Université PSL



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Multicolor superresolution imaging using volumetric multifocus microscopy
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
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
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.
ß-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network
Deleglise B, Magnifico S, Duplus E, Vaur P, Soubeyre V, Belle M, Vignes M, Viovy JL, Jacotot E, Peyrin JM and Brugg B
Acta Neuropathologica - 2 145 - DOI: 10.1186/s40478-014-0145-3 - 2014
INTRODUCTION: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level.

RESULTS: In this study, we have grown primary cortical mouse neurons in microfluidic (μFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied β-amyloid (Aβ) peptides locally to the different cellular compartments. We observed that Aβ application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aβ deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aβ peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aβ stress at the network level we developed new μFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aβ application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration.

CONCLUSION: Thanks to μFD-based reconstructed neuronal networks we evaluated the distant effects of local Aβ stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aβ production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aβ can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.
High Spatiotemporal Control of Spontaneous Reactions Using Ultrasound-Triggered Composite Droplets
M. Bezagu, C. Errico, V. Chaulot-Talmon, F. Monti, M. Tanter, . Tabeling, J. Cossy, T. Arseniyadis and O. Couture
JACS - 136 (20) 7205–7208 - DOI: 10.1021/ja5019354 - 2014
Achieving high spatial and temporal control over a spontaneous reaction is a particularly challenging task with potential breakthroughs in various fields of research including surface patterning and drug delivery. We report here an exceptionally effective method that allows attaining such control. This method relies on a remotely triggered ultrasound-induced release of a reactant encapsulated in a composite microdroplet of liquid perfluorohexane. More specifically, the demonstration was achieved by locally applying a focused 2.25 MHz transducer onto a microfluidic channel in which were injected composite microdroplets containing a solution of an azidocoumarin and an external flow containing a reactive alkyne.
Recent progress in the physics of microfluidics and related biotechnological applications
Patrick Tabeling
Curr. Opin. Biotechnol. - -25 129-34 - 10.1016/j.copbio.2013.11.009 - 2014
Since the mid-nineties, the physical understanding of microfluidic flows has reached a level sufficiently elaborate for envisaging applications in all sorts of domains. As the domain expanded, the existence of new situations where fluid dynamics at small or moderate Reynolds numbers combines with confinement, interfaces, transport, particles along with disordered substrates raised new challenges. The present review is restricted to three domains in which progress in the physical description has been made recently (droplet-based, inertial and paper-based microfluidics) and for which biotechnological applications are foreseeable.
Physics and technological aspects of nanofluidics
Lydéric Bocquet et Patrick Tabeling
Lab. Chip - 14 3143–3158 - DOI: 10.1039/c4lc00325j - 2014
From a physical perspective, nanofluidics represents an extremely rich domain. It hosts many mechanisms acting on the nanoscale, which combine together or interact with the confinement to generate new phenomena. Superfast flows in carbon nanotubes, nonlinear electrokinetic transport, slippage over smooth surfaces, nanobubble stability, etc. are the most striking phenomena that have been unveiled over the past few years, and some of them are still awaiting an explanation. One may anticipate that new nanofluidic effects will be discovered in the future, but at the moment, the technological barrier is high. Fabrication of nanochannels is most often a tour de force, slow and costly. However, with the accumulation of technological skills along with the use of new nanofluidic materials (like nanotubes), nanofluidics is becoming increasingly accessible to experimentalists. Among the technological challenges faced by the field, fabricating devices mimicking natural nanometric systems, such as aquaporins, ionic pumps or kidney osmotic filtering, seems the most demanding in terms of groundbreaking ideas. Nanoflow characterization remains delicate, although considerable progress has been achieved over the past years. The targeted application of nanofluidics is not only in the field of genomics and membrane science - with disruptive developments to be expected for water purification, desalination, and energy harvesting - but also for oil and gas production from unconventional reservoirs. Today, in view of the markets that are targeted, nanofluidics may well impact the industry more than microfluidics; this would represent an unexpected paradox. These successes rely on using a variety of materials and technologies, using state-of-the-art nanofabrication, or low-tech inexpensive approaches. As a whole, nanofluidics is a fascinating field that is facing considerable challenges today. It possesses a formidable potential and offers much space for creative groundbreaking ideas.
Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells
M. Reffay, M. C. Parrini, O. Cochet-Escartin, B. Ladoux, A. Buguin, S. Coscoy, F. Amblard, J. Camonis and P. Silberzan
Nat. Cell Biol. - 16 (2014) 217–223 - DOI:10.1038/ncb2917 - 2014
The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.
How to control proteins with light in living systems
Gautier A., Gauron C., Volovitch M., Bensimon D., Jullien L. and Vriz S.
Nature Chemical Biology - 10 (2014) 33–541 - DOI::10.1038/nchembio.1534 - 2014
The possibility offered by photocontrolling the activity of biomolecules in vivo while recording physiological parameters is opening up new opportunities for the study of physiological processes at the single-cell level in a living organism. For the last decade, such tools have been mainly used in neuroscience, and their application in freely moving animals has revolutionized this field. New photochemical approaches enable the control of various cellular processes by manipulating a wide range of protein functions in a noninvasive way and with unprecedented spatiotemporal resolution. We are at a pivotal moment where biologists can adapt these cutting-edge technologies to their system of study. This user-oriented review presents the state of the art and highlights technical issues to be resolved in the near future for wide and easy use of these powerful approaches.
Photosensitive Polyamines for High-Performance Photocontrol of DNA Higher-Order Structure Venancio-Marques
Venancio-Marques, Anna, Bergen Anna, Rossi-Gendron Caroline, Rudiuk Sergii, and Baigl Damien
American Chemical Society Nano (ACS Nano) - Volume 8 (4) 3654–3663 - DOI: 10.1021/nn500266b - 2014
Polyamines are small, ubiquitous, positively charged molecules that play an essential role in numerous biological processes such as DNA packaging, gene regulation, neuron activity, and cell proliferation. Here, we synthesize the first series of photosensitive polyamines (PPAs) and demonstrate their ability to photoreversibly control nanoscale DNA higher-order structure with high efficiency. We show with fluorescence microscopy imaging that the efficiency of the PPAs as DNA-compacting agents is directly correlated to their molecular charge. Micromolar concentration of the most efficient molecule described here, a PPA containing three charges at neutral pH, compacts DNA molecules from a few kilobase pairs to a few hundred kilobase pairs, while subsequent 3 min UV illuminations at 365 nm triggers complete unfolding of DNA molecules. Additional application of blue light (440 nm for 3 min) induces the refolding of DNA into the compact state. Atomic force microscopy reveals that the compaction involves a global folding of the whole DNA molecule, whereas UV-induced unfolding is a modification initiated from the periphery of the compacted DNA, resulting in the occurrence of intermediate flower-like structures prior to the fully unfolded state.

Keywords: polyamines; DNA compaction; photocontrol; DNA; AFM; light

278 publications.