Biology subjects

Simoncelli, S.

Publications and source records attributed to Simoncelli, S..

9 recordsLinked to original sources

bioRxiv2026

Gag proteolysis drives HIV-1 envelope conformational closure through the gp41 cytoplasmic tail

HIV-1 must present a fusion-competent envelope glycoprotein (Env) to enter host cells while concealing the very conformation targeted by broadly neutralising antibodies (bnAbs). How the virus resolves this conflict has remained unclear. Using a minimally invasive nanobody-labelling strategy targeting Env's V4 loop, we combine single-virus FRET, fluorescence lifetime cross-correlation spectroscopy, and DNA-PAINT super-resolution microscopy to resolve Env conformation on individual, intact virions from the primary isolate JR-FL. We show that immature virions predominantly display an open Env conformation, while Gag maturation drives acquisition of the tightly closed, fusion-competent State 1 that harbours the principal bnAb epitope. Truncating the Env cytoplasmic tail abolishes this regulation, identifying the gp41 tail as the conduit linking Gag lattice maturation to Env's conformational fate. We further show that bnAb b12 preferentially binds open Env on immature virions, supporting a decoy mechanism that shields mature virions from immune recognition, with direct implications for HIV-1 vaccine design.

biophysics↗

bioRxiv2025

Ephrin inhibition disrupts stromal-cancer crosstalk and reduces metastasis in pancreatic cancer

Cancer associated fibroblasts (CAFs) are critical drivers of disease progression and metastasis within the pancreatic tumour microenvironment. Using a 3D spheroid model of CAF-led invasion, we identified complementary expression of ephrin family receptors (EPHB2) and ligands (EPHRINB2) between cancer cells and CAFs, implicating this bidirectional signalling family in tumour progression. Through pancreatic stellate cell-derived CAFs isolated from genetically modified mouse models, where EphrinB was either lacking or modified by a gain-of-function mutation, we identified both forward and reverse signalling to be required for invasion. In a syngeneic murine orthotopic model of pancreatic cancer, we show that first-in-class tetramerisation inhibitors of Eph/Ephrin interactions can reduce local invasion in primary tumours, and significantly decrease metastatic tumour spread across multiple organ sites. Our data highlight Ephrin signalling as a critical nexus for CAF-cancer crosstalk and establish a foundation for the clinical development of targeted EPH-EPHRIN inhibitors to counter metastatic invasion.

cancer biology↗

bioRxiv2025

Early HIV-1 maturation drives Env clustering and fusion competence

Human immunodeficiency virus type 1 (HIV-1) particles are initially released from the cell in an immature state that cannot cause infection. The proteolytic processing of the Gag structural polyprotein into its individual components triggers a dramatic structural reorganisation that produces fully mature and infectious virions. HIV-1 maturation remodels the Gag lattice to enable membrane fusion, but how early proteolytic steps regulate the envelope glycoprotein (Env) organisation is unknown. Although cryo-electron tomography has defined structural intermediates of maturation, it has not resolved how Env conformers relate to specific early maturation states. Here, we show that partial Gag cleavage, prior to capsid formation, allosterically reorganises the membrane-proximal lattice, promoting Env clustering and fusion-competent conformations. Using native virus preparations combined with cryogenic fluorescence-lifetime imaging correlated to cryo-electron tomography, we map Env conformational signatures onto defined Gag-processing intermediates. This reveals an unrecognised early maturation stage in which incomplete Gag processing primes Env for entry, shifting current models by positioning functional Env activation upstream of core maturation. These results establish that early lattice remodelling is a key determinant of HIV-1 fusion competence and expose a previously inaccessible, intervention-sensitive step with implications for therapeutic and vaccine strategies targeting maturation-entry coupling.

microbiology↗

bioRxiv2025

PTPN22 Regulates T Cell Synapse Formation through PSTPIP1-Dependent Actin Remodeling

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a critical regulator of T cell signaling, working in concert with C-terminal SRC kinase (Csk) to dephosphorylate key signaling proteins and suppress lymphocyte activation. The R620W variant of PTPN22, one of the most prevalent mutations associated with autoimmune diseases, has been implicated in altered T cell responses, although its broader effects on T cell activation are not fully understood. Recent studies have uncovered a novel interaction between PTPN22 and proline-serine-threonine phosphatase interacting protein 1 (PSTPIP-1), a cytoskeletal adaptor protein involved in F-actin remodeling. PSTPIP-1 recruits Wiskott-Aldrich Syndrome Protein (WASp) to facilitate actin foci formation, a process integral to stabilizing TCR microclusters and amplifying downstream signaling. Given that mutations in PSTPIP-1 impair actin remodeling and localize within the PTPN22 binding domain, we hypothesized that PTPN22 modulates actin dynamics through its interaction with PSTPIP-1. Using live and fixed multi-color super-resolution fluorescence imaging, we demonstrate that PTPN22 deficiency or inhibition of its phosphatase activity leads to aberrant Arp2/3-dependent actin remodeling and exaggerated calcium signaling, particularly under low- affinity TCR stimulation. Single-protein resolution imaging via DNA-PAINT further revealed disrupted nanoscale clustering of PSTPIP-1 and TCR in PTPN22-deficient T cells, uncovering a previously unrecognized PSTPIP-1-TCR interaction in the absence of PTPN22. These findings highlight a novel PTPN22-PSTPIP-1 signaling axis, offering new insights into the molecular mechanisms that may contribute to autoimmune disease susceptibility. One Sentence SummaryPTPN22 and PSTPIP-1 control actin remodeling upon T cell synapse formation, modulating TCR clustering and activation.

immunology↗

bioRxiv2025

Alpha-BET: Functional labeling of envelope glycoproteins with single domain antibodies for in-virus single molecule imaging

We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFA-tag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and NiV G, Alpha-BET preserves structural integrity and function. For HIV-1 Env, we demonstrate super-resolution DNA-PAINT MINFLUX 3D imaging enabled by tag insertion, showcasing its power for visualizing native trimers in single virions and potential for broader applications in virus research.

biophysics↗

bioRxiv2024

Super-Resolution Simplified: Sub-10nm Imaging Over Large Areas and Deep Penetration via SDC-OPR and DNA-PAINT

Single Molecule Localization Microscopy (SMLM) has traditionally faced challenges to optimize signal-to-noise ratio, penetration depth, field-of-view (FOV), and spatial resolution simultaneously. Here, we show that DNA-PAINT imaging on a Spinning Disk Confocal with Optical Photon Reassignment (SDC-OPR) system overcomes these trade-offs, enabling high-resolution imaging across multiple cellular layers and large FOVs. We demonstrate the systems capability with DNA origami constructs and biological samples, including nuclear pore complexes, mitochondria, and microtubules, achieving a spatial resolution of 6 nm in the basal plane and sub-10 nm localization precision at depths of 9 {micro}m within a 53 x 53 {micro}m{superscript 2} FOV. Additionally, imaging of the developing Drosophila eye epithelium at depths up to 9 {micro}m with sub-13 nm average localization precision, reveals distinct E-cadherin populations in adherens junctions. Quantitative analysis of Collagen IV deposition in this epithelium indicated an average of 46 {+/-} 27 molecules per secretory vesicle. These results underscore the versatility of DNA-PAINT on an SDC-OPR for advancing super-resolution imaging in complex biological systems.

biophysics↗

bioRxiv2022

Extracellular ATP drives pancreatic cancer cell invasion via purinergic receptor-integrin interactions

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet clinical need. Given the elevated ATP levels seen in PDAC, the purinergic axis represents an attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, it plays essential roles in fibrosis, inflammation response and immune function. We have analysed the PDAC purinome using publicly available databases to discern which members may impact patient survival. We identified P2RY2 to be the purinergic gene with the strongest association to hypoxia, the highest cancer cell specific expression and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y2 to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification or pharmacological strategies we identify the mechanism of this ATP-driven invasion to require direct protein-protein interactions between P2Y2 and V integrins. Using DNA-PAINT super-resolution fluorescence microscopy, we found that P2Y2 regulates the amount and distribution of integrin V in the plasma membrane. This work highlights a novel GPCR-integrin interaction in cancer invasion and its potential for therapeutic targeting.

cancer biology↗

bioRxiv2019

Multi-colour DNA-qPAINT reveals how Csk nano-clusters regulate T-cell receptor signalling

Phosphorylation of the negative regulatory element of the tyrosine kinase Lck by Csk down-modulates T-cell receptor induced signalling. Being constitutively active, Csk spatial organization is responsible for regulating this signalling interaction. Here, we used stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-qPAINT) to image the nanoscale spatial architecture of Csk and two binding partners implicated in its membrane association - PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we provide a powerful resource for dissecting signalling pathways regulated by spatio-temporal organisation. We found that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and re-recruited at later time points. Unexpectedly, these clusters do not directly co-localise with PAG at the membrane, but instead provide a ready pool of monomers to down-regulate signalling. By generating CRISPR/Cas9 knock-out T-cells, our data also identify that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population.

immunology↗

bioRxiv2019

Three-dimensional total internal reflection fluorescence nanoscopy with sub-10 nm resolution

Single-molecule localization microscopy (SMLM) enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single molecules image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present SIMPLER, a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence (TIRF) microscope. SIMPLER requires no hardware modification whatsoever to a conventional TIRF microscope, and complements any 2D SMLM method to deliver 3D images with nearly isotropic nanometric resolution. Examples of the performance of SIMPLER include the visualization of the nuclear pore complex through dSTORM with sub-20 nm resolution and of microtubule cross-sections resolved with sub-10 nm through DNA-PAINT.

biophysics↗