Biology subjects

Mandal, T.

Publications and source records attributed to Mandal, T..

14 recordsLinked to original sources

bioRxiv2025

Fluorescence lifetime estimation: a practical approach using Flipper-TR FLIM

Flipper-TR is a membrane dye sensitive to lipid packing widely used to probe membrane tension in live cells via fluorescence lifetime imaging microscopy (FLIM). However, no consensus currently exists on the optimal strategy for extracting lifetime values, particularly across varying experimental setups and biological systems. Here, we systematically compare multiple approaches to estimate Flipper-TR lifetime, including multi-exponential reconvolution fitting, tail fitting, mean photon arrival time (first moment), and phasor analysis. These estimators are tested against changes in photon budget, sample characteristics, microscope manufacturer, and laser frequency. This offers a comprehensive benchmark and decision-making framework for quantitative FLIM analysis of Flipper dyes in various contexts.

biophysics↗

bioRxiv2025

Spatio-temporal regulation of ligand trafficking and TLR9 activation involves PIEZO1 mechanosensing in human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are specialized innate immune cells which play a pivotal role in antiviral immunity by producing large quantities of type I interferons (IFNs) upon sensing nucleic acids via Toll-like receptor 9 (TLR9). Synthetic oligodeoxynucleotides (ODNs) such as CpGA and CpGB, both containing unmethylated CpG motifs, are commonly used experimental TLR9 agonists. Interestingly, CpGA and CpGB elicit markedly different responses in pDCs - CpGA induces robust IFN- production, whereas CpGB does not. The mechanistic basis underlying this ligand-specific functional divergence has remained unclear. Here, we identify PIEZO1, a mechanosensory ion channel, as a critical determinant of ligand-specific IFN responses in human primary pDCs. We demonstrate that unlike CpGB, CpGA self-associates into larger aggregates that generate membrane tension during cellular uptake, leading to activation of PIEZO1. This activation triggers localized calcium influx and cytoskeletal remodeling, resulting in the formation of local F-actin structures that retain CpGA within early endosomes enabling sustained IRF7 nuclear translocation and robust type I IFN production. Disruption of PIEZO1 or actin polymerization abrogates CpGA-induced IFN production, while pharmacological activation of Piezo1 enhances IFN production in response to CpGB. Thus, these findings uncover a previously unrecognized biophysical checkpoint in nucleic acid sensing, wherein membrane tension is transduced via PIEZO1 into spatially controlled TLR9 signaling. Overall, our study establishes PIEZO1 mechanosensing at the plasma membrane as a key regulatory event in nucleic acid-induced immunity, with different functional outcomes based on the cargo structure, opening potential new avenues for modulating type I IFN responses in infection and autoimmunity.

immunology↗

bioRxiv2025

Anionic Phospholipids Modulate Hepatitis C Virus Core Protein Assembly on Lipid Membranes

Hepatitis C virus (HCV) genome codes for various proteins essential to its replication cycle. Among these, the core protein (HCC) forms the capsid via organised multimerization, thereby guiding viral assembly. This process depends on the dynamic localisation of HCC between the endoplasmic reticulum bilayer membrane and the lipid droplet monolayer. While past studies have examined the role of some HCC structural properties and other viral and host proteins in the assembly process, the contribution of lipid molecules was not thoroughly investigated yet. Since specific lipids are upregulated during HCV infection, it is reasonable that these molecules might play an important role for virus fitness and infection progression. In this study, we have addressed this topic and, specifically, how HCC-lipid interactions might impact HCC-HCC interactions. Using in vitro models and quantitative fluorescence microscopy techniques, we investigated HCC binding affinity to lipid membranes, multimerization and lateral diffusivity. Our results reveal the effect of HCC interactions with anionic phospholipids (PLs) on the binding to monolayers and bilayers. Furthermore, our analysis shows that PI(4)P enhances HCC multimerization and, therefore, might drive lateral assembly for capsid formation. These results not only provide insights into the lipid-driven regulation of HCV assembly and identify anionic PLs as potential modulators of the viral replications cycle, but also highlight a previously unrecognised role of PL composition in HCC-membrane interaction. SignificanceHepatitis C virus (HCV) remains a major global health challenge and is associated with substantial mortality. In certain regions, HCV infection reaches epidemic proportions. Despite its prevalence, targeted and cost-effective therapeutic options remain limited, largely due to an incomplete understanding of the viral life cycle. A critical - yet poorly characterized - step in this cycle is the assembly of the HCV core (HCC) protein into the viral capsid, which is essential for replication. Here, we use quantitative fluorescence microscopy to investigate the role of specific lipid species in HCC assembly on model membranes. Our findings show that distinct lipid environments modulate HCC assembly and that HCC, in turn, induces measurable changes in the biophysical properties of the host membrane.

biophysics↗

bioRxiv2024

Challenges in the Accurate Modelling of Lipid Dynamics in Monolayers and Bilayers

Recent advances in hydrodynamic theory have revealed the severe effect of periodic boundary conditions (PBCs) on the diffusive dynamics of lipid membranes in molecular dynamics simulations. Even when accounting for PBC effects, the corrected lipid diffusion coefficients often severely overshoot the experimental estimates. Here, we investigate the underlying reasons for the exaggerated dynamics, and suggest potential ways for improvement. To this end, we examine the diffusion of four lipid types in both bilayers and monolayers using the CHARMM36 force field. We account for PBC effects using the full hydrodynamic treatment: for bilayers we use non-equilibrium simulations to extract the interleaflet friction parameter used in the correction; whereas monolayer hydrodynamics are treated by setting this parameter to zero. Our results suggest that the dynamics of bilayers are too fast, even if interleaflet friction is accounted for. However, the change of the water model to OPC leads to an excellent agreement with experiments. For monolayers, the dynamics with the TIP3P water model agree well with experiments, whereas they are undershot with OPC. As OPC and TIP3P differ in both shear viscosity and surface tension, we develop two new mass-scaled water models to clarify the roles of the thermodynamic and kinetic properties of the water model on lipid dynamics. Our results indicate that both of these quantities play a major role in lipid dynamics. Moreover, it seems that the accurate description of diffusion in both lipid bilayers and monolayers cannot be accounted for by changes in the water model alone, but likely also requires modifications in the lipid model.

biophysics↗

bioRxiv2024

Dynamins combine mechano-constriction and membrane remodeling to enable two-step mitochondrial fission via a snap-through instability.

Mitochondrial fission is controlled by dynamin proteins, the dysregulation of which is correlated with diverse diseases. Fission dynamins are GTP hydrolysis-driven mechanoenzymes that self-oligomerize into helical structures that constrict membrane to achieve fission, but details are not well understood. However, dynamins can also remodel membranes by inducing negative Gaussian curvature, the type of curvature required for completion of fission. Here, we examine how these drastically different mechanisms synergistically exert their influences on a membrane, via a mechanical model calibrated with small-angle X-ray scattering structural data. We find that free dynamin can trigger a "snap-through instability" that enforces a shape transition from an oligomer-confined cylindrical membrane to a drastically narrower catenoid-shaped neck within the spontaneous hemi-fission regime, in a manner that depends critically on the length of the confined tube. These results indicate how the combination of dynamin assembly, and paradoxically disassembly, can lead to diverse pathways to scission. TeaserDynamin mechano-constriction by assembly and curvature-driven instability by free monomers synergistically drive mitochondrial fission.

biophysics↗

bioRxiv2024

Transcriptional noise sets fundamental limits to spatially resolved cell-state decoding of the circadian clock.

The rhythmic circadian clock generates temporal variation of critical physiological processes in tissues, making accurate measurement of clock phase or tissue time a fundamentally important problem. Recent advances in single-sample time inference provides a potentially powerful alternative to traditional time-series based approaches. Single-sample techniques typically leverage population-level RNA rhythms, but the feasibility of single-cell phase detection remains an open question. Combining multiplexed smFISH to simultaneously measure up to 6 mouse fibroblast genes, with a novel inference algorithm using Gaussian Processes, here we demonstrate that even when technical drop-outs are minimized, transcriptional noise in core-clock genes precludes single-cell phase inference. Simulations predict that above 50 clock-like genes would make single-cell phase inference possible. Remarkably however, just 3 core-clock genes are sufficient if RNA levels of [~] 70 cells are averaged. Further, we demonstrate how averaging allows detecting spatially-resolved, heterogeneous clock phases in desynchronized cells. Our work provides a conceptual framework for achieving high-resolution phase detection with a minimal set of core-clock genes, with implications for probing the origins of clock dysfunction, otherwise unresolvable using population-based approaches.

cell biology↗

bioRxiv2024

A comparison of lipid diffusive dynamics in monolayers and bilayers in the context of interleaflet coupling

Cellular membranes are composed of lipids typically organized in a double-leaflet structure. Interactions between these two leaflets - often referred to as interleaflet coupling - play a crucial role in various cellular processes. Despite extensive study, the mechanisms governing such interactions remain incompletely understood. Here, we investigate the effects of interleaflet coupling from a specific point of view, i.e. by comparing diffusive dynamics in bilayers and monolayers, focusing on potential lipid-specific interactions between opposing leaflets. Through quantitative fluorescence microscopy techniques, we characterize lipid diffusion and mean molecular area in monolayers and bilayers composed of different lipids. Our results suggest that the observed decrease in bilayer lipid diffusion compared to monolayers depends on lipid identity. Furthermore, our analysis suggests that lipid acyl chain structure and spatial configuration at the bilayer may strongly influence interleaflet interactions and dynamics in bilayers. These findings provide insights into the role of lipid structure in mediating interleaflet coupling and underscore the need for further experimental investigations to elucidate the underlying mechanisms.

biophysics↗

bioRxiv2024

Comparing multifunctional viral and eukaryotic proteins for generating scission necks in membranes

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is the M2 viroporin, a proton pump from the influenza A virus which is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging Small-Angle X-ray Scattering (SAXS) structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemi-fission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.

biophysics↗

bioRxiv2023

How cell penetrating peptides behave differently from pore forming peptides: structure and stability of induced transmembrane pores

Peptide induced trans-membrane pore formation is commonplace in biology. Examples of transmembrane pores include pores formed by antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) in bacterial membranes and eukaryotic membranes, respectively. In general, however, transmembrane pore formation depends on peptide sequences, lipid compositions and intensive thermodynamic variables and is difficult to observe directly under realistic solution conditions, with structures that are challenging to measure directly. In contrast, the structure and phase behavior of peptide-lipid systems are relatively straightforward to map out experimentally for a broad range of conditions. Cubic phases are often observed in systems involving pore forming peptides; however, it is not clear how the structural tendency to induce negative Gaussian curvature (NGC) in such phases is quantitatively related to the geometry of biological pores. Here, we leverage the theory of anisotropic inclusions and devise a facile method to estimate transmembrane pore sizes from geometric parameters of cubic phases measured from small angle X-ray scattering (SAXS) and show that such estimates compare well with known pore sizes. Moreover, our model suggests that whereas AMPs can induce stable transmembrane pores for membranes with a broad range of conditions, pores formed by CPPs are highly labile, consistent with atomistic simulations.

biophysics↗

bioRxiv2023

Formation of clathrin-pits and ATP-independent cholesterol-dependent tubules initiates mechano-regulation on de-adhesion.

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis is not well understood. To investigate this, we study the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical-trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether-force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by a saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes as well as transferrin-labelled pits close to the basal plasma membrane also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked but proceeded uncontrolled. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested on depleting both. The data strongly supports pit-formation to be central to the reduction in fluctuations whether in normal or ATP depleted condition. Furthermore, while in normal conditions the contribution of clathrin-mediated endocytosis is clear, under ATP-depleted conditions we propose that cholesterol-dependent pits spontaneously regulate tension. SummaryWe show that during de-adhesion, cell edges retract, creating membrane folds and increasing fluctuations. Cells increase the rate of endocytosis to regulate back their membrane fluctuations. This is achieved by forming invaginations. Dynamin-dependent pathways are majorly involved, while cholesterol-dependent ATP-independent mechanisms also contribute.

biophysics↗

bioRxiv2022

Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells

T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, play a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 play any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension on chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn are crucial for the integrin LFA-1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the outside-in signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.

immunology↗

bioRxiv2022

Synaptotagmin 7 outperforms synaptotagmin 1 to open and stabilize nascent fusion pores via robust membrane penetration

Synaptotagmin-1 and synaptotagmin-7 are two prominent Ca2+ sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding Ca2+, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examined the biophysical properties of these two synaptotagmin isoforms and compared their interactions with phospholipid membranes. We discovered that synaptotagmin-1*membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a "super-penetrator". We exploited these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to Ca2+.

neuroscience↗

bioRxiv2019

Interplay of bacterial membrane vesicle and host lipid physicochemical parameters during host-bacterial membrane vesicle interaction

Bacterial membrane vesicles (MVs) facilitate long-distance delivery of virulence factors crucial for pathogenicity. The entry and trafficking mechanisms of virulence factors inside host cells are recently emerging, however, if bacterial MVs can fuse and modulate the physicochemical properties of the host lipid membrane and membrane lipid parameter for fusion remains unknown. Here we reconstitute the interaction of bacterial MV with host cell lipid membranes and quantitatively show that bacterial MV interaction increases the fluidity, dipole potential, and Compressibility of a biologically relevant multi-component host membrane upon fusion. The presence of cylindrical lipids such as phosphatidylcholine and a moderate acyl chain length of C16 helps the MV interaction. While significant binding of bacterial MVs to the raft-like lipid membranes with phase-separated regions of the membrane was observed, however, MVs prefer binding to the liquid-disordered regions of the membrane. Further, the elevated levels of cholesterol tend to hinder the interaction of bacterial MVs as evident from the favorable excess Gibbs free energy of mixing of bacterial MVs with host lipid membranes. The findings provide new insights that might have implications for the regulation of host machinery by bacterial pathogens through the manipulation of host membrane properties.

biochemistry↗

bioRxiv2019

Photo-Disassembly of Membrane Microdomains Revives Conventional Antibiotics against MRSA

Confronted with the rapid evolution and dissemination of antibiotic resistance, there is an urgent need to develop alternative treatment strategies for drug-resistant pathogens. Here, we present an unconventional approach to restore the susceptibility of methicillin-resistant S. aureus (MRSA) to a broad spectrum of conventional antibiotics via photo-disassembly of functional membrane microdomains. The photo-disassembly of microdomains is based on effective photolysis of staphyloxanthin, the golden carotenoid pigment that gives its name. Upon pulsed laser treatment, cell membranes are found severely disorganized and malfunctioned to defense antibiotics, as unveiled by membrane permeabilization, membrane fluidification, and detachment of membrane protein, PBP2a. Consequently, our photolysis approach increases susceptibility and inhibits development of resistance to a broad spectrum of antibiotics including penicillins, quinolones, tetracyclines, aminoglycosides, lipopeptides, and oxazolidinones.\n\nOne Sentence SummaryUsing photons to crash S. aureus cell membrane and its formidable defense against a broad spectrum of antibiotics.

microbiology↗