Membrane Repair Assay for Human Skeletal Muscle Cells

Carmeille, Romain; Croissant, Coralie; Bouvet, Flora; Bouter, Anthony

doi:10.1007/978-1-4939-7283-8_14

Cited by 12 publications

(15 citation statements)

References 23 publications

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“…Post-injury images were taken every 5 sec for a total of 150 sec. Specific settings used as described in Carmeille et al [27]. At least three different myotubes were selected to be injured in each dish.…”

Section: Laser Injury Assaymentioning

confidence: 99%

Treatment with galectin-1 improves myogenic potential and membrane repair in dysferlin-deficient models

et al. 2020

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Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity and myogenic potential of dysferlin-deficient muscle cells and muscle fibers. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, myoblast migration, and membrane repair capacity in dysferlin-deficient myotubes and myofibers. Furthermore, increased membrane repair capacity of dysferlin-deficient myotubes, independent of increased myogenic maturation is apparent and co-localizes on the membrane of myotubes after a brief 10min treatment with labeled rHsGal-1. We show the carbohydrate recognition domain of Gal-1 is necessary for observed membrane repair. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggest mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-1 shows calcium-independent membrane repair in dysferlin-deficient and wild-type myotubes and myofibers. Together our novel results reveal Gal-1 mediates disease pathologies through both changes in integral myogenic protein expression and mechanical membrane stabilization.

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Section: Laser Injury Assaymentioning

confidence: 99%

Treatment with galectin-1 improves myogenic potential and membrane repair in dysferlin-deficient models

et al. 2020

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“…In order to investigate the membrane repair ability of MDA-MB-231 cells, plasma membrane was damaged by laser ablation and membrane repair was assayed by monitoring the kinetics of cell entry of membrane-impermeable FM1-43 dye, using a standard protocol (Carmeille et al, 2017;Bouter et al, 2011). Membrane resealing leads to a stop in the entry of FM1-43 molecules into the cytosol and therefore to a stop in the increase of the intracellular fluorescence intensity, whereas the absence of membrane resealing leads to a continuous entry of FM1-43 into the cytosol and a continuous and strong increase in the fluorescence intensity.…”

Section: Mda-mb-231 Cells Exhibit a Highly Efficient Membrane Repair mentioning

confidence: 99%

“…512 x 512 images were acquired at 1.3 s intervals with pinhole set to 1 Airy unit. Membrane rupture and repair processes were monitored by measuring variations in fluorescence intensity of FM1-43 as previously described (Carmeille et al, 2017). FM1-43 was excited by the 488-nm laser line (intensity set at 30% of maximal power) and fluorescence emission was measured between 520 nm and 650 nm.…”

Section: Membrane Rupture and Repair Assaymentioning

confidence: 99%

Defective membrane repair machinery impairs survival of invasive cancer cells

Bouvet

Ros

Bonedeau

et al. 2020

Preprint

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Cancer cells are able to reach distant tissues by migration and invasion processes. This study shows that inhibition of the plasma membrane repair machinery may represent a promising avenue for annihilating cancer metastasis. AbstractCancer cells are able to reach distant tissues by migration and invasion processes. Enhanced ability to cope with physical stresses leading to cell membrane damages may offer to cancer cells high survival rate during metastasis. Consequently, down-regulation of the membrane repair machinery may be a therapeutic avenue for inhibiting metastasis. We show that migration of MDA-MB-231 cells on collagen I fibrils induces disruptions of plasma membrane and pullout of membrane fragments in the wake of cells. These cells are able to reseal membrane damages thanks to annexins (Anx) that are highly expressed in invasive cancer cells. In vitro membrane repair assays reveal that MDA-MB-231 cells respond heterogeneously to membrane injury and some of them possess very efficient repair machinery. Finally, we show that silencing of AnxA5 and AnxA6 leads to major defect of the membrane repair machinery responsible for the death of migrating MDA-MB-231 cells. Inhibition of membrane repair machinery may therefore represent a promising avenue for annihilating cancer metastasis.

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“…This method was successfully applied to demonstrate that Annexin-A5 (AnxA5) binds to the edges of the torn membrane and is present on the surface of lipid vesicles accumulating at the site of membrane injury in human skeletal muscle cells (Carmeille et al, 2016). Laser irradiation induces membrane injury of varying sizes in a highly controlled and reproducible way, depending mainly on the laser power (Bouter et al, 2011;Carmeille et al, 2017). The maximum size of the membrane rupture that can be repaired is likely to depend on the cell type.…”

Section: Introductionmentioning

confidence: 99%

“…Irradiation consists of three successive scans (1.6 sec per scan) of the ROI with a power of 110 mW ( Fig. 3B-D, see also Video 1), as previously described (Carmeille et al, 2017).…”

mentioning

confidence: 99%

Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy

et al. 2018

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Many cells possess the ability to repair plasma membrane disruption in physiological conditions. Growing evidence indicates a correlation between membrane repair and many human diseases. For example, a negative correlation is observed in muscle where failure to reseal sarcolemma may contribute to the development of muscular dystrophies. Instead, a positive correlation is observed in cancer cells where membrane repair may be exacerbated during metastasis. Here we describe a protocol that combines laser technology for membrane damage, immunostaining with gold nanoparticles and imaging by fluorescence microscopy and transmission electron microscopy (TEM), which allows the characterization of the molecular machinery involved in membrane repair. Fluorescence microscopy enables to determine the subcellular localization of candidate proteins in damaged cells while TEM offers high-resolution ultrastructural analysis of the µm²-disruption site, which enables to decipher the membrane repair mechanism. Here we focus on the study of human skeletal muscle cells, for obvious clinical interest, but this protocol is also suitable for other cell types. © 2018 by John Wiley & Sons, Inc.

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Membrane Repair Assay for Human Skeletal Muscle Cells

Cited by 12 publications

References 23 publications

Treatment with galectin-1 improves myogenic potential and membrane repair in dysferlin-deficient models

Treatment with galectin-1 improves myogenic potential and membrane repair in dysferlin-deficient models

Defective membrane repair machinery impairs survival of invasive cancer cells

Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy

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