Biology subjects

Sainlos, M.

Publications and source records attributed to Sainlos, M..

13 recordsLinked to original sources

bioRxiv2026

Adaptive-optics lattice light-sheet microscopy enables correlative single-molecule imaging of endogenous proteins in native environments

Resolving molecular organization and dynamics in native environments remains challenging, as single-molecule imaging requires high sensitivity and spatiotemporal resolution while preserving 3D cellular context. Here, we present ALPINE (Adaptive-optics Lattice light-sheet microscopy for endogenous Protein Imaging in Native Environments), a modular framework for quantitative single-molecule imaging into complex biological samples. ALPINE combines adaptive-optics lattice light-sheet microscopy, programmable PSF shaping and acquisition stabilization, an engineered cell-specific labelling toolkit for endogenous cell-surface proteins and analysis pipelines assigning molecular localizations and trajectories to 3D cellular compartments. Labelling endogenous AMPA receptors in intact brain tissue, we demonstrate aberration-corrected 3D single-molecule localization and nanoscale organization within neuronal compartments. Importantly, fast optical sectioning of lattice light-sheet illumination further enables 3D single-molecule tracking in live tissue and quantification of molecular dynamics within native cellular environment. Together, ALPINE provides a modular approach to investigate how endogenous molecules are organized and dynamically regulated in complex biological systems, with components adaptable to diverse biological questions and imaging modalities.

neuroscience↗

bioRxiv2026

Dishevelled2 Condensates control Axon Initial Segment (AIS) assembly and function through MTCL1-Dependent microtubule recruitment.

In the epithelium, Planar Cell Polarity (PCP) signaling coordinates cellular organization along the plane of the tissue, perpendicular to the apicobasal axis. In neurons, polarity is instead expressed through the structural and molecular asymmetry that separates the axon from the somatodendritic compartment. Core PCP proteins are essential for neuronal development and function, and have been linked to neurodevelopmental disorders, including autism spectrum disorders and epilepsy. However, their precise roles in the establishment of neuronal polarity remain incompletely understood. Here, we identify Dishevelled2 (Dvl2), a central PCP signaling scaffold, as a novel component of the axon initial segment (AIS), the proximal axonal domain that maintains neuronal polarity and initiates action potentials. We show that Dvl2 defines, both in vivo and in cultured hippocampal neurons, a proximal subdomain positioned upstream of the major AIS marker Ankyrin-G (Ank-G) and is closely associated with microtubule bundles. Dvl2 is already detected during early axon specification, suggesting that it participates in the initial organization of the proximal axon. Functional depletion of Dvl proteins disrupts AIS architecture, reduces the accumulation of Ankyrin-G and other core AIS components, and impairs the organization of AIS microtubule bundles. These structural defects translate into altered neuronal excitability, with a reduced AIS-dependent component of the action potential. Mechanistically, we identify MTCL1 as a microtubule-associated partner that recruits Dvl2 from biomolecular condensate assemblies onto microtubules. This recruitment requires the DIX and PDZ domains of Dvl2. Notably, selective intracellular binders targeting the Dvl2 PDZ domain perturb Dvl2 condensate organization and compromise AIS formation. Together, our data identify Dvl2 as an organizer of the proximal AIS and reveal a Dvl2-MTCL1-dependent mechanism coupling condensate scaffolding, microtubule organization, and neuronal excitability.

neuroscience↗

bioRxiv2026

βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

Neuron connectivity and signal processing rely on a complex, dynamic morphology regulated by the cytoskeleton. The membrane-associated periodic skeleton (MPS), a 190-nm periodic actin-spectrin lattice, is a conserved feature of neurons. Here, we show that the dendritic MPS is built from a dual {beta}-spectrin system in which {beta}II- and {beta}III-spectrins are co-expressed and interleaved at the nanoscale in shafts and spine necks. 3D MINFLUX nanoscopy suggests that these paralogues form both homotypic ({beta}II- or {beta}III-only) and heterotypic ({beta}II/{beta}III) tetramers with an approximately 100-nm radial periodicity. Either paralogue alone is sufficient to maintain lattice architecture, whereas their combined loss disrupts MPS integrity. Using targeted mutagenesis, we show that actin binding is required to stabilize both paralogues within the MPS, whereas {beta}III-spectrin additionally depends on phosphoinositide interactions. Our findings reveal that, unlike the axonal MPS, the dendritic MPS is a composite scaffold in which structural redundancy coexists with paralogue-specific regulatory mechanisms, with potential consequences for synaptic function.

neuroscience↗

bioRxiv2025

Synchrotron XRF imaging reveals manganese accumulation in the Golgi and post-synapses of neurons and enhanced uptake in astrocytes

Manganese is an essential trace metal for humans, but excessive exposure can cause neurotoxicity, including parkinsonian syndromes, cognitive deficits, and has also been implicated in the pathogenesis of neurodegenerative diseases. Globally, tens of millions of people are exposed to elevated manganese levels through drinking water, exceeding the WHO recommended guideline. Despite its public health significance, the cellular and subcellular mechanisms underlying manganese neurotoxicity remain poorly defined, particularly its distribution across brain cell types and its specific intracellular targets. In this study, we investigated manganese accumulation in primary cultures of hippocampal neurons and astrocytes. Using a correlative imaging approach that combined cryo-fluorescence light microscopy with synchrotron X-ray fluorescence imaging, we mapped and quantified manganese at subcellular resolution. Our analysis revealed that manganese preferentially accumulates in the Golgi apparatus of both neurons and astrocytes. In neurons, manganese was also detected in dendrites at the postsynaptic density, suggesting a role in synaptic vulnerability. Quantitative elemental analysis showed that astrocytes accumulated 3 times more manganese than neurons. Furthermore, when neurons were co-cultured with astrocytes, their manganese uptake was significantly reduced, indicating a possible protective or buffering role of astrocytes. These findings identify key cellular and subcellular targets of manganese and highlight the Golgi apparatus as a major regulator of manganese neurotoxicity. This work provides a foundation for understanding cell type-specific responses to manganese exposure and may inform the development of targeted neuroprotection strategies. Significance statementManganese neurotoxicity impact millions of people worldwide. Understanding how manganese distributes within brain cells is critical for addressing its neurotoxic effects. This study identifies the Golgi apparatus and postsynaptic density as key sites of manganese accumulation and reveals astrocytes as major regulators of neuronal manganese uptake. These findings provide a cellular framework for developing targeted strategies to mitigate manganese-induced neurotoxicity.

neuroscience↗

bioRxiv2025

Paralog-specific intrabodies for PSD-93 and SAP102 expand the molecular toolkit to resolve excitatory synapse organization

A scarcity of live, paralog-specific tools has limited analysis of PSD-MAGUKs at excitatory synapses. To address this gap, we engineered small, 10FN3-derived binders that selectively recognize PSD-93 and SAP102 -alongside an enhanced PSD-95 reagent- and converted them into regulated, gene-encoded intrabodies for endogenous imaging. Through sequence-guided selection and targeted optimization, we obtained high-specificity reagents that label their native targets in neurons with minimal perturbation and support multiplexed live-cell and advanced imaging modalities. This toolkit enables differential visualization of MAGUK paralogs at native levels and provides a practical route to dissect their distinct contributions to synapse organization and plasticity.

bioengineering↗

bioRxiv2024

Synapse specific and plasticity-regulated AMPAR mobility tunes synaptic integration

Synaptic responses adapt to fast repetitive inputs during bursts of neuronal network activity over timescales of milliseconds to seconds, either transiently facilitating or depressing. This high-frequency stimulus-dependent short-term synaptic plasticity (HF-STP) relies on a number of molecular processes that collectively endow synapses with filtering properties for information processing, optimized for the transmission of certain input frequencies and patterns in distinct circuits 1-3. Changes in HF-STP are traditionally thought to stem from changes in pre-synaptic transmitter release 1,2, but post-synaptic modifications in receptor biophysical properties or surface diffusion also regulate HF-STP 4-11. A major challenge in understanding synapse function is to decipher how pre- and post-synaptic mechanisms synergistically tune synaptic transmission efficacy during HF-STP, and to determine how neuronal activity modifies post-synaptic signal computation and integration to diversify neuronal circuit function. Here, taking advantage of new molecular tools to directly visualize glutamate release 12 and specifically manipulate the surface diffusion of endogenous AMPAR in intact circuits 13, we define the respective contributions of pre-synaptic glutamate release, AMPAR desensitization and surface mobility to frequency-dependent synaptic adaptation. We demonstrate that post-synaptic gain control and signal integration capacity in synaptic networks is influenced by synapse-specific differences in AMPAR desensitization and diffusion-trapping characteristics that are shaped by molecular signaling events recruited during LTP.

neuroscience↗

bioRxiv2024

Dissecting the binding mechanisms of synaptic membrane adhesion complexes using a micropattern based cellular model

The formation of adhesive cell-cell contacts is based on the intrinsic binding properties between specific transmembrane ligand-receptor pairs. In neurons, synaptic adhesion molecules provide a physical linkage between pre- and post-synaptic compartments, but the strength and the dynamic of these complexes in their actual membrane environments remain essentially unknown. To access such information, we developed a versatile assay to measure the affinity and binding kinetics of synaptic ligand-receptor interactions, based on the immobilization of Fc-tagged ligands on micropatterned substrates combined with live imaging of fluorescently-tagged counter receptors in heterologous cells. We applied this strategy to study the heterophilic complex formed between neurexin-1{beta} (Nrx1{beta}) and neuroligin-1 (Nlg1), compared to the homophilic SynCAM1 complex. First, the control of ligand density combined to the measurement of steady-state receptor enrichment at micropatterns demonstrates the high specificity of the matching molecular interactions and allows for the quantification of the two-dimensional affinity of the interaction in a membrane environment. Second, long-term FRAP experiments performed on the two molecular complexes and fitted with analytical models, demonstrate a diffusion-limited regime for SynCAM1 and a reaction-limited regime for Nlg1. This analysis provides a very long bond lifetime of the Nrx1{beta}-Nlg1 complex, which by comparison with a monomeric mutant of Nlg1, can be attributed to the constitutive dimerization of Nlg1. Finally, we used the stable Nrx1{beta}-Nlg1 complex as a pseudo-synaptic platform to analyze the rapid binding kinetics between the scaffolding protein PSD-95 and the intracellular domain of Nlg1, dissecting the contribution of the different PDZ domains through the use of specific PSD-95 point mutants.

neuroscience↗

bioRxiv2021

High-resolution imaging and manipulation of endogenous AMPA receptor surface mobility during synaptic plasticity and learning

Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPAR) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminus. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP-GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing for target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

neuroscience↗

bioRxiv2021

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

neuroscience↗

bioRxiv2021

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior

MDGAs are molecules that can bind neuroligins in cis and interfere with trans-synaptic neurexin-neuroligin interactions, thereby impairing synapse development. However, the sub-cellular localization and dynamics of MDGAs, as well as their specific mode of action in neurons are still unclear. Here, using both surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons, we show that MDGA1 and MDGA2 molecules are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation in culture Using shRNAs and CRISPR/Cas9 strategies to knock-down/out MDGA1 or MDGA2, we demonstrate an increase in the density of excitatory synapses accompanied by enhanced membrane immobilization and an increase in the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, we show that decreasing MDGA expression level reduces the mobility of AMPA receptors and increases the frequency of AMPA receptor mediated mEPSCs. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.

neuroscience↗

bioRxiv2018

Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

During clathrin mediated endocytosis (CME), membrane scission is achieved by the concerted action of dynamin and its interacting partners. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamins efficient recruitment and function. First, we show in dynamin triple knock-out cells that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, are partially capable of rescuing CME. However, mutating two motifs largely prevents that ability. To support this observation, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME 2 to 3 times more effectively than with monovalent ones. Finally, the frequency of endocytic events decreases with competing peptides or hypomorphic rescue mutants but the kinetics of dynamin recruitment is unaffected. This suggests that PRD-SH3 interactions act upstream of dynamin accumulation at the neck of nascent vesicles. We conclude from these data that dynamin drives vesicle scission via multivalent interactions in vivo.

cell biology↗

bioRxiv2018

Pharmacological modulation of AMPA receptor surface diffusion restores hippocampal synaptic plasticity and memory in Huntington’s disease

Impaired hippocampal synaptic plasticity is increasingly considered to play an important role in cognitive impairment in Huntingtons disease (HD). However, the molecular basis of synaptic plasticity defects is not fully understood. Combining live-cell nanoparticle tracking and super-resolution imaging, we show that dysregulation of AMPA receptors (AMPARs) surface diffusion represents a molecular basis underlying the aberrant hippocampal synaptic plasticity during HD. AMPARs surface diffusion is increased in various HD neuronal models, which results in the failure of AMPARs surface stabilization after long-term potentiation (LTP) stimuli. This appears to result from a defective brain-derived neurotrophic factor (BDNF) - tyrosine receptor kinase B (TrkB) - Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling pathway that impacts the interaction between the AMPAR auxiliary subunit stargazin and postsynaptic density protein 95 (PSD-95). Notably, the disturbed AMPAR surface diffusion is rescued, via BDNF signaling pathway and by the antidepressant tianeptine. Tianeptine also restores the impaired LTP and hippocampus-dependent memory as well as anxiety/depression-like behavior in different HD mouse models. We thus unveil a mechanistic framework underlying hippocampal synaptic and memory dysfunction and propose a new perspective in HD treatment by targeting AMPAR surface diffusion.

neuroscience↗

bioRxiv2017

Pre-post synaptic alignment through neuroligin tunes synaptic transmission efficiency

The nanoscale organization of neurotransmitter receptors relative to pre-synaptic release sites is a fundamental determinant of both the amplitude and reliability of synaptic transmission. How modifications in the alignment between pre- and post-synaptic machineries affect synaptic current properties has only been addressed with computer modeling, and therefore remains hypothetical. Using dual-color single molecule based super-resolution microscopy, we found a strong spatial correlation between AMPA receptor (AMPAR) nanodomains and the post-synaptic adhesion protein neuroligin-1 (NLG1). Expression of a C-terminal truncated form of NLG1 disrupted this correlation without affecting the intrinsic organization of AMPAR nanodomains. Moreover, this NLG1 dominant-negative mutant significantly shifted the pre-synaptic release machinery from AMPAR synaptic clusters. Electrophysiology and computer modeling show that this physical shift is sufficient to induce a significant decrease in synaptic transmission. Thus, our results suggest the necessity for synapses to release glutamate in front of AMPAR nanodomains, to maintain a high efficiency of synaptic responses.

neuroscience↗