Biology subjects

Popovic, B.

Publications and source records attributed to Popovic, B..

12 recordsLinked to original sources

bioRxiv2026

Naturalistic face-directed attention predicts face recognition ability

Recognising people precedes and informs most social interactions, yet it is not clear why individuals differ so markedly in face recognition ability. Exposure-based accounts propose that it is shaped by visual experience with faces, known as 'face diet', but most prior work treats exposure as passively acquired, thereby masking the active and dynamic nature of visual attention in natural settings. Here we examined whether spontaneous attention to faces in everyday environments predicts face recognition ability. Eighty participants wore mobile eye-tracking glasses while walking through a busy university campus and during a face-to-face interaction, before completing standard measures of face recognition ability. Fixations directed to faces showed stable individual differences in both settings. People who looked more at faces scored higher on our composite measure of face recognition ability, and gaze in the two settings predicted ability independently, together accounting for 22% of its variance. These findings suggest that differences in how people actively sample visual information from their environments may be an overlooked source of variance in perceptual expertise.

animal behavior and cognition↗

bioRxiv2026

Decoding translational control by cis-regulatory elements and RNA binding proteins in human effector T cells

T cells are central players in killing virally infected and malignant cells. To achieve this, T cells depend on dynamic, tightly regulated alterations of the proteome. To decipher the rules instructing translation in T cells, we performed transcriptome and proteome analysis of polysome fractions from human effector CD8+ T cells. Transcriptome analysis informed on ribosomal occupancy of RNAs and uncovered the rapid and RNA-specific redistribution upon T cell activation. With machine learning, we identified RNA binding motifs that predict the RNA (re)distribution among the polysome fractions. Using matched proteome analysis, we identified polysome-associated RBPs and their swift shuttling across polysome fractions upon T cell activation. Integrating sequence feature analysis with polysome RBP localization uncovered PTBP1 as a positive translation regulator, through its interactions with cis-regulatory elements in 3'UTRs of target mRNAs. In conclusion, the multi-level analysis presented here identifies rules of selective translation control which shape the T cell proteome.

immunology↗

bioRxiv2026

Teaching Diffusion Models Physics: Reinforcement Learning for Physically Valid Diffusion-Based Docking

Molecular docking aims to predict the binding conformation of a small molecule to its protein target. Recent work has proposed diffusion models for this task, from rigid-body docking that diffuses over ligand degrees of freedom to co-folding approaches that jointly generate protein structure and ligand pose. However, diffusion-based docking models have been shown to frequently produce physically implausible poses and fail to consistently recover key protein-ligand interactions. To address this, we introduce a reinforcement learning framework for training diffusion-based docking models directly on non-differentiable objectives. Fine-tuning DiffDock-Pocket for physical validity with our approach substantially increases the number of generated poses that are physically valid and interaction-preserving, with no increase in inference-time compute. Importantly, this comes without sacrificing structural accuracy; in fact, our approach increases the proportion of structures with near-native poses. These effects are most pronounced for protein targets that are dissimilar to the training data. Our fine-tuned DiffDock-Pocket model outperforms both classical docking algorithms and machine learning-based approaches on the PoseBusters set. Our results demonstrate that reinforcement learning can teach diffusion-based docking models to better respect physical constraints and recover key interactions, without the requirement to rely on inference-time corrections.

bioinformatics↗

bioRxiv2025

Proximity labeling reveals ZFP36L1 as a central hub for post-transcriptional regulation networks in T cells

Effective T cell responses against pathogens require a rapid yet tightly controlled remodeling of the proteome, and RNA binding proteins (RBPs) are key in this process. For instance, the RBP ZFP36L1 prevents excessive protein production and thereby limits immunopathology. ZFP36L1 is primarily known to mediate mRNA decay, but it can also regulate other processes. How its mode of action relates to its interaction partners is, however, not well-understood. Here, we mapped the ZFP36L1 interactome in primary human T cells. Using proximity labeling, we identified known and new interactors that regulate 3UTR-mediated RNA degradation, deadenylation, stress granule/p-body formation, as well as 5UTR-mediated translation repression and mRNA decapping. Snapshot analysis uncovered the ZFP36L1 interactome dynamics and RNA (in)dependency throughout T cell activation. Intriguingly, proximity labeling also uncovered regulators of ZFP36L1 protein expression: This included the helicase UPF1, which not only interacts with ZFP36L1 protein but also promotes its protein expression. Altogether, this comprehensive interactome map underlines the versatility of interactions with ZFP36L1 and their possible role in cellular function.

molecular biology↗

bioRxiv2024

Combined deletion of ZFP36L1 and ZFP36L2 drives superior cytokine production in T cells at the cost of cell fitness

A key feature of cytotoxic CD8+ T cells for eliminating pathogens and malignant cells is their capacity to produce pro-inflammatory cytokines, which includes TNF and IFN{gamma}. Provided that these cytokines are highly toxic, a tight control of their production is imperative. RNA-binding proteins (RBPs) are essential for the fine-tuning of cytokine production. The role of the RBP ZFP36L1 and its sister protein ZFP36L2 herein has been established, however, their relative contribution to cytokine production is not well known. We here compared the effect of ZFP36L1 and ZFP36L2 single and double deficiency in murine effector CD8+ T cells. Whereas single deficient T cells significantly increased cytokine production, double deficiency completely unleashed the cytokine production. Not only the TNF production was substantially prolonged in double-deficient T cells. Also, the production of IFN{gamma} reached unprecedented levels with >90% IFN{gamma}-producing T cells compared to 3% in WT T cells, even after 3 days of continuous activation. This continuous cytokine production by double-deficient T cells was also observed in tumor-infiltrating lymphocytes in vivo, however, with no effect on tumor growth. Rather, ZFP36L1 and ZFP36L2 double deficiency resulted in decreased cell viability, impaired STAT5 signaling, and dysregulated cell cycle progression. In conclusion, while combined deletion in ZFP36L1 and ZFP36L2 can drive continuous cytokine production even under chronic activation, safeguards are in place to counteract such super-cytokine producers.

immunology↗

bioRxiv2024

Single molecule imaging of transcription dynamics, RNA localization and fate in T cells

T cells are critical effector cells against infections and malignancies. To achieve this, they produce pro-inflammatory cytokines, including IFN-{gamma} and TNF. Cytokine production is a tightly regulated process. The relative contribution of transcriptional and post-transcriptional regulation to mRNA expression is, however, unknown. We therefore optimized single-molecule FISH for primary human T cells (T-cell smFISH) to simultaneously quantify nascent RNA, mature mRNA levels and its localization with single-cell resolution. T-cell smFISH uncovered heterogeneous cytokine mRNA levels, with high cytokine producers displaying biallelic IFNG/TNF RNA transcription activity. Throughout activation, nuclear cytokine mRNAs accumulated, whereas cytoplasmic cytokine mRNA was degraded through translation-dependent decay. Lastly, T-cell smFISH uncovered cytokine-specific regulation by the RNA-binding protein HuR. Thus, T-cell smFISH provides novel insights in the intricate (post)-transcriptional processes in T cells.

immunology↗

bioRxiv2024

mTOR signaling promotes cytokine production in T cells through 3UTR-mediated translation control

T cells are key contributors to clear our body from infected and malignant cells. When T cells respond to target cells, they undergo profound translational alterations. The evolutionary and highly conserved kinase mammalian target of rapamycin (mTOR) is a central mediator of T cell differentiation, homeostasis, and T cell activation, including the production of the key pro-inflammatory cytokines TNF, IL2, and IFN{gamma}. mTOR was shown to execute its translation activity through TOP motifs located in the 5 Untranslated region (5UTR) of its target genes. Here, we uncovered a distinct mechanism of mTOR signaling on cytokine production in T cells, which is under control of the 3UTR. Even though non-classical TOP motifs are present in cytokine 3UTRs, they do not contribute to mTOR-mediated translation regulation. Rather, AU-rich elements (AREs) are required for mTOR-mediated cytokine production. Furthermore, we discovered that the RNA binding protein DDX21 binds to 3UTR AREs and confers the mTOR-mediated translation control. In conclusion, we here present a previously unappreciated ARE-dependent, 3UTR-mediated mode of action that mTOR employs to regulate cytokine production.

immunology↗

bioRxiv2024

AutoMorFi: Automated Whole-image Morphometry in Fiji/ImageJ for Diverse Image Analysis Needs

Running on the highly popular and accessible ImageJ/Fiji platform for biological image analysis, we have established AutoMorFi as a streamlined interface for automated whole-image morphometric analysis that generates at least 47 measurements per cell or object in under 1 minute. We performed multiple validated cluster and principal component analyses on nonredundant morphometric parameters derived from AutoMorFi for various cell types, objects, and organisms. We used images of rodent macrophages, human postmortem brain tissues from multiple sclerosis (MS) and Alzheimers disease (AD) patients, iPSC/animal models for Downs syndrome and autism spectrum disorder (ASD), and organisms such as sea anemone and corals. AutoMorFis adaptability extends across diverse imaging modalities including brightfield, confocal, or widefield fluorescence microscopy as well as underwater photography. Due to its unlimited and unbiased sampling across any image and high potential for modification and customization, using AutoMorFi has led to the discovery of new distinguishing features in previously studied cell types and organisms as well as the development of rapid diagnostic approaches. AutoMorFi represents a transformative tool that will accelerate morphometric analysis and offer broad relevance in biological studies.

cell biology↗

bioRxiv2024

Regulation of IFNgamma production by ZFP36L2 in T cells is context-dependent

CD8+ T cells kill target cells by releasing cytotoxic molecules and pro-inflammatory cytokines, such as TNF and IFN{gamma}. The magnitude and duration of cytokine production is defined by post-transcriptional regulation, and a critical regulator herein are RNA-binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production is not well understood. Previously, we showed that the RBP ZFP36L2 blocks translation of pre-formed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a context-dependent manner. T cell-specific deletion of ZFP36L2 (CD4-cre) had no effect on T cell development, or on cytokine production during early time points (2-6h) of T cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN{gamma} during prolonged T cell activation (20-48h). ZFP36L2 deficiency also resulted in increased production of IFN{gamma} production in tumour-infiltrating T cells that are chronically exposed to antigen. Mechanistically, ZFP36L2 regulates IFN{gamma} production at late time points of activation by destabilizing Ifng mRNA in an AU-rich element-dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production.

immunology↗

bioRxiv2021

Time-dependent regulation of cytokine production by RNA binding proteins defines T cell effector function

Potent T cell responses against infections and malignancies depend on the release of effector molecules, such as pro-inflammatory cytokines. Because effector molecules can be toxic, their production is tightly regulated through post-transcriptional events at 3 Untranslated Regions (3UTRs). RNA binding proteins (RBPs) were shown to be key regulators herein. With an RNA aptamer-based capture assay from human T cells, we identified >130 RBPs interacting with the IFNG, TNF and IL2 3UTRs in human T cells. T cell activation altered RBP-RNA interactions, revealing that RBP-target mRNA interactions rapidly respond to stimulation. Furthermore, we uncovered the intricate and time-dependent regulation of cytokine production by RBPs: whereas HuR supports early cytokine production, ZFP36L1, ATXN2L and ZC3HAV1 dampen and shorten the production duration, each at different time points. Strikingly, even though ZFP36L1 deletion did not phenotypically rescue T cell dysfunction in tumors, the increased production of cytokines and cytotoxic molecules resulted in superior anti-tumoral T cell responses in vivo. Our findings thus show that identifying RBP-RNA interactions reveals key modulators of T cell responses in health and disease.

immunology↗

bioRxiv2020

In vitro Evolution of Antibody Affinity via Insertional Mutagenesis Scanning of an Entire Antibody Variable Region

We report the first systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD was used to generate large libraries with random in-frame InDels across the entire scFv gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (ISM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach and suggests that the results of this InDel approach and conventional exploration of point mutations can synergize to generate antibodies with higher affinity. SignificanceInsertion/deletion (InDel) mutations play key roles in genome and protein evolution. Despite their prominence in evolutionary history, the potential of InDels for changing function in protein engineering by directed evolution remains unexplored. Instead point mutagenesis is widely used. Here we create antibody libraries containing InDels and demonstrate that affinity maturation can be achieved in this way, establishing an alternative to the point mutation strategies employed in all previous in vitro selections. These InDels mirror the observation of considerable length variation in loops of natural antibodies originating from the same germline genes and be combined with point mutations, making both natural sources of functional innovation available for artificial evolution in the test tube.

biochemistry↗

bioRxiv2019

Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production

Long-lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-{gamma} is a key effector molecule herein. We recently showed that in murine T cells, the production of IFN-{gamma} is tightly regulated through AU-rich elements (AREs) that are located in the 3 Untranslated Region (UTR). Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of sustained IFN-{gamma} protein-producing T cells. Importantly, this was also true for tumor antigen-specific T cells. MART-1 TCR engineered T cells that were gene-edited for ARE-deletion showed increased percentages of IFN-{gamma} producing MART-1-specific ARE-Del T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated post-transcriptional regulation is highly conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be exploited for therapeutical purposes.

immunology↗