Biology subjects

Fritzsche, M.

Publications and source records attributed to Fritzsche, M..

8 recordsLinked to original sources

bioRxiv2025

Isolation of functional supramolecular attack particles (SMAPs)

High density cultures of the GMP compatible NK-92 natural killer cell line release heterogeneous extracellular particles (EP), notably extracellular vesicles (EV) and non-vesicular extracellular particles (NVEP). The NVEP include a small proportion of supramolecular attack particles (SMAPs), the direct cytotoxic potential of which suggests therapeutic promise, yet scalable enrichment and functional evaluation alongside other extracellular particles (EPs) have been lacking. Here, we develop a high-resolution serial size-exclusion liquid chromatography (SELC) workflow that resolves EPs into 4 fractions F1-F4, in which EV are enriched in F2 and SMAPs are enriched in F3. Multi-modal characterization using Nanoparticle Tracking Analysis (NTA), Nano Flow Cytometry (Nano FCM), Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRFM) and Proteomics) demonstrated that F2 NK-92 EVs are [~]130-150 nm particles enriched for complement proteins, whereas SMAPs (F3) are 100-110 nm particles enriched for cytotoxic proteins (PRF1, GZMB) and SMAP shell components (e.g., THBS1, THBS4). Functionally, Ca2+-stabilized SMAPs (F3) trigger caspase-3-dependent apoptosis in tumor cell lines with robust dose responses, while F2 lacks direct cytotoxicity in vitro. Ex vivo, F3 SMAPs kill patient-derived breast cancer and chronic lymphocytic leukaemia cells (CLL) in a concentration-dependent manner. In NSG mice, intra-tumoral treatment with SMAPs (F3) restrains the growth of aggressive B16F10 melanoma and PANC-1 pancreatic cancer, confirming in vivo functionality. These results establish a scalable method to isolate and compare NK-derived particle classes and provide a foundation for targeted SMAP engineering as a promising approach for treatment of solid tumours. Significance StatementCell therapies face barriers in solid tumours, including lack of entry and suppression. NK cells release extracellular particles (EPs), including supramolecular attack particles (SMAPs), that can traffic into tumour tissue. We define a practical, scalable chromatography process that resolves NK-derived SMAPs from other EPs and demonstrate that SMAPs induce caspase mediated apoptosis in vitro and engage in cytotoxic lymphocyte independent anti-tumour activity in vivo. These insights open a route to engineering and testing SMAP-based therapeutics to overcome limitations of whole-cell approaches in solid cancers.

immunology↗

bioRxiv2025

Actin waves guide an outward movement of microclusters in the lymphocyte immunological synapse

The lymphocyte immune response begins with antigen recognition on antigen-presenting cells, leading to the formation of the immunological synapse--a specialized interface for biochemical and biophysical exchange. At the synapse, most antigen-engaged receptor microclusters move inward toward the central supramolecular activation cluster (cSMAC) via retrograde F-actin flow, eventually clearing from the cell surface. This retrograde movement and receptor downregulation maintain antigen receptor homeostasis, critical for adaptive immunity, though its regulation remains unclear. Using live T cells, we identified a significant pool of antigen-engaged microclusters moving anterogradely toward the cell periphery, rather than the cSMAC. This movement was driven by actin waves propagating outward and coupling to microclusters through the Wiskott-Aldrich Syndrome Protein. These findings reveal a previously unrecognized mode of actin dynamics--anterograde actin waves--that co-exist with retrograde flow and direct microclusters away from the downregulation zone. This dual actin behavior underscores the complex cytoskeletal mechanisms T cells employ to regulate receptor distribution and maintain signaling homeostasis during immune activation.

immunology↗

bioRxiv2023

Single-cell phenomics reveals behavioural and mechanical heterogeneities underpinning collective migration during mouse anterior patterning

Distal Visceral Endoderm (DVE) cells show a stereotypic unidirectional migration essential for correct orientation of the anterior-posterior axis. They migrate within a simple epithelium, the Visceral Endoderm (VE). It is unknown how DVE cells negotiate their way amongst the surrounding VE cells, what determines the bounds of DVE migration within the VE, and the relative contributions of different cell behaviours to this migration. To address these questions, we used lightsheet microscopy to generate a multi-embryo, singlecell resolution, longitudinal dataset of cell behaviour and morphology. We developed a machine learning based pipeline to segment cells and a data-informed systematic computational framework to extract and compare select morphological, behavioural and molecular parameters of all VE cells in a unified coordinate space. Unbiased clustering of this single-cell phenomic dataset reveals considerable patterned phenotypic heterogeneity within the VE and a previously unknown sub-grouping within the DVE. While migrating, DVE cells retain regular morphology, do not exchange neighbours and are crowded, all hallmarks of the jammed state. In contrast, VE cells immediately ahead of them deform and undergo neighbour exchange. We show that DVE cells are characterised by higher levels of apical F-actin and elevated tension relative to the VE cells immediately ahead of them through which they migrate, but stop migrating upon reaching a region of the VE with matching elevated tension. Lefty1 mutants, known to show abnormal over-migration of DVE cells, show disruption to this patterned tension in the VE. Our findings provide novel insights into the control of cell behaviour during the remodelling of curved epithelia, indicating that the collective migration of sub-sets of cells can be circumscribed by modulating the mechanical properties of surrounding cells and that migrating cells in this context remain as a jammed solid flock, with surrounding cells facilitating their movement by becoming unjammed. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/534937v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@10a7153org.highwire.dtl.DTLVardef@963a1forg.highwire.dtl.DTLVardef@c1fcd0org.highwire.dtl.DTLVardef@1bcdded_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

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↗

bioRxiv2021

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

bioRxiv2019

Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells

Semaphorin-3A (Sema3A) regulates tumor angiogenesis, but its role in modulating anti-tumor immunity is unclear. We demonstrate that Sema3A secreted within the tumor microenvironment (TME) suppresses tumor-specific CD8+ T cell function via Neuropilin-1 (NRP1), a receptor that is upregulated upon activation with T cells cognate antigen. Sema3A inhibits T cell migration, assembly of the immunological synapse, and tumor killing. It achieves these functional effects through hyper-activating the acto-myosin system in T cells leading to cellular paralysis. Finally, using a clear cell renal cell carcinoma patient cohort, we demonstrate that human tumor-specific CD8+ T cells express NRP1 and are trapped in Sema3A rich regions of tumors. Our study establishes Sema3A as a potent inhibitor of anti-tumor immunity.

immunology↗

bioRxiv2019

Missing-In-Metastasis / Metastasis Suppressor 1 modulates B cell receptor signaling, B cell metabolic potential and T cell-independent immune responses.

Efficient generation of antibodies by B cells is one of the prerequisites of protective immunity. B cell activation by cognate antigens via B cell receptors (BCR), or pathogen-associated molecules through pattern-recognition receptors, such as Toll-like receptors (TLRs), leads to transcriptional and metabolic changes that ultimately transform B cells into antibody producing plasma cells or memory cells. BCR signaling and a number of steps downstream of it rely on coordinated action of cellular membranes and the actin cytoskeleton, tightly controlled by concerted action of multiple regulatory proteins, some of them exclusive to B cells. Here, we dissect the role of Missing-In-Metastasis (MIM), or Metastasis suppressor 1 (MTSS1), a cancer-associated membrane and actin cytoskeleton regulating protein, in B cell-mediated immunity by taking advantage of MIM knockout mouse strain. We show undisturbed B cell development and largely normal composition of B cell compartments in the periphery. Interestingly, we found that MIM-/- B cells are defected in BCR signaling in response to surface-bound antigens but, on the other hand, show increased metabolic activity after stimulation with LPS or CpG. In vivo, MIM knockout animals exhibit impaired IgM antibody responses to immunization with T cell-independent antigen. This study provides the first comprehensive characterization of MIM in B cells, demonstrates its regulatory role for B cell-mediated immunity, as well as proposes new functions for MIM in tuning receptor signaling and cellular metabolism, processes which may also contribute to the poorly understood functions of MIM in cancer.

immunology↗

bioRxiv2018

Precise tuning of gene expression output levels in mammalian cells

Precise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. Here we report on a method for the precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. To demonstrate the value of this technology, we used a panel of miSFITs to tune the expression of a peptide antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression output levels with applications in research and cellular engineering.

synthetic biology↗