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ABSTRACT: Spindle orientation plays a pivotal role in tissue morphogenesis. An asymmetric anthrax receptor cap is revealed to promote activation of a formin to orient the spindle along the planar cell polarity (PCP) axis in zebrafish dorsal epiblast cells.
Nature Cell Biology 12/2012; 15(1):11-3. · 19.49 Impact Factor
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ABSTRACT: A paradigm of cytokinesis in animal cells is that the actomyosin contractile ring provides the primary force to divide the cell [1]. In the fission yeast Schizosaccharomyces pombe, cytokinesis also involves a conserved cytokinetic ring, which has been generally assumed to provide the force for cleavage [2-4] (see also [5]). However, in contrast to animal cells, cytokinesis in yeast cells also requires the assembly of a cell wall septum [6], which grows centripetally inward as the ring closes. Fission yeast, like other walled cells, also possess high (MPa) turgor pressure [7-9]. Here, we show that turgor pressure is an important factor in the mechanics of cytokinesis. Decreasing effective turgor pressure leads to an increase in cleavage rate, suggesting that the inward force generated by the division apparatus opposes turgor pressure. The contractile ring, which is predicted to provide only a tiny fraction of the mechanical stress required to overcome turgor, is largely dispensable for ingression; once septation has started, cleavage can continue in the absence of the contractile ring. Scaling arguments and modeling suggest that the large forces for cytokinesis are not produced by the contractile ring but are driven by the assembly of cell wall polymers in the growing septum.
Current biology: CB 07/2012; 22(17):1601-8. · 10.99 Impact Factor
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ABSTRACT: Predicting cellular behavior is a major challenge in cell and developmental biology. Since the late nineteenth century, empirical rules have been formulated to predict the position and orientation of mitotic cleavage planes in plant and animal cells. Here, we review the history of division plane orientation rules and discuss recent experimental and theoretical studies that refine these rules and provide mechanistic insights into how division can be predicted. We describe why some of these rules may better apply to certain cell types and developmental contexts and discuss how they could be integrated in the future to allow the prediction of division positioning in tissues.
Trends in cell biology 02/2012; 22(4):193-200. · 12.12 Impact Factor
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ABSTRACT: The spatial organization of cells depends on their ability to sense their own shape and size. Here, we investigate how cell shape affects the positioning of the nucleus, spindle and subsequent cell division plane. To manipulate geometrical parameters in a systematic manner, we place individual sea urchin eggs into microfabricated chambers of defined geometry (e.g., triangles, rectangles, and ellipses). In each shape, the nucleus is positioned at the center of mass and is stretched by microtubules along an axis maintained through mitosis and predictive of the future division plane. We develop a simple computational model that posits that microtubules sense cell geometry by probing cellular space and orient the nucleus by exerting pulling forces that scale to microtubule length. This model quantitatively predicts division-axis orientation probability for a wide variety of cell shapes, even in multicellular contexts, and estimates scaling exponents for length-dependent microtubule forces.
Cell 02/2011; 144(3):414-26. · 32.40 Impact Factor
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Antoine-Emmanuel Saliba,
Laure Saias,
Eleni Psychari, Nicolas Minc,
Damien Simon,
François-Clément Bidard,
Claire Mathiot,
Jean-Yves Pierga,
Vincent Fraisier,
Jean Salamero,
Véronique Saada,
Françoise Farace,
Philippe Vielh,
Laurent Malaquin,
Jean-Louis Viovy
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ABSTRACT: We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.
Proceedings of the National Academy of Sciences 08/2010; 107(33):14524-9. · 9.68 Impact Factor
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ABSTRACT: Electric signals surround tissues and cells and have been proposed to participate in directing cell polarity in processes such as development, wound healing, and host invasion [1, 2]. The application of exogenous electric fields (EFs) can direct cell polarization in cell types ranging from bacteria and fungi to neurons and neutrophils [3-7]. The mechanisms by which EFs modulate cell polarity, however, remain poorly understood. Here we introduce the fission yeast Schizosaccharomyces pombe as a model organism to elucidate the mechanisms underlying this process. In these rod-shaped cells, an exogenous EF reorients cell growth in a direction orthogonal to the field, producing cells with a bent morphology. A candidate genetic screen identifies conserved factors involved in this process: an integral membrane proton ATPase pma1p that regulates intracellular pH, the small GTPase cdc42p, and the formin for3p that assembles actin cables. Interestingly, mutants in these genes still respond to the EF but orient in a different direction, toward the anode. In addition, EFs also cause electrophoretic movement of cell wall synthase complex proteins toward the anode. These data suggest molecular models for how the EF reorients cell polarization by modulating intracellular pH and steering cell polarity factors in multiple directions.
Current biology: CB 03/2010; 20(8):710-6. · 10.99 Impact Factor
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ABSTRACT: Mechanical properties contribute to the control of cell size, morphogenesis, development, and lifestyle of fungal cells. Tip growth can be understood by a viscoplastic model, in which growth is derived by high internal turgor pressure and cell-wall elasticity. To understand how these properties regulate growth in the rod-shaped fission yeast Schizosaccaromyces pombe, we devised femtoliter cylindrical polydimethylsiloxane (PDMS) microchambers with varying elasticity as force sensors for single cells. By buckling cells in these chambers, we determine the elastic surface modulus of the cell wall to be 20.2 +/- 6.1 N.m(-1). By analyzing the growth of the cells as they push against the walls of the chamber, we derive force-velocity relationships and values for internal effective turgor pressure of 0.85 +/- 0.15 MPa and a growth-stalling force of 11 +/- 3 muN. The behavior of cells buckling under the force of their own growth provides an independent test of this model and parameters. Force generation is dependent on turgor pressure and a glycerol synthesis gene, gpd1(+) (glycerol-3-phosphate dehydrogenase), and is independent of actin cables. This study develops a quantitative framework for tip cell growth and characterizes mechanisms of force generation that contribute to fungal invasion into host tissues.
Current biology: CB 07/2009; 19(13):1096-101. · 10.99 Impact Factor
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ABSTRACT: Microtubules (MTs) participate in the spatial regulation of actin-based processes such as cytokinesis and cell polarization. The fission yeast Schizosaccharomyces pombe is a rod-shaped cell that exhibits polarized cell growth at cell tips. MT plus ends contact and shrink from the cell tips and contribute to polarity regulation.
Here, we investigate the effects of changing cell shape on MTs and cell-polarization machinery. We physically bend fission yeast cells by forcing them into microfabricated femtoliter chambers. In these bent cells, MTs maintain a straight axis and contact and shrink from cortical sites at the sides of cells. At these ectopic sites, polarity factors such as bud6p, for3p (formin), and cdc42p are recruited and assemble actin cables in a MT-dependent manner. MT contact at the cortex induces the appearance of a bud6p dot within seconds. The accumulation of polarity factors leads to cell growth at these sites, when the MT-associated polarity factor tea1p is absent. This process is dependent on MTs, mal3p (EB1), moe1p (an EB1-binding protein), and for3p but, surprisingly, is independent of the tea1p-tea4p pathway.
These studies provide a direct demonstration for how MTs induce actin assembly at specific locations on the cell cortex and begin to identify a new pathway involved in this process. MT interactions with the cortex may be regulated by cortical-attachment sites. These findings highlight the crosstalk between cell shape, polarity mechanisms, and MTs responsible for cell morphogenesis.
Current biology: CB 02/2009; 19(2):83-94. · 10.99 Impact Factor
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ABSTRACT: A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactor's performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated.
Lab on a Chip 03/2008; 8(2):294-301. · 5.67 Impact Factor
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Zuzana Bílková,
Marcela Slováková, Nicolas Minc,
Claus Fütterer,
Roxana Cecal,
Daniel Horák,
Milan Benes,
Isabelle le Potier,
Jana Krenková,
Michael Przybylski,
Jean-Louis Viovy
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ABSTRACT: The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.
Electrophoresis 06/2006; 27(9):1811-24. · 3.30 Impact Factor
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Zuzana Bílková Dr,
Marcela Slováková, Nicolas Minc,
Claus Fütterer,
Roxana Cecal,
Daniel Horák,
Milan Beneš,
Isabelle le Potier,
Jana Křenková,
Michael Przybylski,
Jean-Louis Viovy
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ABSTRACT: The preparation of an easily replaceable protease microreactor for μ-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in μ-chip capillary (μ-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the μ-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic μ-chip devices.
Electrophoresis 04/2006; 27(9):1811 - 1824. · 3.30 Impact Factor
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ABSTRACT: The potential of the commercially available dye sypro orange for in-capillary derivatization was evaluated for the detection of insulin and one gastrointestinal peptide (Arg-Arg-gastrin) by capillary electrophoresis with laser induced fluorescence (CE-LIF). The fluorescent emission intensity (lambda(ex) = 488 nm, lambda(em) = 610 nm) of this probe is very low in aqueous medium, and increases strongly in less polar solvent, e.g. methanol. The hydrophobic character of the two analyzed peptides is too low to induce sufficient interaction with the fluorescent probe for good sensitivity when the latter is alone in the background electrolyte. Thus, the potential of several neutral, zwitterionic, cationic and anionic surfactants to favor probe/peptide interactions has been evaluated. It was demonstrated that a borate buffer (pH 8.5) containing tetradecyltrimethylammonium bromide (TTAB) in sub-micellar conditions can be considered as the most suitable buffer for insulin CE-LIF analysis. In addition, the method showed a good linearity between insulin concentration and the peak area of the labeled insulin, allowing quantitative measurements. The sensitivity achieved so far is comparable with that achieved with UV absorption detection, but even at this level it is interesting for microchip analysis, in which fluorescence detection is much more commonly available than UV absorption detection.
Journal of Chromatography 10/2005; 1087(1-2):203-9. · 4.53 Impact Factor
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ABSTRACT: The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.
Lab on a Chip 10/2005; 5(9):935-42. · 5.67 Impact Factor
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ABSTRACT: We present an analytically solvable model for the transport of long DNA through microfluidic arrays of posts. The motion is a repetitive three-part cycle: (i) collision with the post and extension of the arms; (ii) rope-over-pulley post disengagement; and (iii) a random period of uniform translation before the next collision. This cycle, inspired by geometration, is a nonseparable (Scher-Lax) continuous-time random walk on a lattice defined by the posts. Upon adopting a simple model for the transition probability density on the lattice, we analytically compute the mean DNA velocity and dispersivity in the long-time limit without any adjustable parameters. The results compare favorably with the limited amount of experimental data on separations in self-assembled arrays of magnetic beads. The Scher-Lax formalism provides a template for incorporating more sophisticated microscale models.
Physical Review Letters 06/2005; 94(19):198105. · 7.37 Impact Factor
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ABSTRACT: We present a videomicroscopy study of T4 DNA (169 kbp) in microfluidic arrays of posts formed by the self-assembly of magnetic beads. We observe DNA moving through an area of 10 000 microm(2), typically containing 100-600 posts. We determine the distribution of the contact times with the posts and the distribution of passage times across the field of view for hundreds of DNA per experiment. The contact time is well approximated by a Poisson process, scaling like the inverse of the field strength, independent of the density of the array. The distribution of passage times allows us to estimate the mean velocity and dispersivity of the DNA during its motion over distances long compared to our field of view. We compare these values with those computed from a lattice Monte Carlo model and geometration theory. We find reasonable quantitative agreement between the lattice Monte Carlo model and experiment, with the error increasing with increasing post density. The deviation between theory and experiment is attributed to the high mobility of DNA after disengaging from the posts, which leads to a difference between the contact time and the total time lost by colliding. Classical geometration theory furnishes surprisingly good agreement for the dispersivity, while geometration theory with a mean free path significantly overestimates the dispersivity.
Electrophoresis 02/2005; 26(2):362-75. · 3.30 Impact Factor
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ABSTRACT: The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.
Journal of Chromatography B 09/2004; 808(1):15-24. · 2.89 Impact Factor
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ABSTRACT: We present an experimental study of the microfluidic electrophoresis of long DNA in self-assembling matrixes of magnetic bead columns. Results are presented for the rapid separation of lambda-phage, 2lambda-DNA, and bacteriophage T4 DNA, where separation resolutions greater than 2 between lambda and T4 are achieved in times as short as 150 s. The use of a computer-piloted flow control system and injection results in high reproducibility between separations. We compare the experimentally measured mobility and dispersion with an exactly solvable lattice Monte Carlo model. The theory predicts that the mean velocity scales linearly with the field, the band broadening scales with the inverse of the field, and the resolution is independent of the field for intermediate fields-all of which are in accord with the experimental results. Moreover, reasonable quantitative agreement is achieved for band broadening for longer DNA (2lambda and T4) when the average postengagement time is measured experimentally. This work demonstrates the possibility of achieving fast microfluidic separation of large DNA on a routine basis.
Analytical Chemistry 08/2004; 76(13):3770-6. · 5.86 Impact Factor
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ABSTRACT: When placed in a Hele-Shaw cell and submitted to a uniform magnetic field, a suspension of superparamagnetic particles self-organizes into a 2D array of columns with hexagonal order on short distances and glasslike order on long distances. Such arrays were usefully used for fast electrophoretic separation of long DNA molecules. Although self-assembled matrices have the advantages of simplicity and tunability, it is impossible to have the precisely controlled order and pore size obtainable in a microlithographic array. We address the latter challenge here. A magnetic nickel template is prepared on one cell surface by lift-off. Under a uniform external field, the nickel dots create local magnetic field gradients that attracts the beads, imposing a predefined order. We demonstrate the ability to separate long DNA molecules with these matrices and achieve the first steps of studying the resolution of the DNA separation over a wide range of posts density.
http://dx.doi.org/10.1051/lhb:2007079.
