Biology subjects

Becherer, U.

Publications and source records attributed to Becherer, U..

6 recordsLinked to original sources

bioRxiv2026

Interferon-γ promotes SMAP production by cytotoxic T lymphocytes in a thrombospondin 4 dependent manner

Cytotoxic T lymphocytes (CTLs) eliminate infected and cancerous cells by exocytosing cytotoxic granules, either as single-core granules (SCGs) releasing diffusible Granzyme B and Perforin, or as multi-core granules (MCGs) releasing these effectors as thrombospondin-1/4-encapsulated supramolecular attack particles (SMAPs). How CTLs differentially deploy these granule types remains unclear. We demonstrate that prolonged in vitro expansion and restimulation selectively enhance SMAP release, correlating with increased MCG maturation and thrombospondin-4 expression. Using high-resolution imaging, we identify fusion-competent MCG intermediates lacking SMAPs but releasing granzyme B diffusively. Mechanistically, interferon-{gamma} upregulates thrombospondin-4, driving MCG maturation and SMAP biogenesis, enhancing late-phase CTL killing efficiency against resistant targets. Consistent with this, THBS1 and THBS4 transcript levels appear elevated in melanoma-infiltrating CTLs compared with those during acute adenovirus infection. These findings define a stimulus-dependent, interferon-{gamma}-driven pathway tailoring CTL responses to chronic pathology and highlight opportunities for SMAP-targeted immunotherapies.

immunology↗

bioRxiv2025

Stenocytosis: a mechanism for supramolecular attack particle transfer at the CTL lytic synapse

Supramolecular attack particles (SMAPs) are recently discovered key components of the cytotoxic T lymphocytes (CTLs) lytic arsenal exhibiting autonomous cytotoxic behavior. Yet, how these particles might serve as synaptic weapons transferred from CTL into target cells within dynamic lytic synapses remains to be elucidated. In CTL interacting with immobilized stimuli, total internal reflection fluorescence microscopy (TIRFM) and rapid live 3D cell imaging showed that granules containing SMAPs navigate through narrow cortical actin cytoskeleton depletion areas to reach plasma membrane secretion hot-spots where SMAPs are released. In CTLs interacting with cognate target cells, correlative light-electron microscopy (CLEM) and structured illumination 3D imaging showed SMAP release into the synaptic cleft and penetration into the target cell trough an equally narrow gap in the target cell cortical actin cytoskeleton mirroring that formed in the CTL cytoskeleton, and just large enough to admit the SMAP into an early endosome. Our results reveal a previously undescribed process, stenocytosis (from the ancient Greek {sigma}{tau}{varepsilon}{nu}o{varsigma}, "narrow"), which allows hundred-nanometer-scale lytic particles to exit CTLs and enter target cells while evading synaptic defenses.

immunology↗

bioRxiv2025

Lytic IFNγ is stored in granzyme B-containing cytotoxic granules and co-secreted by effector CD8⁺ T cells

Cytotoxic CD8 T cells form immunological synapses with target cells and release effector molecules, including IFN{gamma}, to mediate antitumor immunity. However, the mechanisms by which IFN{gamma} contributes to cytotoxicity remain incompletely understood. Here, we identify a subset of IFN{gamma} stored within GzmB cytotoxic granules (CGs) in activated mouse and human CD8 T cells, termed lytic IFN{gamma}. Lytic IFN{gamma} is polarized to the synapse and co-secreted with GzmB in both soluble and supramolecular attack particle (SMAP)-associated forms. Mouse CD8 T cells lacking the vesicle priming factor Munc13-4 exhibit impaired both CG and early IFN{gamma} release at the immunological synapse, while prolonged synaptic engagement restores IFN{gamma} secretion. Super-resolution imaging demonstrates that sustained synaptic interactions drive IFN{gamma} secretion at distal membrane sites, suggesting the existence of distinct IFN{gamma} populations with potentially diverse functions beyond lytic IFN{gamma}. These findings uncover an unrecognized mechanism of IFN{gamma} storage and release, underscoring its pivotal role in CD8 T cell-mediated tumor elimination.

immunology↗

bioRxiv2024

Highly adaptable deep-learning platform for automated detection and analysis of vesicle exocytosis

Vesicle exocytosis is a fundamental component of intercellular communication, in all organisms. It has been studied for decades, using various imaging tools. Nevertheless, exocytosis research is still limited by the lack of reliable automated analysis procedures. To address this, we developed the Intelligent Vesicle Exocytosis Analysis Platform (IVEA), a nearly universal solution for analyzing exocytosis acquired with live cell imaging. IVEA is applicable to a wide variety of experimental model systems, microscopes and reporter fluorophores. IVEA combines state-of-the-art deep-learning and computer vision regimes to enable fully automated analysis of large data. IVEA runs as a FIJI plugin and does not require prior training or human intervention. IVEA is 60 times faster than manual analysis and is able to detect rare events often missed by the human eye. Overall, IVEA represents a breakthrough in the analysis of cellular secretory mechanisms and has a transformative potential for the exocytosis imaging field.

bioengineering↗

bioRxiv2024

Thrombospondins 1 and 4 undergo coordinated transport to multicore cytotoxic granules to regulate SMAP biogenesis and function in CTL-mediatedcytotoxicity

Supramolecular Attack Particles (SMAPs) are particulate entities, characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a glycoproteic shell, released by CTLs and NK cells. Prior proteomic analysis identified thrombospondin-1 (TSP-1) and thrombospondin-4 (TSP-4) as putative components of SMAPs. While TSP-1 has been validated as a component of the SMAP shell and shown to contribute significantly to CTL-mediated killing, the expression and function of TSP-4 in CTLs, and its interplay with TSP-1 in SMAP biogenesis and function, has not been investigated as yet. Here we demonstrate that TSP-4 and TSP-1 have a complementary expression profile during in vitro human CD8+ T cell differentiation to CTLs and sequentially localize to lytic granules (LG), with TSP-4 being required for TSP-1 association with LGs. Correlative light microscopy identified the TSP-enriched LGs as the SMAP-containing multicore granules. We show by STED microscopy a heterogeneity among TSP-enriched LGs, the most abundant population being positive for both TSP-4 and TSP-1. We also show that TSP-1 and TSP-4 are co-released in association with SMAPs at immune synapses formed on planar supported lipid bilayers, as assessed by dSTORM imaging. Finally, we provide evidence that TSP-4 is required for CTL- and SMAP-mediated cell killing. Of note, we found that chronic lymphocytic leukemia (CLL) cell supernatants, which suppress CTL mediated killing, also suppress expression of TSP-4 as well as of cytolytic effectors and impair SMAP biogenesis. These results identify TSP-4 as a key player in SMAP structure and activity and suggest that SMAPs may be a new target for immune suppression by CLL.

immunology↗

bioRxiv2022

Expansion microscopy at one nanometer resolution

Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of one-step nanoscale expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7 to enable the description of cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the [~]17 kDa protein calmodulin upon Ca2+ binding were readily observable. We also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinsons Disease (PD) patients, which represents a promising new development towards improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

biophysics↗