Docking of Syk to FcεRI is enhanced by Lyn but limited in duration by SHIP1.

The high-affinity immunoglobulin E (IgE) receptor, FcεRI, is the primary immune receptor found on mast cells and basophils. Signal initiation is classically attributed to phosphorylation of FcεRI ß- and γ-subunits by the Src family kinase (SFK) Lyn, followed by the recruitment and activation of the tyrosine kinase Syk. FcεRI signaling is tuned by the balance between Syk-driven positive signaling and the engagement of inhibitory molecules, including SHIP1. Here, we investigate the mechanistic contributions of Lyn, Syk, and SHIP1 to the formation of the FcεRI signalosome. Using Lyn-deficient RBL-2H3 mast cells, we found that another SFK can weakly monophosphorylate the γ-subunit, yet Syk still binds the incompletely phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs). Once recruited, Syk further enhances γ-phosphorylation to propagate signaling. In contrast, the loss of SHIP1 recruitment indicates that Lyn is required for phosphorylation of the ß-subunit. We demonstrate two noncanonical Syk binding modes, trans γ-bridging and direct ß-binding, that can support signaling when SHIP1 is absent. Using single particle tracking, we reveal a novel role of SHIP1 in regulating Syk activity, where the presence of SHIP1 in the signaling complex acts to increase the Syk:receptor off-rate. These data suggest that the composition and dynamics of the signalosome modulate immunoreceptor signaling activities.

Anti-Glucosylsphingosine Autoimmunity, JAK2V617F-Dependent Interleukin-1ß and JAK2V617F-Independent Cytokines in Myeloproliferative Neoplasms.

Inflammatory cytokines play a major role in myeloproliferative neoplasms (MPNs) as regulators of the MPN clone and as mediators of clinical symptoms and complications. Firstly, we investigated the effect of JAK2V617F on 42 molecules linked to inflammation. For JAK2V617F-mutated patients, the JAK2V617F allele burden (%JAK2V617F) correlated with the levels of IL-1ß, IL-1Rα, IP-10 and leptin in polycythemia vera (PV), and with IL-33 in ET; for all other molecules, no correlation was found. Cytokine production was also studied in the human megakaryocytic cell line UT-7. Wild-type UT-7 cells secreted 27/42 cytokines measured. UT-7 clones expressing 50% or 75% JAK2V617F were generated, in which the production of IL-1ß, IP-10 and RANTES was increased; other cytokines were not affected. Secondly, we searched for causes of chronic inflammation in MPNs other than driver mutations. Since antigen-driven selection is increasingly implicated in the pathogenesis of blood malignancies, we investigated whether proinflammatory glucosylsphingosine (GlcSph) may play a role in MPNs. We report that 20% (15/75) of MPN patients presented with anti-GlcSph IgGs, distinguished by elevated levels of 11 cytokines. In summary, only IL-1ß and IP-10 were linked to JAK2V617F both in patients and in UT-7 cells; other inflammation-linked cytokines in excess in MPNs were not. For subsets of MPN patients, a possible cause of inflammation may be auto-immunity against glucolipids.

Combinatorial diversity of Syk recruitment driven by its multivalent engagement with FcεRIγ.

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as tetrameric (αßγ2) FcεRI. Syk activation is generally attributed to cis binding of its tandem SH2 domains to dual phosphotyrosines within FcεRIγ-ITAMs (immunoreceptor tyrosine-based activation motifs). However, the mechanistic details of Syk docking on γ homodimers are unresolved. Here, we estimate that multivalent interactions for WT Syk improve cis-oriented binding by three orders of magnitude. We applied molecular dynamics (MD), hybrid MD/worm-like chain polymer modeling, and live cell imaging to evaluate relative binding and signaling output for all possible cis and trans Syk-FcεRIγ configurations. Syk binding is likely modulated during signaling by autophosphorylation on Y130 in interdomain A, since a Y130E phosphomimetic form of Syk is predicted to lead to reduced helicity of interdomain A and alter Syk's bias for cis binding. Experiments in reconstituted γ-KO cells, whose γ subunits are linked by disulfide bonds, as well as in cells expressing monomeric ITAM or hemITAM γ-chimeras, support model predictions that short distances between γ ITAM pairs are required for trans docking. We propose that the full range of docking configurations improves signaling efficiency by expanding the combinatorial possibilities for Syk recruitment, particularly under conditions of incomplete ITAM phosphorylation.

Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics.

Cross-linking of immunoglobulin E-bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single-molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (Syk-Y130E) led to an increased Syk-FcεRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. Genome edited cells expressing only Syk-Y130E were deficient in antigen-stimulated calcium release, degranulation, and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FcεRI signaling pathway occurs at the level of Syk-FcεRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.

Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

Ag-mediated crosslinking of IgE-FcεRI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat-based gene editing, human RBLrαKO cells were created that exclusively express the human FcεRIα subunit. Pen a 1-specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1-specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on FcεRI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ∼2700 IgE-FcεRI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of ≥8), although measurable responses were achieved when only a few hundred FcεRI were occupied. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60-79 aa), which together span the entire length of tropomyosin, were weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE-FcεRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia.

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34+ cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

Three dimensional time-gated tracking of non-blinking quantum dots in live cells.

Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.

Secretory autophagy.

Autophagy, once viewed exclusively as a cytoplasmic auto-digestive process, has its less intuitive but biologically distinct non-degradative roles. One manifestation of these functions of the autophagic machinery is the process termed secretory autophagy. Secretory autophagy facilitates unconventional secretion of the cytosolic cargo such as leaderless cytosolic proteins, which unlike proteins endowed with the leader (N-terminal signal) peptides cannot enter the conventional secretory pathway normally operating via the endoplasmic reticulum and the Golgi apparatus. Secretory autophagy may also export more complex cytoplasmic cargo and help excrete particulate substrates. Autophagic machinery and autophagy as a process also affect conventional secretory pathways, including the constitutive and regulated secretion, as well as promote alternative routes for trafficking of integral membrane proteins to the plasma membrane. Thus, autophagy and autophagic factors are intimately intertwined at many levels with secretion and polarized sorting in eukaryotic cells.

Mpl traffics to the cell surface through conventional and unconventional routes.

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER-tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling.

The architectural relationship of components controlling mast cell endocytosis.

Eukaryotic cells use multiple routes for receptor internalization. Here, we examine the topographical relationships of clathrin-dependent and clathrin-independent endocytic structures on the plasma membranes of leukemia-derived mast cells. The high affinity IgE receptor (FcεRI) utilizes both pathways, whereas transferrin receptor serves as a marker for the classical clathrin-mediated endocytosis pathway. Both receptors were tracked by live-cell imaging in the presence or absence of inhibitors that established their differential dependence on specific endocytic adaptor proteins. The topology of antigen-bound FcεRI, clathrin, dynamin, Arf6 and Eps15-positive structures were analyzed by 2D and 3D immunoelectron microscopy techniques, revealing their remarkable spatial relationships and unique geometry. We conclude that the mast cell plasma membrane has multiple specialized domains for endocytosis. Their close proximity might reflect shared components, such as lipids and adaptor proteins, that facilitate inward membrane curvature. Intersections between these specialized domains might represent sorting stations that direct cargo to specific endocytic pathways.

Nested high-resolution melting curve analysis a highly sensitive, reliable, and simple method for detection of JAK2 exon 12 mutations--clinical relevance in the monitoring of polycythemia.

JAK2 exon 12 mutations are found in myeloproliferative disorders characterized by erythrocytosis. Lying in a 33-bp region and conserving the open reading frame, they often present a low allelic burden (<10%), which excludes screening with techniques such as allele-specific PCR or different sequencing protocols. High-resolution melting (HRM), a fast in-tube method, seems the most accurate routine technique for that. We describe a reliable and powerful nested HRM technique, independent of DNA preparation and with technical sensitivity of 100% (95% CI, 93% to 100%) and specificity of 96.7% (95% CI, 89.7% to 96.7%). Screening a cohort of 10 idiopathic erythrocytosis, 28 polycythemia vera, and 7 secondary erythrocytosis cases allowed the detection of 15 mutants, including 9 different mutations, of which 3 were unreported, all in the polycythemia vera group, and presented a characteristic profile: pure erythrocytosis associated with low serum erythropoietin. Threshold detection level ranged from 1% to 3% allelic burden, depending on the mutation. All of the HRM positive signals were found mutated by sequencing. Six of them (40%), however, required cloning before sequencing, because of low allelic burden. Classic techniques such as genomic sequencing may therefore miss patients with mutations. Given its sensitivity, HRM (and nested HRM) can be used in routine diagnosis and seems to be the most efficient of current techniques for detection of JAK2 exon 12 mutations.

Frequent reduction or absence of detection of the JAK2-mutated clone in JAK2V617F-positive patients within the first years of hydroxyurea therapy.

We analyzed the effect of hydroxyurea on the JAK2V617F allelic ratio (%JAK2V617F), measured in purified blood granulocytes, of patients with polycythemia vera and essential thrombocythemia. Thirty-six patients were examined sequentially prior to and after start of hydroxy-urea therapy (8 polycythemia vera, 17 essential thrombocythemia), or while remaining untreated (2 polycythemia vera, 9 essential thrombocythemia). Hydroxyurea therapy (median duration: 15 months) reduced the %JAK2V617F by >30% in 13/25 patients (4 polycythemia vera, 9 essential thrombocythemia). For 3 patients, JAK2V617F remained undetectable for 3-27 months. In addition, a single time point study of two large cohorts of patients, examined either at the time of diagnosis (99 polycythemia vera, 178 essential thrombocythemia) or while receiving hydroxyurea (36 polycythemia vera, 98 essential thrombocythemia; median length of therapy: 32 months), confirmed reduction of %JAK2V617F in the hydroxyurea-treated group (24% vs. 33% JAK2V617F at diagnosis, p<0.01). Prospective studies are needed to determine the prognostic value of reduced JAK2V617F allele burden under cytoreductive therapy.