Biology subjects

Lidke, D. S.

Publications and source records attributed to Lidke, D. S..

8 recordsLinked to original sources

bioRxiv2025

IgE occupancy and antigen valency cooperate to control FcϵRI aggregation geometry and signaling efficiency

The crosslinking of IgE-bound Fc{varepsilon}RI by multivalent allergens initiates mast cell and basophil signaling underlying Type 1 allergy. Yet, how allergen properties and IgE occupancy impact receptor aggregation and downstream signaling remain unclear. We used Phl p 1-specific IgE and recombinant fusion proteins presenting a Phl p 1-derived peptide in defined valencies and positions (MB1N, MB2N, MB4N, MB1N1C) to probe antigen-dependent signaling. Tetravalent MB4N evoked stronger degranulation and Ca2+ response than bivalent antigens, MB2N and MB1N1C. MB4N was also capable of signaling at low IgE occupancy and in Lyn-deficient cells. Monte Carlo simulations predicted that MB4N forms larger, complex receptor aggregates, while MB2N and MB1N1C produce dimers and linear chains. Consistently, addition of MB4N showed larger aggregates by electron microscopy and slower mobility by single particle tracking, compared to bivalent antigens. Thus, allergen valency and epitope spatial arrangement dictate Fc{varepsilon}RI aggregate organization and subsequent effector cell activation.

immunology↗

bioRxiv2025

The Influence of Macrophages within the Tumor Microenvironment on Ovarian Cancer Growth and Response to Therapies

While the majority of ovarian cancer (OC) patients respond to front-line carboplatin/paclitaxel chemotherapy and surgical debulking, nearly all patients will develop platinum-resistance and recur. Our study investigates how tumor-associated macrophages (TAMs) within the tumor microenvironment (TME) affect chemotherapy outcomes using OC patient-derived organoids and humanized patient-derived xenografts (huPDX). In vitro macrophage migration assays demonstrate the selective recruitment of M2 polarized macrophages to OC organoids. M2 macrophages, but not M1 macrophages, increase OC organoid viability and reduce their sensitivity to paclitaxel in co-culture assays. Furthermore, we identified BMS777607, a receptor tyrosine kinase inhibitor, that is capable of repolarizing M2 macrophages in vitro and reduces organoid viability by a macrophage-dependent mechanism. In a platinum-sensitive humanized patient-derived xenograft (huPDX) model, the presence of human immune cells increased between-mouse variability in response to paclitaxel with two of four mice demonstrating tumor regrowth after two weeks of treatment. A TAM-targeted CSF-1R inhibitor, BLZ945 significantly reduced the total number of human immune cells within the ascites fluid of huPDX3 mice, but did not reduce tumor burden. However BLZ945 in combination with paclitaxel reduced tumor burden with no sign of regrowth. Our study demonstrates that patient-derived OC organoids and huPDX, are useful for evaluating the immunomodulatory effects of therapies and the influence of TAMs on response. huPDX models could serve as a robust platform for preclinical testing of novel anti-cancer treatments, providing insights into the complex interplay between immune cells and cancer therapeutics.

cancer biology↗

bioRxiv2023

Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcϵRI Aggregation andStructural Changes

Fc receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs) are critical components of the innate immune system that bridge adaptive antibody recognition to cellular effector responses. In allergic responses, the high-affinity IgE receptor, Fc{varepsilon}RI, is activated when multivalent antigens crosslink receptor-bound IgE, yet the molecular mechanisms linking antigen structure to signaling output remain incompletely understood. Here, we compare two antigens presenting identical IgE-binding haptens but differing in geometry: the high-valency, heterogeneous DNP-BSA and the defined trivalent antigen DF3. We find that these ligands elicit distinct patterns of degranulation and Fc{varepsilon}RI {gamma}-chain phosphorylation, correlating with differences in the recruitment of the inhibitory lipid phosphatase SHIP1. Monte Carlo simulations predicted that each antigen generates receptor aggregates with distinct size, complexity, and inter-receptor spacing. Using direct stochastic optical reconstruction microscopy (dSTORM) and Bayesian Grouping of Localizations (BaGoL) analysis, we directly visualized the nanoscale aggregate geometry and found that DF3 induced smaller, more linear aggregates with tighter receptor spacing than DNP-BSA. Together, our results show that antigen properties, including size, valency, and epitope spacing, modulate Fc{varepsilon}RI aggregate architecture and tune the balance of positive and negative signaling to ultimately shape mast cell outcomes. Statement of SignificanceAllergic immune responses are initiated when multivalent antigens aggregate IgE-bound Fc{varepsilon}RI on mast cells, yet the relationship between antigen structure and signaling strength remains unclear. This study combines biochemical assays, Monte Carlo modeling, and super-resolution imaging to show that allergen properties, specifically valency and nanoscale epitope spacing, govern the geometry of Fc{varepsilon}RI aggregates and the balance of activating and inhibitory signals. These findings establish a direct mechanistic link between antigen structure and immune receptor output, providing new insight into how physical antigen features encode mast cell responses.

biophysics↗

bioRxiv2020

Characterization of the signalling modalities of prostaglandin E2 receptors EP2 and EP4 reveals crosstalk and a role for microtubules

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signalling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gs protein, EP4 also couples to the inhibitory Gi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signalling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signalling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signalling in myeloid cells. Considering that modulation of PGE2 signalling is regarded as an important therapeutic possibility in anti-tumour immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

immunology↗

bioRxiv2020

EGFR transactivates RON to drive oncogenic crosstalk

SUMMARYCrosstalk between disparate membrane receptors is thought to drive oncogenic signaling and allow for therapeutic resistance. EGFR and RON are members of two unique receptor tyrosine kinase (RTK) subfamilies that engage in crosstalk through unknown mechanisms. We combined high resolution imaging with biochemical studies and structural mutants to understand how EGFR and RON communicate. We found that EGF stimulation results in EGFR-dependent RON phosphorylation. Crosstalk is unidirectional, since MSP stimulation of RON does not trigger EGFR phosphorylation. Two-color single particle tracking captured the formation of complexes between RON and EGFR, supporting a role for direct interactions in propagating crosstalk. We further show that RON is a substrate for EGFR kinase, and transactivation of RON requires the formation of a signaling competent EGFR dimer. These results identify critical structural features of EGFR/RON crosstalk and provide new mechanistic insights into therapeutic resistance.

cancer biology↗

bioRxiv2018

Molecular aspects of multivalent engagement between Syk and FcϵRIγ

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as the tetrameric ({beta}{gamma}2) Fc{varepsilon}RI The simplest model assumes that Syk activation occurs through cis binding of its tandem SH2 domains to dual phosphotyrosines within immunoreceptor tyrosine-based activation motifs of individual {gamma} chains. In this model, Syk activity is modulated by phosphorylation occurring between adjacent Syk molecules docked on {gamma} homodimers and by Lyn molecules bound to Fc{varepsilon}RI{beta}. However, the mechanistic details of Syk docking on {gamma} homodimers are not fully resolved, particularly the possibility of trans binding orientations and the impact of Y130 autophosphorylation within Syk interdomain A. Analytical modeling shows that multivalent interactions lead to increased WT Syk cis-oriented binding by three orders of magnitude. Molecular dynamics (MD) simulations show increased inter-SH2 flexibility in a Y130E phosphomimetic form of Syk, associated with reduced overall helicity of interdomain A. Hybrid MD/worm-like chain polymer models show that the Y130E substitution reduces cis binding of Syk. We report computational models and estimates of relative binding for all possible cis and trans 2:2 Syk:Fc{varepsilon}RI{gamma} complexes. Calcium imaging experiments confirm model predictions that cis binding of WT Syk is strongly preferred for efficient signaling, while trans conformations trigger weak but measurable responses.

biophysics↗

bioRxiv2017

Sequential Super-Resolution Imaging using DNA Strand Displacement

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.

bioengineering↗

bioRxiv2017

Single Molecule Pull-down for Quantification of Membrane Receptor Multisite Phosphorylation

The Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1) plays an important role in both physiological and cancer-related processes. To study the factors that influence receptor phosphorylation, we have coupled Single Molecule Pull-down (SiMPull) measurements with rule-based modeling of EGFR signaling. Using SiMPull, we quantified the phosphorylation state of thousands of individual receptors. These measurements enabled the first direct detection of multisite phosphorylation on full-length EGFR and revealed that the extent of phosphorylation varies by tyrosine site and is dependent on the relative abundance of signaling partners that limit access by tyrosine phosphatases. We also evaluated the impact of oncogenic mutations and ligands with varying affinity on phosphorylation kinetics. Simulations highlight the importance of dimer lifetimes on EGFR phosphorylation and signaling output.

cell biology↗