Biology subjects

Thiam, H. R.

Publications and source records attributed to Thiam, H. R..

7 recordsLinked to original sources

bioRxiv2026

Nuclear remodeling and optimal migration emerge early, and constriction-passage progressively improves during hematopoietic stem cells to neutrophils differentiation

Neutrophils ability to rapidly and efficiently migrate through narrow pores in tissues is essential for host defense and is proposed to depend on their multilobulated and deformable nucleus. When during neutrophil differentiation does optimal migration and its proposed nuclear determinants emerge, and whether they are intrinsic to progenitors, is unclear in part because of the scarcity of tractable models of human neutrophils and their progenitors. Here, we optimized a CD34+ hematopoietic stem cell (HSC) to neutrophil differentiation pipeline to generate millions of mature neutrophils (HSC-neutrophils) that recapitulate the surface markers, proteome, ROS production and NETosis of primary human blood-derived neutrophils, better than the widely used HL60-derived neutrophils. Comparative proteomics across differentiation showed that HSC-neutrophils become translationally repressed while acquiring immune functions and actin-related processes. Quantitative microscopy and proteomics showed that nuclear multilobulation occurs at the granulocyte progenitors-early neutrophils transition and is accompanied by drastic remodeling of nuclear envelope composition (increasing LBR, decreasing lamin A/C, B1/B2 and NUPs). Single nuclear envelope proteins only weakly corelate with nuclear multilobularity suggesting that an ensemble envelope state, rather than any one protein, sets nuclear shape. Using microfabricated devices with constrictions, we show that migration speed increases the most in early neutrophils; that the capacity to cross nucleus-deforming pores is continuously enhanced during differentiation; and that early neutrophils recover the best from such migration. We show that while mature neutrophils most effectively cross pores, they remain impaired. Our work resolves three features of neutrophils migration - speed, deformation through constrictions, and recovery from deformation - and maps when each emerges, opening the door to future mechanistic and engineering studies for modulating neutrophil migration in tissue-like microenvironments.

cell biology↗

bioRxiv2026

Benchmarking three simple DNA staining-based image metrics for live-cell tracking of chromatin organization

Quantifying chromatin-state dynamics in living cells remains challenging, in part because most methods require fixation or cell lysis. Here, we benchmark and introduce three simple live-cell image-derived metrics computed from routine DNA staining - the coefficient of variation (CV), 1-Gini, and the Diffuse Signal Index (DSI), introduced here - as fixation-free readouts of chromatin organization. Using HL60-derived neutrophils (dHL-60) undergoing NETosis as a model system with a pronounced compact-to-decompact chromatin transition, we show that all three metrics track progressive chromatin reorganization in live-cell trajectories, with DSI providing the strongest trajectory-level discrimination between NETing and non-NETing cells. Comparison with Tn5-based chromatin accessibility measurements in fixed cells shows that all three metrics correlate with chromatin accessibility, supporting their biological interpretability. We further show that all three metrics track chromatin reorganization in a second, mechanistically distinct biological process, capturing mitotic chromatin compaction and post-mitotic chromatin decompaction in U2OS cells. Together, our results provide a practical framework for extracting chromatin-organization readouts from routine live-cell DNA staining and highlight that metric performance is task- and context-dependent. To make our metrics accessible to a broad audience, we built NucMetrics, an open-source ImageJ/Fiji macro toolset that computes all three metrics without requiring programming.

cell biology↗

bioRxiv2026

Investigation of Regulation and Binding Patterns of the Human Cathelicidin Peptide LL-37 in Complexation with Nucleic Acids, and its Impact on Neutrophil Extracellular Traps

The human cathelicidin host defense peptide LL-37 forms complexes with nucleic acids that can have either beneficial or detrimental health effects. We suggest that these differential impacts are directly connected to dsDNA binding by LL-37 and to complex formation between protomers. Here, we show using phage {lambda} DNA that LL-37 binds non-specifically to dsDNA, condensing it, followed by complex formation between LL-37 peptides. We find that complex formation is concentration-dependent, with low LL-37 amounts yielding loosely aggregated DNA structures, while higher LL-37 concentrations lead to well-defined, disc-like structures of about 150 nm in diameter. The condensation of the nucleic acids, which causes a loss of the characteristic B-DNA features, results from interactions of the phosphodiester backbone with protonated amino acid side chains of the peptide at physiological pH, predominantly in A-T rich sequences of the nucleic acid. However, in our studies, electrostatic interactions did not appear to be the driving force for complexation, but rather we found the -helical structure of the peptide with its amphipathic and hydrophobic surfaces to be essential. Further, we show that LL-37 also interacts with nucleic acids from neutrophil extracellular traps (NETs) in a concentration-dependent way, causing a reduction in NET aggregate area, which may offer new biophysical insights into diseases such as systemic lupus erythematosus (SLE), which involve slower-than-normal NET clearance. Our results indicate the key importance of LL-37 expression levels for regulation of the innate immune system for optimal human health, since the relative amounts of expressed LL-37 present to interact with extracellular DNA will determine the extent to which the DNA can be condensed, which in turn will affect the ability of the body to clear the NETs before they can cause inflammatory conditions.

biophysics↗

bioRxiv2025

Chromatin is a long-range force generator that regulates plasma membrane tension and cell integrity independently of gene expression

Primarily studied for its role in gene expression, chromatin organization is emerging as an important regulator of nuclear mechanics. Although the nucleus is in mechanical equilibrium with the cell, we do not know whether and how chromatin reorganization actively regulates the mechanical properties and downstream behaviors of cells. Here, we tested the hypothesis that as a dynamic crosslinked polymer, chromatin directly impacts cell mechanics independently of transcription by studying NETosis: a transcription-independent process where chromatin decompacts and the plasma membrane (PM) ruptures. Using high resolution microscopy and ATAC-see, we found that chromatin accessibility progressively increases during NETosis suggesting that chromatin binding proteins (CBPs) dissociate from chromatin during NETosis. To determine the identity and dynamics of these dissociated CBPs, we used fluorescent recovery after photobleaching to measure the mobility and localization of the linker histone H1, the nucleosomal histone H3 and the heterochromatin binding protein HP1. We found that the mobile fraction of nuclear H1 increases during NETosis while fractions of HP1 and H3 diffuse outside of the nucleus suggesting that they become cytosolic osmolytes and potentially alter the mechanical state of cells. Consistently, we found that plasma membrane tension and cell volume increase as chromatin decompacts during NETosis. In non-NETing U2OS cells, we found that inducing chromatin decompaction increases plasma membrane tension, independently of the cytoskeleton, indicating a causal relationship between chromatin organization, cell volume and plasma membrane tension. Our findings reveal a novel non- genetic role of chromatin in cellular biophysics: regulating cell volume, PM tension, and thus, overall cell mechanics. Considering the critical role of cell mechanics in biological processes such as cell migration, proliferation and pathogen killing, our work broadens our understanding of how chromatin regulates cell physiology and pathology. Significance StatementChromatin organizes our DNA inside the nucleus and is important for gene expression. However, chromatin is also a polymer which can passively regulate the rigidity of the nucleus, but whether and how chromatin can actively regulate the mechanical properties of the whole cell remains unknown. Here, we leverage the immune process of NETosis to show that the organization of chromatin inside the nucleus actively regulates the volume and tension of cells. Our work establishes chromatin as a long-range force generator in cells, broadening our understanding of the roles of this crucial polymer network in cells and opening the door to new strategies for controlling the mechanical properties of cells as needed by their physiology.

cell biology↗

bioRxiv2025

Autoantibody hotspots reveal origin and impact of immunogenic XIST ribonucleoprotein complex

Four out of five patients with autoimmune diseases are women. The XIST ribonucleoprotein (RNP) complex, comprising the female-specific long noncoding RNA XIST and over 100 associated proteins, may drive several autoimmune diseases that disproportionately affect women, who have elevated levels of autoantibodies against the XIST RNP. However, the structural distribution, potential origin, and clinical significance of XIST RNP autoantibodies remained unexplored. Here, we find that XIST RNP is associated with autoantigens associated with six female-biased autoimmune conditions. Mapping autoantibody targets to their occupancy sites on XIST shows that these autoantigens are concentrated at discrete "hotspots" along the XIST lncRNA, notably the A-repeat. Cell type-specific protein expression data nominated neutrophils as a predominant source of hotspot antigens, and we confirmed the presence of both XIST and hotspot antigens in neutrophil extracellular traps during NETosis, an immunogenic programmed cell death pathway triggered by neutrophil activation upon which neutrophils extrude their nuclear content. Furthermore, we found that levels of autoantibodies against a top hotspot antigen, SPEN, that binds the A-repeat, correlate with severe digital ischemia in systemic sclerosis in two independent cohorts. Together, these data show a plausible mechanism for the origin of AXA, guided by RNA structure and RNA-protein interactions, and show that antibodies to XIST RNP holds promise for disease endotyping and prognostication in female-biased autoimmune conditions. One Sentence SummaryNovel autoantibodies target hotspots on XIST ribonucleoprotein complex in female-biased autoimmune diseases.

immunology↗

bioRxiv2020

Calculation of the force field required for nucleus deformation during cell migration through constrictions

During cell migration in confinement, the nucleus has to deform for a cell to pass through small constrictions. Such nuclear deformations require significant forces. A direct experimental measure of the deformation force field is extremely challenging. However, experimental images of nuclear shape are relatively easy to obtain. Therefore, here we present a method to calculate predictions of the deformation force field based purely on analysis of experimental images of nuclei before and after deformation. Such an inverse calculation is technically non-trivial and relies on a mechanical model for the nucleus. Here we compare two simple continuum elastic models of a cell nucleus undergoing deformation. In the first, we treat the nucleus as a homogeneous elastic solid and, in the second, as an elastic shell. For each of these models we calculate the force field required to produce the deformation given by experimental images of nuclei in dendritic cells migrating in microchannels with constrictions of controlled dimensions [1]. These microfabricated channels provide a simplified confined environment mimicking that experienced by cells in tissues. We extract the nuclear shape from the boundary of the fluorescently stained region in each consecutive image over time. From this we calculate the deformation field between images and use our elastic models to calculate the traction force field. Our calculations therefore predict the forces felt by a deforming nucleus as a migrating cell encounters a constriction. Since a direct experimental measure of the deformation force field is very challenging and has not yet been achieved, our numerical approaches can make important predictions motivating further experiments, even though all the parameters are not yet available. In addition, the algorithm we have developed could be adapted to analyse experimental images of deformation in other situations. Author summaryMany cell types are able to migrate and squeeze through constrictions that are narrower than the cells resting radius. For example, both immune cells and metastatic cancer cells change their shape to migrate through small holes in the complex tissue media they move in. During migration the cell nucleus is more difficult to deform than the cell cytoplasm and therefore significant forces are required for a cell to pass through spaces that are smaller than the resting size of the nucleus. Experimental measurements of these forces are extremely challenging but experimental images of nuclear deformation are regularly obtained in many labs. Therefore we present a computational method to analyse experimental images of nuclear deformation to deduce the forces required to produce such deformations. A mechanical model of the nucleus is necessary for this analysis and here we present two different models. The first treats the nucleus as a homogeneous elastic solid and the second treats the nucleus as an elastic shell. Our computational tool enables us to obtain detailed information about forces causing deformation from microscopy images.

biophysics↗

bioRxiv2019

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture

Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high resolution time-lapse microscopy of mouse and human neutrophils (PMN) and differentiated HL-60 neutrophil-like cells (dHL-60) labelled with fluorescent markers of organelles and stimulated with ionomycin or lipopolysaccharides to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin de-condensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin de-condensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a step-wise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.\n\nSignificance StatementNeutrophils are white blood cells specialized as the first line of host defense in the immune system. One way they protect organisms is through NETosis, in which they expel their DNA to form a web-like trap that ensnares pathogens and promotes clotting. However, NETs also mediate sterile inflammation, causing damage to the body. We used high-resolution live-cell microscopy to perform the first systematic characterization of the timing of dynamic cellular events leading to NETosis in human and mouse neutrophils and a neutrophil-like cell line. We discovered that NETosis proceeds by a step-wise sequence of cellular events that is conserved across species, and requires the activity of the PAD4 enzyme for DNA to be released from the nucleus and cell membrane.

immunology↗