Université PSL

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Liquid Crystal Coacervates Composed of Short Double-Stranded DNA and Cationic Peptides
LABORATOIRE AUTO-ASSEMBLAGE MOLÉCULAIRE - Tommaso P. Fraccia and Tony Z. Jia
ACS Nano - 14, 11 15071–15082 - doi.org/10.1021/acsnano.0c05083 - 2020
Phase separation of nucleic acids and proteins is a ubiquitous phenomenon regulating subcellular compartment structure and function. While complex coacervation of flexible single-stranded nucleic acids is broadly investigated, coacervation of double-stranded DNA (dsDNA) is less studied because of its propensity to generate solid precipitates. Here, we reverse this perspective by showing that short dsDNA and poly-l-lysine coacervates can escape precipitation while displaying a surprisingly complex phase diagram, including the full set of liquid crystal (LC) mesophases observed to date in bulk dsDNA. Short dsDNA supramolecular aggregation and packing in the dense coacervate phase are the main parameters regulating the global LC-coacervate phase behavior. LC-coacervate structure was characterized upon variations in temperature and monovalent salt, DNA, and peptide concentrations, which allow continuous reversible transitions between all accessible phases. A deeper understanding of LC-coacervates can gain insights to decipher structures and phase transition mechanisms within biomolecular condensates, to design stimuli-responsive multiphase synthetic compartments with different degrees of order and to exploit self-assembly driven cooperative prebiotic evolution of nucleic acids and peptides.
Elasticity and Viscosity of DNA Liquid Crystals
LABORATOIRE AUTO-ASSEMBLAGE MOLÉCULAIRE - Liana Lucchetti, Tommaso P. Fraccia, Giovanni Nava, Taras TurivTaras Turiv Advanced Materials and Liquid Crystal Institute, Chemical Physics Interdisciplinary Program, Kent State University, Kent, Ohio 44242, United States More by Taras Turiv , Fabrizio
ACS Nano - 9, 7 1034–1039 - doi.org/10.1021/acsmacrolett.0c00394 - 2020
Concentrated solutions of blunt-ended DNA oligomer duplexes self-assemble in living polymers and order into lyotropic nematic liquid crystal phase. Using the optical torque provided by three distinct illumination geometries, we induce independent splay, twist, and bend deformations of the DNA nematic and measure the corresponding elastic coefficients K1, K2, and K3, and viscosities ηsplay, ηtwist, and ηbend. We find the viscoelasticity of the system to be remarkably soft, as the viscoelastic coefficients are smaller than in other lyotropic liquid crystals. We find K1 > K3 > K2, in agreement with the elasticity of the nematic phase of flexible polymers, and ηbend > ηsplay > ηtwist a behavior that is nonconventional in the context of chromonic, polymeric, and thermotropic liquid crystals, indicating a possible role of the weakness and reversibility of the DNA aggregates.
Liquid Crystal ordering of DNA Dickerson Dodecamer duplexes with different 5’- Phosphate terminations
LABORATOIRE AUTO-ASSEMBLAGE MOLÉCULAIRE - Marco Todisco Gregory P. Smith Tommaso Pietro Fraccia
Molecular Crystals and Liquid Crystals - 683(1) 69-80 - DOI: 10.1080/15421406.2019.1581706 - 2020
The onset of liquid crystal (LC) phases in concentrated aqueous solutions of DNA oligomers crucially depends on the end-to-end interaction between the DNA duplexes, which can be provided by the aromatic stacking of the terminal base-pairs or by the pairing of complementary dangling-ends. Here we investigated the LC behavior of three blunt-end 12-base-long DNA duplexes synthesized with hydroxyl, phosphate and triphosphate 5’-termini. We experimentally characterized the concentration-temperature phase diagrams and we quantitatively estimated the end-to-end stacking free energy, by comparing the empirical data with the predictions of coarse-grained linear aggregation models. The preservation of LC ordering, even in presence of the bulky and highly charged triphosphate group, indicates that attractive stacking interactions are still present and capable of induce linear aggregation of the DNA duplexes. This finding strengthens the potential role of chromonic like self-assembly for the prebiotic formation of linear polymeric nucleic acids.
Polarization of Myosin II Refines Tissue Material Properties to Buffer Mechanical Stress
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Maria Duda, Natalie J Kirkland, Nargess Khalilgharibi, Melda Tozluoglu , Alice C Yuen , Nicolas Carpi , Anna Bove , Matthieu Piel , Guillaume Charras , Buzz Baum , Yanlan Mao
Dev Cell - 48(2) 245-260.e7 - DOI: 10.1016/j.devcel.2018.12.020 - 2020
mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.
mTOR and S6K1 drive polycystic kidney by the control of Afadin-dependent oriented cell division
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Martina Bonucci, Nicolas Kuperwasser, Serena Barbe, Vonda Koka, Delphine de Villeneuve, Chi Zhang, Nishit Srivastava, Xiaoying Jia, Matthew P Stokes, Frank Bienaimé, Virginie Verkarre, Jean Baptiste Lopez, Fanny Jaulin, Marco Pontoglio, Fabiola Terzi, Be
Nature Communications - - DOI : 10.1038/s41467-020-16978-z - 2020
mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.
Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle.
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Andrea Dimitracopoulos, Pragya Srivastava, Agathe Chaigne, Zaw Win, Roie Shlomovitz, Oscar M Lancaster, Maël Le Berre, Matthieu Piel, Kristian Franze, Guillaume Salbreux, Buzz Baum
Current biology - - DOI : S0960-9822(20)30984-2 - 2020
Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.
ATR is essential for preservation of cell mechanics and nuclear integrity during interstitial migration
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Gururaj Rao Kidiyoor, Qingsen Li, Giulia Bastianello, Christopher Bruhn, Irene Giovannetti, Adhil Mohamood, Galina V. Beznoussenko, Alexandre Mironov, Matthew Raab, Matthieu Piel, Umberto Restuccia, Vittoria Matafora, Angela Bachi, Sara
Nature Communications - 11 4828 - https://doi.org/10.1038/s41467-020-18580-9 - 2020
ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.

The nucleus acts as a ruler tailoring cell responses to spatial constraints
Laboratoire Biologie cellulaire systémique de la polarité et de la division - A. J. Lomakin, C. J. Cattin, D. Cuvelier, Z. Alraies, M. Molina. Nader, N. Sri
Science - 6514 370 - DOI: 10.1126/science.aba2894 - 2020
Single cells continuously experience and react to mechanical challenges in three-dimensional tissues. Spatial constraints in dense tissues, physical activity, and injury all impose changes in cell shape. How cells can measure shape deformations to ensure correct tissue development and homeostasis remains largely unknown (see the Perspective by Shen and Niethammer). Working independently, Venturini et al. and Lomakin et al. now show that the nucleus can act as an intracellular ruler to measure cellular shape variations. The nuclear envelope provides a gauge of cell deformation and activates a mechanotransduction pathway that controls actomyosin contractility and migration plasticity. The cell nucleus thereby allows cells to adapt their behavior to the local tissue microenvironment.
Sliding walls: a new paradigm for fluidic actuation and protocol implementation in microfluidics
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Venzac, Bastien; Liu, Yang; Ferrante, Ivan; Vargas, Pablo; Yamada, Ayako; Courson, Rémi; Verhulsel, Marine; Malaquin, Laurent; Viovy, Jean-Louis; Descroix, Stéphanie
Microsystems & Nanoengineering - 6(1) 18 - DOI: 10.1038/s41378-019-0125-7 - 2020
Currently, fluidic control in microdevices is mainly achieved either by external pumps and valves, which are expensive and bulky, or by valves integrated in the chip. Numerous types of internal valves or actuation methods have been proposed, but they generally impose difficult compromises between performance and fabrication complexity. We propose here a new paradigm for actuation in microfluidic devices based on rigid or semi-rigid walls with transversal dimensions of hundreds of micrometres that are able to slide within a microfluidic chip and to intersect microchannels with hand-driven or translation stage-based actuation. With this new concept for reconfigurable microfluidics, the implementation of a wide range of functionalities was facilitated and allowed for no or limited dead volume, low cost and low footprint. We demonstrate here several fluidic operations, including on/off or switch valving, where channels are blocked or reconfigured depending on the sliding wall geometry. The valves sustain pressures up to 30 kPa. Pumping and reversible compartmentalisation of large microfluidic chambers were also demonstrated. This last possibility was applied to a "4D" migration assay of dendritic cells in a collagen gel. Finally, sliding walls containing a hydrogel-based membrane were developed and used to concentrate, purify and transport biomolecules from one channel to another, such functionality involving complex fluidic transport patterns not possible in earlier microfluidic devices. Overall, this toolbox is compatible with "soft lithography" technology, allowing easy implementation within usual fabrication workflows for polydimethylsiloxane chips. This new technology opens the route to a variety of microfluidic applications, with a focus on simple, hand-driven devices for point-of-care or biological laboratories with low or limited equipment and resources.
Oncogenic Signaling Alters Cell Shape and Mechanics to Facilitate Cell Division under Confinement
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Helen K.Matthews, Sushila Ganguli, KatarzynaPlak, Anna V.Taubenberger, Zaw Win, Max Williamson, Matthieu Piel, Jochen Guck, Buzz Baum
Dev Cell - 52(5) 563-573.e3 - doi.org/10.1016/j.devcel.2020.01.004 - 2020
To divide in a tissue, both normal and cancer cells become spherical and mechanically stiffen as they enter mitosis. We investigated the effect of oncogene activation on this process in normal epithelial cells. We found that short-term induction of oncogenic RasV12 activates downstream mitogen-activated protein kinase (MEK-ERK) signaling to alter cell mechanics and enhance mitotic rounding, so that RasV12-expressing cells are softer in interphase but stiffen more upon entry into mitosis. These RasV12-dependent changes allow cells to round up and divide faithfully when confined underneath a stiff hydrogel, conditions in which normal cells and cells with reduced levels of Ras-ERK signaling suffer multiple spindle assembly and chromosome segregation errors. Thus, by promoting cell rounding and stiffening in mitosis, oncogenic RasV12 enables cells to proliferate under conditions of mechanical confinement like those experienced by cells in crowded tumors.
The nucleus acts as a ruler tailoring cell responses to spatial constraints
Laboratoire Biologie cellulaire systémique de la polarité et de la division - A. J. Lomakin, C. J. Cattin, D. Cuvelier, Z. Alraies, M. Molina, G. P. F. Nader, N. Srivastava, P. J. Saez, J. M. Garcia-Arcos, I. Y. Zhitnyak, A. Bhargava, M. K. Driscoll, E. S. Welf, R. Fiolka, R. J. Petrie, N. S. De Silva, J. M. González-Grana
Science - 370 6514 - DOI: 10.1126/science.aba2894 - 2020
The human body is a crowded place. This crowding is even more acute when the regulation of cell growth and proliferation fails during the formation of a tumor. Dealing with the lack of space in crowded environments presents cells with a challenge. This is especially true for immune cells, whose task is to patrol tissues, causing them to experience both acute and sustained deformation as they move. Although changes in tissue crowding and associated cell shape alterations have been known by pathologists to be key diagnostic traits of late-stage tumors since the 19th century, the impact of these changes on the biology of cancer and immune cells remains unclear. Moreover, it is not known whether cells can detect and adaptively respond to deformations in densely packed spaces.
A statistical inference approach to reconstruct intercellular interactions in cell migration experiments
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Elena Agliari1, Pablo J. Sáez, Adriano Barra, Matthieu Piel, Pablo Vargas
Science Advances - 6 11 - DOI: 10.1126/sciadv.aay2103 - 2020
Migration of cells can be characterized by two prototypical types of motion: individual and collective migration. We propose a statistical inference approach designed to detect the presence of cell-cell interactions that give rise to collective behaviors in cell motility experiments. This inference method has been first successfully tested on synthetic motional data and then applied to two experiments. In the first experiment, cells migrate in a wound-healing model: When applied to this experiment, the inference method predicts the existence of cell-cell interactions, correctly mirroring the strong intercellular contacts that are present in the experiment. In the second experiment, dendritic cells migrate in a chemokine gradient. Our inference analysis does not provide evidence for interactions, indicating that cells migrate by sensing independently the chemokine source. According to this prediction, we speculate that mature dendritic cells disregard intercellular signals that could otherwise delay their arrival to lymph vessels.
Deterministic actin waves as generators of cell polarization cues
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Luiza Stankevicins, Nicolas Ecker, Emmanuel Terriac, Paolo Maiuri, Rouven Schoppmeyer, Pablo Vargas, Ana-Maria Lennon-Duménil, Matthieu Piel, Bin Qu, Markus Hoth, Karsten Kruse, and Franziska Lautenschläger
PNAS - 117 (2) 826-835 - doi.org/10.1073/pnas.1907845117 - 2020
Migration of cells can be characterized by two prototypical types of motion: individual and collective migration. We propose a statistical inference approach designed to detect the presence of cell-cell interactions that give rise to collective behaviors in cell motility experiments. This inference method has been first successfully tested on synthetic motional data and then applied to two experiments. In the first experiment, cells migrate in a wound-healing model: When applied to this experiment, the inference method predicts the existence of cell-cell interactions, correctly mirroring the strong intercellular contacts that are present in the experiment. In the second experiment, dendritic cells migrate in a chemokine gradient. Our inference analysis does not provide evidence for interactions, indicating that cells migrate by sensing independently the chemokine source. According to this prediction, we speculate that mature dendritic cells disregard intercellular signals that could otherwise delay their arrival to lymph vessels.
Pressure sensing through Piezo channels controls whether cells migrate with blebs or pseudopods
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Nishit Srivastava, David Traynor, Matthieu Piel, Alexandre J. Kabla, and Robert R. Kay
PNAS - 117 (5) 2506-2512 - doi.org/10.1073/pnas.1905730117 - 2020
Cells migrating within the body perform vital functions in development and for defense and repair of tissues. In this dense environment, cells encounter mechanical forces and constraints not experienced when moving under buffer, and, accordingly, many change how they move. We find that gentle squashing, which mimics mechanical resistance, causes cells to move using blebs—a form of projection driven by fluid pressure—rather than pseudopods. This behavior depends on the Piezo stretch-operated ion channel in the cell membrane and calcium fluxes into the cell. Piezo is highly conserved and is required for light touch sensation; this work extends its functions into migrating cells.
Predicting Confined 1D Cell Migration from Parameters Calibrated to a 2D Motor-Clutch Model
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Prahl LS, Stanslaski MR, Vargas P, Piel M, Odde DJ
Biophys. J. - 118(7) 1709-1720 - DOI: 10.1016/j.bpj.2020.01.048 - 2020
Biological tissues contain micrometer-scale gaps and pores, including those found within extracellular matrix fiber networks, between tightly packed cells, and between blood vessels or nerve bundles and their associated basement membranes. These spaces restrict cell motion to a single-spatial dimension (1D), a feature that is not captured in traditional in vitro cell migration assays performed on flat, unconfined two-dimensional (2D) substrates. Mechanical confinement can variably influence cell migration behaviors, and it is presently unclear whether the mechanisms used for migration in 2D unconfined environments are relevant in 1D confined environments. Here, we assessed whether a cell migration simulator and associated parameters previously measured for cells on 2D unconfined compliant hydrogels could predict 1D confined cell migration in microfluidic channels. We manufactured microfluidic devices with narrow channels (60-μm2 rectangular cross-sectional area) and tracked human glioma cells that spontaneously migrated within channels. Cell velocities (vexp = 0.51 ± 0.02 μm min-1) were comparable to brain tumor expansion rates measured in the clinic. Using motor-clutch model parameters estimated from cells on unconfined 2D planar hydrogel substrates, simulations predicted similar migration velocities (vsim = 0.37 ± 0.04 μm min-1) and also predicted the effects of drugs targeting the motor-clutch system or cytoskeletal assembly. These results are consistent with glioma cells utilizing a motor-clutch system to migrate in confined environments.
Centrosome–nuclear axis repositioning drives the assembly of a bipolar spindle scaffold to ensure mitotic fidelity
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Vanessa Nunes, Margarida Dantas, Domingos Castro, Elisa Vitiello,e Irène Wang,e Nicolas Carpi, Martial Balland, Matthieu Piel, Paulo Aguiar, Helder Maiato, and Jorge G. Ferreira
Mol Biol Cell - 31(16) 1675–1690 - doi: 10.1091/mbc.E20-01-0047 - 2020
During the initial stages of cell division, the cytoskeleton is extensively reorganized so that a bipolar mitotic spindle can be correctly assembled. This process occurs through the action of molecular motors, cytoskeletal networks, and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. To investigate how cell shape, cytoskeletal organization, and molecular motors cross-talk to regulate initial spindle assembly, we use a combination of micropatterning with high-resolution imaging and 3D cellular reconstruction. We show that during prophase, centrosomes and nucleus reorient so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. We also find that this orientation depends on a combination of centrosome movement controlled by Arp2/3-mediated regulation of microtubule dynamics and Dynein-generated forces on the NE that regulate nuclear reorientation. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation.
Cellular locomotion using environmental topography
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Anne Reversat, Florian Gaertner, Jack Merrin, Julian Stopp, Saren Tasciyan, Juan Aguilera, Ingrid de Vries, Robert Hauschild, Miroslav Hons, Matthieu Piel, Andrew Callan-Jones, Raphael Voituriez, Michael Sixt
Nature - 582(7813) 582-585 - doi: 10.1038/s41586-020-2283-z. - 2020
Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces1. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour.
Cellular locomotion using environmental topography
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Anne Reversat, Florian Gaertner, Jack Merrin, Julian Stopp, Saren Tasciyan, Juan Aguilera, Ingrid de Vries, Robert Hauschild, Miroslav Hons, Matthieu Piel, Andrew Callan-Jones, Raphael Voituriez, Michael Sixt
Nature - 582(7813) 582-585 - doi: 10.1038/s41586-020-2283-z. - 2020
Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces1. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour.
The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire
Laboratoire Colloïdes et Matériaux Divisés - Author links open overlay panelMagdaRybczynskaaJeanBaudryaEyerKlaus
Vaccine - 38(33) 5337-5342 - https://doi.org/10.1016/j.vaccine.2020.05.066 - 2020
Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Laboratoire Colloïdes et Matériaux Divisés - Yacine Bounab, Klaus Eyer, Sophie Dixneuf, Magda Rybczynska, Cécile Chauvel, Maxime Mistretta, Trang Tran, Nathan Aymerich, Guilhem Chenon, Jean-François Llitjos, Fabienne Venet, Guillaume Monneret, Iain A. Gillespie, Pierre Cortez, Virginie Moucadel, Al
Nature Protocols - 15 2920–2955 - https://www.nature.com/articles/s41596-020-0354-0 - 2020
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.

578 publications.