Biology subjects

Manzo, C.

Publications and source records attributed to Manzo, C..

4 recordsLinked to original sources

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↗