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Interference and crosstalk in double optical tweezers using a single laser source
P. Mangeol and U. Bockelmann
Rev Sci Instrum - 79(8) :083103 - DOI:10.1063/1.2957652 - 2008
Experimental studies of single molecule mechanics require high force sensitivity and low drift, which can be achieved with optical tweezers. We built an optical tweezer setup for force measurements in a two bead assay. A cw infrared laser beam is split by polarization and focused by a high numerical aperture objective to create two traps. The same laser is used to form both traps and to measure the force by back focal plane interferometry. We show that although the two beams entering the microscope are designed to exhibit orthogonal polarization, interference and a significant parasitic force signal occur. Comparing the experimental results with a ray optics model, we show that the interference patterns are caused by the rotation of polarization on microscope lens surfaces and slides. The model qualitatively describes the pattern and the dependence of the parasitic force signal on the experimental parameters. We present two different approaches to experimentally reduce the crosstalk, namely, polarization rectification and frequency shifting.
In situ quantitative measurement of concentration profiles in a microreactor with submicron resolution using multiplex CARS microscopy
Dawn Schafer, Jeff A. Squier, Jan van Maarseveen, Daniel Bon, Mischa Bonn, Michiel Mu¨ller
JACS - 130(35) :11592-3 - DOI:10.1021/ja804158n - 2008
In situ quantitative imaging of concentration profiles of reactants and products inside a microfluidic reactor is achieved, with submicron spatial resolution with mM sensitivity and on ms time scales, for a given position. The label-free approach relies on quantitative vibrational spectroscopy, using Coherent Anti-Stokes Raman scattering microscopy in a spectrally resolved fashion, and is demonstrated on an elementary acid-base reaction.
Determination of nanoparticle diffusion coefficients by Taylor dispersion analysis using a capillary electrophoresis instrument
d'Orlye F, Varenne A, Gareil P.
J. Chrom. A - 1204(2) :226-32 - DOI:10.1016/j.chroma.2008.08.008 - 2008
The collective diffusion coefficient D(C) of diluted suspensions of positively charged iron oxide maghemite particles was experimentally investigated using a capillary electrophoresis instrument on the grounds of Taylor dispersion theory. Conditions for this approach to be applicable to nanoparticles of mean solid diameter below 10nm were set in this work, enabling precisions on D(C) determination of less than 2% relative standard deviation (RSD). Significantly different D(C) values were thus measured for particle populations differing in solid number mean diameter by only 2 nm. The obtained values were compared to the z-average diffusion coefficient derived from dynamic light scattering (DLS) experiments and used for the calculation of the Stokes radius. The measured diffusion coefficients appeared to be dependent on particle volume fraction and electrolyte ionic strength. These observations were eventually discussed in terms of particle interactions.
Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain
Faure-André G, Vargas P, Yuseff MI, Heuzé M, Diaz J, Lankar D, Steri V, Manry J, Hugues S, Vascotto F, Boulanger J, Raposo G, Bono MR, Rosemblatt M, Piel M, Lennon-Duménil AM
Science - 322(5908) :1705-10 - DOI:10.1126/science.1159894 - 2008
Dendritic cells (DCs) sample peripheral tissues of the body in search of antigens to present to T cells. This requires two processes, antigen processing and cell motility, originally thought to occur independently. We found that the major histocompatibility complex II-associated invariant chain (Ii or CD74), a known regulator of antigen processing, negatively regulates DC motility in vivo. By using microfabricated channels to mimic the confined environment of peripheral tissues, we found that wild-type DCs alternate between high and low motility, whereas Ii-deficient cells moved in a faster and more uniform manner. The regulation of cell motility by Ii depended on the actin-based motor protein myosin II. Coupling antigen processing and cell motility may enable DCs to more efficiently detect and process antigens within a defined space.
Physical mechanisms redirecting cell polarity and cell shape in fission yeast
Terenna CR, Makushok T, Velve-Casquillas G, Baigl D, Chen Y, Bornens M, Paoletti A, Piel M, Tran PT
Curr Biol. - 18(22) :1748-53 - DOI:10.1016/j.cub.2008.09.047. - 2008
The cylindrical rod shape of the fission yeast Schizosaccharomyces pombe is organized and maintained by interactions between the microtubule, cell membrane, and actin cytoskeleton [1]. Mutations affecting any components in this pathway lead to bent, branched, or round cells [2]. In this context, the cytoskeleton controls cell polarity and thus dictates cell shape. Here, we use soft-lithography techniques to construct microfluidic channels to control cell shape. We show that when wild-type rod-shaped cells are physically forced to grow in a bent fashion, they will reorganize their cytoskeleton and redirect cell polarity to make new ectopic cell tips. Moreover, when bent or round mutant cells are physically forced to conform to the wild-type rod-shape, they will reverse their mutational phenotypes by reorganizing their cytoskeleton to maintain proper wild-type-like localization of microtubules, cell-membrane proteins, and actin. Our study provides direct evidence that the cytoskeleton controls cell polarity and cell shape and demonstrates that cell shape also controls the organization of the cytoskeleton in a feedback loop. We present a model of the feedback loop to explain how fission yeast maintain a rod shape and how perturbation of specific parameters of the loop can lead to different cell shapes.
Specific Wetting Probed With Biomimetic Droplets
J. Fattaccioli, J. Baudry, F. Brochard-Wyart, N. Henry and J. Bibette
Soft Matter - 4(12) :2334-40 - DOI:10.1039/B806635C - 2008
We have produced emulsion droplets of controlled size and composition coated by ligands, and studied the adhesion of these drops on a solid substrate coated by receptors and polymers. Using transmission, RICM and fluorescence microscopy we assess the size, contact angle and ligand density for each drop. We first show that non-specific interactions significantly enhance the proteins density within the adhesive patch. Then we show that binding within the patch is partially inhibited in good agreement with the hypothesis of an absence of translational diffusion. We confirm that the density of specific bonds sets the adhesive energy and therefore the final contact angle, and finally show that specific binding in our system is always associated with the existence of a positive line tension, which linearly increases with the density of receptors. These experiments describe a new scenario for specific wetting which raises the importance of the coupling between non-specific interactions and specific binding.
Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
T. Lionnet, M. M. Spiering, S. J. Benkovic, D. Bensimon and V. Croquette
Proc. Nat. Acad. Sci. USA - 104(50) :19790–95 - DOI:10.1073/pnas.0709793104 - 2007
Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (approximate to 400 bp/s), its unwinding rate extrapolated to zero force is much slower (approximate to 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.
Time-course analysis of mouse serum proteome changes following exposure of the skin to ionizing radiation
Guipaud O, Holler V, Buard V, Tarlet G, Royer N, Vinh J, Benderitter M.
Proteomics - 7(21) :3992-4002 - PMID:17960731 - 2007
Radiation-induced lesion outcomes of normal tissues are difficult to predict. In particular, radiotherapy or local exposure to a radioactive source by accident can trigger strong injury to the skin. The finding of biomarkers is of fundamental relevance for the prediction of lesion apparition and its evolution, and for the settlement of therapeutic strategies. In order to study radiation-induced cutaneous lesions, we developed a mouse model in which the dorsal skin was selectively exposed to ionizing radiation (IR). 2-D difference gel electrophoresis (2-D DIGE) coupled with MS was used to investigate proteins altered in expression and/or PTM in serum. Proteome changes were monitored from 1 day to 1 month postirradiation, at a dose of 40 Gy, in this specific model developing reproducible clinical symptoms ranging from erythema to skin ulceration with wound healing. About 60 proteins (including some isoforms and likely post-translational variants), representing 20 different proteins, that exhibited significant and reproducible kinetic expression changes, were identified using MS and database searches. Several proteins, down- or up-regulated from day one, could prove to be good candidates to prognosticate the evolution of a skin lesion such as necrosis. In addition, we observed shifts in pI of several spot trains, revealing potential PTM changes, which could also serve as indicators of irradiation or as predictors of lesion severity.
Polyadenylation of a functional mRNA controls gene expression in Escherichia coli
Joanny G, Le Derout J, Bréchemier-Baey D, Labas V, Vinh J, Régnier P, Hajnsdorf E.
Nucleic Acids Res. - 35(8) :2494-502 - PMID:17395638 - 2007
Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine-6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5' monophosphate and a 3' stable hairpin is highly susceptible to poly(A)-dependent degradation.
Collective migration of an epithelial monolayer in response to a model wound
Poujade M., Grasland-Mongrain E., Hertzog A., Jouanneau J., Chavrier P., Ladoux B., Buguin A., Silberzan P.
Proc. Nat. Acad. Sci. USA - 104(41) :15988-93 - DOI:10.1073/pnas.0705062104 - 2007
Using an original microfabrication-based technique, we experimentally study situations in which a virgin surface is presented to a confluent epithelium with no damage made to the cells. Although inspired by wound-healing experiments, the situation is markedly different from classical scratch wounding because it focuses on the influence of the free surface and uncouples it from the other possible contributions such as cell damage and/or permeabilization. Dealing with Madin–Darby canine kidney cells on various surfaces, we found that a sudden release of the available surface is sufficient to trigger collective motility. This migration is independent of the proliferation of the cells that mainly takes place on the fraction of the surface initially covered. We find that this motility is characterized by a duality between collective and individual behaviors. On the one hand, the velocity fields within the monolayer are very long range and involve many cells in a coordinated way. On the other hand, we have identified very active “leader cells” that precede a small cohort and destabilize the border by a fingering instability. The sides of the fingers reveal a pluricellular actin “belt” that may be at the origin of a mechanical signaling between the leader and the followers. Experiments performed with autocrine cells constitutively expressing hepatocyte growth factor (HGF) or in the presence of exogenous HGF show a higher average velocity of the border and no leader.

325 publications.