growth and stability of this architecture. Single crystal XRD, IR and elemental analysis confirmed the formation of compound (1). Morphology and thermal stability were evaluated by SEM and TG respectively. Electrochemical studies were carried out in a three-electrode system using 1 coated FTO as a working electrode using 0.1 M KOH solution. The electrochemical investigation showed that 1 produced a current density of 10 mAcm−2 at a relatively low overpotential (η = 404 mV) while a maximum current density of 62 mAcm−2 was achieved. A catalytic onset potential for OER was observed at 1.5 V (vs RHE) and needed overpotential of 258 mV. The catalyst offered a turnover frequency (TOF) of 0.06 s−1 for OER at 404 mV. Long-term stability measurements of 1 were recorded by using chronoamperometry, IR analysis and LS voltammograms for the pristine and post-catalytic 1 coated FTO electrodes that confirmed the durability of the catalyst. The fascinating electrochemical behaviour of 1 toward oxygen evolution reaction can be attributed to its 3D polymeric structure and represents 1 as a robust and efficient catalyst towards OER.

Transforming growth factor beta orchestrates PD-L1 enrichment in tumor-derived exosomes and mediates CD8 T-cell dysfunction regulating early phosphorylation of TCR signalome in breast cancer.

Tumor cells promote immune evasion through upregulation of programmed death-ligand 1 (PD-L1) that binds with programmed cell death protein 1 (PD1) on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti-PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer (BC). Hence, more details understanding of PD-L1-mediated immune evasion is necessary. Here, we report BC cells secrete extracellular vesicles in form of exosomes carry PD-L1 and are highly immunosuppressive. Transforming growth factor beta (TGF-β) present in tumor microenvironment orchestrates BC cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-β level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T-cell phenotype. Recombinant TGF-β1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-β dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on T-cell receptor signaling, we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, linker for activation of T cells and phosphoinositide phospholipase Cγ of CD8 T cells more than PD-L1 exosomes. In vivo inhibition of exosome release and TGF-β synergistically attenuates tumor burden by promoting Granzyme and interferon gamma release in tumor tissue depicting rejuvenation of exhausted T cells. Thus, we establish TGF-β as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T-cell dysfunction.

Contextual Reprogramming of CAR-T Cells for Treatment of HER2+ Cancers

Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. The conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17–11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

5-Bromo-3,4-dihydroxybenzaldehyde from Polysiphonia morrowii attenuate IgE/BSA-stimulated mast cell activation and passive cutaneous anaphylaxis in mice

The present study investigates the anti-allergic activity of the marine algal bromophenol, 3-bromo-4,5-dihydroxybenzaldehyde (BDB), isolated from Polysiphonia morrowii Harvey in immunoglobulin (Ig)E/bovine serum albumin (BSA)-stimulated mouse bone marrow-derived cultured mast cells (BMCMCs) and a passive cutaneous anaphylaxis (PCA) mice ear model. BDB effectively inhibited β-hexosaminidase release (IC50 = 80.12 µM), in IgE/BSA-stimulated BMCMCs without a cytotoxic response. Also, BDB down-regulated the expression or secretion of cytokines, interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α and the chemokine (thymus and activation-regulated chemokine (TARC). The above effects could be attributed to the dose-dependent decrease of FcεRI expression on the surface of BMCMCs and its stable IgE binding. Moreover, BDB suppressed the nuclear factor (NF)-κB and spleen tyrosine kinase (SYK)-linker for T-cell activation (LAT)-GRB2 associated binding protein 2 (Gab2) signaling axis activated by IgE/BSA stimulation. Furthermore, oral administration of BDB to IgE-sensitized mice effectively attenuated IgE-triggered PCA reaction. Collectively, the anti-allergic effects of BDB suggest its potential applicability as a candidate for in-depth test trials.

ERM-Dependent Assembly of T Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli prior to Activation

T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαβ and TCRζ, as well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human Tcells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of Tcells), are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, and moesin) family colocalize with TCRαβ as well as with actin filaments, implying a role for one or more ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are preassembled prior to activation in an ERM-dependent manner, facilitating initial antigen sensing.

IL-15 and CD155 expression regulate LAT expression in murine DNAM1+ NK cells, enhancing their effectors functions.

NK cells are innate immune cells characterized by their ability to spontaneously lyse tumor and virally infected cells. We have recently demonstrated that IL-15-sufficient DC regulate NK cell effector functions in mice. Here, we established that among ITAM-proximal signaling molecules, the expression levels of the scaffold molecule Linker for Activation of T cells (LAT) and its transcription factor ELF-1 were reduced 4 days after in vivo depletion of DC. Addition of IL-15, a cytokine presented by DC to NK cells, regulates LAT expression in NK cells with a significant effect on the DNAM1+ subset compared to DNAM1- cells. We also found that LAT expression is regulated via interaction of the DNAM1 receptor with its ligand CD155 in both immature and mature NK cells, independently of NK cell education. Finally, we found that LAT expression within DNAM1+ NK cells might be responsible for enhanced calcium mobilization following the triggering of activating receptors on NK cells. Altogether, we found that LAT expression is tightly regulated in DNAM1+ NK cells, via interaction(s) with DC, which express CD155 and IL-15, resulting in rapid activation of the DNAM1+ subset during activating receptor triggering.

A common signaling pathway leading to degranulation in mast cells and its regulation by CCR1-ligand.

Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.

Molecular Mechanisms for the Stochastic Condensation of LAT Assemblies in T Cells

T cells discriminate between self and agonist by requiring long dwelling binding events between peptide-MHC (pMHC) and the T Cell Receptor (TCR). A consequence of successful signal transduction is the formation of a multi-molecular assembly of downstream molecules that is crosslinked by the phosphorylated scaffold protein Linker for Activation of T cells (LAT). While it is known that after ligand binding Zap70 kinase recruits to the TCR and begins phosphorylating LAT, the early molecular events precipitating LAT condensation in relation to a single ligated TCR is unknown. Using supported lipid bilayer technology and TIRF microscopy, we directly measure the intracellular responses to single pMHC-TCR binding events in living primary T cells with high spatiotemporal, multi-color resolution. This provides the ability to accurately determine the order of events in TCR signal transduction in relation to a single TCR, as opposed to bulk stimulation methods. We observe that after a pMHC binds to a TCR that LAT condensation occurs after an extended delay. While this timing is likely set, in part, by a competition of kinase/phosphatase pressure and LAT diffusion, the phosphorylation of LAT has a similarly extended delay. This suggests that diffusive phospho-LAT requires an additional event to trigger aggregation. These data demonstrate novel roles for key signaling proteins and, in contrast to ensemble methods, are the first to visualize the decisive steps leading to LAT condensation and completion of kinetic proofreading.