Biology subjects

Manzo, C.

Publications and source records attributed to Manzo, C..

9 recordsLinked to original sources

bioRxiv2026

ExoFILT: Transfer learning for robust and accelerated analysis of exocytosis single-particle tracking data

MotivationUnderstanding constitutive exocytosis at the molecular level requires quantitative characterization of protein dynamics during the process. Single-particle tracking allows the measurement of protein dynamics in living cells. However, identifying bona fide exocytic events requires extensive manual annotation, limiting throughput and introducing personal biases that affect reproducibility. ResultsWe present ExoFILT, a deep learning-based classifier designed to identify exocytic events in single-particle tracking data, using the exocyst complex as a reference. Trained via transfer learning on simulated and experimental data, ExoFILT reduces the time required for manual annotation by ten-fold while improving measurement consistency across researchers. When applied to simultaneous dual-color time-lapse movies, ExoFILT enabled the systematic quantification of temporal relationships between exocytic proteins. The increased throughput uncovered distinct subpopulations of exocytic events with differential molecular composition (e.g., events with and without detectable levels of Sec1), underscoring the potential of ExoFILT to reveal mechanistic insights into exocytosis.

bioinformatics↗

bioRxiv2025

Continuum architecture dynamics of vesicle tethering in exocytosis

Essential for eukaryotic organisms, multiple copies of the exocyst complex tether each secretory vesicle to the plasma membrane (PM) in constitutive exocytosis. The exocyst higher-order structure (ExHOS) that coordinates the action of these multiple exocysts remains unexplored. We integrated particle-tracking, super-resolution microscopy and cryo-electron tomography in a model that time-resolves the continuum conformational landscape of the ExHOS and functionally annotates its different conformations. We found that 7 exocysts form flexible ring-shaped ExHOS that tether vesicles at <45 nm from the PM. The ExHOS, initially 19 nm in radius, rapidly expands while it pulls the vesicle towards the PM. Subsequently, the ExHOS stabilizes, securing the vesicle at [~]4 nm from the PM. After fusion, Sec18 mediates the ExHOS disassembly when its radius is 38 nm. By resolving the fundamental biophysical principles of tethering we bridged the gap between static isolated structures and the dynamic and multimeric nature of exocytosis.

cell biology↗

bioRxiv2024

Learning Continuous 2D Diffusion Maps from Particle Trajectories without Data Binning

Diffusion coefficients often vary across regions, such as cellular membranes, and quantifying their variation can provide valuable insight into local membrane properties such as composition and stiffness. Toward quantifying diffusion coefficient spatial maps and uncertainties from particle tracks, we use a Bayesian method and place Gaussian Process (GP) Priors on the maps. For the sake of computational efficiency, we leverage inducing point methods on GPs arising from the mathematical structure of the data giving rise to non-conjugate likelihood-prior pairs. We analyze both synthetic data, where ground truth is known, as well as data drawn from live-cell singlemolecule imaging of membrane proteins. The resulting tool provides an unsupervised method to rigorously map diffusion coefficients continuously across membranes without data binning.

biophysics↗

bioRxiv2023

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

Epithelial Intercellular Adhesion Molecule (ICAM)-1 is apically polarized, interacts with and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures (BCs). We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

cell biology↗

bioRxiv2022

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DC) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

cell biology↗

bioRxiv2020

Shear flow-driven actin re-organization induces ICAM-1 nanoclustering on endothelial cells that impact T-cell migration

The leukocyte specific {beta}2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial cells (ECs), are involved in the arrest, adhesion and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1, has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear-flow induces a global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear-forces promoted ICAM-1 nanoclustering prior to LFA-1 engagement. This spatial nanoscale organization was driven by actin cytoskeleton re-arrangements induced by shear-force. We further assessed the impact of prolonged shear-stress EC stimulation on T cell migration. T cells adhered to mechanically pre-stimulated ECs developed a more pro-migratory phenotype, migrated faster and exhibited shorter EC interactions than when adhered to non-mechanically stimulated ECs. Together, our results indicate that shear-forces increase the number of ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening adhesion and thereby reducing actin retrograde flow of T-cells, leading to their increased migration speed. Our data also underscores the importance of mechanical forces regulating the spatial organization of cell membrane receptors and their contribution to adhesion regulation, regardless of integrin activation. Summary statementWe show that shear forces promote ICAM-1 spatial re-arrangement and actin-dependent nanoclustering on ECs prior to integrin engagement. This mechanism might be important for firm leukocyte adhesion and migration during the immune response.

cell biology↗

bioRxiv2020

Membrane receptor MerTK is a newly identified transcriptional regulator that associates to chromatin as nanoclusters during human DC differentiation

MerTK is a transmembrane receptor tyrosine kinase (RTK) mainly expressed in dendritic cells (DCs) and macrophages where it plays an important role in immunotolerance, but also in activating oncogenic signalling pathways. Albeit MerTK is exploited as clinical target in cancer and auto-immune disorders, the mechanisms that regulate its diverse functions are poorly understood. Here, we identified a remarkably high pool of the full receptor in the nucleus of human DCs. Nuclear translocation was ligand-dependent. Importantly, MerTK nuclear levels correlated to DC differentiation and were spatiotemporally regulated by the transmembrane receptor LRP-1. Using dual-colour super-resolution nanoscopy we discovered that nuclear MerTK forms nanoclusters, whose strength strongly depends on chromatin accessibility during DC differentiation. We finally revealed high transcription capacity of MerTK. Overall, our work indicates that nuclear MerTK acts as a transcription factor regulating DC differentiation, thus implicating for the first time a physiological function for RTK nuclear translocation in immunity.

immunology↗

bioRxiv2019

Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern.

cell biology↗

bioRxiv2018

Quantifying motor protein copy number in super-resolution using an imaging invariant calibration

Motor proteins are nanoscale machines that convert the energy of ATP hydrolysis into the mechanical motion of walking along cytoskeletal filaments. In doing so, they transport organelles and help maintain sub-cellular organization. We previously developed a DNA origami-based calibration approach to extract protein copy number from super-resolution images. Using this approach, we show here that the retrograde motor protein dynein is mostly present as a single motor in the cytosol, whereas a small population of dynein along the microtubule cytoskeleton forms higher-order multimers organized into nano-sized domains. We further demonstrate, using dynein as a test sample, that the DNA origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any GFP-fused protein of interest. Our results have implications for motor coordination during intracellular trafficking as well as for using super-resolution as a quantitative method to determine protein copy number at the nanoscale level.

biophysics↗