Biology subjects

Sibarita, J.-B.

Publications and source records attributed to Sibarita, J.-B..

18 recordsLinked to original sources

bioRxiv2026

Neurospheres from primary rodent brain cells to probe the 3D organization and function of synapses

To improve our understanding of synapse assembly, there is a need for robust, easy-to-use, and physiologically relevant in-vitro models allowing the controllable formation of neuronal contacts in a reasonable time, whose structure and function can be investigated using advanced microscopy. To address this challenge, we engineered 3D cultures from rodent dissociated hippocampal cells, that spontaneously assemble in low attachment U-bottom wells into compact spheroids of reproducible dimensions (100-300 microns), determined by the number of seeded cells. These neurospheres contain a mix of neurons and glial cells and grow over time in culture, through the combination of cell proliferation and neurite extension. Neurospheres were immunostained in fluid phase, and/or sparsely electroporated for the multi-color visualization of synaptic proteins. Neurons extend an elaborate network of axons and dendrites, forming within 2 weeks numerous excitatory and inhibitory synapses identified at the structural level by confocal and electron microscopy, and at the functional level by electrophysiology. Periodic calcium oscillations throughout neurospheres further highlight network activity. Finally, we demonstrate the potential of neurospheres to study synaptogenesis by modulating and visualizing the adhesion protein neuroligin-1. Overall, neurospheres represent a standardized and cost-effective system to study synapse structure and function at high resolution in 3D, that should be quite appealing to the cellular neurobiology community.

neuroscience↗

bioRxiv2025

PacL-organized membrane-associated effluxosomes coordinate multi-metal resistance in Mycobacterium tuberculosis

Metal ion homeostasis is crucial for bacterial pathogens to withstand metal-induced stress during infection. However, the mechanisms underlying bacterial resistance to metal stress remain incompletely understood, particularly how bacteria coordinate responses to simultaneous exposure to multiple metals. Here, we uncover a previously unrecognized mechanism by which Mycobacterium tuberculosis, the causative agent of tuberculosis, orchestrates a coordinated response to multi-metal stress. We demonstrate that M. tuberculosis assembles dynamic, membrane-associated platforms, organized by PacL proteins, that confer resistance to multiple metals simultaneously. PacL proteins function as scaffolds, clustering multiple P-type ATPase (P-ATPase) pumps, CtpC, CtpG, and CtpV, into functional complexes we term "effluxosomes". Our findings show that PacL proteins are critical for stabilizing CtpG within membrane-associated clusters, conferring cadmium tolerance, while CtpC serves as a backup, promoting cross-resistance to both zinc and cadmium. Using super-resolution microscopy and single-particle tracking, we elucidate the 3D structure and dynamics of effluxosomes in the mycobacterial membrane. We further demonstrate that conserved residues within the transmembrane domain of PacL proteins are crucial for the assembly of dynamic effluxosomes, which are essential for P-ATPase activity. Additionally, we reveal that PacL1 exhibits metallochaperone activity, binding zinc, cadmium, and copper via a conserved C-terminal motif. Proximity labeling further identifies an extensive PacL1 interaction network, encompassing multiple proteins involved in stress adaptation. Our findings introduce effluxosomes as dynamic, membrane-associated efflux machineries that mediate coordinated multi-metal resistance in M. tuberculosis, providing new insights into bacterial metal homeostasis and unveiling potential antimicrobial targets.

microbiology↗

bioRxiv2025

Extracellular matrix deposition controls early differentiation patterns in human cardiac gastruloids

Amongst the various factors that affect differentiation and tissue organization, the autonomous deposition of extracellular matrix (ECM) has hardly been considered in the context of gastruloids. Using biofunctionalized colloidal particles as artificial organizing centers (aOCs), we patterned differentiation loci within 3D embryonic bodies. Our findings reveal that the spatial distribution of the aOCs induces various pattern organizations, mediated by endogenous deposition of ECM. We investigated how the interplay between meso-endoderm cell epithelial-to-mesenchymal transition, migration into germ layers, and the prolonged maintenance of local stem-cell niches orchestrates gastruloid structure and composition. These factors collectively regulate the complexity of emergent cardiac structures observed at later time points. This work uncovers a critical feedback loop between cell differentiation rates and endogenous ECM deposition patterns, which governs the differentiation paths in scaffolded cardiac gastruloids.

cell biology↗

bioRxiv2024

Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy

Fluorescence lifetime imaging (FLIM) is widely used for functional and multiplexed bioimaging. The lifetime of autofluorescence or fluorescent sensors encodes physiologically relevant parameters. Thus, FLIM is especially relevant for the investigation of living systems. However, application of FLIM to live specimen is hampered by its slow speed and high phototoxicity. To enable faster and gentler FLIM, we integrated single-objective light-sheet microscopy with pulsed excitation and time-resolved detection on a novel SPAD array detector. We achieved 10-100-fold acceleration compared to confocal FLIM, down to 100 ms acquisition time per image, with excellent quantitative agreement. The massively enhanced speed enables volumetric FLIM acquisitions on live multicellular specimens, which we demonstrate with lifetime-based multiplexing in 3D and time-lapse FLIM of tension probes on living embryonic organoids. We benchmark both scanned and static light-sheet modalities to facilitate adding FLIM capability to a large variety of light-sheet microscopes.

biophysics↗

bioRxiv2022

Selective volumetric excitation and imaging for single molecule localization microscopy in multicellular systems

Light sheet fluorescence microscopy (LSFM) has become a leading standard in high-resolution imaging of living samples in 2- and 3-dimensions. Biological samples are however not restricted to a single observation plane and several molecular processes evolve rapidly in 3D. The conventional mechanical scanning required in LSFM limits the range of observable dynamics and are usually restricted in resolution. Here we introduce a new strategy for instantaneous volumetric excitation and volumetric imaging of single-molecules in cell aggregates. The technique combines, for the first time, the use of light sheet microscopy and multifocus microscopy (MFM) and enables a volumetric 4D imaging of biological samples with single-molecule resolution. We engineered the excitation beam to yield a modular and uniform excitation matching the observable detection range of MFM. The strength of the method is highlighted with examples of single-molecule 3D tracking and 3D super-resolution imaging in multicellular samples.

biophysics↗

bioRxiv2022

SalienceNet: an unsupervised Image-to-Image translation method for nuclei saliency enhancement in microscopy images

Automatic segmentation of nuclei in low-light microscopy images remains a difficult task, especially for high-throughput experiments where need for automation is strong. Low saliency of nuclei with respect to the background, variability of their intensity together with low signal-to-noise ratio in these images constitute a major challenge for mainstream algorithms of nuclei segmentation. In this work we introduce SalienceNet, an unsupervised deep learning-based method that uses the style transfer properties of cycleGAN to transform low saliency images into high saliency images, thus enabling accurate segmentation by downstream analysis methods, and that without need for any parameter tuning. We have acquired a novel dataset of organoid images with soSPIM, a microscopy technique that enables the acquisition of images in low-light conditions. Our experiments show that SalienceNet increased the saliency of these images up to the desired level. Moreover, we evaluated the impact of SalienceNet on segmentation for both Otsu thresholding and StarDist and have shown that enhancing nuclei with SalienceNet improved segmentation results using Otsu thresholding by 30% and using StarDist by 26% in terms of IOU when compared to segmentation of non-enhanced images. Together these results show that SalienceNet can be used as a common preprocessing step to automate nuclei segmentation pipelines for low-light microscopy images.

bioinformatics↗

bioRxiv2022

Multi-synaptic boutons are a feature of CA1 hippocampal connections that may underlie network synchrony

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from P21 to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared to neighbouring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favour synchronous activity in CA1 networks.

neuroscience↗

bioRxiv2022

EndophilinA-dependent coupling between activity-dependent calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation

Neuronal activity and neurotransmitter release cause use-dependent decline in protein function. However, it is unclear how this is coupled to local protein turnover and quality control mechanisms. Here we show that the endocytic protein Endophilin-A (EndoA/ENDOA1) couples activity-induced calcium influx to synaptic autophagy and neuronal survival. We identify single mutations in the EndoA flexible region that either increases EndoA diffusion and promotes autophagosome formation in the absence of calcium, or immobilizes EndoA and blocks autophagy, even in the presence of calcium. Hence, the EndoA flexible region is a switch that responds to calcium, regulating EndoA nanoscale synaptic organization and association with autophagosomes driving their formation. Interestingly, a pathogenic variant in the human ENDOA1 variable region that confers risk to Parkinsons disease (PD), also confines ENDOA1 to the synaptic plasma membrane and equally blocks autophagy in flies in vivo and in induced human neurons. Thus, our work reveals a mechanism neurons use to connect neuronal activity to local protein turnover by autophagy, which is critical for neuronal survival.

neuroscience↗

bioRxiv2022

Single-molecule imaging reveals Tau trapping at nanometer-sized dynamic hot spots near the plasma membrane

Accumulation of Tau aggregates is a pathological hallmark of Alzheimers disease, but little is known about where these aggregates assemble and how stable these assemblies are. Using quantitative, single-molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane, resulting in the formation of nanometer-sized hot spots smaller than the diffraction limit. The hot spots lasted tens of seconds, much longer than the short dwell time (~40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not affect hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions.

neuroscience↗

bioRxiv2021

A framework for evaluating the performance of SMLM cluster analysis algorithms

Single molecule localisation microscopy (SMLM) generates data in the form of Cartesian coordinates of localised fluorophores. Cluster analysis is an attractive route for extracting biologically meaningful information from such data and has been widely applied. Despite the range of developed cluster analysis algorithms, there exists no consensus framework for the evaluation of their performance. Here, we use a systematic approach based on two metrics, the Adjusted Rand Index (ARI) and Intersection over Union (IoU), to score the success of clustering algorithms in diverse simulated clustering scenarios mimicking experimental data. We demonstrate the framework using three analysis algorithms: DBSCAN, ToMATo and KDE, show how to deduce optimal analysis parameters and how they are affected by fluorophore multiple blinking. We propose that these standard conditions and metrics become the basis for future analysis algorithm development and evaluation.

biophysics↗

bioRxiv2021

High content 3D imaging method for quantitative characterization of organoid development and phenotype.

Quantitative analysis on a large number of organoids can provide meaningful information from the morphological variability observed in 3D organotypic cultures, called organoids. Yet, gathering statistics of growing organoids is currently limited by existing imaging methods and subsequent image analysis workflows that are either restricted to 2D, limited in resolution, or with a low throughput. Here, we present an automated high content imaging platform synergizing high density organoid cultures with 3D live light-sheet imaging. The platform is an add-on to a standard inverted microscope. We demonstrate our capacity to collect libraries of 3D images at a rate of 300 organoids per hour, enabling training of artificial intelligence-based algorithms to quantify the organoid morphogenetic organization at multiple scales with subcellular resolution. We validate our approach on different organotypic cell cultures (stem, primary, and cancer), and quantify the development of hundreds of neuroectoderm organoids (from human Embryonic Stem Cells) at cellular, multicellular and whole organoid scales.

biophysics↗

bioRxiv2020

CaMKII activation triggers persistent formation and segregation of postsynaptic liquid phase

Transient information input to brain leads to persistent changes in synaptic circuit, thereby forming memory engrams. Synapse undergoes coordinated functional and structural changes during this process but how such changes are achieved by its component molecules still largely remain enigmatic. We found that activated CaMKII, the central player of synaptic plasticity, undergoes liquid-liquid phase separation (LLPS) with NMDAR subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B co-segregates AMPAR/neuroligin (NLGN) into a phase-in-phase assembly. Because postsynaptic NLGN clusters presynaptic neurexin and other active zone proteins thereby increasing the release probability of synaptic vesicles, this ensures efficient synaptic transmission. In this way, Ca2+-induced and persistent formation of LLPS by CaMKII serves as molecular basis of memory by functioning as an activity-dependent crosslinker for postsynaptic proteins and segregating trans-synaptic nanocolumns.

neuroscience↗

bioRxiv2020

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

AO_SCPLOWBSTRACTC_SCPLOWFocal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

cell biology↗

bioRxiv2019

SpineJ: A software tool for quantitative analysis of nanoscale spine morphology

Super-resolution microscopy provides diffraction-unlimited optical access to the intricate morphology of neurons in living brain tissue, resolving their finest structural details, which are critical for neuronal function. However, as existing image analysis software tools have been developed for diffraction-limited images, they are generally not well suited for quantifying nanoscale structures like dendritic spines. We present SpineJ, a semi-automatic ImageJ plugin that is specifically designed for this purpose. SpineJ comes with an intuitive and user-friendly graphical user interface, facilitating fast, accurate, and unbiased analysis of spine morphology.

bioinformatics↗

bioRxiv2019

Specific nanoscale synaptic reshuffling and control of short-term plasticity following NMDAR- and P2XR-dependent Long-Term Depression

Long-Term Potentiation (LTP) and Long-Term Depression (LTD) of excitatory synaptic transmission are considered as cellular basis of learning and memory. These two forms of synaptic plasticity have been mainly attributed to global changes in the number of synaptic AMPA-type glutamate receptor (AMPAR) through a regulation of the diffusion/trapping balance at the PSD, exocytosis and endocytosis. While the precise molecular mechanisms at the base of LTP have been intensively investigated, the ones involved in LTD remains elusive. Here we combined super-resolution imaging technique, electrophysiology and modeling to describe the various modifications of AMPAR nanoscale organization and their effect on synaptic transmission in response to two different LTD protocols, based on the activation of either NMDA receptors or P2X receptors. While both type of LTD are associated with a decrease in synaptic AMPAR clustering, only NMDAR-dependent LTD is associated with a reorganization of PSD-95 at the nanoscale. This change increases the pool of diffusive AMPAR improving synaptic short-term facilitation through a post-synaptic mechanism. These results demonstrate that specific dynamic reorganization of synapses at the nanoscale during specific LTD paradigm allows to improve the responsiveness of depressed synapses.

neuroscience↗

bioRxiv2018

Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities - 2D, astigmatic 3D, biplane 3D, and double helix 3D - and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

biophysics↗

bioRxiv2018

Live single molecule microscopy of HIV-1 assembly in host T cells reveals a spatio-temporal effect of the viral genome.

Monitoring virus assembly dynamic at the nanoscale level in host cells remains a major challenge. Human Immunodeficiency Virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100nm in diameter. HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble immature virus. Here, we monitored Gag assembly in host CD4 T lymphocytes using single molecule dynamics microscopy and energy mapping. A workflow allowing long time recordings of single Gag molecule localization, diffusion and effective energy maps was developed for robust quantitative analysis of HIV assembly and budding. Comparison of numerous cell plasma membrane assembling platforms in cells expressing wild type or assembly-defective Gag proteins showed that VLP formation last 15 minutes, with an assembly time of 5 minutes, and that the nucleocapsid domain is mandatory. Importantly, it reveals that the viral genome coordinates spatio-temporally HIV-1 assembly.

biophysics↗

bioRxiv2017

Fast Activation Cycles Of Rac1 At The Lamellipodium Tip Trigger Membrane Protrusion

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE complex, effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Yet, correlation of Rho GTPases activation with cycles of membrane protrusions, suggested that Rac1 activation is not synchronized with membrane protrusion and occurs behind the lamellipodium. However, RhoA activation is maximal at the cell edge and synchronized with edge progression. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function of Rho GTPases mutants, we demonstrate that Rac1 immobilizations at the lamellipodium tip are correlated with Rac1 activation, on the contrary to RhoA. We show that Rac1 effector WAVE and Rac1 regulator IRSp53 accumulate at the lamellipodium tip by membrane free-diffusion and trapping. Nevertheless, wild-type Rac1, which directly interacts with WAVE and IRSp53, only displays slower diffusion at the lamellipodium tip, suggesting fast local activation/inactivation cycles. Local optogenetic activation of Rac1, triggered by Tiam1 membrane recruitment, proves that Rac1 activation must occur at the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. Furthermore, coupling tracking with optogenetic activation of Rac1 demonstrates that Rac1-WT diffusive properties are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model where Rac1 is rapidly switching between activation and inhibition at the lamellipodium tip, ensuring a local and fast control of Rac1 actions on its targets.\n\nSignificanceRac1 and RhoA GTPases are molecular switches controlling the actin cytoskeletal during cell migration. WAVE, Rac1 effector during cell protrusion, is concentrated at the lamellipodium tip. But, recent biosensor imaging studies suggested that Rac1 activation occurs behind the lamellipodium, while RhoA activation is maximal at the cell edge. Using single-molecule imaging and optogentics Rac1 activation we solved this apparent contradiction. We revealed a strong correlation between Rac1 activation and transient immobilizations at the lamellipodium tip, unlike RhoA. Furthermore, we demonstrated that Rac1 must be activated at the lamellipodium tip and not away from it to stimulate protrusion. Thus, fast cycling between activation and inhibition at the proximity of Rac1 targets ensures a local and fast control over Rac1 actions.\n\nAbbreviations

cell biology↗