Biology subjects

Rizzoli, S.

Publications and source records attributed to Rizzoli, S..

18 recordsLinked to original sources

bioRxiv2026

Thousandfold Expansion Microscopy

Biological macromolecules, such as proteins, are made of concatenated building blocks. We hypothesized that individual protein residues could be imaged by anchoring their side chains to a swellable polymer, cleaving backbone amide bonds, and expanding residues away from each other to a degree that enables them to be visualized separately. We introduce thousandfold expansion microscopy (1000ExM), a four-network interpenetrating hydrogel architecture that enables successive expansion from [~]18-fold to >1000-fold (one billion-fold in volume). Protein and peptide structures are maintained across these expansion factors, as verified by analyses of proteins with known structures (nanobodies, GFP) and a well-studied peptide (mCLING). Computational analysis indicates that 1000ExM resolves adjacent amino acid residues, thereby achieving sub-nanometer precision on conventional light microscopes. We anticipate that 1000ExM will find wide utility in protein visualization and identification, potentially even in intact cells and tissues.

molecular biology↗

bioRxiv2026

Nanoscale imaging of expanded cells and proteins with spontaneously blinking dyes

Expansion microscopy (ExM) enables nanoscale imaging on standard microscopes, but combining ExM with single-molecule localization microscopy (SMLM) remains difficult, owing to the incompatibility of expanded hydrogels with photoswitching buffers. Here, we introduce a single-step expansion microscopy method that allows SMLM with spontaneously blinking dyes in 6-14x expanded samples, without re-embedding. We demonstrate nanometer-resolution imaging by resolving the organization of the nuclear pore complex (NPC) and the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen (PCNA).

biophysics↗

bioRxiv2026

Push-and-pull protein dynamics leads to log-normal synaptic sizes and probabilistic multi-spine plasticity

A typical neuron receives thousands of inputs and is able to adapt the strength of its synapses to store new information and meet ongoing computational demands. The synaptic response to plasticity induction is stochastic and spatially structured but is traditionally described by deterministic models representing the "average" dynamics. Growing experimental evidence indicates that not only the stimulation protocol determines the plasticity outcome but that the initial synaptic sizes, their fluctuations, and the spatial competition for the plasticity-relevant proteins play a decisive role. This probabilistic perspective makes it hard to predict the fate of a given synapse and requires a conceptual shift from a single synapse view to a probabilistic multi-spine competitive process where the plasticity needs and the available resources are considered together. Here, we propose a data-driven modeling framework able to predict collective plasticity outcomes along a dendrite based on the initial size, the number, and the spatial distance between simultaneously stimulated synapses. Our data analysis reveals a log-normal distribution of protein numbers for many plasticity-mediating proteins and shows that this log-normal protein allocation constrains and controls the collective plasticity outcome across multiple stimulated and non-stimulated synapses while preserving a global size distribution. Our findings highlight how local stochastic processes and global protein allocation rules give rise to synaptic plasticity outcomes, offering a new framework to understand and predict dendritic computation.

neuroscience↗

bioRxiv2025

A correlative workflow streamlines synaptic imaging by cryo-electron tomography

Despite decades of intense research, the molecular organization of the synapse is not well understood. To address this issue, we sought to develop a method for systematic imaging of synapses by cryo-electron tomography (cryo-ET), a technology capable of mapping cellular architecture at molecular resolution. Thinning of cellular samples by cryo-focused ion beam milling is a prerequisite for high-quality cryo-ET imaging, but this process needs to be guided to the structures of interest. To allow robust synaptic targeting, we established a correlative cryo-light/electron microscopy approach by which synapses are fluorescently labeled in a minimally invasive manner, using a synthetic binder of the postsynaptic scaffold PSD-95 and antibodies against the presynaptic protein Synaptotagmin-1. Cryo-ET imaging at sites of colocalization consistently revealed excitatory synapses. Our method allows structural studies of synaptic protein complexes in situ, facilitating investigations of the molecular architecture of synapses.

neuroscience↗

bioRxiv2025

Stable Actin As Synaptic Tag

According to the tagging and capture hypothesis, long-lasting long-term potentiation (LTP) requires protein synthesis and a synaptic tag, which is a synapse specific memory of the synapse with a so far unclear molecular or biophysical identity. Here we use an interdisciplinary approach to explore the hypothesis that interaction between the dynamics of actin and the spine geometry can provide such a memory. Using a mathematical model, we demonstrate that this implementation of the tag requires an increase in the stable, cross-linked pool of actin filaments, and is not possible without this stable pool. Using FRAP experiments, we show that such an increase in stable actin can indeed be observed after cLTP in vitro. Thus, the interaction between actin dynamics and spine geometry could indeed serve as a synaptic tag for LTP.

neuroscience↗

bioRxiv2025

Geometry effects on protein mobility in a synapse

It is generally assumed that synaptic function requires a tight regulation of the mobility and localisation of synaptic proteins. Evidence for this hypothesis has been difficult to gather. Protein mobility can be measured via fluorescence recovery after photobleaching (FRAP), but the inter- pretation of the results remains challenging. In this study, we perform in silico FRAP experiments to study the effects of the synaptic geometry and/or protein binding to synaptic vesicles on protein mobility. We matched simulations with published FRAP data for 40 different synaptic proteins, to obtain diffusion coefficients, vesicle binding rates, and binding times. Importantly, we identify two mechanisms that govern the obtained recovery times: redistribution of material inside the synaptic bouton and inflow through the axon. We show that their dissection is crucial for the correct interpretation of FRAP experiments, especially for proteins binding to synaptic vesicles.

biophysics↗

bioRxiv2024

Neddylation regulates the development and function of excitatory neurons

The development and function of neurons is orchestrated by a plethora of regulatory mechanisms that control the abundance, localization, interactions, and function of proteins. A key role in this regard is assumed by post-translational protein modifications (PTMs). While some PTM types, such as phosphorylation or ubiquitination, have been explored comprehensively, PTMs involving ubiquitin-like modifiers (Ubls) have remained comparably enigmatic (Ubls). This is particularly true for the Ubl Nedd8 and its conjugation to proteins, i.e. neddylation, in nerve cells. In the present study, we generated a conditional Nedd8 knock-out mouse line and examined the consequences of Nedd8-deletion in cultured postmitotic glutamatergic neurons. Our findings reveal that Nedd8-ablation in young glutamatergic neurons causes alterations in the expression of developmental transcription factors that control neuronal differentiation, ultimately leading to defects in the development of a mature glutamatergic neuronal phenotype. Apparent manifestations of these defects include increased vGlut2 expression levels, reduced vGlut1 and endophilin 1 expression levels, reduced dendrite complexity, and increased transmitter release probability. Collectively, our results highlight a pivotal role for neddylation in controlling the fate of glutamatergic neurons and excitatory synaptic transmission. HighlightsO_LIReduced dendrite complexity in Nedd8-deficient neurons C_LIO_LIIncreased synaptic vesicle fusion probability in Nedd8-deficient neurons C_LIO_LIAltered synaptic vesicle cycling in Nedd8-deficient neurons C_LIO_LINeddylation controls the maturation of glutamatergic neurons C_LI Graphical AbstractResearch Topic

neuroscience↗

bioRxiv2024

Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture

ABSTRACTSynaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification, but requires fixation. The latter enables monitoring of proteins in live microscopy, but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed neurons and brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 here as a highly-efficient dye for SNAP-tag labeling of extracellular epitopes, and of intracellular proteins in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal- and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for the analysis of presynaptic nanoarchitectural dynamics, with a potential for wide adoption.

neuroscience↗

bioRxiv2024

Power-law adaptation in the presynaptic vesicle cycle

After synaptic transmission, fused synaptic vesicles are recycled, enabling the synapse to recover its capacity for renewed release. The recovery steps, which range from endocytosis to vesicle docking and priming, have been studied individually, but it is not clear what their impact on the overall dynamics of synaptic recycling is, and how they influence signal transmission. Here we model the dynamics of vesicle recycling and find that the multiple timescales of the recycling steps are reflected in synaptic recovery. This leads to multi-timescale synapse dynamics, which can be described by a simplified synaptic model with power-law adaptation. Using cultured hippocampal neurons, we test this model experimentally, and show that the duration of synaptic exhaustion changes the effective synaptic recovery timescale, as predicted by the model. Finally, we show that this adaptation could implement a specific function in the hippocampus, namely enabling efficient communication between neurons through the temporal whitening of hippocampal spike trains.

neuroscience↗

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

NanoPlex: a universal strategy for fluorescence microscopy multiplexing using nanobodies with erasable signals

Fluorescence microscopy has long been a transformative technique in biological sciences. Nevertheless, most implementations are limited to a few targets, revealed using primary antibodies (1.Abs) and fluorescently conjugated secondary antibodies. Super-resolution techniques such as Exchange-PAINT and, more recently, SUM-PAINT have increased multiplexing capabilities, but they require specialized equipment, software, and knowledge. To enable multiplexing for any imaging technique in any laboratory, we developed NanoPlex, a streamlined method based on conventional 1.Abs revealed by engineered secondary nanobodies (2.Nbs) that allow to selectively erase the fluorescence signals. We developed three complementary signal removal strategies: OptoPlex (light-induced), EnzyPlex (enzymatic), and ChemiPlex (chemical). We showcase NanoPlex reaching 21 targets for 3D confocal analyses and 5-8 targets for dSTORM and STED super-resolution imaging. NanoPlex has the potential to revolutionize multi-target fluorescent imaging methods, potentially redefining the multiplexing capabilities of antibody-based assays.

neuroscience↗

bioRxiv2023

Erythropoietin restrains the inhibitory potential of interneurons in the mouse hippocampus

Erythropoietin (EPO) aids in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, thereby mitigating mood and cognition-associated disorders. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing [~] 12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.

neuroscience↗

bioRxiv2023

Dynamic Regulation of Vesicle Pools in a Detailed Spatial Model of the Complete Synaptic Vesicle Cycle

Synaptic transmission is driven by a complex cycle of synaptic vesicle docking, release, and recycling, and maintained by distinct vesicle pools with differing propensities for docking and release. However, the partitioning of vesicles into distinct pools and the conditions under which they are recruited from the reserve to the recycling pool remain poorly understood. Whilst computational modeling at the molecular level has progressed massively in recent years, modeling voluminous, dynamic, and heterogenous structures such as synaptic vesicles remains an unsolved problem. Here, we use a novel vesicle modeling technology to model the complete synaptic vesicle cycle in unprecedented molecular and spatial detail at a hippocampal en passant synapse, incorporating vesicle diffusion, the accumulation and diffusion of proteins on the vesicle surface, vesicle clustering, tethering, and docking, and regulated vesicle fusion and recycling. Our model demonstrates highly dynamic and robust recycling of synaptic vesicles able to maintain stable and consistent synaptic release over time, even during high frequency and sustained firing. We also reveal how the cytosolic proteins synapsin-1 and tomosyn-1 can cooperate to regulate the recruitment of vesicles from the reserve pool during sustained periods of synaptic activity in order to maintain transmission, as well as the potential of selective vesicle tethering close to the active zone to ensure rapid vesicle replenishment and enhance the efficiency of the vesicle cycle by minimizing the recruitment of vesicles from the reserve pool.

neuroscience↗

bioRxiv2022

Expansion microscopy at one nanometer resolution

Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of one-step nanoscale expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7 to enable the description of cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the [~]17 kDa protein calmodulin upon Ca2+ binding were readily observable. We also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinsons Disease (PD) patients, which represents a promising new development towards improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

biophysics↗

bioRxiv2021

IGF1 Receptor Regulates Upward Firing Rate Homeostasis via the Mitochondrial Calcium Uniporter

Regulation of firing rate homeostasis constitutes a fundamental property of central neural circuits. While intracellular Ca2+ has long been hypothesized to be a feedback control signal, the molecular machinery enabling network-wide homeostatic response remains largely unknown. Here we show that deletion of insulin-like growth factor-1 receptor (IGF1R), a well-known regulator of neurodevelopment and ageing, limits firing rate homeostasis in response to inactivity, without altering the baseline firing rate distribution. Disruption of both synaptic and intrinsic homeostatic plasticity contributed to deficient firing rate homeostatic response. At the cellular level, a fraction of IGF1Rs was localized in mitochondria with the mitochondrial calcium uniporter complex (MCUc). IGF1R deletion suppressed spike burst-evoked mitochondrial Ca2+ (mitoCa2+) by weakening mitochondria-to-cytosol Ca2+ coupling. MCUc overexpression in IGF1R-deficient neurons rescued the deficits in spike-to-mitoCa2+ coupling and firing rate homeostasis. Our findings highlight IGF1R as a key regulator of the integrated homeostatic response by tuning mitochondrial temporal filtering. Decline in mitochondrial reliability for burst transfer may drive dysregulation of firing rate homeostasis in ageing and brain disorders associated with aberrant IGF1R / MCUc signaling.

neuroscience↗

bioRxiv2020

Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging

Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.

cell biology↗

bioRxiv2020

White matter integrity requires continuous myelin synthesis at the inner tongue

Myelin, the electrically insulating axonal sheath, is composed of lipids and proteins with exceptionally long lifetime. This raises the question how myelin function is affected by myelin turnover. We have studied the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survived recombination, continued expressing myelin genes, but failed to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualized myelin-like membranes that failed to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicated degradation of existing myelin at the abaxonal side and at the inner tongue of the sheath. Compacted myelin thinning and shortening of internodes, with about 50% myelin lost after 20 weeks (=5 months), ultimately led to axonal pathology and neurological disease. These data reveal that functional axon-myelin units require the continuous incorporation of new myelin membranes.

neuroscience↗

bioRxiv2018

An in vivo knockdown strategy reveals multiple functions for circMbl

Circular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. circRNAs are enriched in the brain and accumulate with age in flies, worms and mice. Despite their abundance, little is known about their functions, especially in the context of whole organisms. Here we report the development and use of shRNAs to knock down and study the function of circMbl, the most abundant circRNA in Drosophila. This circRNA is highly conserved through evolution and is generated from the locus of the essential splicing factor muscleblind (mbl). Briefly, we generated flies in which circMbl is reduced more than 90% without measurable off-target effects in the hosting gene as well as in other RNAs. These flies display specific defects that suggest roles of circMbl in muscle and neural tissues during development and in adult flies. More specifically, whole organism downregulation of circMbl leads to male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. Moreover, these phenotypes are recapitulated by a second shRNA targeting circMbl. Importantly, knockdown and overexpression of circMbl affect mostly the same genes but in the opposite direction. Last but not least, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level likely by acting in multiple tissues. Moreover, here we provide the first proof of functionality of circRNAs in Drosophila as well as a methodological approach that enables the comprehensive study of circRNAs in vivo.\n\nSIGNIFICANCE STATEMENTCircular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. Here we report the development and use of a shRNA-based system to knockdown specific circRNAs in vivo. We generated flies in which circMbl, the most abundant circRNA is reduced more than 90% without measurable off-target effects. These flies display male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. These phenotypes are recapitulated by a second shRNA targeting circMbl. Moreover, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level and provide a methodological approach that enables the comprehensive study of circRNAs in vivo.

molecular biology↗