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Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein
Stan Perchepied, Nicolas Eskenazi, Chiara Giangrande, Julien Camperi, Thierry Fournier, Joëlle Vinh, Nathalie Delaunay, Valérie Pichon
Biosci Rep - 206 120171 - doi.org/10.1016/j.talanta.2019.120171 - 2019
The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl
Contribution of proteases and cellulases produced by solid-state fermentation to the improvement of corn ethanol production
Anaïs Guillaume, Aurore Thorigné, Yoann Carré, Joëlle Vinh and Loïc Levavasseur
Biosci Rep - 6 7 - doi.org/10.1186/s40643-019-0241-0 - 2019
By cultivating a strain of Aspergillus tubingensis on agro-industrial by-products using solid-state fermentation technology, a biocatalyst containing more than 130 different enzymes was obtained. The enzymatic complex was composed mainly of hydrolases, among which a protease, an aspergillopepsin, accounted for more than half of the total proteins. Cell-wall-degrading enzymes such as pectinases, cellulases and hemicellulases were also highly represented. Adding the biocatalyst to corn mash at 1 kg/T corn allowed to significantly improve ethanol production performances. The final ethanol concentration was increased by 6.8% and the kinetics was accelerated by 14 h. The aim of this study was to identify the enzymes implicated in the effect on corn ethanol production. By fractionating the biocatalyst, the particular effect of the major enzymes was investigated. Experiments revealed that, together, the protease and two cellulolytic enzymes (an endoglucanase and a β-glucosidase) were responsible for 80% of the overall effect of the biocatalyst. Nevertheless, the crude extract of the biocatalyst showed greater impact than the combination of up to seven purified enzymes, demonstrating the complementary enzymatic complex obtained by solid-state fermentation. This technology could, therefore, be a relevant natural alternative to the use of GMO-derived enzymes in the ethanol industry.
Global host molecular perturbations upon in situ loss of bacterial endosymbionts in the deep-sea mussel Bathymodiolus azoricus assessed using proteomics and transcriptomics
Détrée C1, Haddad I, Demey-Thomas E, Vinh J, Lallier FH, Tanguy A, Mary J
BMC Genomics - 20(1) 109 - doi: 10.1186/s12864-019-5456-0. - 2019
BACKGROUND:
Colonization of deep-sea hydrothermal vents by most invertebrates was made efficient through their adaptation to a symbiotic lifestyle with chemosynthetic bacteria, the primary producers in these ecosystems. Anatomical adaptations such as the establishment of specialized cells or organs have been evidenced in numerous deep-sea invertebrates. However, very few studies detailed global inter-dependencies between host and symbionts in these ecosystems. In this study, we proposed to describe, using a proteo-transcriptomic approach, the effects of symbionts loss on the deep-sea mussel Bathymodiolus azoricus' molecular biology. We induced an in situ depletion of symbionts and compared the proteo-transcriptome of the gills of mussels in three conditions: symbiotic mussels (natural population), symbiont-depleted mussels and aposymbiotic mussels.

RESULTS:
Global proteomic and transcriptomic results evidenced a global disruption of host machinery in aposymbiotic organisms. We observed that the total number of proteins identified decreased from 1118 in symbiotic mussels to 790 in partially depleted mussels and 761 in aposymbiotic mussels. Using microarrays we identified 4300 transcripts differentially expressed between symbiont-depleted and symbiotic mussels. Among these transcripts, 799 were found differentially expressed in aposymbiotic mussels and almost twice as many in symbiont-depleted mussels as compared to symbiotic mussels. Regarding apoptotic and immune system processes - known to be largely involved in symbiotic interactions - an overall up-regulation of associated proteins and transcripts was observed in symbiont-depleted mussels.

CONCLUSION:
Overall, our study showed a global impairment of host machinery and an activation of both the immune and apoptotic system following symbiont-depletion. One of the main assumptions is the involvement of symbiotic bacteria in the inhibition and regulation of immune and apoptotic systems. As such, symbiotic bacteria may increase their lifespan in gill cells while managing the defense of the holobiont against putative pathogens.
Topological defects in confined populations of spindle-shaped cells
Guillaume Duclos, Christoph Erlenkämper, Jean-François Joanny & Pascal Silberzan
Nature Physics - 16 (2014) 217–223 - DOI:10.1038/nphys3876 - 2019
Most spindle-shaped cells (including smooth muscles and sarcomas) organize in vivo into well-aligned ‘nematic’ domains1, 2, 3, creating intrinsic topological defects that may be used to probe the behaviour of these active nematic systems. Active non-cellular nematics have been shown to be dominated by activity, yielding complex chaotic flows4, 5. However, the regime in which live spindle-shaped cells operate, and the importance of cell–substrate friction in particular, remains largely unexplored. Using in vitro experiments, we show that these active cellular nematics operate in a regime in which activity is effectively damped by friction, and that the interaction between defects is controlled by the system’s elastic nematic energy. Due to the activity of the cells, these defects behave as self-propelled particles and pairwise annihilate until all displacements freeze as cell crowding increases6, 7. When confined in mesoscopic circular domains, the system evolves towards two identical +1/2 disclinations facing each other. The most likely reduced positions of these defects are independent of the size of the disk, the cells’ activity or even the cell type, but are well described by equilibrium liquid crystal theory. These cell-based systems thus operate in a regime more stable than other active nematics, which may be necessary for their biological function.
Active cargo positioning in antiparallel transport networks.
Mathieu Richard, Carles Blanch-Mercader, Hajer Ennomani, Wenxiang Cao, Enrique M De La Cruz, Jean-François Joanny, Frank Jülicher, Laurent Blanchoin, Pascal Martin
Green Processing and Synthesis - - DOI : 10.1073/pnas.1900416116 - 2019
Cytoskeletal filaments assemble into dense parallel, antiparallel, or disordered networks, providing a complex environment for active cargo transport and positioning by molecular motors. The interplay between the network architecture and intrinsic motor properties clearly affects transport properties but remains poorly understood. Here, by using surface micropatterns of actin polymerization, we investigate stochastic transport properties of colloidal beads in antiparallel networks of overlapping actin filaments. We found that 200-nm beads coated with myosin Va motors displayed directed movements toward positions where the net polarity of the actin network vanished, accumulating there. The bead distribution was dictated by the spatial profiles of local bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Remarkably, beads coated with heavy-mero-myosin II motors showed a similar behavior. However, although velocity gradients were steeper with myosin II, the much larger bead diffusion observed with this motor resulted in less precise positioning. Our observations are well described by a 3-state model, in which active beads locally sense the net polarity of the network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk. The precision of bead positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. Our results unveiled physical rules for cargo transport and positioning in networks of mixed polarity.
Stiffness and tension gradients of the hair cell’s tip-link complex in the mammalian cochlea
Mélanie Tobin, Atitheb Chaiyasitdhi, Vincent Michel, Nicolas Michalski, Pascal Martin
Nature Communications - - doi: 10.7554/eLife.43473 - 2019
Frequency analysis of sound by the cochlea relies on sharp frequency tuning of mechanosensory hair cells along a tonotopic axis. To clarify the underlying biophysical mechanism, we have investigated the micromechanical properties of the hair cell’s mechanoreceptive hair bundle in the rat cochlea. We studied both inner and outer hair cells, which send nervous signals to the brain and amplify cochlear vibrations, respectively. We find that tonotopy is associated with gradients of stiffness and resting mechanical tension, with steeper gradients for outer hair cells, emphasizing the division of labor between the two hair-cell types. We demonstrate that tension in the tip links that convey force to the mechano-electrical transduction channels increases at reduced Ca2+. Finally, we reveal tonotopic gradients in stiffness and tension at the level of a single tip link. We conclude that mechanical gradients of the tip-link complex may help specify the characteristic frequency of the hair cell.

SIGNIFICANCE STATEMENT The tip-link complex of the hair cell is mechanically tuned along the tonotopic axis of the cochlea.

An intrinsically disordered region in OSBP acts as an entropic barrier to control protein dynamics and orientation at membrane contact sites
Jamecna D, Polidori DJ, Mesmin B, Dezi M, Lévy D, Bigay J, Antonny B
Dev Cell - - DOI : 10.1016/j.devcel.2019.02.021 - 2019
Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g. ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈ 90 aa intrinsically unfolded sequence at the N-terminus of oxysterol binding protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N-terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein orientation, limits protein density and facilitates protein mobility in the narrow and crowded MCS environment.

Actin dynamics drive cell-like membrane deformation
Simon C*, Kusters R*, Caorsi V*, Allard A, Abou-Ghali M, Manzi J, Di Cicco A, Lévy D, Lenz M, Joanny J-F, Campillo C, Plastino J, Sens P, Sykes C
Nature Physics - 15 602–609 - DOI : 10.1038/s41567-019-0464-1 - 2019
Cell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that the cell uses respectively to uptake external substances or probe the environment. The assembly and dynamics of the actin cytoskeleton are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient to initiate both inward and outward membrane deformation. The polymerization of a dense actin network at the membrane of liposomes produces inward membrane bending at low tension, while outward deformations are robustly generated regardless of tension. Our results shed light on the mechanism cells use to internalize material, both in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where those forces are necessary to overcome the opposing turgor pressure. By combining experimental observations with physical modelling, we propose a mechanism that explains how membrane tension and the architecture of the actin network regulate cell-like membrane deformations.
HLH-2/E2A Expression Links Stochastic and Deterministic Elements of a Cell Fate Decision during C. elegans Gonadogenesis
Attner MA, Keil W, Benavidez JM, Greenwald I.
Curr Biol. - - DOI: 10.1016/j.cub.2019.07.062 - 2019
Stochastic mechanisms diversify cell fate in organisms ranging from bacteria to humans [1-4]. In the anchor cell/ventral uterine precursor cell (AC/VU) fate decision during C. elegans gonadogenesis, two "α cells," each with equal potential to be an AC or a VU, interact via LIN-12/Notch and its ligand LAG-2/DSL [5, 6]. This LIN-12/Notch-mediated interaction engages feedback mechanisms that amplify a stochastic initial difference between the two α cells, ensuring that the cell with higher lin-12 activity becomes the VU while the other becomes the AC [7-9]. The initial difference between the α cells was originally envisaged as a random imbalance from "noise" in lin-12 expression/activity [6]. However, subsequent evidence that the relative birth order of the α cells biases their fates suggested other factors may be operating [7]. Here, we investigate the nature of the initial difference using high-throughput lineage analysis [10]; GFP-tagged endogenous LIN-12, LAG-2, and HLH-2, a conserved transcription factor that orchestrates AC/VU development [7, 11]; and tissue-specific hlh-2 null alleles. We identify two stochastic elements: relative birth order, which largely originates at the beginning of the somatic gonad lineage three generations earlier, and onset of HLH-2 expression, such that the α cell whose parent expressed HLH-2 first is biased toward the VU fate. We find that these elements are interrelated, because initiation of HLH-2 expression is linked to the birth of the parent cell. Finally, we provide a potential deterministic mechanism for the HLH-2 expression bias by showing that hlh-2 is required for LIN-12 expression in the α cells.
A tuneable microfluidic system for long duration chemotaxis experiments in a 3D collagen matrix
Aizel K, Clark AG, Simon A, Geraldo S, Funfak A, Vargas P, Bibette J, Vignjevic DM, Bremond N.
Lab. Chip - 7;17(22): 3851-3861 - DOI: 10.1039/c7lc00649g - 2019
In many cell types, migration can be oriented towards a chemical stimulus. In mammals, for example, embryonic cells migrate to follow developmental cues, immune cells migrate toward sites of inflammation, and cancer cells migrate away from the primary tumour and toward blood vessels during metastasis. Understanding how cells migrate in 3D environments in response to chemical cues is thus crucial to understanding directed migration in normal and disease states. To date, chemotaxis in mammalian cells has been primarily studied using 2D migration models. However, it is becoming increasingly clear that the mechanisms by which cells migrate in 2D and 3D environments dramatically differ, and cells in their native environments are confronted with a complex chemical milieu. To address these issues, we developed a microfluidic device to monitor the behaviour of cells embedded in a 3D collagen matrix in the presence of complex concentration fields of chemoattractants. This tuneable microsystem enables the generation of (1) homogeneous, stationary gradients set by a purely diffusive mechanism, or (2) spatially evolving, stationary gradients, set by a convection-diffusion mechanism. The device allows for stable gradients over several days and is large enough to study the behaviour of large cell aggregates. We observe that primary mature dendritic cells respond uniformly to homogeneous diffusion gradients, while cell behaviour is highly position-dependent in spatially variable convection-diffusion gradients. In addition, we demonstrate a directed response of cancer cells migrating away from tumour-like aggregates in the presence of soluble chemokine gradients. Together, this microfluidic device is a powerful system to observe the response of different cells and aggregates to tuneable chemical gradients.

414 publications.