Biology subjects

Milovanovic, D.

Publications and source records attributed to Milovanovic, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Reverse engineering Lewy bodies suggests alpha-synuclein pathology as condensatopathy

Understanding how soluble monomeric alpha-synuclein transitions into pathological alpha-synuclein (Lewy bodies) has remained a central challenge in neurodegeneration. Existing models, largely based on treating cells and animals with pre-formed alpha-synuclein fibrils cannot account for the emergence of the first inclusion without pre-existing seeds. Here, we combine in vitro reconstitutions, computational modeling and a minimal cellular system to show that phase separation of alpha-synuclein with an interacting scaffold can recapitulate Lewy body-like structures. An unbiased proteomics screen, live-cell imaging, correlative light-electron microscopy, and soft X-ray tomography show that Lewy body-like structures are associated with redistribution of actin and membrane-bound organelles, with mitochondria accumulating at their periphery in an actin-dependent manner. Structural analyses further indicate that alpha-synuclein condensates provide local environments in which the insoluble beta-sheets can form. Together, these findings support a seed-independent trajectory from soluble alpha-synuclein through condensation and progressive immobilization to Lewy body-like assemblies and, subsequently, insoluble beta-sheet fibrils. This framework offers a new mechanistic route for understanding the disease progression and promises to guide the design of innovative therapeutic interventions.

neuroscience↗

bioRxiv2026

Synapsin-1 and actin form ordered nanoscale assemblies

Liquid-liquid phase separation is a vital and ubiquitous principle of subcellular organization. Several actin-binding proteins have recently been shown to undergo liquid-liquid phase separation, forming micron-sized droplets that assemble actin into distinct network shapes. However, our knowledge of how these phase-separated proteins organize actin filaments at the nanoscale is rather limited. Here, we seek to address this knowledge gap by investigating synapsin-1 condensates through a combination of computational simulations and nanoscale imaging experiments. Our results show that while synapsin-1 condensates by themselves lack any special structural organization, the addition of actin filaments results in the generation of a regularly organized actin scaffold within the condensates. The organized scaffold forms both when actin and synapsin-1 are added simultaneously and when actin is added after the formation of synapsin condensates. Dissolving the condensates, by synapsin-1 removal, leaves the actin scaffold largely unaffected in both simulations and experiments, implying that this scaffold should guide droplet reformation when synapsin-1 is added back. Taken together, our findings suggest that the actin-rich component of the condensate retains a structural memory that promotes and guides the reassembly of synapsin-actin condensates. This structural memory may be important in the long-term maintenance of synaptic function in vivo.

biophysics↗

bioRxiv2026

Biomolecular condensates provide a unique environment for redox-mediated protein crosslinking

Biomolecular condensates, often formed through liquid-liquid phase separation, are dynamic cellular compartments. Here, we demonstrate that a wide range of fluorescently tagged proteins undergo inadvertent, condensate-mediated crosslinking, resulting in rapid solidification of condensates under common fluorescence imaging conditions. The process is driven by excitation-induced, short-lived reactive oxygen species (ROS), whose otherwise limited crosslinking potential becomes uniquely enabled in the dense phase. In live cells, excitation-induced ROS potently trigger stress granule formation, while the ROS-driven solidification of condensates is modulated by compartment-dependent antioxidant buffering. Our findings demonstrate that condensates create a distinct environment that enables ROS chemistry unlikely to occur in the bulk cytosol. Furthermore, the cellular redox level can be a general regulator of condensate rheology. Beyond biological insights, our findings underscore the need for scrutiny when examining fluorophore-labeled condensates.

biophysics↗

bioRxiv2025

Tissue-specific restriction of TE-derived regulatory elements safeguards cell-type identity

Transposable elements (TEs) have extensively reshaped the cis-regulatory landscape of mammalian genomes, yet the mechanisms that govern their context-specific activity remain incompletely understood. KRAB zinc finger proteins (KZFPs), a large family of transcription factors specialized in TE recognition, are known repressors of TE-derived regulatory activity through TRIM28-mediated H3K9me3 deposition. Here, we expand this paradigm by uncovering noncanonical relationships between TEs and KZFPs. By generating a comprehensive epigenomic map of KZFP-bound TEs, we find that the regulatory activity of ancient mammalian L2/MIR elements is broadly delineated by KZFP binding patterns, despite low H3K9me3 enrichment. We further dissect this relationship by investigating the function of ZNF436, a non-canonical KZFP, highly expressed during in vivo human fetal heart development. Using loss-of-function approaches, we show that ZNF436 preserves cardiomyocyte function by promoting cardiac gene expression while restricting the activation of alternative lineage programs. Mechanistically, ZNF436 recruits specialized SWI/SNF chromatin remodeling complexes to limit the accessibility of L2/MIR-derived enhancers, many of which are active in non-cardiac tissues. These findings reveal a noncanonical, TRIM28-independent role for KZFPs in shaping cell-type-specific regulatory landscapes and emphasize the importance of repressing alternative regulatory programs alongside activating lineage-specific ones to safeguard cell identity.

genomics↗

bioRxiv2025

Brain-derived synaptic vesicles have an intrinsic ability to sequester tubulin

The presence and function of microtubules within the synaptic bouton has long been under investigation. In recent years evidence has accumulated that connects the synaptic vesicle cluster to the local dynamics of microtubule ends. Nonetheless, one question remains open, namely whether the vesicles influence the availability of tubulin within the synaptic compartment. An analysis of previously published live imaging experiments indicates that tubulin is strongly enriched in the synaptic vesicle cluster. To analyze the vesicle-tubulin interaction directly, we isolated vesicles from the mouse brain and imaged them together with fluorescent tubulin in vitro. We found that soluble tubulin is collected by synaptic vesicles in physiological buffers, resulting in the formation of tubulin-rich regions (TRRs) on the respective vesicle clusters. We conclude that the synaptic vesicle cluster is indeed able to recruit soluble tubulin.

neuroscience↗

bioRxiv2025

SynGAP forms biocondensates at sub-micromolar concentrations and recruits PSD95 and receptor oligomers, functioning as a key initiator of PSD formation

A key issue in neuronal circuit regulation is how synapse formation is initiated. Synapse formation could start when one or more synaptic scaffold proteins that can initiate synapse formation reach certain threshold concentrations in the dendritic shaft, which might lead to their oligomerization or even liquid-liquid phase separation (LLPS). By combining in vitro reconstitution of purified proteins with live-cell single-molecule and confocal imaging, we demonstrated that SynGAP alone forms assemblies of nanoscale clusters containing several to several tens of molecules at 10-nM order concentrations and micron-scale LLPS hydrogel-like condensates at submicromolar concentrations. The trimers of SynGAP's intrinsically disordered region (IDR) induced by its coiled-coil domain are responsible for SynGAP condensation. CaMKII-mediated phosphorylation moderately suppresses SynGAP condensation, and also increases condensate liquidity. While PSD95 fails to form assemblies under these conditions, it is recruited to SynGAP condensates by specifically binding to the PDZ-binding motif of SynGAP. SynGAP[PSD95] condensates selectively immobilize postsynaptic transmembrane proteins, Neuroligin1 and AMPAR-TARP2 complexes, in a manner dependent on their oligomerization state, indicating cooperative recruitment dynamics among SynGAP, PSD95, and transmembrane components, which might mimic initial PSD assembly. These findings suggest that SynGAP may act as a primary nucleator of postsynaptic density assembly, challenging the PSD95-centered models.

neuroscience↗

bioRxiv2025

Transposable element co-option drives transcription factor neofunctionalization

Gene duplication and domain acquisition are key mechanisms driving protein diversification. However, the impact of these processes on the function and evolution of transcription factors (TFs) remains poorly characterized. Here, we show that the DUF3669 domain, found in a subset of KRAB zinc-finger proteins (KZFPs), originated from the co-option of a fragment of a LINE-1 transposable element ORF1p. Similar to its LINE-1-encoded ancestor, the DUF3669 domain promotes KZFPs trimerization and enables the formation of nuclear condensates with ribonucleoparticle properties, which critically influence the genomic recruitment and action of these TFs. Thus, our study uncovers a direct link between TE co-option and TF neofunctionalization, highlighting how mobile genetic elements shape the evolution of protein functionality.

genetics↗

bioRxiv2025

Identification of the Lipid Oxygen Radical Defense pathway and its epigenetic control

Membrane phospholipids are vulnerable to oxidative radicals, and uncontrolled lipid peroxidation affects cell viability. Cells have evolved quality control and defense mechanisms, of which the genetic regulation is not fully understood. Here, we identify what we have coined the Lipid Oxygen Radical Defense (LORD) pathway. It is epigenetically repressed by a complex comprising the KRAB-zinc finger protein ZNF354A, the scaffold protein KAP1/TRIM28, the histone methyltransferase SETDB1, and the transcriptional activator ATF2. Upon lipid peroxide accumulation, p38- and JNK-dependent phosphorylation of ATF2, KAP1/TRIM28, and ZNF354A leads to disassembly of the repressive complex, releasing ZNF354A from specific DNA loci and activating a network of protective genes, including NRF2 targets. The pathway affects the cellular sensitivity to oxidative stress and ferroptosis, revealing a novel layer of epigenetic control in lipid quality control and damage repair. This positions the LORD pathway as a promising therapeutic target for diseases linked to chronic inflammation, neurodegeneration and cancer.

cell biology↗

bioRxiv2025

RNA promotes synapsin coacervation and modulates local translation

Condensates at synapses organize synaptic vesicle (SV) clusters and are essential for efficient neurotransmitter release. While it is established that RNA granules traffic along the axons, the function of RNA at the presynapse remains unclear. Here, we uncover a direct structural role of coding RNAs in organizing presynaptic condensates by focusing on SV clusters, condensates between synapsin-1 and lipid vesicles, a defining feature of nerve terminals. Using in vitro reconstitution systems, we show that RNA drives synapsin-1 coacervation, with bias toward structured RNAs being more effective at promoting phase transitions. The importance of RNA was confirmed in living synapses, where acute disruption of native RNA induces a dispersion of SVs and synapsin. Conversely, ectopically expressed SV-like condensates have the ability to recruit the translational machinery. The microscopy-based in vitro translation assay demonstrates increased translation efficiency within synapsin-1/RNA condensates. Together, our work indicates a novel structural role of RNAs in modulating SV condensates.

cell biology↗

bioRxiv2024

Driving forces for condensation of synapsin are governed by sequence-encoded molecular grammars

Multiple biomolecular condensates coexist at the pre- and post-synapse to enable vesicle dynamics and controlled neurotransmitter release in the brain. In pre-synapses, intrinsically disordered regions (IDRs) of synaptic proteins are drivers of condensation that enable clustering of synaptic vesicles (SVs). Using computational analysis, we show that the IDRs of SV proteins feature evolutionarily conserved non-random compositional biases and sequence patterns. Synapsin-1 is essential for condensation of SVs, and its C-terminal IDR has been shown to be a key driver of condensation. Focusing on this IDR, we dissected the contributions of two conserved features namely the segregation of polar and proline residues along the linear sequence, and the compositional preference for arginine over lysine. Scrambling the blocks of polar and proline residues weakens the driving forces for forming micron-scale condensates. However, the extent of clustering in subsaturated solutions remains equivalent to that of the wild-type synapsin-1. In contrast, substituting arginine with lysine significantly weakens both the driving forces for condensation and the extent of clustering in subsaturated solutions. Co-expression of the scrambled variant of synapsin-1 with synaptophysin results in a gain-of-function phenotype in cells, whereas arginine to lysine substitutions eliminate condensation. We report an emergent consequence of synapsin-1 condensation, which is the generation of interphase pH gradients realized via differential partitioning of protons between coexisting phases. This pH gradient is likely to be directly relevant for vesicular ATPase functions and the loading of neurotransmitters. Our study highlights how conserved IDR grammars serve as drivers of synapsin-1 condensation. Research HighlightsO_LIDistinct biomolecular condensates formed by pre-synaptic proteins contain a unique bias of sequence grammars within their IDRs. C_LIO_LIThe IDR of synapsin-1, the essential protein for the clustering of synaptic vesicles, has a conserved compositional bias for Arg and blockiness of proline/polar residues. C_LIO_LIUsing sequence designs, we uncovered how conserved sequence features of the driver IDR of synapsin-1 affect condensation in vitro and in cells. C_LIO_LISynapsin-1 condensates are defined by a measurable interphase pH gradient. C_LI

biochemistry↗

bioRxiv2024

Direct quantification of condensate material properties in live cells reveals the regulation of synapsin condensates viscoelasticity by alpha-synuclein

Synapsin and -synuclein represent a growing list of condensate-forming proteins where the material states of condensates are directly linked to cellular functions (e.g., neurotransmission) and pathology (e.g., neurodegeneration). However, quantifying condensate material properties in living systems has been a significant challenge. To address this, we develop MAPAC (micropipette aspiration and whole-cell patch clamp), a platform that allows direct material quantification of condensates in live cells. We find 10,000-fold variations in the viscoelasticity of synapsin condensates, regulated by the partitioning of -synuclein, a marker for synucleinopathies. Through in vitro reconstitutions, we identify 4 molecular factors that distinctly regulate the viscosity and interfacial tension of synapsin condensates, verifying the cellular effects of -synuclein. Overall, our study provides unprecedented quantitative insights into the material properties of neuronal condensates and reveals a crucial role of -synuclein in regulating condensate viscoelasticity. Furthermore, we envision MAPAC applicable to study a broad range of condensates in vivo.

cell biology↗

bioRxiv2024

CONDENSATES OF SYNAPTIC VESICLES AND SYNAPSIN ARE MOLECULAR BEACONS FOR ACTIN SEQUESTERING AND POLYMERIZATION

Neuronal communication relies on precisely maintained synaptic vesicle (SV) clusters, which assemble via liquid-liquid phase separation (LLPS). This process requires synapsins, the major synaptic phosphoproteins, which are known to bind actin. The reorganization of SVs, synapsins and actin is a hallmark of synaptic activity, but their interplay is still unclear. Here, we combined the reconstitution approaches, expansion microscopy, super-resolution imaging and cryo-electron tomography to dissect the roles of synapsin-SV condensates in the organization of the presynaptic actin cytoskeleton. Our data indicate that LLPS of synapsin initiates actin polymerization, allowing for SV:synapsin:actin assemblies to facilitate the mesoscale organization of SV clusters along axons mimicking the native presynaptic organization in both lamprey and mammalian synapses. Understanding the relationship between the actin network and synapsin-SVs condensates is an essential building block on a roadmap to unravel how coordinated neurotransmission along the axon enables circuit function and behavior.

cell biology↗

bioRxiv2024

Synphilin-1 as a modulator of aSyn assembly

Alpha-synuclein (aSyn) is an intrinsically disordered protein that undergoes phase-separation and is associated with several neurodegenerative conditions. However, the function and the pathological role of aSyn are still elusive. Here, we modeled different types of aSyn assemblies in living cells, and developed a model that reports on gel and solid-like inclusions based on the coexpression of aSyn and synphilin-1 (Sph1). We identified striking morphological differences between aSyn-aSyn and Sph1-aSyn assemblies, characterized by distinct antibody recognition patterns, resistance to Proteinase K treatment, and protein mobilities. Importantly, we showed that the interaction between Sph1-aSyn can be manipulated, altering inclusion size and number. Sph1-aSyn interactions were central for inclusion formation and localization, and that inclusions include lysosomes and AP-1 vesicles, consistent with previous studies in human brain tissue. In total, we provide novel insight into the biology of protein aggregation, shedding light on potential therapeutic strategies that extend beyond conventional targets. Deciphering the role of Sph1 and other aSyn-interacting proteins on aSyn biology and pathobiology will be essential for treating synucleinopathies.

molecular biology↗

bioRxiv2023

Protein thiol alterations drive aberrant phase separation in aging

Cellular homeostasis relies on precise regulation through chemical processes, such as protein posttranslational modifications (PTM) and physical processes, such as biomolecular condensation. Aging disrupts this balance, increasing susceptibility to diseases and death. However, the mechanisms behind age-related pathogenesis remain elusive. In this study, we dissected various cysteine PTMs and their impact on protein-mediated biomolecular condensation in aging brain. Our findings reveal that aging is associated with significant remodeling of cysteine PTMs, which impacts protein ability to participate in liquid-liquid phase separation (LLPS). Specifically, aging leads to an increase in protein sulfenylation and sulfonylation, which promotes LLPS and through conformational change increases the propensity of proteins to aggregate. Protein persulfidation, a protective thiol modification, prevents this by causing condensate dissolution. We demonstrate that age-induced alterations in cysteine PTMs influence the LLPS properties of synapsin-1 and G3BP2, resulting in disruptions in neurotransmitter release and stress granule formation, respectively. Additionally, our study uncovers that GAPDH is susceptible to LLPS and cysteine sulfonylation exacerbates its transition from condensates to aggregates. Mice deficient in cystathionine gamma-lyase, a pro-longevity gene that regulates intracellular persulfide levels, exhibit a shorter lifespan and spontaneous development of neurofibrillary tangles.

biochemistry↗

bioRxiv2023

Intersectin and Endophilin condensates prime synaptic vesicles for release site replenishment

Neurotransmitter is released from dedicated sites of synaptic vesicle fusion within a synapse. Following fusion, the vacated sites are replenished immediately by new vesicles for subsequent neurotransmission. These replacement vesicles are assumed to be located near release sites and used by chance. Here, we find that replacement vesicles are clustered around this region by Intersectin-1. Specifically, Intersectin-1 forms dynamic molecular condensates with Endophilin A1 near release sites and sequesters vesicles around this region. In the absence of Intersectin-1, vesicles within 20 nm of the plasma membrane are reduced, and consequently, vacated sites cannot be replenished rapidly, leading to depression of synaptic transmission. Similarly, mutations in Intersectin-1 that disrupt Endophilin A1 binding result in similar phenotypes. However, in the absence of Endophilin, this replacement pool of vesicles is available but cannot be accessed, suggesting that Endophilin A1 is needed to mobilize these vesicles. Thus, our work describes a distinct physical region within a synapse where replacement vesicles are harbored for release site replenishment.

neuroscience↗

bioRxiv2023

The synapsin-dependent vesicle cluster is crucial for presynaptic plasticity at a glutamatergic synapse in male mice

Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III isoform is usually downregulated after development, but in hippocampal mossy fiber boutons it persists in adulthood. Mossy fiber boutons express presynaptic forms of short- and long-term plasticity, which are thought to underlie different forms of learning. Previous research on synapsins at this synapse focused on synapsin isoforms I and II. Thus, a complete picture regarding the role of synapsins in mossy fiber plasticity is still missing. Here, we investigated presynaptic plasticity at hippocampal mossy fiber boutons by combining electrophysiological field recordings and transmission electron microscopy in a mouse model lacking all synapsin isoforms. We found decreased short-term plasticity - i.e. decreased facilitation and post-tetanic potentiation - but increased long-term potentiation in male synapsin triple knockout mice. At the ultrastructural level, we observed more dispersed vesicles and a higher density of active zones in mossy fiber boutons from knockout animals. Our results indicate that all synapsin isoforms, including synapsin III, are required for fine regulation of short- and long-term presynaptic plasticity at the mossy fiber synapse. Significance statementSynapsins cluster vesicles at presynaptic terminals and shape presynaptic plasticity at giant hippocampal mossy fiber boutons. Deletion of all synapsin isoforms results in decreased short- but increased long-term plasticity.

neuroscience↗

bioRxiv2021

iTRVZ: liquid nano-platform for signal integration on the plasma membrane

Cell survival in immune-competent organisms relies on both growth and immune evasion, yet these processes have been studied as independent cellular programs. Here, using super-resolution single-molecule imaging, we identify GEM (Growth and Evasion Metastable hub), a {approx}34-nm, metastable, nano-liquid, plasma membrane hub that physically integrates receptor-type tyrosine kinase (RTK) inputs with CD59-mediated immune-evasion signaling within the same assembly. GEM is formed by LLPS-like protein clustering on PI(4,5)P2-containing raft-nanodomains. Upon concurrent RTK and CD59 stimulations, GEM recruits both receptors and downstream kinases, enabling reciprocal activation via nano-liquid confinement that supralinearly amplifies PLC{gamma}-IP3-Ca2+ and PI3K-Akt survival signaling outputs. In mice, disrupting GEM suppresses tumor growth in vivo, establishing nano-liquid GEM as an integrator that physically couples growth and CD59-mediated immune-evasion signaling for cell survival.

cell biology↗

bioRxiv2021

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model

Deposition of amyloid beta (A{beta}) along with glia cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimers disease (AD). In recent years, impairment of autophagy has been found to be another important feature, contributing to AD progression and aging. Therefore, we assessed the effect of the autophagy activator Spermidine, a small body-endogenous polyamine often used as dietary supplement and known to promote longevity, on glia cell-mediated neuroinflammation. Spermidine reduced TLR3- and TLR4- mediated inflammatory processes in microglia and astrocytes by decreasing cytotoxicity, inflammasome activity and NF-{kappa}B signaling. In line with these anti-inflammatory effects, oral treatment of the amyloid prone AD-like APPPS1 mice with Spermidine reduced neuroinflammation and neurotoxic soluble A{beta}. Mechanistically, single nuclei sequencing revealed microglia as one of the main targets of Spermidine treatment, with increased expression of genes implicated in cell motility and phagocytosis. Thus, Spermidine provides a promising therapeutic potential to target glia cells in AD progression.

neuroscience↗