Biology subjects

Ghosh, A.

Publications and source records attributed to Ghosh, A..

At least 127 records · Page 7Linked to original sources

bioRxiv2024

Age-related behavioral resilience in smartphone touchscreen interaction dynamics

We experience a life that is full of ups and downs. The ability to bounce back after adverse life events such as the loss of a loved one or serious illness declines with age, and such isolated events can even trigger accelerated aging. How humans respond to common day-to-day perturbations is less clear. Here, we infer the aging status from smartphone behavior by using a decision tree regression model trained to accurately estimate the chronological age based on the dynamics of touchscreen interactions. Individuals (N = 280, 21 to 83 years of age) expressed smartphone behavior that appeared younger on certain days and older on other days through the observation period that lasted up to [~]4 years. We captured the essence of these fluctuations by leveraging the mathematical concept of critical transitions and tipping points in complex systems. In most individuals, we find one or more alternative stable aging states separated by tipping points. The older the individual, the lower the resilience to forces that push the behavior across the tipping point into an older state. Traditional accounts of aging based on sparse longitudinal data spanning decades suggest a gradual behavioral decline with age. Taken together with our current results, we propose that the gradual age-related changes are interleaved with more complex dynamics at shorter timescales where the same individual may navigate distinct behavioral aging states from one day to the next. Real-world behavioral data modeled as a complex system can transform how we view and study aging.

animal behavior and cognition↗

bioRxiv2024

A genetic and physiological model of renal dysfunction in Lowe syndrome

Lowe syndrome (LS) is an X-linked recessive genetic disorder characterized by renal dysfunction, neurodevelopmental defects, and cataract. The affected gene, OCRL encodes for a polyphosphoinositide 5-phosphatase. OCRL is localized to multiple sub-cellular locations in the endolysosomal system and defects in these organelles have been described in human cells depleted of OCRL. However, the relationship of the endolysosomal defects in OCRL depleted cells to the altered physiology of kidney cells of LS patients has not been completely determined. Here we model the kidney phenotypes of LS using a Drosophila nephrocyte model. Using this model system, we demonstrate that OCRL plays a cell-autonomous role in nephrocyte function. Deletion of the only OCRL ortholog in Drosophila (dOCRL) leads to cell-autonomous defects in larval nephrocyte structure and function. Null mutants of dOCRL (dOCRLKO) show defects in the endolysosomal system of larval nephrocytes that are associated with physiological defects in nephrocyte function. These defects could be rescued by reconstitution with a human OCRL transgene but not with a phosphatase dead version or a human LS patient derived mutation. Overall, this work provides a model system to understand the mechanisms by which the sub-cellular changes from loss of OCRL leads to defects in kidney function in human patients.

cell biology↗

bioRxiv2024

Functional diversity in the Hsp60 of Sulfolobus acidocaldarius: mosaic of Group I and Group II chaperonin

External stress can disrupt protein homeostasis in organisms, necessitating the involvement of heat shock proteins (Hsps) to restore equilibrium and ensure survival. Unlike other organisms, the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius lacks Hsp100, Hsp90, and Hsp70, possessing only two small heat shock proteins (Hsp14 and Hsp20) and one group II chaperonin, Hsp60. This raises questions about how protein substrates are protected and transferred to Hsp60 for refolding without other chaperones. Our study focused on ATP-dependent Hsp60 in S. acidocaldarius, revealing its formation of oligomeric structures in the presence of ATP. While ATP hydrolysis is not essential for oligomer formation and lid closure, it is crucial for Hsp60s chaperone activity, effectively folding stress-denatured substrate proteins by stabilizing their folded conformations. The mechanism involves hydrophobic recognition of unfolded substrates, encapsulating and releasing them in a more folded state. Negatively charged inner surface of the ring seems to be responsible for driving the folding of the substrate. Importantly, Hsp14 was found to transfer substrate proteins to Hsp60{beta}, orchestrating their refolding into an active state. Beyond protein folding, Hsp60{beta} protects the membrane under stress, contributing to maintaining membrane rigidity. Hsp60 exhibits nested cooperativity in ATPase activity, adapting to ATP concentration changes and interestingly Hsp60{beta} and Hsp60{beta} complex shows a mosaic behaviour during ATP hydrolysis belonging to both group I and group II chaperonin respectively. In conclusion, our study provides insights into the intricate mechanisms employed by Hsp60 in S. acidocaldarius to maintain protein homeostasis. It offers a comprehensive understanding of Hsp60s role in the heat shock response pathway, shedding light on fundamental cellular processes in extremophilic archaea.

biochemistry↗

bioRxiv2024

Translation initiation or elongation inhibition triggers contrasting effects on Caenorhabditis elegans survival during pathogen infection

Diverse microbial pathogens are known to attenuate host protein synthesis. Consequently, the host mounts a defense response against protein translation inhibition, leading to increased transcript levels of immune genes. The seemingly paradoxical upregulation of immune gene transcripts in response to blocked protein synthesis suggests that the defense mechanism against translation inhibition may not universally benefit host survival. However, a comprehensive assessment of host survival on pathogens upon blockage of different stages of protein synthesis is currently lacking. Here, we investigate the impact of knockdown of various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, we observe opposing effects on C. elegans survival depending on whether translation initiation or elongation is inhibited. While translation initiation inhibition enhances survival, elongation inhibition decreases it. Transcriptomic studies reveal that translation initiation inhibition activates a bZIP transcription factor ZIP-2-dependent innate immune response that protects C. elegans from P. aeruginosa infection. In contrast, inhibiting translation elongation triggers both ZIP-2-dependent and ZIP-2-independent immune responses that, while effective in clearing the infection, are detrimental to the host. Thus, our findings reveal the opposing roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection, highlighting distinct transcriptional reprogramming that may underlie these differences.

immunology↗

bioRxiv2023

Prognostic relevance of correlated co-expression of coding and noncoding RNAs in cervical cancers

Human papillomavirus (HPV) drives cervical cancer (CaCx) pathogenesis and viral oncoproteins jeopardize global gene expression in such cancers. We aimed to identify differentially expressed coding (DEcGs) and long noncoding (sense intronic and Natural Antisense Transcripts) RNA genes (DElncGs) in HPV16-positive CaCx patients (N=44) compared to HPV-negative normal individuals (N=34). Thereby, employing strand-specific RNA-seq, we determined the relationships between DEcGs and DElncGs and their clinical implications. Gene set enrichment and protein-protein interaction analyses of DEcGs revealed enrichment of processes crucial for abortive virus life cycle and cancer progression. The DEcGs formed 16 gene clusters, portraying cancer-related functions. We recorded significantly correlated co-expression of 79 DElncGs with DEcGs at proximal genomic loci, and 24 such pairs portrayed significantly altered correlation coefficients among patients, compared to normal individuals. Of these, 6 DEcGs belonged to 5 gene clusters, one of which was survival-associated. Out of the 24 correlated DEcG: DElncG pairs, 3 pairs were identified, where expression of both members was significantly associated with patient overall survival. Besides being prognostically relevant, disruption of the significant correlative relationships of such gene pairs in CaCx bears immense potential for patient-targeted therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/573593v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@19091c8org.highwire.dtl.DTLVardef@52fbeaorg.highwire.dtl.DTLVardef@18ecf3org.highwire.dtl.DTLVardef@6c5e30_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO A schematic representation of the analyses undertaken to draw insights on the biological relevance of joint analysis of co-expressed and significantly correlated DElncGs and DEcGs considering the ncNATs and sense intronic DElncGs, in HPV16-positive CaCx patients compared with HPV-negative normal individuals. C_FIG

cancer biology↗

bioRxiv2023

Regions of Inflammation in mouse asthma correspond to regions of heme-free soluble guanylyl cyclase and can be tracked by marked expression of heme-oxygenase-1

Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the Nitric Oxide (NO)-soluble Guanylyl Cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However this bronchodilation is dysfunctional in asthma due to high NO levels which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using models of mouse asthma (OVA, CFA/HDM) that the inflammed segments of the mouse asthma lungs can be tracked by upregulated expression of HO1 and these regions in-turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-1{beta}1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770 relative to naive uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact in developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in-turn becomes heme-free. Recognition of these specific lung segments enhance our understanding of the inflammed lungs in asthma with the ultimate aim to evaluate potential therapies and suggests that regional and not global inflammation impacts lung function in asthma.

immunology↗

bioRxiv2023

A novel vitamin C analog acts as a potent bio-enhancer to augment the activities of anti-tuberculosis drugs against Mycobacterium tuberculosis

Mycobacterium tuberculosis is a deadly pathogen that claims millions of lives every year. Current research focuses on finding new anti-tuberculosis drugs that are safe and effective, with lesser side effects and toxicity. One important approach is to identify bio-enhancers that can improve the effectiveness of anti-tuberculosis drugs, resulting in reduced doses and shortened treatment times. We investigated the use of vitamin C-derived isotetrones as bio- enhancer agents. In this context, our results revealed that the lead compound C11 inhibits growth, improves MIC/MBC, and enhances the killing of M. tuberculosis H37Rv strain when used in combination with first-line and injectable anti-TB drugs in a dose-dependent manner. The combination of C11 and rifampicin also reduced the generation of spontaneous mutants against rifampicin and reached a mutation prevention concentration (MPC) with moderate rifampicin concentrations. The identified compounds were proven to be effective against the MDR strain of M. tuberculosis and non-cytotoxic in HepG2 cells. We also found that C11 induced the generation of reactive oxygen species (ROS) inside macrophages and within bacteria, resulting in better efficacy.

microbiology↗

bioRxiv2023

Control of Biofilm Formation by an Agrobacterium tumefaciens Pterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in A. tumefaciens and is found in multiple additional bacterial pathogens. SIGNIFICANCEBiofilms are bacterial communities attached to surfaces, physiologically distinct from free-living cells, and a common cause of persistent infections. Here we define the mechanism of a novel biofilm regulatory system based on excreted metabolites called pterins, that is conserved within a wide range of Gram-negative bacteria, including multiple pathogens of animals and plants. The molecular mechanism of pterin-dependent regulation is reported including structural determination of several members of a new family of pterin-binding proteins. Pterins are produced across all domains of life and mechanistic insights into this regulatory circuit could lead to new advances in antibiofilm treatments.

microbiology↗

bioRxiv2023

Computationally Driven Discovery and Characterization of SIRT3 Activating Compounds that Fully Recover Catalytic Activity under NAD+ Depletion

Mammalian sirtuins (SIRT1-SIRT7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play critical roles in lifespan and age-related diseases. The physiological importance of sirtuins has stimulated intense interest in designing sirtuin activating compounds. However, except for allosteric activators of SIRT1-catalyzed reactions that are limited to specific substrates, methodologies for the rational design of sirtuin activating compounds -- including compounds that activate mitochondrial sirtuins implicated in the age-related decline of cellular metabolism -- have been lacking. Here, we use computational high-throughput screening methodologies and a biophysical model for activation of the major mitochondrial sirtuin SIRT3 to identify novel small molecule activators of the human SIRT3 enzyme from a 1.2 million compound library. Unlike previously reported SIRT3 activators like Honokiol, which only transiently upregulate SIRT3 under non-steady state conditions and reduce the steady state catalytic efficiency of the enzyme, several of the novel compounds identified here are potent SIRT3 activators in both the steady and non-steady states. Two such compounds can almost double the catalytic efficiency of the enzyme with respect to NAD+, which would be sufficient to almost entirely compensate for the loss in SIRT3 activity that occurs due to the reduction in mitochondrial coenzyme concentration associated with aging, and display AC50s (concentrations of half-maximal activation) as low as 100 nM. The current work thus reports first-in-class, non-allosteric steady state activators that activate SIRT3 through a novel, mechanism-based mode of activation and that may be developed further for therapeutic applications.

biochemistry↗

bioRxiv2023

Enzalutamide Induces Cytotoxicity in Desmoplastic Small Round Cell Tumor Independent of the Androgen Receptor

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, pediatric cancer caused by the EWSR1::WT1 fusion protein. DSRCT predominantly occurs in males, which comprise 80-90% of the patient population. While the reason for this male predominance remains unknown, one hypothesis is that the androgen receptor (AR) plays a critical role in DSRCT and elevated testosterone levels in males help drive tumor growth. Here, we demonstrate that AR is highly expressed in DSRCT relative to other fusion-driven sarcomas and that the AR antagonists enzalutamide and flutamide reduce DSRCT growth. However, despite these findings, which suggest an important role for AR in DSRCT, we show that DSRCT cell lines form xenografts in female mice at the same rate as male mice and AR depletion does not significantly alter DSRCT growth in vitro. Further, we find that AR antagonists reduce DSRCT growth in cells depleted of AR, establishing an AR-independent mechanism of action. These findings suggest that AR dependence is not the reason for male predominance in DSRCT and that AR-targeted therapies may provide therapeutic benefit primarily through an AR-independent mechanism that requires further elucidation.

cancer biology↗

bioRxiv2023

The feature landscape of visual cortex

Understanding computations in the visual system requires a characterization of the distinct feature preferences of neurons in different visual cortical areas. However, we know little about how feature preferences of neurons within a given area relate to that areas role within the global organization of visual cortex. To address this, we recorded from thousands of neurons across six visual cortical areas in mouse and leveraged generative AI methods combined with closed-loop neuronal recordings to identify each neurons visual feature preference. First, we discovered that the mouses visual system is globally organized to encode features in a manner invariant to the types of image transformations induced by self-motion. Second, we found differences in the visual feature preferences of each area and that these differences generalized across animals. Finally, we observed that a given areas collection of preferred stimuli ( own-stimuli) drive neurons from the same area more effectively through their dynamic range compared to preferred stimuli from other areas ( other-stimuli). As a result, feature preferences of neurons within an area are organized to maximally encode differences among own-stimuli while remaining insensitive to differences among other-stimuli. These results reveal how visual areas work together to efficiently encode information about the external world.

neuroscience↗

bioRxiv2023

The Distal-Proximal Relationships Among the Human Moonlighting Proteins: Evolutionary hotspots and Darwinian checkpoints

Moonlighting proteins, known for their ability to perform multiple, often unrelated functions within a single polypeptide chain, challenge the traditional "one gene, one protein, one function" paradigm. As organisms evolved, their genomes remained relatively stable in size, but the introduction of post-translational modifications and sub-strategies like protein promiscuity and intrinsic disorder enabled multifunctionality. Enzymes, in particular, exemplify this phenomenon, engaging in unrelated processes alongside their primary catalytic roles. This study employs a systematic, quantitative informatics approach to shed light on human moonlighting protein sequences. Phylogenetic analyses of human moonlighting proteins are presented, elucidating the distal-proximal relationships among these proteins based on sequence-derived quantitative features. The findings unveil the captivating world of human moonlighting proteins, urging further investigations in the emerging field of moonlighting proteomics, with the potential for significant contributions to our understanding of multifunctional proteins and their roles in diverse cellular processes and diseases.

genomics↗

bioRxiv2023

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

CRISPR-Cas9 is the newest technology available for targeted genome editing. It is very efficient and cheap compared to other genome editing techniques. However, its therapeutic application is limited due to its off-target activity. To have a better understanding of this off-target effect, we concentrated our efforts on its mismatch-prone PAM distal end. Current off-target prediction algorithms use RNA-DNA complementation derived energy as a major factor in predicting off-target effect. RNA-DNA complementation derived energy drives Cas9 conformational change, which in turn drives its functional activity. In the case of lower RNA-DNA complementarity, a partial conformational change occurs resulting in a slower reaction rate and partial activity. However, extensive mismatches are often tolerated despite lower complementation derived energy available from RNA: DNA duplex formation. Thus, the off-target activity of Cas9 depends directly on the nature of mismatches which in turn result in deviation of the active site of the enzyme due to structural instability in the duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites and performed in vitro and cell line-based experiments to assess the effects of PAM distal mismatches in Cas9 activity. For further mechanistic validation, Molecular dynamics simulation was performed and it revealed that certain mismatch mutations induced pronounced conformational instability within the RNA-DNA duplex, leading to elevated root mean square deviation (RMSD) values. We found that, target sites having mismatches in the 18th to 16th position upstream of the PAM showed no to little activity.

genomics↗

bioRxiv2023

Fast, whole-cell DNA-PAINT imaging with dual-labeled self-quenched imager probes

Elucidating the interaction between membrane proteins and antibodies requires fast whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-PAINT achieves molecular resolution but is practically restricted to two-dimensional imaging due to long acquisition times. Here, we introduce two-dye imager (TDI) probes, manifesting negligible background and amplified fluorescence signal upon transient binding, enabling [~]15-fold faster imaging. Using a combination of TDI-DNA-PAINT and LLS microscopy on B cells, we reveal the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibody rituximab (RTX), unperturbed by surface effects. Our results demonstrate that B cells become polarized, and microvilli stabilized by RTX binding. These findings, we believe, will aid rational design of improved immunotherapies targeting tumor-associated antigens.

biophysics↗

bioRxiv2023

Bayesian model discovery for reverse-engineering biochemical networks from data

The reverse engineering of gene regulatory networks based on gene expression data is a challenging inference task. A related problem in computational systems biology lies in identifying signalling networks that perform particular functions, such as adaptation. Indeed, for many research questions, there is an ongoing search for efficient inference algorithms that can identify the simplest model among a larger set of related models. To this end, in this paper, we introduce SLIO_SCPLOWNC_SCPLOWG, a Bayesian sparse likelihood-free inference method using Gibbs sampling. We demonstrate that SLIO_SCPLOWNC_SCPLOWG can reverse engineer stochastic gene regulatory networks from single-cell data with high accuracy, outperforming state-of-the-art correlation-based methods. Furthermore, we show that SLIO_SCPLOWNC_SCPLOWG can successfully identify signalling networks that execute adaptation. Sparse hierarchical Bayesian inference thus provides a versatile tool for model discovery in systems biology and beyond.

systems biology↗

bioRxiv2023

A novel glideosome-associated protein S14 coordinates sporozoite gliding motility and infectivity in mosquito and mammalian hosts

Plasmodium sporozoites are the infective forms of the malaria parasite in the vertebrate host. Gliding motility allows sporozoites to migrate and invade the salivary gland and hepatocytes. Invasion is powered by an actin-myosin motor complex linked to glideosome. However, the gliding complex and the role of several glideosome-associated proteins (GAPs) are poorly understood. In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins. We confirmed S14 expression in sporozoites using real-time PCR. Further, the S14 gene was endogenously tagged with 3XHA-mCherry to study expression and localization. We found its expression and localization on the inner membrane of sporozoites. By targeted gene deletion, we demonstrate that S14 is essential for sporozoite gliding motility, salivary gland, and hepatocyte invasion. The gliding and invasion-deficient S14 KO sporozoites showed normal expression and organization of IMC and surface proteins. Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein and plays an essential role in sporozoite gliding motility, which is critical for the invasion of the salivary gland, hepatocyte, and malaria transmission.

microbiology↗

bioRxiv2023

α-Synuclein emulsifies TDP-43 prion-like domain - RNA liquid droplets to promote heterotypic amyloid fibrils

Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and -synuclein (S) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and S synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear whether and how S modulates TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD - RNA liquid droplets as models along with microscopy, nanoscale spatially-resolved spectroscopy, and other biophysical analyses, we uncover the interactions of S with phase-separated droplets. We learn that S acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD - RNA droplets and emulsifying them. The hardening of the droplets that follow by S aggregates on the periphery, nucleates the formation of heterotypic TDP-43PrLD amyloid fibrils with structures distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of S as a Pickering agent in interacting with SGs and unmask the hitherto unknown role of S in modulating TDP-43 proteinopathies.

biophysics↗

bioRxiv2023

CARs are organized in nanodomains in the plasma membrane of T cells that accumulate at tumor contact sites

Chimeric antigen receptors (CARs) are synthetic immune receptors that are expressed in T cells through genetic engineering. CAR-T cells have been successfully used to eradicate very advanced leukemias and lymphomas and their functional properties have been intensively studied. However, relatively little is known about the spatiotemporal expression and organization of CARs on the T-cell membrane and how this influences their efficacy. Here, we applied super-resolution microscopy to visualize CD19-, ROR1-, and ROR2-specific CARs in human CD4+ and CD8+ T cells that were engineered with lentiviral and transposon-mediated gene transfer. Our data show that the majority of CARs is organized in nanodomains virtually independent of the T cell type, CAR construct and expression level. Quantitative analyses revealed a slightly higher CAR density in transposon-engineered T cells correlating with higher antigen sensitivity and faster resolution of anti-tumor functions compared to lentivirally-engineered T cells. Live-cell fluorescence imaging revealed that both, CAR nanodomains and CAR monomers accumulate at tumor contact sites and form multifocal immunological synapses. Our study provides novel insights into the membrane organization of CARs with single-molecule resolution and illustrates the potential of advanced microscopy to inform the rational design of synthetic immune receptors for applications in immune cell therapy.

immunology↗