Publications by authors named "Jean-Baptiste Sibarita"

74 Publications

Advanced imaging and labelling methods to decipher brain cell organization and function.

Nat Rev Neurosci 2021 Apr 12;22(4):237-255. Epub 2021 Mar 12.

University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France.

The brain is arguably the most complex organ. The branched and extended morphology of nerve cells, their subcellular complexity, the multiplicity of brain cell types as well as their intricate connectivity and the scattering properties of brain tissue present formidable challenges to the understanding of brain function. Neuroscientists have often been at the forefront of technological and methodological developments to overcome these hurdles to visualize, quantify and modify cell and network properties. Over the last few decades, the development of advanced imaging methods has revolutionized our approach to explore the brain. Super-resolution microscopy and tissue imaging approaches have recently exploded. These instrumentation-based innovations have occurred in parallel with the development of new molecular approaches to label protein targets, to evolve new biosensors and to target them to appropriate cell types or subcellular compartments. We review the latest developments for labelling and functionalizing proteins with small localization and functionalized reporters. We present how these molecular tools are combined with the development of a wide variety of imaging methods that break either the diffraction barrier or the tissue penetration depth limits. We put these developments in perspective to emphasize how they will enable step changes in our understanding of the brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41583-021-00441-zDOI Listing
April 2021

Distance-dependent regulation of NMDAR nanoscale organization along hippocampal neuron dendrites.

Proc Natl Acad Sci U S A 2020 09 14;117(39):24526-24533. Epub 2020 Sep 14.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, 33076 Bordeaux, France;

Hippocampal pyramidal neurons are characterized by a unique arborization subdivided in segregated dendritic domains receiving distinct excitatory synaptic inputs with specific properties and plasticity rules that shape their respective contributions to synaptic integration and action potential firing. Although the basal regulation and plastic range of proximal and distal synapses are known to be different, the composition and nanoscale organization of key synaptic proteins at these inputs remains largely elusive. Here we used superresolution imaging and single nanoparticle tracking in rat hippocampal neurons to unveil the nanoscale topography of native GluN2A- and GluN2B-NMDA receptors (NMDARs)-which play key roles in the use-dependent adaptation of glutamatergic synapses-along the dendritic arbor. We report significant changes in the nanoscale organization of GluN2B-NMDARs between proximal and distal dendritic segments, whereas the topography of GluN2A-NMDARs remains similar along the dendritic tree. Remarkably, the nanoscale organization of GluN2B-NMDARs at proximal segments depends on their interaction with calcium/calmodulin-dependent protein kinase II (CaMKII), which is not the case at distal segments. Collectively, our data reveal that the nanoscale organization of NMDARs changes along dendritic segments in a subtype-specific manner and is shaped by the interplay with CaMKII at proximal dendritic segments, shedding light on our understanding of the functional diversity of hippocampal glutamatergic synapses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1922477117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533699PMC
September 2020

Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes.

Biophys J 2020 09 12;119(6):1157-1177. Epub 2020 Aug 12.

Aix Marseille University, CNRS, INSERM, LAI, Turing Centre for Living Systems, Marseille, France. Electronic address:

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fiber traction, whereas the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on two-dimensional and in three-dimensional solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimental and computational evidence that leukocytes do swim, and that efficient propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell external membrane. Our model consists of a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. Furthermore, continuous paddling is enabled by a combination of external treadmilling and selective recycling by internal vesicular transport of cortex-bound transmembrane proteins. This mechanism explains observations that swimming is five times slower than the retrograde flow of cortex and also that lymphocytes are motile in nonadherent confined environments. Resultantly, the ubiquitous ability of mammalian amoeboid cells to migrate in two dimensions or three dimensions and with or without adhesion can be explained for lymphocytes by a single machinery of heterogeneous membrane treadmilling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2020.07.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499394PMC
September 2020

Nanoscale co-organization and coactivation of AMPAR, NMDAR, and mGluR at excitatory synapses.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14503-14511. Epub 2020 Jun 8.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, 33077 Bordeaux, France;

The nanoscale co-organization of neurotransmitter receptors facing presynaptic release sites is a fundamental determinant of their coactivation and of synaptic physiology. At excitatory synapses, how endogenous AMPARs, NMDARs, and mGluRs are co-organized inside the synapse and their respective activation during glutamate release are still unclear. Combining single-molecule superresolution microscopy, electrophysiology, and modeling, we determined the average quantity of each glutamate receptor type, their nanoscale organization, and their respective activation. We observed that NMDARs form a unique cluster mainly at the center of the PSD, while AMPARs segregate in clusters surrounding the NMDARs. mGluR5 presents a different organization and is homogenously dispersed at the synaptic surface. From these results, we build a model predicting the synaptic transmission properties of a unitary synapse, allowing better understanding of synaptic physiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1922563117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321977PMC
June 2020

The adaptor protein APS modulates BCR signalling in mature B cells.

Cell Signal 2020 09 26;73:109673. Epub 2020 May 26.

INSERM, U978, UFR SMBH, Bobigny, France; Comue USPC, Labex Inflamex, Université Paris 13, UFR SMBH, Bobigny, France. Electronic address:

Activation process of mature B cell is predominantly driven by specific BCR-mediated pathways, switched on and off all through late B cell differentiation stages. Mice deficient for APS, a member of the Lnk/SH2B family of adaptor proteins, showed that this adaptor plays a BCR-mediated regulatory role in mature B cells. However, the intermediates involved in this adaptor modulating functions in B cells are still unknown. In the present study, we investigated the role of APS in regulating BCR signalling notably through cytoskeleton remodeling in mature B cells. Herein, we showed that APS function is stage specific, as it exclusively intervenes in mature B cells. Upon activation, APS colocalizes with the BCR and associates with important regulators of BCR signalling, such as Syk and Cbl kinase. Importantly, APS interferes, as a scaffold protein, with the stability of Syk kinase by recruiting Cbl. This function is mainly mediated by APS SH2 domain, which regulates BCR-evoked cell dynamics. Our findings thus reveal that APS plays a regulatory role in BCR-induced responses by specifically modulating its interacting partners, which positions APS as a relevant modulator of BCR signalling in mature B cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellsig.2020.109673DOI Listing
September 2020

Real-time nanoscale organization of amyloid precursor protein.

Nanoscale 2020 Apr 7;12(15):8200-8215. Epub 2020 Apr 7.

Centre for Neuroscience, Indian Institute of Science, Bangalore 560012, India.

Despite an intuitive understanding of the role of APP in health and disease, there exist few attempts to dissect its molecular localization at excitatory synapses. Though the biochemistry involved in the enzymatic processing of APP is well understood, there is a void in understanding the nonuniformity of the product formation in vivo. Here, we employed multiple paradigms of single molecules and ensemble based nanoscopic imaging to reveal that APP molecules are organized into regulatory nanodomains that are differentially compartmentalized in the functional zones of an excitatory synapse. Furthermore, with the aid of high density single particle tracking, we show that the lateral diffusion of APP in live cells dictates an equilibrium between these nanodomains and their nano-environment, which is affected in a detrimental variant of APP. Additionally, we incorporate this spatio-temporal detail 'in silico' to generate a realistic nanoscale topography of APP in dendrites and synapses. This approach uncovers a nanoscale heterogeneity in the molecular organization of APP, depicting a locus for differential APP processing. This holistic paradigm, to decipher the real-time heterogeneity of the substrate molecules on the nanoscale, could enable us to better evaluate the molecular constraints overcoming the ensemble approaches used traditionally to understand the kinetics of product formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0nr00052cDOI Listing
April 2020

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

Methods 2020 03 7;174:49-55. Epub 2020 Feb 7.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France; Interdisciplinary Institute for Neuroscience, CNRS, UMR 5297, Bordeaux, France. Electronic address:

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 offers an intuitive and user-friendly graphical user interface, facilitating fast, accurate, and unbiased analysis of spine morphology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2020.01.020DOI Listing
March 2020

A Discrete Presynaptic Vesicle Cycle for Neuromodulator Receptors.

Neuron 2020 02 11;105(4):663-677.e8. Epub 2019 Dec 11.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco School of Medicine, San Francisco, CA 94158, USA; Department of Psychiatry, University of California, San Francisco School of Medicine, San Francisco, CA 94158, USA. Electronic address:

A major function of GPCRs is to inhibit presynaptic neurotransmitter release, requiring ligand-activated receptors to couple locally to effectors at terminals. The current understanding of how this is achieved is through receptor immobilization on the terminal surface. Here, we show that opioid peptide receptors, GPCRs that mediate highly sensitive presynaptic inhibition, are instead dynamic in axons. Opioid receptors diffuse rapidly throughout the axon surface and internalize after ligand-induced activation specifically at presynaptic terminals. We delineate a parallel regulated endocytic cycle for GPCRs operating at the presynapse, separately from the synaptic vesicle cycle, which clears activated receptors from the surface of terminals and locally reinserts them to maintain the diffusible surface pool. We propose an alternate strategy for achieving local control of presynaptic effectors that, opposite to using receptor immobilization and enforced proximity, is based on lateral mobility of receptors and leverages the inherent allostery of GPCR-effector coupling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2019.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035187PMC
February 2020

A super-resolution platform for correlative live single-molecule imaging and STED microscopy.

Nat Methods 2019 12 21;16(12):1263-1268. Epub 2019 Oct 21.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.

Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-019-0611-8DOI Listing
December 2019

Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion.

Curr Biol 2019 09 15;29(17):2852-2866.e5. Epub 2019 Aug 15.

Interdisciplinary Institute for Neuroscience, Université de Bordeaux, UMR 5297, 33000 Bordeaux, France; Interdisciplinary Institute for Neuroscience, CNRS, UMR 5297, 33000 Bordeaux, France. Electronic address:

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2019.07.035DOI Listing
September 2019

A tessellation-based colocalization analysis approach for single-molecule localization microscopy.

Nat Commun 2019 05 30;10(1):2379. Epub 2019 May 30.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, 33076, France.

Multicolor single-molecule localization microscopy (λSMLM) is a powerful technique to reveal the relative nanoscale organization and potential colocalization between different molecular species. While several standard analysis methods exist for pixel-based images, λSMLM still lacks such a standard. Moreover, existing methods only work on 2D data and are usually sensitive to the relative molecular organization, a very important parameter to consider in quantitative SMLM. Here, we present an efficient, parameter-free colocalization analysis method for 2D and 3D λSMLM using tessellation analysis. We demonstrate that our method allows for the efficient computation of several popular colocalization estimators directly from molecular coordinates and illustrate its capability to analyze multicolor SMLM data in a robust and efficient manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-10007-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542817PMC
May 2019

Publisher Correction: Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software.

Nat Methods 2019 Jun;16(6):561

Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK.

In the version of this paper originally published, Figure 4a contained errors that were introduced during typesetting. The bottom 11° ThunderSTORM image is an xz view but was incorrectly labeled as xy, and the low x-axis value in the four line profiles was incorrectly set as -60 instead of -50. These errors have been corrected in the PDF and HTML versions of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-019-0453-4DOI Listing
June 2019

Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software.

Nat Methods 2019 05 8;16(5):387-395. Epub 2019 Apr 8.

Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK.

With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. 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 SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-019-0364-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684258PMC
May 2019

Author Correction: Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells.

Sci Rep 2018 Nov 22;8(1):17426. Epub 2018 Nov 22.

Infectious Disease Research Institute of Montpellier (IRIM), UMR9004 CNRS, University of Montpellier, 1919 route de Mende, 34293, Montpellier, France.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-35954-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250660PMC
November 2018

Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells.

Sci Rep 2018 11 2;8(1):16283. Epub 2018 Nov 2.

Infectious Disease Research Institute of Montpellier (IRIM), UMR9004 CNRS, University of Montpellier, 1919 route de Mende, 34293, Montpellier, France.

Monitoring virus assembly at the nanoscale 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 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4 T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4 T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-34536-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214999PMC
November 2018

Differential Nanoscale Topography and Functional Role of GluN2-NMDA Receptor Subtypes at Glutamatergic Synapses.

Neuron 2018 10 27;100(1):106-119.e7. Epub 2018 Sep 27.

Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33000 Bordeaux, France; CNRS, IINS UMR 5297, Bordeaux, France. Electronic address:

NMDA receptors (NMDARs) play key roles in the use-dependent adaptation of glutamatergic synapses underpinning memory formation. In the forebrain, these plastic processes involve the varied contributions of GluN2A- and GluN2B-containing NMDARs that have different signaling properties. Although the molecular machinery of synaptic NMDAR trafficking has been under scrutiny, the postsynaptic spatial organization of these two receptor subtypes has remained elusive. Here, we used super-resolution imaging of NMDARs in rat hippocampal synapses to unveil the nanoscale topography of native GluN2A- and GluN2B-NMDARs. Both subtypes were found to be organized in separate nanodomains that vary over the course of development. Furthermore, GluN2A- and GluN2B-NMDAR nanoscale organizations relied on distinct regulatory mechanisms. Strikingly, the selective rearrangement of GluN2A- and GluN2B-NMDARs, with no overall change in NMDAR current amplitude, allowed bi-directional tuning of synaptic LTP. Thus, GluN2A- and GluN2B-NMDAR nanoscale organizations are differentially regulated and seem to involve distinct signaling complexes during synaptic adaptation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2018.09.012DOI Listing
October 2018

Bacterial cell wall nanoimaging by autoblinking microscopy.

Sci Rep 2018 09 19;8(1):14038. Epub 2018 Sep 19.

Univ. Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France.

Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this 'autoblinking' phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-32335-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145920PMC
September 2018

Catalytic activation of β-arrestin by GPCRs.

Nature 2018 05 2;557(7705):381-386. Epub 2018 May 2.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco School of Medicine, San Francisco, CA, USA.

β-arrestins are critical regulator and transducer proteins for G-protein-coupled receptors (GPCRs). β-arrestin is widely believed to be activated by forming a stable and stoichiometric GPCR-β-arrestin scaffold complex, which requires and is driven by the phosphorylated tail of the GPCR. Here we demonstrate a distinct and additional mechanism of β-arrestin activation that does not require stable GPCR-β-arrestin scaffolding or the GPCR tail. Instead, it occurs through transient engagement of the GPCR core, which destabilizes a conserved inter-domain charge network in β-arrestin. This promotes capture of β-arrestin at the plasma membrane and its accumulation in clathrin-coated endocytic structures (CCSs) after dissociation from the GPCR, requiring a series of interactions with membrane phosphoinositides and CCS-lattice proteins. β-arrestin clustering in CCSs in the absence of the upstream activating GPCR is associated with a β-arrestin-dependent component of the cellular ERK (extracellular signal-regulated kinase) response. These results delineate a discrete mechanism of cellular β-arrestin function that is activated catalytically by GPCRs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0079-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058965PMC
May 2018

Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion.

Mol Biol Cell 2018 06 18;29(11):1299-1310. Epub 2018 Apr 18.

Université Bordeaux, CNRS, UMR 5095, European Institute of Chemistry and Biology, Pessac 33607, France.

The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatiotemporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters. The Cdc42 nanoclusters were larger at the cell pole than those observed elsewhere in the cell. These features were exacerbated in cells expressing Cdc42-GTP, and were dependent on the scaffold Bem1, which contributed to the range of mobility and nanocluster size exhibited by Cdc42. The lipid environment, in particular phosphatidylserine levels, also played a role in regulating Cdc42 nanoclustering. These studies reveal how the mobility of a Rho GTPase is controlled to counter the depletive effects of diffusion, thus stabilizing Cdc42 on the plasma membrane and sustaining cell polarity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E18-01-0051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994902PMC
June 2018

Localization-based super-resolution imaging meets high-content screening.

Nat Methods 2017 Dec 30;14(12):1184-1190. Epub 2017 Oct 30.

Université de Bordeaux, Institut interdisciplinaire de Neurosciences, Bordeaux, France.

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4486DOI Listing
December 2017

3D Protein Dynamics in the Cell Nucleus.

Biophys J 2017 Jan;112(1):133-142

Mechanobiology Institute, National University of Singapore, Singapore; Department of Biological Sciences, National University of Singapore, Singapore; Institute for Molecular and Cell Biology, A(∗)Star, Singapore. Electronic address:

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2016.11.3196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5232349PMC
January 2017

Special Section Guest Editorial: Super-resolution microscopy of neural structure and function.

Neurophotonics 2016 Oct 21;3(4):041801. Epub 2016 Dec 21.

University of Bordeaux-CNRS , Interdisciplinary Institute for Neuroscience , UMR5297, 33077 Bordeaux, France.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/1.NPh.3.4.041801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5178106PMC
October 2016

Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy.

Neurophotonics 2016 Oct 3;3(4):041810. Epub 2016 Nov 3.

Centre National de la Recherche Scientifique, Interdisciplinary Institute for Neuroscience, UMR 5297, 147 rue Léo-Saignat, Bordeaux Cedex 33077, France; University of Bordeaux, Interdisciplinary Institute for Neuroscience, 147 rue Léo-Saignat, Bordeaux Cedex 33077, France.

The advent of superresolution imaging has created a strong need for both optimized labeling strategies and analysis methods to probe the nanoscale organization of complex biological structures. We present a thorough description of the distribution of synaptic adhesion proteins at the nanoscopic scale, namely presynaptic neurexin-[Formula: see text] ([Formula: see text]), and its two postsynaptic binding partners neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2). We monitored these proteins in the membrane of neurons by direct stochastic optical reconstruction microscopy, after live surface labeling with Alexa647-conjugated monomeric streptavidin. The small probe ([Formula: see text]) efficiently penetrates into crowded synaptic junctions and reduces the distance to target. We quantified the organization of the single-molecule localization data using a tesselation-based analysis technique. We show that Nlg1 exhibits a fairly disperse organization within dendritic spines, while LRRTM2 is organized in compact domains, and [Formula: see text] in presynaptic terminals displays a dual-organization pattern intermediate between that of Nlg1 and LRRTM2. These results suggest that part of [Formula: see text] interacts transsynaptically with Nlg1 and the other part with LRRTM2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/1.NPh.3.4.041810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093229PMC
October 2016

Hunting Down HIV-1 Gag Proteins at the Plasma Membrane of Human T Lymphocytes.

AIDS Res Hum Retroviruses 2016 07 6;32(7):658-9. Epub 2016 Jun 6.

1 Centre des agents Pathogènes et Biotechnologies pour la Santé, CNRS & Montpellier University , Montpellier, France .

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/aid.2016.0052DOI Listing
July 2016

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons.

Neurophotonics 2016 Oct 18;3(4):041804. Epub 2016 May 18.

University of Bielefeld , Cell Biology, Universitätsstr. 25, 33501 Bielefeld, Germany.

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient ([Formula: see text]) in neurites from [Formula: see text] to [Formula: see text] after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from [Formula: see text] to [Formula: see text], whereas a velocity increase from [Formula: see text] to [Formula: see text] was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/1.NPh.3.4.041804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870386PMC
October 2016

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin.

Nat Commun 2016 Mar 16;7:10773. Epub 2016 Mar 16.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, 33077 Bordeaux, France.

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms10773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799371PMC
March 2016

SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data.

Nat Methods 2015 Nov 7;12(11):1065-71. Epub 2015 Sep 7.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.

Localization-based super-resolution techniques open the door to unprecedented analysis of molecular organization. This task often involves complex image processing adapted to the specific topology and quality of the image to be analyzed. Here we present a segmentation framework based on Voronoï tessellation constructed from the coordinates of localized molecules, implemented in freely available and open-source SR-Tesseler software. This method allows precise, robust and automatic quantification of protein organization at different scales, from the cellular level down to clusters of a few fluorescent markers. We validated our method on simulated data and on various biological experimental data of proteins labeled with genetically encoded fluorescent proteins or organic fluorophores. In addition to providing insight into complex protein organization, this polygon-based method should serve as a reference for the development of new types of quantifications, as well as for the optimization of existing ones.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.3579DOI Listing
November 2015

Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones.

Proc Natl Acad Sci U S A 2015 Jun 18;112(22):6997-7002. Epub 2015 May 18.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique (CNRS), University of Bordeaux, 33077 Bordeaux, France;

Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1423455112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460488PMC
June 2015

3D high- and super-resolution imaging using single-objective SPIM.

Nat Methods 2015 Jul 11;12(7):641-4. Epub 2015 May 11.

1] Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France. [2] Centre National de la Recherche Scientifique, Bordeaux, France.

Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam-steering unit installed on a standard inverted microscope. The illumination and detection are done through the same objective. soSPIM can be used with standard sample preparations and features high background rejection and efficient photon collection, allowing for 3D single-molecule-based super-resolution imaging of whole cells or cell aggregates. Using larger mirrors enabled us to broaden the capabilities of our system to image Drosophila embryos.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.3402DOI Listing
July 2015

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis.

Mol Biol Cell 2015 Jul 6;26(13):2519-34. Epub 2015 May 6.

Dynamics of Cell Growth and Division, European Institute of Chemistry and Biology, F-33607 Bordeaux, France Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université Bordeaux, F-33000 Bordeaux, France

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E14-11-1527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571305PMC
July 2015