Background: Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context.Method: To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9.Results: We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (β1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (β1-6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression.Conclusion: MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer.

Aim: With the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following spinal cord injury (SCI) we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature, and the nanobiomechanical behavior of healthy and injured spinal cord tissue we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h, 1week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks’post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 1 week and 6 weeks post injury.

Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide yielding major handicaps and inducing high economical costs. A scar, called glial scar and composed of two main cellular populations i.e. astrocytes and microglia, inhibits axonal regeneration by forming a physical and chemical barrier. Currently, there is no curative treatment on any symptoms associated with SCI. In this context, with the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following SCI, we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature and the nanobiomechanical behavior of healthy and injured spinal cord tissue, we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different key post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h,1 week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 2 weeks and 6 weeks post injury.

Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide (40 000 in France). Handicaps induced by SCI range from minimal sensory motor deficits to complete tetraplegia. Currently, there is no curative treatment on any symptoms associated with SCI. A scar, called glial and composed mainly of astrocytes and microglia, appears at the the injury site and inhibits axonal regeneration. This mechanism is poorly understood. Better monitoring of animal models of SCI in particular through the development of bioimaging translational tools are therefore mandatory.Our main objectives are to pursue and extend a multimodal imaging approach combining multiphoton spectroscopy (MPM), Atomic Force Microscopy (AFM) and Broadband coherent anti-Stokes Raman Spectroscopy (BCARS), a novel technique [1] based on Raman spectroscopy with faster image acquisitions [2], to better understand mechanisms that underlie the glial scar formation and the subsequent absence of spontaneous axonal regeneration following SCI, as well as to evaluate outcomes of therapeutic strategies that favor axonal regeneration in mice and non-human primates. MPM, conceived to specifically reveal non centro-symmetric structures with second harmonic generation (SHG) [3], provided a first evidence of the presence of collagen fibers as a biomarker of the injury site. AFM showed significant differences in Young Modulus between healthy and injured areas. These results must be supplemented with a deeper collagen characterization and axons imaging using MPM, as well as a treatment monitoring by co-localizing drugs within healthy/diseased tissues using BCARS [4].[1]. C. H. Camp Jr, Y. J. Lee, J. M. Heddleston, C. M. Hartshorn, A. R. Hight Walker, J. N. Rich, J. D. Lathia, M. T. Cicerone, Nat. Photonics 2014, 8, 627.[2]. C. M. Hartshorn, Y. J. Lee, C. H. Camp Jr, Z. Liu, J. M. Heddleston, N. Canfield, T. A. Rhodes, A. R. Hight Walker, P. J. Marsac, M. T. Cicerone, Anal. Chem. 2013, 85, 8102[3]. L. Mostaço-Guidolin, N.L. Rosin, T.L. Hackett, Int. J. Mol. 2017, 18(8), 1772. [4]. C. Zhang, D. Zhang, J.-X. Cheng, Annu. Rev. Biomed. Eng. 2015, 17, 415. [4]. C. Zhang, D. Zhang, J.-X. Cheng, Annu. Rev. Biomed. Eng. 2015, 17, 415.

Aim: Our main objective is to implement a multimodal (bio)physical approach to decipher mechanisms that underlie absence of spontaneous axonal regeneration following spinal cord injury (SCI) in mice and nonhuman primates. Method: A multimodal imaging strategy is employed combining state-of-the-art non-linear microscopy techniques (multiphoton - MPM and BCARS microscopy) with force spectroscopy measurements via atomic force microscopy (AFM). This approach enables to assess the relevant structural, morphological and spectral signature as well as the nanobiomechanical behavior of healthy and injured spinal cord tissue at cellular level. Results: We have first imaged excised mice SC tissues in 2-photon excited fluorescence and second harmonic generation (SHG). These MPM studies revealed the apparition of a strong SHG signal due to the formation of collagen I fibers by the injury site in the glial scar. AFM based force spectroscopy measurements have been also performed to map the elastic properties of the healthy and injured spinal cord tissue. Therefore, MPM data could be correlated with Young modulus elasticity results, indicating the presence of a fibrotic process 7 days after injury. As tissue stiffness is a regulator of neuronal growth, such kind of measurements will enable to understand why adult mammalian axons do not regenerate spontaneously after injury. Our next step will be to use a novel, emerging imaging technology that is the broadband coherent anti-Stokes Raman scattering (BCARS) microscopy for fast, label-free, highly selective and specific screening of spinal cord injury and potentially of drugs uptake into excised tissues. Combination of BCARS and MPM will first enable to retrieve information on the biological state of the cells/tissues that may underly the absence of spontaneous axonal regeneration following SCI (Raman neuronal markers: myelin, acetylcholine neurotransmitter, glutamate; MPM markers: collagen, neural axons, mitochondria,…). Second, it will permit to further correlate modification of these structural, morphological and spectral signatures in the context of therapeutic strategies.

Munc13-1 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13-1 form nanoassemblies in active zones of neurons. However, it is not known if such clustering of Munc13-1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13-1 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13-1, and is indeed required for vesicle binding to bilayers containing Munc13-1. Purified Munc13-1 protein reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ~20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters containing a minimum of 6 copies of Munc13-1 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C 2 C domain of Munc13-1 is not required for Munc13-1 clustering, but is required for efficient vesicle capture. This capture is largely due to a combination of electrostatic and hydrophobic interactions between the C 2 C domain and the vesicle membrane.

Motivation Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process. Results For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization. Implementation To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes. Supplementary information Supplementary data are available at Bioinformatics online.