Université PSL



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Gradients of Rac1 nanoclusters support spatial patterns of Rac1 signaling
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Amanda Remorino, Simon De Beco, Fanny Cayrac, Fahima Di Federico, Gaetan Cornilleau, Alexis Gautreau, Maria Carla Parrini, Jean-Baptiste Masson, Maxime Dahan, Mathieu Coppey
Cell Reports - 21(7) 1922-1935 - DOI: 10.1016/j.celrep.2017.10.069 - 2019
Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.
Gradients of Rac1 nanoclusters support spatial patterns of Rac1 signaling
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - El Beheiry, Mohamed Doutreligne, Sébastien Caporal, Clément Ostertag, Cécilia et al.
journal article - 431(7) 1315-1321 - - 2019
Virtual reality (VR) has recently become an affordable technology. A wide range of options are available to access this unique visualization medium, from simple cardboard inserts for smartphones to truly advanced headsets tracked by external sensors. While it is now possible for any research team to gain access to VR, we can still question what it brings to scientific research. Visualization and the ability to navigate complex three-dimensional data are undoubtedly a gateway to many scientific applications; however, we are convinced that data treatment and numerical simulations, especially those mixing interactions with data, human cognition, and automated algorithms will be the future of VR in scientific research. Moreover, VR might soon merit the same level of attention to imaging data as machine learning currently has. In this short perspective, we discuss approaches that employ VR in scientific research based on some concrete examples.
Dynamics of CRISPR-Cas9 genome interrogation in living cells
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, Zhang ET, El Beheiry M, Masson J-B, Dahan M, Liu Z, Doudna JA, Tjian R
Science - 350(6262) 823-6 - DOI: 10.1126/science.aac6572 - 2015
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.
Single-molecule dynamics of enhanceosome assembly in embryonic stem cells
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Chen J., Zhang Z., Li L., Chen B.C, Revyakin A., Hajj B., Legant W., Dahan M., Lionnet T., Betzig E., Tjian R. and Liu Z.
Cell - 156(6) 1274-85 - DOI: 10.1016/j.cell.2014.01.062 - 2014
Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
Magneto-fluorescent core-shell supernanoparticles
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Ou Chen, Lars Riedemann, Fred Etoc, Hendrik Herrmann, Mathieu Coppey, Mariya Barch, Christian T. Farrar, Jing Zhao, Oliver T. Bruns, He Wei, Peng Guo, Jian Cui, Russ Jensen, Yue Chen, Daniel K. Harris, Jose M. Cordero, Zhongwu Wang, Alan Jasanoff, Dai Fu
Nature Communications - 5 Article number:5093 - DOI:10.1038/ncomms6093 - 2014
Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close-packed magnetic nanoparticle ‘core’, which is fully surrounded by a ‘shell’ of fluorescent quantum dots. A thin layer of ​silica coating provides high colloidal stability and biocompatibility, and a versatile surface functionality. We demonstrate that after surface pegylation, these ​silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our ​silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe.
Multicolor superresolution imaging using volumetric multifocus microscopy
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Bassam Hajja, Jan Wisniewskib, Mohamed El Beheiry,, Jiji Chen, Andrey Revyakin, Carl Wu, and Maxime Dahan
Proc. Nat. Acad. Sci. USA - vol.111(n°49) 17480–17485 - DOI: 10.1073/pnas.1412396111 - 2014
Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.
Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Izeddin I, Récamier V, Bosanac L, Cissé II, Boudarene L, Dugast-Darzacq C, Proux F, Bénichou O, Voituriez R, Bensaude O, Dahan M ans Darzacq X
e-Life - -3 e02230 - DOI: 10.7554/eLife.02230 - 2014
Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.
Imaging Cse4 histone fate reveals stable residence at centromeres after de novo replacement in S phase
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Jan Wisniewski, Bassam Hajj, Jiji Chen, Gaku Mizuguchi, Hua Xiao, Debbie Wei, Maxime Dahan, and Carl Wu
e-Life - -3 e02203 - DOI:10.7554/eLife.02203 - 2014
The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.
Real time dynamics of RNA Polymerase II clustering in live human cells
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - Ibrahim Cisse, Ignacio Izeddin, Sebastien Causse, Lydia Boudarene, Adrien Senecal, Leila Muresan, Claire Dugast-Darzacq, Bassam Hajj, Maxime Dahan and Xavier Darzacq
Science - 341(6146) 664-7 - DOI: 10.1126/science.1239053 - 2013
Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA Polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, refuting their notion as statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.
ViSP: tool for visualizing 3D super-resolution data
Laboratoire Imagerie et contrôle optique de l’organisation cellulaire - M. El Beheiry and M. Dahan
Nat. Methods - 10 :689–690 - doi: 10.1093/bioinformatics/btu720 - 2013

16 publications.