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The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire
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
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.
Ultrafast photomechanical transduction through thermophoretic implosion
N. Kavokine, S. Zou, R. Liu, H. Zhong, A. Nigues, B. Zou and L. Bocquet
Nature Communications - 50(11) - doi.org/10.1038/s41467-019-13912-w - 2020
Since the historical experiments of Crookes, the direct manipulation of matter by light has been both a challenge and a source of scientific debate. Here we show that laser illumination allows to displace a vial of nanoparticle solution over centimetre-scale distances. Cantilever-based force measurements show that the movement is due to millisecond-long force spikes, which are synchronised with a sound emission. We observe that the nanoparticles undergo negative thermophoresis, and ultrafast imaging reveals that the force spikes are followed by the explosive growth of a bubble in the solution. We propose a mechanism accounting for the propulsion based on a thermophoretic instability of the nanoparticle cloud, analogous to the Jeans’s instability that occurs in gravitational systems. Our experiments demonstrate a new type of laser propulsion and a remarkably violent actuation of soft matter, reminiscent of the strategy used by certain plants to propel their spores.
Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle.
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.
mTOR and S6K1 drive polycystic kidney by the control of Afadin-dependent oriented cell 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.
Parallelized DNA tethered bead measurements to scrutinize DNA mechanical structure
Allemand JF, Tardin C, Salomé L.
Nat. Methods - 1;169 46-56 - doi: 10.1016/j.ymeth.2019.07.020. - 2019
Tethering beads to DNA offers a panel of single molecule techniques for the refined analysis of the conformational dynamics of DNA and the elucidation of the mechanisms of enzyme activity. Recent developments include the massive parallelization of these techniques achieved by the fabrication of dedicated nanoarrays by soft nanolithography. We focus here on two of these techniques: the Tethered Particle motion and Magnetic Tweezers allowing analysis of the behavior of individual DNA molecules in the absence of force and under the application of a force and/or a torque, respectively. We introduce the experimental protocols for the parallelization and discuss the benefits already gained, and to come, for these single molecule investigations.
Anisotropic cellular forces support mechanical integrity of the Stratum Corneum barrier
Guo S, Domanov Y, Donovan M, Ducos B, Pomeau Y, Gourier C, Perez E, Luengo GS.
Chem. Mater - 92 45231 - doi: 10.1016/j.jmbbm.2018.12.027 - 2019
The protective function of biological surfaces that are exposed to the exterior of living organisms is the result of a complex arrangement and interaction of cellular components. This is the case for the most external cornified layer of skin, the stratum corneum (SC). This layer is made of corneocytes, the elementary 'flat bricks' that are held together through adhesive junctions. Despite the well-known protective role of the SC under high mechanical stresses and rapid cell turnover, the subtleties regarding the adhesion and mechanical interaction among the individual corneocytes are still poorly known. Here, we explore the adhesion of single corneocytes at different depths of the SC, by pulling them using glass microcantilevers, and measuring their detachment forces. We measured their interplanar adhesion between SC layers, and their peripheral adhesion among cells within a SC layer. Both adhesions increased considerably with depth. At the SC surface, with respect to adhesion, the corneocyte population exhibited a strong heterogeneity, where detachment forces differed by more than one order of magnitude for corneocytes located side by side. The measured detachment forces indicated that in the upper-middle layers of SC, the peripheral adhesion was stronger than the interplanar one. We conclude that the stronger peripheral adhesion of corneocytes in the SC favors an efficient barrier which would be able to resist strong stresses.
Anisotropic cellular forces support mechanical integrity of the Stratum Corneum barrier
Guo S, Domanov Y, Donovan M, Ducos B, Pomeau Y, Gourier C, Perez E, Luengo GS.
Chem. Mater - 92 45231 - doi: 10.1016/j.jmbbm.2018.12.027 - 2019
The protective function of biological surfaces that are exposed to the exterior of living organisms is the result of a complex arrangement and interaction of cellular components. This is the case for the most external cornified layer of skin, the stratum corneum (SC). This layer is made of corneocytes, the elementary 'flat bricks' that are held together through adhesive junctions. Despite the well-known protective role of the SC under high mechanical stresses and rapid cell turnover, the subtleties regarding the adhesion and mechanical interaction among the individual corneocytes are still poorly known. Here, we explore the adhesion of single corneocytes at different depths of the SC, by pulling them using glass microcantilevers, and measuring their detachment forces. We measured their interplanar adhesion between SC layers, and their peripheral adhesion among cells within a SC layer. Both adhesions increased considerably with depth. At the SC surface, with respect to adhesion, the corneocyte population exhibited a strong heterogeneity, where detachment forces differed by more than one order of magnitude for corneocytes located side by side. The measured detachment forces indicated that in the upper-middle layers of SC, the peripheral adhesion was stronger than the interplanar one. We conclude that the stronger peripheral adhesion of corneocytes in the SC favors an efficient barrier which would be able to resist strong stresses.
PICH and TOP3A cooperate to induce positive DNA supercoiling
Anna Hélène Bizard, Jean-Francois Allemand, Tue Hassenkam, Manikandan Paramasivam
Nature - 26(4) 1 - DOI: 10.1038/s41594-019-0201-6 - 2019
All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.
Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers
Saurabh Raj, Debjani Bagchi, Jessica Valle Orero, Josette Banroques, N Kyle Tanner, Vincent Croquette
Nucleic Acids Res. - 47(7) 3699–3710 - doi.org/10.1093/nar/gkz057 - 2019
DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.

391 publications.