Biology subjects

Korobchevskaya, K.

Publications and source records attributed to Korobchevskaya, K..

4 recordsLinked to original sources

bioRxiv2022

FCHO controls AP2's critical endocytic roles through a PtdIns4,5P2 membrane-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-localised FCHO. Here, live-cell eTIRF-SIM 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/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. AP2s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCPs outer edge to engage and activate further AP2s to drive CCP growth/maturation. 125 character summaryFCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s

cell biology↗

bioRxiv2022

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

Ligation of the T cell receptor (TCR) to peptide-MHC complexes initiates signaling leading to T cell activation. Regulation of T cell responses also requires mechanisms to stop this signaling and to downregulate surface expression of the receptor. T cells achieve this both by TCR internalization and by releasing TCR loaded vesicles directly from the plasma membrane. How these distinct fates are coordinated is unknown. Here we show that clathrin is recruited to TCR microclusters by HRS and STAM2 and that this process is essential for TCR release. Subsequently, EPN1 recruits clathrin to the remaining antigen conjugated TCRs to enable a late wave of TCR endocytosis. With these results we demonstrate two clathrin-dependent mechanisms and show how the clathrin machinery participates in membrane evagination and invagination depending upon the adaptor recruiting it. These sequential mechanisms mediate bi-directional membrane exchange at the immunological synapse, providing a scaffold for critical communication.

cell biology↗

bioRxiv2019

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

Cell communication through extracellular vesicles is an emerging topic in biology, including communication between cells of the immune system. Planar supported lipid bilayers (PSLBs) presenting T cell receptor (TCR) ligands and intercellular adhesion molecule-1 (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 their ability to incorporate 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. Our results demonstrate selective enrichment of CD40 ligand (CD40L) and inducible T-cell costimulator (ICOS) in SE in response to addition of CD40 and ICOS ligand (ICOSL), respectively, to SLB presenting TCR ligands and ICAM-1. TCR triggering mobilized intracellular CD40L to the T cells surface at the IS, where it engaged CD40 to enable sorting into SE. SEs were enriched in tetraspanins and bone marrow stromal cell antigen 2 (BST-2) by immunofluorescence and TCR signalling and endosomal sorting complexes required for transport by proteomics. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2 microclusters. CD40L in SE retains the capacity to induce dendritic cell (DC) maturation and cytokine production. SE enabled helper T cells to release effectors physically linked to TCR. One Sentence SummaryTCR and CD40L microclusters can be linked in synaptic ectosomes (extracellular vesicles) that are released in the immunological synapse by helper T cells and induce dendritic cell maturation and cytokine production.

immunology↗

bioRxiv2019

CD2 expression acts as a quantitative checkpoint for immunological synapse structure and T-cell activation.

The CD2 receptor has been described as an adhesion and costimulatory receptor on T cells. Here, transcriptional profiling of colorectal cancers (CRC) revealed a negative correlation between CD2 expression and \"exhausted CD8+ T-cells\" gene signatures. Furthermore, we detected reduced surface CD2 levels in exhausted CD127lowPD-1hi CD3+CD8+ tumour infiltrating lymphocytes (TILs) in CRC. We describe a CD2 expression-level-dependent switch in CD2-CD58 localization between central and peripheral domains in the immunological synapse (IS). A peripheral \"CD2 corolla\" formed when CD2 surface expression was sufficiently high and its cytoplasmic domain intact. The corolla recruited other ligated receptors like CD28, boosted recruitment of activated Src-family kinases (pSrc), LAT and PLC-{gamma} in the IS and consequently T-cell activation in response to a tumour antigen. Corolla formation and pSrc in the IS increased linearly with CD2 expression, whereas pSrc signals were reduced by high, \"exhausted-like\" levels of PD-1, which invaded the corolla. These results suggest two levels of inhibition of Src-family kinases in CD3+CD8+ TILs: reduced CD2 expression and high PD-1 expression.

immunology↗